Composition for inducing proliferation or accumulation of regulatory t cells

[Technical Field

0001Described herein is a composition which has an effect of inducing proliferation or accumulation of regulatory T cells, and which comprises, as an active ingredient, bacteria belonging to the genus Clostridium, a physiologically active substance derived from the bacteria, bacterial spores, or the like.

0002Also described is a method for inducing proliferation or accumulation of regulatory T cells, as well as a method for inhibiting such proliferation or accumulation. Also described is a vaccine composition containing at least one strain of bacteria belonging to the genus Clostridium or a spore of bacteria, as well as a method for treating or preventing at least one disease or condition selected from infectious diseases and autoimmune diseases by administering the vaccine composition to an individual in need thereof.

0003Also described is a method for screening for a compound that promotes proliferation or accumulation of regulatory T cells, as well as a non-human mammal which is used in this method, and in which a reporter gene is expressed under control of IL-10 gene expression.

[Background Art]

0004Hundreds of species of commensal microorganisms are harbored in gastrointestinal tracts of mammals, and intimately interact with the host immune systems. Results of researches using germ-free (GF) animals have shown that the commensal microorganisms exert great influences on the development of mucosal immune systems such as histogenesis of Peyer's patches (PPs) and isolated lymphoid follicles (ILFs), secretion of antimicrobial peptides from epithelium, and accumulation of unique lymphocytes in mucosal tissues, the unique lymphocytes including immunoglobulin A-producing plasma cells, intraepithelial lymphocytes, IL-17-producing CD4-positive T cells (Th 17), and IL-22-producingNK-like cells (Non-Patent Documents 1 to 7). Consequently, the presence of intestinal bacteria enhances protective functions of the mucous membranes, providing the hosts with robust immune responses against pathogenic microbes invading the bodies . On the other hand, the mucosal immune systems maintain unresponsiveness to dietary antigens and harmless microbes (Non-Patent Document 3). For this reason, abnormality in the regulation of cross-talk between commensal bacteria and an immune system (intestinal dysbiosis) may lead to overly robust immune response to environmental antigens, so that inflammatory bowel disease (IBD) is caused (Non-Patent Documents 8 to 10) .

0005Results of Recent studies have shown that individual commensal bacteria control differentiation of their specific immune cells in the mucosal immune system. For example, Bacteroides fragilis, which is a commensal bacterium in humans, specifically induces a systemic Th1 cell response and a mucosal IL-10-producing T cell response in mice, and plays a role in protecting the host from colitis, which would otherwise be caused by a pathogen (Non-Patent Document 3). Segmented filamentous bacteria, which are intestinal commensal bacteria in mice, are shown to induce mucosal Th17 cell response and thereby to enhance resistance against infection of gastrointestinal tracts of the host with a pathogen (Non-Patent Documents 11 to 13). In addition, short-chain fatty acids derived from several commensal bacteria are known to suppress intestinal inflammation (Non-Patent Document 14). Moreover, it is presumed that the presence of some species of intestinal microbiota exerts a great influence on the differentiation of regulatory T cells (hereafter referred to as "Treg cells") which maintain homeostasis of the immune system.

0006Meanwhile, regulatory T cells which have been identified as a subset suppressing immunity are CD4⁺ T cells in which a transcription factor Foxp3 is expressed, and are known to play an important role in maintaining immunological homeostasis (Non-Patent Documents 8, 9, 15, and 16). Moreover, it has been known that the Foxp3-expressing cells are present in a large number especially in the colon, and only Treg cells present locally in the colon constantly expresses IL-10, which is an immunosuppressive cytokine, at a high level (Non-Patent Document 17) . It is also known that animals having CD4⁺ Foxp3⁺ cells from which IL-10 is specifically removed develop inflammatory bowel disease (Non-Patent Document 18) .

0007Accordingly, if the mechanism of the induction of Treg cells which produce IL-10 in the colon at a high level is elucidated, immunosuppression can be enhanced, which in turn can be applied to treatment of autoimmune diseases such as inflammatory bowel disease, as well as to organ transplantation.

0008However, mechanisms of how a large number of Treg cells come to be present in the colon and how the Treg cells produce IL-10 in the colon at a high level are still unclear. Moreover, it is also still unclear what species of bacteria constituting the intestinal commensal bacterial flora exerts the influence on the induction of regulatory T cells.

[Citation List]

[Non Patent Literature]

0009<ul id="ul0001" list-style="none" compact="compact"><li>[NPL 1]<nplcit id="ncit0001" npl-type="s"><text>J. J. Cebra, "Am J Clin Nutr", May, 1999, 69, 1046S</text></nplcit></li><li>[NPL 2]<nplcit id="ncit0002" npl-type="s"><text>A. J. Macpherson, N. L. Harris, "Nat Rev Immunol", June 2004, 4, 478</text></nplcit></li><li>[NPL 3]<nplcit id="ncit0003" npl-type="s"><text>J. L. Round, S. K. Mazmanian, "Nat Rev Immunol", May 2009, 9, 313</text></nplcit></li><li>[NPL 4]<nplcit id="ncit0004" npl-type="s"><text>D. Bouskra et al., "Nature", November 27, 2008, 456, 507</text></nplcit></li><li>[NPL 5]<nplcit id="ncit0005" npl-type="s"><text>K. Atarashi et al., "Nature", October 9, 2008, 455, 808</text></nplcit></li><li>[NPL 6]<nplcit id="ncit0006" npl-type="s"><text>Ivanov, II et al., "Cell Host Microbe", October 16, 2008, 4, 337</text></nplcit></li><li>[NPL 7]<nplcit id="ncit0007" npl-type="s"><text>S. L. Sanos et al., "Nat Immunol", January 2009, 10, 83</text></nplcit></li><li>[NPL 8]<nplcit id="ncit0008" npl-type="s"><text>M. A. Curotto de Lafaille, J. J. Lafaille, "Immunity", May 2009, 30, 626</text></nplcit></li><li>[NPL 9]<nplcit id="ncit0009" npl-type="s"><text>M. J. Barnes, F. Powrie, "Immunity", September 18, 2009, 31, 401</text></nplcit></li><li>[NPL 10]<nplcit id="ncit0010" npl-type="s"><text>W. S. Garrett et al., "Cell", October 5, 2007, 131, 33</text></nplcit></li><li>[NPL 11] <nplcit id="ncit0011" npl-type="s"><text>Ivanov, II et al., "Cell", October 30, 2009, 139, 485</text></nplcit>.</li><li>[NPL 12]<nplcit id="ncit0012" npl-type="s"><text>V. Gaboriau-Routhiau et al., "Immunity", October 16, 2009, 31, 677</text></nplcit></li><li>[NPL 13]<nplcit id="ncit0013" npl-type="s"><text>N. H. Salzman et al., "Nat Immunol", 11, 76</text></nplcit>.</li><li>[NPL 14]<nplcit id="ncit0014" npl-type="s"><text>K. M. Maslowski et al., "Nature", October 29, 2009, 461, 1282</text></nplcit></li><li>[NPL 15]<nplcit id="ncit0015" npl-type="s"><text>L. F. Lu, A. Rudensky, "Genes Dev", June 1, 2009, 23, 1270</text></nplcit></li><li>[NPL 16]<nplcit id="ncit0016" npl-type="s"><text>S. Sakaguchi, T. Yamaguchi, T. Nomura, M. Ono, "Cell", May 30, 2008, 133, 775</text></nplcit></li><li>[NPL 17]<nplcit id="ncit0017" npl-type="s"><text>C. L. Maynard et al., "Nat Immunol", September 2007, 8, 931</text></nplcit></li><li>[NPL 18]<nplcit id="ncit0018" npl-type="s"><text>Y. P. Rubtsov et al., "Immunity", April 2008, 28, 546</text></nplcit></li></ul>

0010<nplcit id="ncit0019" npl-type="s"><text>GABORIAU-ROUTHIAU et al. (2009), Immunity 31:677 </text></nplcit>describes Segmented-Filamentous Bacteria (SFBs) which do not belong to the genus Clostridium, and indeed are phylogenetically remote therefrom.

0011<nplcit id="ncit0020" npl-type="s"><text>SOKOL et al. (2008-10-28) "Faecalibacterium prausnitzii is an anti-inflammatory commensal bacterium identified by gut microbiota analysis of Crohn disease patients", Proc Natl Acad Sci USA 105 (43): 16731-16736</text></nplcit>, proposes Faecalibacterium prausnitzii (belonging to Clostridium cluster IV) as candidate probiotic agent in the treatment of Crohn's disease (CD).

0012<nplcit id="ncit0021" npl-type="s"><text>KARIMI et al. (2009), "Lactobacillus reuteri-induced regulatory T cells protect against and allergic airway response in mice" American Journal of Respiratory and Critical Care Medicine 17(3):186-193</text></nplcit>, describes that <i>Lactobacillus reuteri</i> induces Foxp3+ regulatory T cells that have a potential to protect against and allergic airway response in mice.

0013<patcit id="pcit0001" dnum="EP1955706A1"><text>EP 1 955 706 A1</text></patcit> relates to a composition containing interferon alpha and natural bile pigment C-Phycocyanin, for treating autoimmune diseases, allergy and cancer, by inducing regulatory T cells.

0014<nplcit id="ncit0022" npl-type="s"><text>ITOH K et al. "Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice." LABORATORY ANIMALS APR 1985 vol. 19, no. 2, April 1985 (1985-04), pages 111-118</text></nplcit>, discloses the characterization of 115 strains of clostridia accumulated from 3 separate isolations from the faeces of 1 limited flora (LF) mouse produced by inoculation of germ-free mice with chloroform-treated faeces of conventional mice, and the effect on caecal size when associated with germ-free mice was studied.

0015<nplcit id="ncit0023" npl-type="s"><text>BAKKEN (2009) Anaerobe 15:285-289</text></nplcit> relates to Faecal bacteriotherapy as a treatment for recurrent <i>Clostridium difficile</i> infection.

[Summary of Invention]

0016The invention is defined in the claims, and provides a composition for use in a method of treating or preventing an allergic disease by inducing proliferation or accumulation of transcription factor Foxp3-positive regulatory T cells, the composition comprising, as an active ingredient, bacteria belonging to the genus Clostridium of clusters XIVa and/or IV, wherein the bacteria induce said proliferation or accumulation of transcription factor Foxp3-positive regulatory T cells. Other aspects of the invention are also defined in the claims.

0017The present inventors have found that a chloroform-treated fraction and a spore-forming fraction of a fecal sample obtained from a mammal induces accumulation of regulatory T cells (Treg cells) in the colon. Moreover, the present inventors have found that bacteria belonging to the genus Clostridium induce proliferation or accumulation of regulatory T cells in the colon. The present inventors have also found that the regulatory T cells induced by these bacteria suppress proliferation of effector T cells. Furthermore, the present inventors have also found that colonization of bacteria belonging to the genus Clostridium and resultant proliferation or accumulation of Treg cells regulate local and systemic immune responses.

0018From these findings, the present inventors have found that the use of bacteria belonging to the genus Clostridium, spores thereof, or a physiologically active substance derived therefrom makes it possible to induce the proliferation or accumulation of regulatory T cells (Treg cells), and further to suppress immune functions.

[Advantageous Effects]

0019The compositions described herein containing as an active ingredient bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria serves as an excellent composition for inducing the proliferation or accumulation of regulatory T cells (Treg cells). Immunity in a living organism can be suppressed in accordance with the claims through administration of the composition of the present invention as defined in the claims as a pharmaceutical product or ingestion of the composition as a food or beverage. Accordingly, the composition of the present invention can be used, for example, to prevent or treat allergic diseases

0020In addition, if a food or beverage such as a health food comprises the composition of the present invention, healthy individuals can ingest the composition easily and routinely. As a result, it is possible to induce the proliferation or accumulation of regulatory T cells and thereby to improve immune functions,as defined in the claims.

[Brief Description of Drawings]

0021<ul id="ul0002" list-style="none" compact="compact"><li>[<figref idref="f0001">Fig. 1] Fig. 1</figref> is a schematic diagram showing a method of producing <u>Il10</u>^venus mouse.</li><li>[<figref idref="f0002">Fig. 2] Fig. 2</figref> is a diagram showing results of Southern blotting performed for analysis as to whether or not the <u>Il10</u>^venus mice have an <u>Il10</u>^venus allele.</li><li>[<figref idref="f0002">Fig. 3] Fig. 3</figref> is a FACS dot-plot diagram showing results obtained when Venus-positive cells and Venus-negative cells from the <u>Il10</u>^venus mice were sorted.</li><li>[<figref idref="f0003">Fig. 4] Fig. 4</figref> is a graph showing the results obtained when the amounts of IL-10 mRNA expressed in Venus positive-cells and Venus-negative cells of the <u>Il10</u>^venus mice were analyzed by real-time RT-PCR.</li><li>[<figref idref="f0003">Fig. 5] Fig. 5</figref> is a graph showing change in the ratio of Foxp3⁺ cells in CD4⁺ lymphocytes of SPF mice.</li><li>[<figref idref="f0004">Fig. 6] Fig. 6</figref> shows FACS dot-plot diagrams showing analysis results of the ratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the small intestine, the colon, and the peripheral lymph nodes of GF mice and SPF mice.</li><li>[<figref idref="f0004">Fig. 7] Fig. 7</figref> is a graph showing analysis results of the ratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the small intestine, the colon, and the peripheral lymph nodes of GF mice and SPF mice.</li><li>[<figref idref="f0005">Fig. 8] Fig. 8</figref> shows graphs showing analysis results of the numbers of CD4⁺ Foxp3⁺ cells isolated from the small intestine, the colon, and the peripheral lymph nodes of GF mice and SPF mice.</li><li>[<figref idref="f0005">Fig. 9] Fig. 9</figref> is a plot diagram showing analysis results of the ratios of Venus⁺ cells in CD4⁺ cells in various tissues of SPF mice treated with antibiotics.</li><li>[<figref idref="f0006">Fig. 10] Fig. 10</figref> shows FACS dot-plot diagrams showing analysis results of the ratio of Foxp3⁺ cell in CD4⁺ lymphocytes isolated from the colonic lamina propria of GF mice to which a fecal suspension of SPF mice was administered.</li><li>[<figref idref="f0006">Fig. 11] Fig. 11</figref> is a graph showing analysis results of the ratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the lamina propria of the colon and the lamina propria of the small intestine of GF mice to which a fecal suspension of SPF mice was administered.</li><li>[<figref idref="f0007">Fig. 12] Fig. 12</figref> is a graph showing analysis results of the ratio of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the lamina propria of mice deficient in ILFs, PPs, and colonic-patches.</li><li>[<figref idref="f0007">Fig. 13] Fig. 13</figref> shows FACS dot-plot diagrams showing analysis results of the ratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the colonic lamina propria of GF mice to which specific commensal bacteria were administered.</li><li>[<figref idref="f0008">Fig. 14] Fig. 14</figref> shows graphs showing analysis results of the ratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the colonic lamina propria of GF mice to which specific commensal bacteria were administered.</li><li>[<figref idref="f0008">Fig. 15] Fig. 15</figref> is a graph showing analysis results of the ratios of IFN-γ⁺ cells in CD4⁺ lymphocytes isolated from the colonic lamina propria of mice in which specific commensal bacteria were colonized.</li><li>[<figref idref="f0009">Fig. 16] Fig. 16</figref> is a graph showing analysis results of the ratios of IL-17⁺ cells in CD4⁺ lymphocytes isolated from the colonic lamina propria of mice in which specific commensal bacteria were colonized.</li><li>[<figref idref="f0009">Fig. 17] Fig. 17</figref> is a graph showing analysis results of the ratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the colon of kinds of SPF mice each being deficient in a pathogen-associated molecular pattern recognition receptor-associated factor.</li><li>[<figref idref="f0010">Fig. 18] Fig. 18</figref> is a graph showing analysis results of the ratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the colonic lamina propria of Myd88^-/-mice in which the Clostridium was colonized.</li><li>[<figref idref="f0011">Fig. 19] Fig. 19</figref> shows FACS dot-plot diagrams showing analysis results of the ratios of Venus⁺ cells in lymphocytes isolated from various tissues of <u>Il10</u>^venus mice.</li><li>[<figref idref="f0012">Fig. 20] Fig. 20</figref> is a FACS dot-plot diagram showing analysis results of the expression of a T cell receptor β chain on cell surfaces of lymphocytes isolated from the colonic lamina propria of <u>Il10</u>^venus mice.</li><li>[<figref idref="f0012">Fig. 21] Fig. 21</figref> shows FACS dot-plot diagrams showing analysis results of the expression of IL-17, IL-4, and IFN-γ in lymphocytes isolated from the colonic lamina propria of <u>Il10</u>^venus mice.</li><li>[<figref idref="f0013">Fig. 22] Fig. 22</figref> shows graphs showing analysis results of the amounts of mRNAs of IL-10, CTLA4, Foxp3, and GITR expressed in spleen Foxp3^-CD4⁺ cells, spleen Foxp3⁺ CD4⁺ cells, colonic lamina propria Venus⁺ cells, and small intestinal lamina propria Venus⁺ cells.</li><li>[<figref idref="f0013">Fig. 23] Fig. 23</figref> shows FACS dot-plot diagrams showing analysis results of the expression of CD4, Foxp3, and Venus in the lamina propria of the small intestine and the lamina propria of the colon of GF <u>Il10</u>^venus mice and SPF <u>Il10</u>^venus mice.</li><li>[<figref idref="f0014">Fig. 24] Fig. 24</figref> shows FACS dot-plot diagrams showing analysis results of the expression of Venus and Foxp3 of CD4 cells in various tissues of SPF <u>Il10</u>^venus mice.</li><li>[<figref idref="f0015">Fig. 25] Fig. 25</figref> shows FACS dot-plot diagrams showing analysis results of the expression of Foxp3 and Venus in <u>Il10</u>^venus mice in which specific commensal bacteria were colonized.</li><li>[<figref idref="f0015">Fig. 26] Fig. 26</figref> is a graph showing analysis results of the expression of Foxp3 and/or Venus of CD4⁺ cells in the small intestine of <u>Il10</u>^venus mice in which specific commensal bacteria were colonized.</li><li>[<figref idref="f0016">Fig. 27] Fig. 27</figref> is a graph showing analysis results of the expression of Foxp3 and/or Venus of CD4⁺ cells in the colon of <u>Il10</u>^venus mice in which specific commensal bacteria were colonized.</li><li>[<figref idref="f0016">Fig. 28] Fig. 28</figref> is a plot diagram showing analysis results of the ratios of Venus⁺ cells in CD4⁺ cells isolated from various tissues of <u>Il10</u>^venus mice treated with antibiotics.</li><li>[<figref idref="f0017">Fig. 29] Fig. 29</figref> is a graph showing analysis results of immunoregulatory functions of CD4⁺ Venus⁺ cells from the colonic lamina propria of GF <u>Il10</u>^venus mice in which the genus Clostridium was colonized, CD4⁺ Venus⁺ cells from the colonic lamina propria of SPF <u>Il10</u>^venus mice, and CD4⁺ GFP⁺ cells from the spleen of Foxp3^eGFP reporter mice.</li><li>[<figref idref="f0018">Fig. 30] Fig. 30</figref> is a graph showing the results obtained when SPF B6 mice were treated with polymyxin B or vancomycin for 4 weeks, and then analyzed for the ratio of Foxp3⁺ cells in the CD4⁺ cell group.</li><li>[<figref idref="f0018">Fig. 31] Fig. 31</figref> is a graph showing the results obtained when SPF mice-derived chloroform-treated feces were orally administered to GF mice, and then the ratio of Foxp3⁺ cells in the CD4⁺ cell group was analyzed.</li><li>[<figref idref="f0019">Fig. 32] Fig. 32</figref> is a graph showing the general results of flow cytometry analysis on Helios expression in LP lymphocytes in the thymuses or the colons of SPF mice, GF mice, Lactobacillus-colonized mice, or Clostridium-colonized mice.</li><li>[<figref idref="f0020">Fig. 33] Fig. 33</figref> shows plot diagrams showing representative results of flow cytometry analysis on CD4 expression, Foxp3 expression, and Helios expression in the LP lymphocytes in the thymuses or the colons of the SPF mice, the GF mice, the Lactobacillus-colonized mice, or the Clostridium-colonized mice.</li><li>[<figref idref="f0021">Fig. 34] Fig. 34</figref> is a graph showing the results obtained when the whole colons derived from GF mice, Lactobacillus-colonized mice, or Clostridium-colonized mice were cultured, and the culture supernatants thereof were analyzed for the TGF-β1 concentration by ELISA.</li><li>[<figref idref="f0021">Fig. 35] Fig. 35</figref> is a graph showing the results obtained when intestinal epithelial cells (IECs) derived from GF mice or Clostridium-colonized mice were cultured, and the culture supernatants thereof were analyzed for the TGF-β1 concentration by ELISA.</li><li>[<figref idref="f0022">Fig. 36] Fig. 36</figref> is a graph showing the results obtained when splenic CD4⁺ T cells were cultured together with an anti-CD3 antibody and with a culture supernatant of IECs isolated from GF mice or mice colonized with 46 bacterial strains of the genus Clostridium (Clost.) in the presence or absence of an anti-TGF-β antibody, and the T cells were collected on day 5 of the culture and analyzed for Foxp3 expression by real-time RT-PCR.</li><li>[<figref idref="f0022">Fig. 37] Fig. 37</figref> is a graph showing the results obtained when C57BL/6 GF mice were orally inoculated with 46 bacterial strains of the genus Clostridium (Clost.) or three bacterial strains of the genus Lactobacillus (Lacto.), and IECs were collected three weeks after the inoculation and analyzed for the relative mRNA expression level of the MMP2 gene by real-time RT-PCR.</li><li>[<figref idref="f0023">Fig. 38] Fig. 38</figref> is a graph showing the results obtained when C57BL/6 GF mice were orally inoculated with 46 bacterial strains of the genus Clostridium (Clost.) or three bacterial strains of the genus Lactobacillus (Lacto.), and IECs were collected three weeks after the inoculation and analyzed for the relative mRNA expression level of the MMP9 gene by real-time RT-PCR.</li><li>[<figref idref="f0023">Fig. 39] Fig. 39</figref> is a graph showing the results obtained when C57BL/6 GF mice were orally inoculated with 46 bacterial strains of the genus Clostridium (Clost.) or three bacterial strains of the genus Lactobacillus (Lacto.), and IECs were collected three weeks after the inoculation and analyzed for the relative mRNA expression level of the MMP13 gene by real-time RT-PCR.</li><li>[<figref idref="f0024">Fig. 40] Fig. 40</figref> is a graph showing the results obtained when C57BL/6 GF mice were orally inoculated with 46 bacterial strains of the genus Clostridium (Clost.) or three bacterial strains of the genus Lactobacillus (Lacto.), and IECs were collected three weeks after the inoculation and analyzed for the relative mRNA expression level of the IDO gene by real-time RT-PCR.</li><li>[<figref idref="f0024">Fig. 41] Fig. 41</figref> is a graph showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF+Clost.) were treated with 2% DSS, observed and measured for the body weight loss, the hardness of stool, and bleeding for six days, and then evaluated numerically.</li><li>[<figref idref="f0025">Fig. 42] Fig. 42</figref> is a photograph showing the state of the colons collected on day 6 after the control mice (SPF) and the Clostridium-administered mice (SPF+Clost.) were treated with 2% DSS.</li><li>[<figref idref="f0025">Fig. 43] Fig. 43</figref> shows photomicrographs showing the results obtained when the control mice (SPF) and the Clostridium-administered mice (SPF+Clost.) were treated with 2% DSS, and the colons thereof were collected on day 6 and analyzed histologically by HE staining.</li><li>[<figref idref="f0026">Fig. 44] Fig. 44</figref> is a graph showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF+Clost.) were sensitized with oxazolone, and subsequently the inside of each rectum was treated with a 1% oxazolone/50% ethanol solution, and the body weight loss was measured.</li><li>[<figref idref="f0026">Fig. 45] Fig. 45</figref> shows photomicrographs showing the results obtained when the control mice (SPF) and the Clostridium-administered mice (SPF+Clost.) were sensitized with oxazolone, and subsequently the inside of each rectum was treated with a 1% oxazolone/50% ethanol solution, and the colons obtained by the treatment were analyzed histologically by HE staining.</li><li>[<figref idref="f0027">Fig. 46] Fig. 46</figref> is a graph showing the results obtained when control mice (SPF) and Clostridium-administered mice (SPF+Clost.) were immunized by administering alum-absorbed ovalbumin (OVA) twice at a 2-week interval, and the sera were collected therefrom and analyzed for the concentration of OVA-specific IgE in these sera by ELISA.</li><li>[<figref idref="f0027">Fig. 47] Fig. 47</figref> is a graph showing the results obtained when the control mice (SPF) and the Clostridium-administered mice (SPF+Clost.) were immunized by administering the alum-absorbed OVA twice at a 2-week interval, and splenic cells were collected and analyzed for IL-4 production of these splenic cells by in-vitro OVA restimulation.</li><li>[<figref idref="f0028">Fig. 48] Fig. 48</figref> is a graph showing the results obtained when the control mice (SPF) and the Clostridium-administered mice (SPF+Clost.) were immunized by administering the alum-absorbed OVA twice at a 2-week interval, and the splenic cells were collected and analyzed for IL-10 production of these splenic cells by the in-vitro OVA restimulation.</li><li>[<figref idref="f0029">Fig. 49] Fig. 49</figref> is Phylogenetic tree constructed by the neighbor-joining method with the resulting sequences of the 41 strains of Clostridium and those of known bacteria obtained from Genbank database using Mega software.</li><li>[<figref idref="f0030">Fig. 50] Fig. 50</figref> is histograms showing Foxp3 expression gated CD4 cells from GF mice (Germ-free mouse #1 and #2) or GF mice colonized with three strains of Clostridium belonging to cluster IV (3 strains of Clost. mouse #1 and #2).</li><li>[<figref idref="f0031">Fig. 51] Fig. 51</figref> is histograms showing Foxp3 expression by CD4 positive lymphocytes from GF mice (GF) or GF mice gavaged with chloroform-treated human stool (GF+Chloro.) .</li><li>[<figref idref="f0031">Fig. 52] Fig. 52</figref> is a graph showing Foxp3 expression by CD4 positive lymphocytes from GF mice (GF) or GF mice gavaged with chloroform-treated human stool (GF+Chloro.) .</li><li>[<figref idref="f0032">Fig. 53] Fig. 53</figref> is a graph showing amounts of Clostridium and Bacteroides in feces of mice gavaged with chloroform-treated human stool</li></ul>

[Description]

<Composition Having Effect of Inducing Proliferation or Accumulation of Regulatory T cells>

0022Described herein is a composition that induces proliferation or accumulation of regulatory T cells, the composition comprising, as an active ingredient, at least one substance selected from the group consisting of the following (a) to (c): <ol id="ol0001" compact="compact"><li>(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;</li><li>(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction; and</li><li>(c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction.</li></ol>

0023Herein, "regulatory T cells" mean T cells which have a function of suppressing an abnormal or excessive immune response, and which play a role in immune tolerance. The regulatory T cells of the invention are transcription factor Foxp3-positive CD4-positive T cells. However, other regulatory T cells also include transcription factor Foxp3-negative regulatory T cells that are IL-10-producing CD4-positive T cells.

0024The meaning of the " induces proliferation or accumulation of regulatory T cells" in the present invention includes an effect of inducing the differentiation of immature T cells into regulatory T cells, which differentiation leads to the proliferation or the accumulation of regulatory T cells. In addition, the meaning of the " induces proliferation or accumulation of regulatory T cells" in the present invention includes in-vivo effects, in vitro effects, and ex vivo effects. Accordingly, all of the following effects are included: an effect of inducing in vivo proliferation or accumulation of regulatory T cells through administration or ingestion of the bacteria belonging to the genus Clostridium or the physiologically active substance or the like derived from the bacteria; an effect of inducing proliferation or accumulation of cultured regulatory T cells by causing the bacteria belonging to the genus Clostridium or the physiologically active substance or the like derived from the bacteria to act on the cultured regulatory T cells; and an effect of inducing proliferation or accumulation of regulatory T cells which are collected from a living organism and which are intended to be subsequently introduced into a living organism, such as the organism from which they were obtained or another organism, by causing the bacteria belonging to the genus Clostridium or the physiologically active substance or the like derived from the bacteria to act on the regulatory T cells. The effect of inducing proliferation or accumulation of regulatory T cells can be evaluated, for example, as follows. Specifically, the bacteria belonging to the genus Clostridium or the physiologically active substance or the like derived from the bacteria is orally administered to an experimental animal such as a germ-free mouse, then CD4-positive cells in the colon are isolated, and the ratio of regulatory T cells contained in the CD4-positive cells is measured by flow cytometry (refer to Example 7).

0025The regulatory T cells of which proliferation or accumulation is induced by the composition of the present invention are transcription factor Foxp3-positive regulatory T cells.

0026The "bacteria belonging to the genus Clostridium, " which are the active ingredient in the composition of the present invention, are defined in the claims, and have the effect of inducing proliferation or accumulation of regulatory T cells.

0027One strain of the bacteria alone can be used for the composition of the present invention, but two or more strains of the bacteria can be used together for the composition of the present invention. The use of multiple strains of bacteria belonging to the cluster XIVa or the cluster IV in combination can bring about an excellent effect on regulatory T cells. In addition to the bacteria belonging to these clusters, bacteria belonging to other clusters (for example, bacteria belonging to the cluster III) can also be used in combination. If more than one strain of bacteria is used (e.g.,one or more strain belonging to cluster XIVa, one or more strain belonging to cluster IV, one or more strain belonging to a cluster other than cluster XIVa or cluster IV, such as one or more strain belonging to cluster III), the type and number of strains used can vary widely. The type and number to be used can be determined based on a variety of factors (e.g., the desired effect, such as induction or inhibition of proliferation or accumulation of regulatory T cells; the disease or condition to be treated, prevented or reduced in severity; the age or gender of the recipient) The strains can be present in a single composition , in which case they will be consumed or ingested together, or can be present in more than one composition (e.g., each can be in a separate composition), in which case they can be consumed individually or the compositions can be combined and the resulting combination (combined compositions) consumed or ingested. Any number or combination of strains that proves effective (e.g., any number from one to 200, such as 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 5 and any number therebetween) can be administered. In certain embodiments of the present invention, a combination of some or all of the 46 strains described in Document (<nplcit id="ncit0024" npl-type="s"><text>Itoh, K., and Mitsuoka, T. Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice. Lab. Animals 19: 111-118 (1985</text></nplcit>)) is used. For example, at least one, two or more, three, three or more, four, four or more, five, five or more, six, six or more or any other number of the 46 described strains, including 46 strains, can be used. They can be used in combination with one another and in combination with strains not described in the cited reference (e.g, in combination with one or more strains belonging to cluster III). Note that, the cluster of "bacteria belonging to the genus Clostridium" can be identified, for example, as follows. Specifically, the bacteria belonging to the genus Clostridium are classified by PCR using a primer set consisting of SEQ ID NOs 64 and 65 (for Clostridium spp. belonging to the cluster XIVa) or a primer set consisting of SEQ ID NOs 66 and 67 (for Clostridium spp. belonging to the cluster IV) (refer to Example 18). In addition, the bacteria belonging to the genus Clostridium are classified by sequencing of 16S rRNA gene amplified using a primer set consisting of SEQ ID NOs 19 and 20 (refer to Example 7).

0028Viable cells of the bacteria belonging to the genus Clostridium can be used for the composition of the present invention, and killed cells thereof may also be used for the composition. In addition, from the viewpoint of stability to heat, resistance to antibiotics and the like, and long storage period, the bacteria belonging to the genus Clostridium are preferably in the form of spore.

0029The meaning of the "physiologically active substance derived from bacteria belonging to the genus Clostridium" of the present invention includes substances contained in the bacteria, secretion products of the bacteria, and metabolites of the bacteria. Such a physiologically active substance can be identified by purifying an active component from the bacteria, a culture supernatant thereof, or intestinal tract contents in the intestinal tract of a mouse in which only bacteria belonging to the genus Clostridium are colonized by an already known purification method.

0030The active ingredient "spore-forming fraction of a fecal sample obtained from a mammal" in the composition as described herein includes spore-forming bacteria present in feces of a mammal, and has the effect of inducing proliferation or accumulation of regulatory T cells.

0031The active ingredient "chloroform-treated fraction of a fecal sample obtained from a mammal" in the composition as described herein is obtained by treating feces of a mammal with chloroform (for example, 3% chloroform), and has the effect of inducing proliferation or accumulation of regulatory T cells.

0032Note that the "mammal" herein includes humans, mice, rats, cattle, horses, pigs, sheep, monkeys, dogs, and cats.

0033Meanwhile, when the "spore-forming fraction of a fecal sample obtained from a mammal" or the "chloroform-treated fraction of a fecal sample obtained from a mammal" is cultured in a medium, substances contained in the bacteria, secretion products of the bacteria, metabolites of the bacteria are released from the bacteria and the like contained in the fraction. The meaning of the active ingredient "culture supernatant of the fraction" in the composition described herein includes such substances, secretion products, and metabolites. The culture supernatant is not particularly limited, as long as the culture supernatant has the effect of inducing proliferation or accumulation of regulatory T cells. Examples of the culture supernatant include a protein fraction of the culture supernatant, a polysaccharide fraction of the culture supernatant, a lipid fraction of the culture supernatant, and a low-molecular weight metabolite fraction of the culture supernatant.

0034The composition of the present invention may be in the form of a pharmaceutical composition, a food or beverage (which may also be an animal feed), or a reagent used for an animal model experiment, the pharmaceutical composition, the food or beverage, and the reagent having the effect of inducing proliferation or accumulation of regulatory T cells. An example herein revealed that regulatory T cells (Treg cells) induced by bacteria or the like belonging to the genus Clostridium suppressed the proliferation of effector T-cells. Accordingly, the composition of the present invention can be used suitably as defined in the claims as a composition having an immunosuppressive effect. The immunosuppressive effect can be evaluated, for example, as follows. Specifically, regulatory T cells isolated from an experimental animal, such as a mouse, to which the composition of the present invention is orally administered are caused to act on effector T-cells (CD4⁺ CD25^- cells) isolated from the spleen, and then proliferation ability thereof is measured by using the intake amount of [³H]-thymidine as an index (refer to Example 14).

0035The composition of the present invention can be used, for example, as claimed as a pharmaceutical composition for preventing or treating an allergic disease such as pollenosis or asthma a food or beverage for improving immune functions; or a reagent for suppressing the proliferation or function of effector T-cells.

0036Autoimmune diseases, allergic diseases, and rejection in organ transplantations and the like include inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, sprue, autoimmune arthritis, rheumatoid arthritis, Type I diabetes, multiple sclerosis, graft vs. host disease following bone marrow transplantation, osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis, spondy loarthropathy, systemic lupus erythematosus, insulin dependent diabetes mellitus, thyroiditis,. asthma, psoriasis, dermatitis scleroderma, atopic dermatitis, graft versus host disease, acute or chronic immune disease associated with organ transplantation, sarcoidosis, atherosclerosis, disseminated intravascular coagulation, Kawasaki's disease, Grave's disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis, Henoch-Schoenlejn purpurea, microscopic vasculitis of the kidneys, chronic active hepatitis, uveitis, septic shock, toxic shock syndrome, sepsis syndrome, cachexia, acquired immunodeficiency syndrome, acute transverse myelitis, Huntington's chorea, Parkinson's disease, Alzheimer's disease, stroke, primary biliary cirrhosis, hemolytic anemia, polyglandular deficiency type I syndrome and polyglandular deficiency type II syndrome, Schmidt's syndrorme, adult (acute) respiratory distress syndrome, alopecia, alopecia areata, seronegative arthopathy, arthropathy, Reiter's disease, psoriatic arthropathy, chlamydia, yersinia and salmonella associated arthropathy spondyloarhopathy, atheromatous disease/arteriosclerosis, atopic allergy, food allergies, autoimmune bullous disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmune haemolytic anaemia, Coombs positive haemolytic anaemia, acquired pernicious anaemia, juvenile pernicious anaemia, myalgic encephalitis/Royal Free Disease, chronic mucocutaneous candidiasis, giant cell arteritis, primary sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome, Acquired Immunodeficiency Related Diseases, Hepatitis C, common varied immunodeficiency (common variable hypogammaglobulinaemia), dilated cardiomyopathy, fibrotic lung disease, cryptogenic fibrosing alveolitis, postinflammatory interstitial lung disease, interstitial pneumonitis, connective tissue disease associated interstitial lung disease, mixed connective tissue disease associated lung disease, systemic sclerosis associated interstitial lung disease, rheumatoid arthritis associated interstitial lung disease, systemic lupus erythematosus associated lung disease, dermatomyositis/polymyositis associated lung disease, Sjogren's disease associated lung disease, ankylosing spondy litis associated lung disease, vasculitic diffuse lung disease, haemosiderosis associated lung disease, drug-induced interstitial lung disease, radiation fibrosis, bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocytic infiltrative lung disease, postinfectious interstitial lung disease, gouty arthritis, autoimmune hepatitis, type-l autoimmune hepatitis (classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis (anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type B insulin resistance with acanthosis nigricans, hypoparathyroidism, acute immune disease associated with organ transplantation, chronic immune disease associated with organ transplantation, osteoarthrosis, primary sclerosing cholangitis, idiopathic leucopenia, autoimmune neutropenia, renal disease NOS, glomerulonephritides, microscopic vasulitis of the kidneys, discoid lupus, erythematosus, male infertility idiopathic or NOS, sperm autoimmunity, multiple sclerosis (all subtypes), insulindependent diabetes mellitus, sympathetic ophthalmia, pulmonary hypertension secondary to connective tissue disease, Goodpasture's syndrome, pulmonary manifestation of polyarteritis nodosa, acute rheumatio fever, rheumatoid spondylitis, Still's disease, systemic sclerosis, Takayasu's disease/arteritis, autoimmune thrombocytopenia, idiopathic thrombocytopenia, autoimmune thyroid disease, hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto's disease), atrophic autoimmune hypothyroidism, primary myxoedema, phacogenic uveitis, primary vasculitis, vitiligo, allergic rhinitis (pollen allergies), , anaphylaxis, pet allergies, latex allergies, drug allergies, allergic rhinoconjuctivitis, eosinophilic esophagitis, hypereosinophilic syndrome, eosinophilic gastroenteritis cutaneous lupus erythematosus, eosinophilic esophagitis, hypereosinophilic syndrome ,and eosinophilic gastroenteritis.

0037Pharmaceutical preparations can be formulated from the composition of the present invention by already known drug formulation methods. For example, the composition of the present invention can be used orally or parenterally in the forms of capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewables, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, cataplasms, transdermal absorption systems, lotions, inhalations, aerosols, injections, suppositories, and the like.

0038For formulating these preparations, the composition of the present invention can be used in appropriate combination with carriers acceptable pharmacologically or acceptable for a food or beverage, specifically, with sterile water, physiological saline, vegetable oil, solvent, a base material, an emulsifier, a suspending agent, a surfactant, a stabilizer, a flavoring agent, an aromatic, an excipient, a vehicle, a preservative, a binder, a diluent, a tonicity adjusting agent, a soothing agent, a bulking agent, a disintegrating agent, a buffer agent, a coating agent, a lubricant, a colorant, a sweetener, a thickening agent, a flavor corrigent, a solubilizer, other additives, or the like.

0039Meanwhile, for formulating a pharmaceutical preparation thereof, and particularly for formulating a pharmaceutical preparation for oral administration, it is preferable to use in combination a composition which enables an efficient delivery of the composition of the present invention to the colon, from the viewpoint of more efficiently inducing the proliferation or accumulation of regulatory T cells in the colon.

0040Such a composition or method which enables the delivery to the colon is not particularly limited, and known compositions or methods can be employed as appropriate. Examples thereof include pH sensitive compositions, more specifically, enteric polymers which release their contents when the pH becomes alkaline after the enteric polymers pass through the stomach. When a pH sensitive composition is used for formulating the pharmaceutical preparation, the pH sensitive composition is preferably a polymer whose pH threshold of the decomposition of the composition is 6.8 to 7.5. Such a numeric value range is a range where the pH shifts toward the alkaline side at a distal portion of the stomach, and hence is a suitable range for use in the delivery to the colon.

0041Moreover, another example of the composition enabling the delivery to the colon is a composition which ensures the delivery to the colon by delaying the release of the contents by approximately 3 to 5 hours, which corresponds to the small intestinal transit time. In an example of formulating a pharmaceutical preparation using the composition for delaying the release, a hydrogel is used as a shell. The hydrogel is hydrated and swells upon contact with gastrointestinal fluid, so that the contents are effectively released. Furthermore the delayed release dosage units include drug-containing compositions having a material which coats or selectively coats a drug. Examples of such a selective coating material include in vivo degradable polymers, gradually hydrolyzable polymers, gradually water-soluble polymers, and/or enzyme degradable polymers. A preferred coating material for efficiently delaying the release is not particularly limited, and examples thereof include cellulose-based polymers such as hydroxypropyl cellulose, acrylic acid polymers and copolymers such as methacrylic acid polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.

0042Examples of the composition enabling the delivery to the colon further include bioadhesive compositions which specifically adhere to the colonic mucosal membrane (for example, a polymer described in the specification of <patcit id="pcit0002" dnum="US6368586B"><text>US Patent No. 6.368.586</text></patcit>), and compositions into which a protease inhibitor is incorporated for protecting particularly a biopharmaceutical preparation in the gastrointestinal tracts from decomposition due to an activity of a protease.

0043An example of a system enabling the delivery to the colon is a system of delivering a composition to the colon by pressure change in such a way that the contents are released by utilizing pressure change caused by generation of gas in bacterial fermentation at a distal portion of the stomach. Such a system is not particularly limited, and a more specific example thereof is a capsule which has contents dispersed in a suppository base and which is coated with a hydrophobic polymer (for example, ethyl cellulose).

0044Another example of the system enabling the delivery to the colon is a system of delivering a composition to the colon, the system being specifically decomposed by an enzyme (for example, a carbohydrate hydrolase or a carbohydrate reductase) present in the colon. Such a system is not particularly limited, and more specific examples thereof include systems which use food components such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.

0045When used as a pharmaceutical composition, the composition of the present invention may be used in combination with an already known pharmaceutical composition for use in immunosuppression. Such a known pharmaceutical composition is not particularly limited, and may be at least one therapeutic composition selected from the group consisting of corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines (preferably vaccines used for vaccination where the amount of an allergen is gradually increased), and combinations thereof. It is preferable to use these therapeutic compositions in combination with the composition of the present invention.

0046When the composition of the present invention is used as a food or beverage, the food or beverage can be, for example, a health food, a functional food, a food for specified health use, a dietary supplement, a food for patients, or an animal feed. The food or beverage of the present invention can be ingested in the forms of the compositions as described above, and also can be ingested in the forms of various foods and beverages. Specific examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products such as Western confectionery products including biscuits, cookies, and the like, Japanese confectionery products including steamed bean-jam buns, soft adzuki-bean jellies, and the like, candies, chewing gums, gummies, cold desserts including jellies, creme caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies. The composition of the present invention can be used for animals including humans. The animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like. Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.

0047Without wishing to be bound by theory, individuals in which the relative abundance of bacteria belonging to the group Firmicutes (the group to which the Clostridium clusters IV and XIVa belong) is large gain more body weight than individuals in which the relative abundance of bacteria belonging to the group Bacteroidetes is large. Accordingly, the composition of the present disclosure is capable of conditioning absorption of nutrients and improving feed efficiency. From such a viewpoint, the composition of the present disclosure can be used for promoting body weight gain, or for an animal feed good in feed efficiency.

0048Moreover, the addition of the composition of the present disclosure to an antibiotic-free animal feed makes it possible to increase the body weight of a subject that ingests the animal feed to a level equal to or higher than those achieved by antibiotic-containing animal feeds, and also makes it possible to reduce pathogenic bacteria in the stomach to a level equal to those achieved by typical antibiotic-containing animal feeds. Accordingly, the composition of the present disclosure can be used for an animal feed which does not need the addition of antibiotics.

0049In addition, unlike conventional bacteria (Lactobacillus and Bifidobacteria) in commercial use which are not easy to incorporate into the livestock production, the composition of the present invention in the spore form can be pelletized, sprayed, or easily mixed with an animal feed, and also can be added to drinking water.

0050The feeding of such an animal feed using the composition described herein is not particularly limited, and the animal feed may be fed to a subject at regular intervals in a selective manner, or may be fed for a certain period (for example, at its birth, during weaning, or when the subject to be fed is relocated or shipped).

0051Moreover, from the above-described viewpoint, the composition of the present disclosure can be preferably used for malnourished humans. In other words, also when the subject who ingests the composition is a human, the composition can be used for promoting the body weight gain, and enhancing the energy absorption from foods.

0052Food or beverage can be manufactured by a manufacturing technique which is well known in the technical field. To the food or beverage, one or more components (for example, a nutrient) which are effective for the improvement of an immune function by the immunosuppressive effect may be added. In addition, the food or beverage may be combined with another component or another functional food exhibiting a function other than the function of the improvement of an immune function to thereby serve as a multi-functional food or beverage.

0053Moreover, the composition described herein can be incorporated into foods requiring a processing step which may destroy ordinary probiotic strains. Specifically, most commercially usable probiotic strains cannot be incorporated into foods which need to be processed by any one of a heat treatment, long term storage, a freezing treatment, a mechanical stress treatment, and a high-pressure treatment (for example, extrusion forming or roll forming). On the other hand, because of an advantageous nature of forming spores, the composition of the present invention can be easily incorporated into such processed foods.

0054For example, compositions in the form of spore can survive even in a dried food, and can remain living even after being ingested. Likewise, the composition described herein can withstand low-temperature sterilization processes, typically processes at a temperature in a range from 70°C to the boiling point, both inclusive. Thus, the composition described herein can be incorporated into all kinds of dairy products. Furthermore, the composition described herein can withstand long-term storage of many years; high-temperature processing such as baking and boiling; low-temperature processing such as freezing and cold storage; and high-pressure treatments such as extrusion forming and roll forming.

0055The foods which need to be processed under such harsh conditions are not particularly limited, and examples thereof include foods which need to be processed in a microwave oven to be edible (for example, oatmeal), foods which need to be baked to be edible (for example, muffin), foods which need to be subjected to a sterilization high-temperature treatment for a short period of time to be edible (for example, milk), and foods which need to be heated to be drinkable (for example, hot tea).

0056When the composition is administered or ingested, the amount thereof for the administration or ingestion is selected as appropriate depending on the age, body weight, symptoms, health conditions, of a subject, the kind of the composition (a pharmaceutical product, a food or beverage, or the like), and the like. For example, the amount per administration or ingestion is generally 0.01 mg/kg body weight to 100 mg/kg body weight, and preferably 1 mg/kg body weight to 10 mg/kg body weight. The present disclosure also includes a method for suppressing the immunity of a subject, the method being characterized in that the bacteria belonging to the genus Clostridium or the physiologically active substance derived from the bacteria is administered into or ingested by the subject as described above.

0057A product of the composition of the present invention (a pharmaceutical product, a food or beverage, or a reagent) or a manual thereof may be provided with a note stating that the product can be used to suppress the immunity (including a note stating that the product has an immunosuppressive effect, and a note stating that the product has an effect of suppressing the proliferation or function of effector T-cells). Here, the "provision to the product or the manual thereof with the note" means that the note is provided to a main body, a container, a package, or the like of the product, or the note is provided to a manual, a package insert, a leaflet, or other printed matters, which disclose information on the product.

<Method for Inducing Proliferation or Accumulation of Regulatory T Cells>

0058As described above, and as will be shown in Examples, the administration of the composition described herein to an individual makes it possible to induce proliferation or accumulation of regulatory T cells in the individual. Thus, the present disclosure includes a method for inducing proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering, to the individual, at least one substance selected from the group consisting of the following (a) to (c): <ol id="ol0002" compact="compact"><li>(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;</li><li>(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction; and</li><li>(c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction.</li></ol>

0059Note that, the "individual" in the present disclosure is not particularly limited, and examples thereof include humans, various kinds of livestock, poultry, pets, experimental animals, and the like. The "individual" may be in a healthy state or a diseased state.

0060Moreover, as will be shown in Example 5 to be described later, Gram-positive commensal bacteria play principal roles in the proliferation or accumulation of regulatory T cells. Accordingly, the present disclosure includes a method for inducing proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering an antibiotic against Gram-negative bacteria to the individual.

0061As used herein, the "antibiotic against Gram-negative bacteria" is not particularly limited, and examples thereof include aminoglycoside antibiotics (amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, and paromomycin), cephalosporin antibiotics (cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, and cefoxotin), sulfonamides, ampicillin, and streptomycin. Without wishing to be bound by theory, the "antibiotic against Gram-negative bacteria" according to the present invention is preferably one which reduces Gram-negative bacteria, and contributes to the colonization of Gram-positive bacteria.

0062Moreover, a prebiotic composition such as almond skin, inulin, oligofructose, raffinose, lactulose, pectin, hemicellulose (such as xyloglucan and alpha-glucans), amylopectin, and resistant starch which are not decomposed in the upper gastrointestinal tract and promote the growth of intestinal microbes in the intestinal tract, as well as growth factors such as acetyl-Co A, biotin, beet molasses, and yeast extracts, contribute to the proliferation of bacteria belonging to the genus Clostridium. Accordingly, the present disclosure includes a method for inducing proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering, to the individual, at least one substance selected from the group consisting of these substances.

0063Meanwhile, in a "method for inducing proliferation or accumulation of regulatory T cells", the composition of the present disclosure, the above-described "antibiotic against Gram-negative bacteria," and the above-described "prebiotic composition or growth factor" may be used in combination. Such combined use is not particularly limited, and examples of the combined use are as follows: the "antibiotic against Gram-negative bacteria" is administered to an individual in advance, and then the composition of the present invention is administered; the "antibiotic against Gram-negative bacteria" and the composition of the present invention are simultaneously administered to an individual; the "prebiotic composition or growth factor" is administered to an individual in advance, and then the composition of the present invention is administered; the "prebiotic composition or growth factor" and the composition of the present invention are simultaneously administered to an individual; the composition of the present invention, the "antibiotic against Gram-negative bacteria," and the "prebiotic composition or growth factor" are administered to an individual simultaneously or individually at any appropriate time.

0064Moreover, a therapeutic composition may be administered to an individual together with at least one substance selected from the group consisting of the composition of the present disclosure, the "antibiotic against Gram-negative bacteria," and the "prebiotic composition or growth factor."

0065Such a therapeutic composition is not particularly limited, and may be at least one therapeutic composition selected from the group consisting of corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines (preferably, vaccines used for vaccination where the amount of an allergen is gradually increased), and combinations thereof. It is preferable to use these therapeutic compositions in combination with the above-described substance.

0066Moreover, there is no particular limitation imposed on the combined use of the therapeutic composition with at least one substance selected from the group consisting of the composition of the present disclosure, the "antibiotic against Gram-negative bacteria," and the "prebiotic composition or growth factor". For example, the "one substance" and the therapeutic composition are administered orally or parenterally to an individual simultaneously or individually at any appropriate time.

0067Moreover, in the above-described "method for inducing proliferation or accumulation of regulatory T cells," whether or not the administration of the composition described herein or the like actually induces the proliferation or accumulation of regulatory T cells can be determined by using, as an index, increase or reinforcement of at least one selected from the group consisting of the number of regulatory T cells, the ratio of regulatory T cells in the T cell group of the colon, a function of regulatory T cells, and expression of a marker of regulatory T cells. It is preferable to use one measurement selected from the group consisting of promotion of IL-10 expression, promotion of CTLA4 expression, promotion of IDO expression, and suppression of IL-4 expression, as the index of the induction of proliferation or accumulation of regulatory T cells.

0068Note that examples of a method for detecting such expression include the northern blotting, the RT-PCR, and the dot blotting for detection of gene expression at the transcription level; and the ELISA, the radioimmunoassay, the immunoblotting, the immunoprecipitation, and the flow cytometry for detection of gene expression at the translation level.

0069Meanwhile, a sample used for measuring such an index is not particularly limited, and examples thereof include blood sampled from an individual and tissue pieces obtained in a biopsy.

<Method for Predicting Response of Individual to Composition of Present Invention and/or Prognosis of Individual>

0070Described herein is a method in which the absolute amount or the ratio of bacteria belonging to the genus Clostridium in a microbiota of an individual is determined, and, when the ratio or the absolute value of the bacteria belonging to the genus Clostridium is reduced in comparison with a base line value obtained by performing a similar determination on an individual in a typical health state, it is determined that the individual is possibly responsive to the composition described herein.

0071Also described is a method to predict a subject's response to a substance and/or the subject's prognosis.

0072The method comprises measuring the percentage or absolute amounts of Clostridium clusters IV and XIV in the microbiota of the subject and comparing them to a baseline value of the same measurements in a prototypical healthy subject, wherein a decreased absolute amount or percentage level of Clostridium clusters IV and/or XIV indicates that the subject may respond favorably to the compositions described herein.

0073The method may further comprise measuring the composition of the microbiota of the subject after administration of the substance, wherein an increase in the percentage or absolute number of Clostridium spp. belonging to clusters IV, XIV after administration of the compositions of the present invention relative to prior to the administering is a positive indicator of enhanced immunosuppression (or immunoregulation). The measurement of the composition of the subject's microbiota can be made with techniques known in the art, such as 16srRNA sequencing.

0074Note that, in these methods, the substance is at least one substance selected from the group consisting of the following (a) to (e): <ol id="ol0003" compact="compact"><li>(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;</li><li>(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction;</li><li>(c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction;</li><li>(d) an antibiotic against Gram-negative bacteria; and</li><li>(e) at least one substance selected from the group consisting of almond skin, inulin, oligofructose, raffinose, lactulose, pectin, hemicellulose (such as xyloglucan and alpha-glucans), amylopectin, acetyl-Co A, biotin, beet molasses, yeast extracts, and resistant starch.</li></ol>

<Method for Inhibiting Proliferation or Accumulation of Regulatory T Cells>

0075As will be shown in Example 5 to be described later, Gram-positive commensal bacteria have principal roles in the proliferation or accumulation of regulatory T cells. Accordingly, the present disclosure also includes a method for inhibiting proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering an antibiotic against Gram-positive bacteria to the individual.

0076As used herein, the term "antibiotic against Gram-positive bacteria" is not particularly limited, and examples thereof include cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole) ; fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).

0077As described above, the term "individual" is not particularly limited, and examples thereof include humans, various kinds of livestock, poultry, pets, experimental animals, and the like. The "individual" may be in a healthy state or a diseased state. Such a diseased state is not particularly limited, and examples thereof include states of being subjected to cancer immunotherapy and of suffering from an infectious disease.

0078Moreover, as another mode of the "method for inhibiting proliferation or accumulation of regulatory T cells," the present disclosure includes a method for inhibiting proliferation or accumulation of regulatory T cells in an individual, the method comprising a step of administering, to the individual, any one of an antibody, an antibody fragment, and a peptide, which are against an antigen that is at least one substance selected from the group consisting of the following (a) to (c): <ol id="ol0004" compact="compact"><li>(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;</li><li>(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction; and</li><li>(c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction.</li></ol>

<Vaccine Composition and Method for Treating or Preventing Infectious Disease or Autoimmune Disease by Using the Vaccine Composition>

0079As described above, and as will be shown in Example 15 to be described later, the induction of Treg cells in the colon by the Clostridium has an important role in local and systemic immune responses. Accordingly, the present disclosure includes a "vaccine composition comprising at least one substance selected from the group consisting of the following (a) to (c) : (a) bacteria belonging to the genus Clostridium; (b) a spore of bacteria in a spore-forming fraction of a fecal sample obtained from a mammal; and (c) bacteria in a chloroform-treated fraction of a fecal sample obtained from a mammal" and a "method for treating, aiding in treating, reducing the severity of, or preventing at least one disease selected from infectious diseases and autoimmune diseases in an individual, the method comprising administering the vaccine composition to the individual."

0080Note that such "autoimmune diseases" are not particularly limited, and examples thereof include those described as the "specific examples of target diseases" in <Composition Having Effect of Inducing Proliferation or Accumulation of Regulatory T cells>. The "infectious diseases" are also not particularly limited, and examples thereof include infectious diseases associated with "infectious pathogens" described as the "example of infectious pathogens" in <Composition Having Effect of Inducing Proliferation or Accumulation of Regulatory T cells>.

<Method for Screening for Compound Having Activity to Promote Proliferation or Accumulation of Regulatory T Cells>

0081Also described is a method for screening for a compound having an activity to promote proliferation or accumulation of regulatory T cells, the method comprising: <ol id="ol0005" compact="compact"><li>(1) preparing a test substance from at least one substance selected from the group consisting of the following (a) to (c): <ol id="ol0006" compact="compact"><li>(a) bacteria belonging to the genus Clostridium or a physiologically active substance derived from the bacteria;</li><li>(b) a spore-forming fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction; and</li><li>(c) a chloroform-treated fraction of a fecal sample obtained from a mammal or a culture supernatant of the fraction.</li></ol></li><li>(2) preparing non-human mammals in which a reporter gene is to be expressed under control of IL-10 gene expression;</li><li>(3) bringing the test substance into contact with the non-human mammal;</li><li>(4) after the contact with the test substance, detecting cells expressing the reporter gene in a CD4⁺ Foxp3⁺ cell group of the non-human mammal, and determining the number of cells in the CD4⁺ Foxp3⁺ cell group expressing the reporter gene or a ratio of cells in the CD4⁺ Foxp3⁺ cell group expressing the reporter gene to cells in the CD4⁺ Foxp3⁺ cell group not expressing the reporter gene;</li><li>(5) detecting cells expressing the reporter gene in a CD4⁺ Foxp3⁺ cell group of the non-human mammal which has not been in contact with the test substance, and determining the number of cells in the CD4⁺ Foxp3⁺ cell group expressing the reporter gene or a ratio of cells in the CD4⁺ Foxp3⁺ cell group expressing the reporter gene to cells in the CD4⁺ Foxp3⁺ cell group not expressing the reporter gene; and</li><li>(6) comparing the absolute numbers or the ratios determined in steps (4) with the number or the ratio determined in (5), and determining, when the number or the ratio determined in (4) is greater than that determined in (5), that the test substance is a compound that promotes proliferation or accumulation of Treg cells.</li></ol>

0082The term "test substance" as used herein is not particularly limited, as long as the test substance is a substance prepared from at least one substance selected from the group consisting of the substances (a) to (c). Examples of the test substance include proteins, polysaccharides, lipids, and nucleic acids which are derived from at least one substance selected from the group consisting of the above described substances (a) to (c).

0083The term "non-human mammal in which a reporter gene is to be expressed under control of IL-10 gene expression" as used herein is not particularly limited, as long as the non-human mammal is a non-human mammal having a reporter gene whose expression is controlled by an IL-10 gene expression control region (for example, a promoter, or an enhancer). Examples of such a reporter gene include genes encoding fluorescent proteins (for example, GFP), and genes encoding luciferase. As the "non-human mammal in which a reporter gene is to be expressed under control of IL-10 gene expression" according to the present invention, an <u>I110</u>^venus mouse to be shown later in Examples can be preferably used.

0084The term "contact" as used herein is not particularly limited, and examples thereof include administration of the test substance to the non-human mammal orally or parenterally (for example, intraperitoneal injection, or intravenous injection).

0085Also described is a non-human mammal which is used for the method, and in which the reporter gene is to be expressed under the control of the IL-10 gene expression.

0086Furthermore, described herein is a method for isolating, from a sample of bacteria belonging to the genus Clostridium, a compound having an activity to promote proliferation or accumulation of regulatory T cells, the method comprising the following steps (1) to (3) : <ol id="ol0007" compact="compact"><li>(1) preparing a genomic DNA from the sample of bacteria belonging to the genus Clostridium;</li><li>(2) inserting the genomic DNA into a cloning system, and preparing a gene library derived from the sample of bacteria belonging to the genus Clostridium; and</li><li>(3) isolating a compound having an activity to promote proliferation or accumulation of regulatory T cells, by use of the gene library obtained in step (2).</li></ol>

0087In such steps, methods for the preparation and the isolation are not particularly limited, and known techniques for an in-vitro or in-vivo system can be used as appropriate. Moreover, the compound isolated by this method is not particularly limited, and examples thereof include nucleic acids (for example, a DNA, a mRNA, and a rRNA) derived from bacteria belonging to the genus Clostridium, as well as polypeptides and proteins derived from the bacteria belonging to the genus Clostridium.

0088Also described is a method for determining the composition of a microbiota in an individual, wherein the increase in the ratio or the absolute number of bacteria belonging to the genus Clostridium after the administration of the composition of the present invention to the individual with respect to the ratio or the absolute number before the administration is used as an index of increased immunosuppression. In such a method, the method for determining the composition of the microbiota is not particularly limited, and known techniques (for example, 16S rRNA sequencing) can be used as appropriate.

0089Also described is a method for measuring differentiation of Treg cells, wherein the increase in differentiation of Treg cells in an individual after administration of the composition described herein to the individual with respect to that before the administration is used as an index of increased immunosuppression (or immunoregulation).

0090Moreover, the composition described herein can also be administered to an individual under an antibiotic treatment. The timing of the administration is not particularly limited, and the composition can be administered before or simultaneously with the antibiotic treatment, for example. Meanwhile, the composition is preferably administered in the spore form from the viewpoint of resistance to antibiotics.

0091Moreover, in a preferred mode of such administration, the composition is administered after or simultaneously with administration of an antibiotic against Gram-positive bacteria, for example. Note that such an " antibiotic against Gram-positive bacteria" is not particularly limited, and examples thereof include cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and cef tobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).

0092Meanwhile, in another preferred mode of such administration, the composition is administered after (or simultaneously with) a treatment using vancomycin, metronidazole, linezolid, ramoplanin, or fidaxomicin, for example.

[Examples]

0093Hereinafter, the present invention is described more specifically on the basis of Examples. However, the present invention is defined in the claims.

0094Note that mice used in Examples were prepared or produced as follows. In the following description, mice may be referred to with "SPF" or "GF" attached in front thereof. These "SPF" and "GF" indicate that the mice were maintained in the absence of specific pathogenic bacteria (specific pathogen-free, SPF), and that the mice were maintained under Germ-Free (GF) conditions, respectively.

<Mice>

0095C57BL/6, Balb/c, and IQI mice maintained under SPF or GF conditions were purchased from Sankyo Labo Service Corporation, Inc. (Japan), JAPAN SLC, INC. (Japan), CLEA Japan, Inc. (Japan), or The Jackson Laboratory (USA) . GF mice and gnotobiotic mice were bread and maintained within the gnotobiotic facility of The University of Tokyo, Yakult Central Institute for Microbiological Research, or Sankyo Labo Service Corporation, Inc. <u>Myd88</u>^-/-, <u>Rip2</u>^-/-, and <u>Card9</u>^-/-mice were produced as described in Non-Patent Documents 1 to 3, and backcrossed for 8 generations or more, so that a C57BL/6 genetic background was achieved. Foxp3^eGFPmice were purchased from the Jackson Laboratory.

<

Il10

venus

mice>

0096To form a bicistronic locus encoding both Il10 and Venus under control of an Il10 promoter, a targeting construct was first created. Specifically, a cassette (IRES-Venus-SV40 polyA signal cassette, refer to Non-Patent Document 4) which was made of an internal ribosome entry site (IRES), a yellow fluorescent protein (Venus), and a SV40 polyA signal (SV40 polyA) and which was arranged next to a neomycin-resistant gene (neo), was inserted between a stop codon and a polyA signal (Exon 5) of a Il10 gene. Next, the obtained targeting construct was used to cause homologous recombination with the Il10 gene region in the genome of mice. Thus, <u>Il10</u>^venus mice having an <u>Il10</u>^venus alleles were produced (refer to <figref idref="f0001">Fig. 1</figref>). Note that in <figref idref="f0001">Fig. 1</figref> "tk" represents a gene coding thymidine kinase, "neo" represents the neomycin-resistant gene, and "BamH1" represents a cleavage site by the restriction enzyme BamH1.

0097Genomic DNAs were extracted from the <u>Il10</u>^venus mice, treated with BamH1, and Southern blotted by use of a probe shown in <figref idref="f0001">Fig. 1</figref>. <figref idref="f0002">Fig. 2</figref> shows the obtained results. Wild-type and <u>Il10</u>^venus alleles were detected as bands having sizes of 19 kb and 5.5 kb, respectively. Hence, as is apparent from the results shown in <figref idref="f0002">Fig. 2</figref>, it was found that the homologous recombination shown in <figref idref="f0001">Fig. 1</figref> occurred in the genome of the <u>Il10</u>^venus mice.

0098Further, CD4⁺ Venus^- cells or CD4⁺ Venus⁺ cells in the colonic lamina propria of the <u>Il10</u>^venus mice were sorted by use of a FACSAria. Then, real-time RT-PCR was carried out on an ABI 7300 system by a method to be described later, to determine the amount of IL-10 mRNA expressed. <figref idref="f0002">Figs. 3</figref> and <figref idref="f0003">4</figref> show the obtained results. As is apparent from the results shown in <figref idref="f0002">Figs. 3</figref> and <figref idref="f0003">4</figref>, it was found that, since the development of the IL-10 mRNA was detected only in the CD4⁺ Venus⁺ cells, the expression of IL-10 mRNA in the <u>Il10</u>^venus mice was correctly reflected in the expression of Venus. Note that the germ-free states of such <u>Il10</u>^venus mice were established in Central Institute for Experimental Animals (Kawasaki, Japan). The <u>Il10</u>^venus mice in the germ-free states were maintained in vinyl isolators in Sankyo Labo Service Corporation, Inc. (Tokyo, Japan), and used in the following Examples.

0099Meanwhile, experiments and analyses in Examples were carried out as follows.

<Method for Colonization of Mice with Bacteria and Analysis Thereof>

0100According to the description in Non-Patent Documents 5 and 6, mice in which SFB or Clostridium were colonized were produced. Cecal contents or feces of the obtained gnotobiotic mice were dissolved in sterile water or an anaerobic dilution solution. The dissolved cecal contents or feces as they were or after a chloroform treatment were orally administered to GF mice. Three strains of the Lactobacillus and 16 strains of the Bacteroides were cultured separately from each other in a BL or EG agar medium in an anaerobic manner. The cultured bacteria were harvested, suspended in an anaerobic TS broth, and orally administrated forcibly to GF mice. The state of the colonization of the bacteria in the mice was assessed by microscopic observation conducted on a smear preparation of fecal pellets.

<Cell Separation and Flow Cytometry>

0101In order to isolate lymphocytes from the colonic lamina propria and the small intestinal lamina propria, the small intestine and the colon were collected, and cut open longitudinally. Then, fecal content and the like thereinside were washed to remove. Subsequently, the small intestine and the colon were shaken in HBSS containing 5 mM of EDTA at 37°C for 20 minutes. After removal of epithelium and fat tissue, the intestinal tissues were cut into small pieces. To the small pieces, RPMI 1640 (4% fetal bovine serum (FBS), 1 mg/ml of collagenase D, 0.5 mg/ml of dispase, and 40 µg/ml of DNaseI (all of which were manufactured by Roche Diagnostics K.K.)) were added, and the mixture was shaken in a water bath kept at 37°C for 1 hour. The digested tissues were washed with HBSS containing 5 mM of EDTA, and resuspended in 5 ml of 40% percoll (GE Healthcare). The suspension was overlayered on 2.5 ml of 80% percoll in a 15-ml Falcon tube. Then, centrifugation was carried out at room temperature and at 2000 rpm for 20 minutes to conduct cell separation by percoll density gradient centrifugation. Cells at the interface were collected, and used as lamina propria lymphocytes. The collected cells were suspend in a staining buffer (PBS, 2% FBS, 2 mM EDTA, and 0.09% NaN₃), and stained by use of an anti-CD4 antibody (RM4-5, BD Biosciences) labeled with PE or PE-Cy7. After the staining of CD4, Foxp3 in the cells were stained by use of Cytofix/Cytoperm Kit Plus with Golgistop (BD Biosciences) or Foxp3 Staining Buffer Set (eBioscience), as well as an anti-Foxp3 antibody (FJK-16s, eBioscience) labeled with Alexa647. Flow cytometry was performed by use of a FACScant II, and the data were analyzed by FlowJo software (TreeStar Inc.). The sorting of the cells were performed by use of a FACSAria.

<Real-Time RT-PCR>

0102From an RNA prepared by using RNeasy Mini Kit (Qiagen), a cDNA was synthesized by use of a MMV reverse transcriptase (Promega KK). The obtained cDNA was analyzed by real-time RT-PCR using Power SYBR Green PCR Master Mix (Applied Biosystems) and ABI 7300 real time PCR system (Applied Biosystems),or real-time RT-PCR using SYBR Premix Ex Taq (TAKARA) and Light Cycler 480. For each sample, a value obtained was normalized for the amount of GAPDH. A primer set was designed by using Primer Express Version 3.0 (Applied Biosystems), and those exhibiting a 90% or higher sequence identity at an initial evaluation were selected. The primer set used was as follows: <ul id="ul0003" list-style="none" compact="compact"><li>Foxp3 <ul id="ul0004" list-style="none" compact="compact"><li>5'-GGCAATAGTTCCTTCCCAGAGTT-3' (SEQ ID NO: 1)</li><li>5'-GGGTCGCATATTGTGGTACTTG-3' (SEQ ID NO: 2)</li></ul></li><li>CTLA4 <ul id="ul0005" list-style="none" compact="compact"><li>5'-CCTTTTGTAGCCCTGCTCACTCT-3' (SEQ ID NO: 3)</li><li>5'-GGGTCACCTGTATGGCTTCAG-3' (SEQ ID NO: 4)</li></ul></li><li>GITR <ul id="ul0006" list-style="none" compact="compact"><li>5'-TCAGTGCAAGATCTGCAAGCA-3' (SEQ ID NO: 5)</li><li>5'-ACACCGGAAGCCAAACACA-3' (SEQ ID NO: 6)</li></ul></li><li>IL-10 <ul id="ul0007" list-style="none" compact="compact"><li>5'-GATTTTAATAAGCTCCAAGACCAAGGT-3' (SEQ ID NO: 7)</li><li>5'-CTTCTATGCAGTTGATGAAGATGTCAA-3' (SEQ ID NO: 8)</li></ul></li><li>GAPDH <ul id="ul0008" list-style="none" compact="compact"><li>5'-CCTCGTCCCGTAGACAAAATG-3' (SEQ ID NO: 9)</li><li>5'-TCTCCACTTTGCCACTGCAA-3' (SEQ ID NO: 10)</li></ul></li><li>Mmp2 <ul id="ul0009" list-style="none" compact="compact"><li>5'-GGACATTGTCTTTGATGGCA-3' (SEQ ID NO: 11)</li><li>5'-CTTGTCACGTGGTGTCACTG-3' (SEQ ID NO: 12)</li></ul></li><li>Mmp9 <ul id="ul0010" list-style="none" compact="compact"><li>5'-TCTCTGGACGTCAAATGTGG-3' (SEQ ID NO: 13)</li><li>5'-GCTGAACAGCAGAGCCTTC-3' (SEQ ID NO: 14)</li></ul></li><li>Mmp13 <ul id="ul0011" list-style="none" compact="compact"><li>5'-AGGTCTGGATCACTCCAAGG-3' (SEQ ID NO: 15)</li><li>5'-TCGCCTGGACCATAAAGAA-3' (SEQ ID NO: 16)</li></ul></li><li>Idol <ul id="ul0012" list-style="none" compact="compact"><li>5'-AGAGGATGCGTGACTTTGTG-3' (SEQ ID NO: 17)</li><li>5'-ATACAGCAGACCTTCTGGCA-3' (SEQ ID NO: 18).</li></ul></li></ul>

<Preparation and Culturing of Large Intestinal Epithelial Cells (IECs)>

0103First, the colon was collected, cut open longitudinally, and rinsed with PBS. Subsequently, the colon was treated with 1mM dithiothreitol (DTT) at 37°C for 30 minutes on a shaker, and then vortexed for one minute to disrupt the epithelial integrity. The released IECs were collected, and suspended in 5 ml of 20% percoll. The suspension was overlayered on 2.5 ml of 80% percoll in a 15-ml Falcon tube. Then, the tube was centrifuged at 25°C and 780 g for 20 minutes to conduct cell separation by percoll density gradient centrifugation. Cells at the interface were collected, and used as colonic IECs (purity: 90% or higher, viability: 95%). The obtained IECs thus collected were suspended in RPMI containing 10% FBS, and 1×10⁵ cells of the IECs were cultured in a 24-well plate for 24 hours. Thereafter, the culture supernatant was collected, and measured for active TGF-β1 level by ELISA (Promega).

0104Meanwhile, for culturing T cells in vitro, 1.5×10⁵ MACS-purified splenic CD4⁺ T cells were cultured in each well of a round-bottomed 96-well plate, together with a 50% conditioned medium in which IECs isolated from GFmice or Clostridium-colonized mice were cultured, and with 25 ng/ml of hIL-2 (Peprotech), in the presence or absence of 25 µg/ml of an anti-TGF-β antibody (R&amp;D). Note that 10 µg/ml of an anti-CD3 antibody and an anti-CD28 antibody (BD Bioscience) were bound to the round-bottomed plate. After a 5-day culture, the CD4⁺ T cells were collected, and subjected to a real-time PCR.

<Colitis Experimental Model>

0105A fecal suspension of Clostridium-colonized mice was orally administered to C57BL/6 mice (2-week old), and grown in a conventional environment for six weeks.

0106For preparing a DSS-induced colitis model, 2% (wt/vol) DSS (reagent grade, DSS salt, molecular weight = 36 to 50 kD, manufactured by MP Biomedicals), together with drinking water, was given to the mice for six days.

0107Meanwhile, for preparing an oxazolone-induced colitis model, the mice were presensitized by transdermally applying, onto the mice, 150 µl of a 3% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one, Sigma-Aldrich)/100% ethanol solution. Five days after that, 150 µl of a 1% oxazolone/50% ethanol solution was intrarectally administered again to the presensitized mice under a light anesthesia. Note that the intrarectal administration was conducted by using a 3.5F catheter.

0108Each mouse was analyzed daily for body weight, occult blood, bleeding visible with the naked eyes (gross blood), and the hardness of stool. Moreover, the body weight loss percentage, intestinal bleeding (no bleeding, occult blood (hemoccult+), or bleeding visible with the naked eyes), and the hardness of stool (normal stool, loose stool, or diarrhea) were evaluated numerically, and the disease activity index (DAI) was calculated in accordance with the description in "<nplcit id="ncit0025" npl-type="s"><text>S. Wirtz, C. Neufert, B. Weigmann, M. F. Neurath, Nat Protoc 2, 541 (2007</text></nplcit>)."

<OVA Specific IgE Reaction>

0109BALB/c SPF mice were inoculated with a fecal suspension of Clostridium-colonized mice (2-week old), and grown in a conventional environment. Then, 1 µg of OVA (grade V, Sigma) and 2 mg of alum (Thermo Scientific), 0.2 ml in total, were intraperitoneally injected to the mice (at their ages of 4 weeks and 6 weeks). Sera were collected every week from the mice at the root of their tail, and OVA-specific IgE was measured by ELISA (Chondrex). Then, at their ages of 8 weeks, splenic cells were collected, inoculated in a 96-well plate at 1×10⁶ cells per well, and stimulated with OVA (100 µg/ml) for three days. Thereafter, the culture supernatant was collected, and measured for IL-4 and IL-10 levels by ELISA (R&amp;D) .

<Statistical Analysis>

0110The difference between control and experimental groups was evaluated by the Student's t-test.

(Example 1)

0111First, it was investigated whether or not accumulation of regulatory T cells (Treg cells) in the colonic lamina propria was dependent on commensal bacteria. Specifically, lymphocytes were isolated from peripheral lymph nodes (pLN) of Balb/c mice bred in the absence of specific pathogenic bacteria (SPF) or from lamina propria of the colon or the small intestine (SI) of the mice. The CD4 and Foxp3 were stained by antibodies. Then, the ratio of Foxp3⁺ cells in CD4⁺ lymphocytes was analyzed by flow cytometry. <figref idref="f0003">Fig. 5</figref> shows the obtained results. As is apparent from the results shown in <figref idref="f0003">Fig. 5</figref>, it was found that Foxp3⁺ Treg cells were present at a high frequency in the lamina propria of the gastrointestinal tracts, especially in the colonic lamina propria, of the mice kept under the environment free from specific pathogenic microorganisms (SPF) . In addition, it was also found that the number of the Foxp3⁺ Treg cells in the colonic lamina propria gradually increased up to three months after their birth, whereas the number of the Foxp3⁺ Treg cells in the peripheral lymph nodes was basically constant from the time of two weeks after their birth.

(Example 2)

0112Next, it was investigated whether or not the temporal accumulation of the Treg cells in the colon as found in Example 1 had a relationship with the colonization of intestinal commensal microbiota. Specifically, the expression of CD4 and the expression of Foxp3 in lymphocytes isolated from the small intestine, the colon, and the peripheral lymph nodes of mice bred under a germ-free (GF) or SPF environment (8 weeks old: Balb/c mice, IQI mice, and C57BL/6 mice) were analyzed. Similar results were obtained in three or more independent experiments. <figref idref="f0004">Figs. 6 and 7</figref> show the obtained results. Note that, in <figref idref="f0004">Fig. 7</figref>, each white circle represents the absolute number of CD4⁺Foxp3⁺ cells in an individual mouse, and the error bars represent standard deviations (SDs).

0113In addition, lamina propria lymphocytes were collected from SPF mice and GF mice (Balb/c mice or C57BL/6 mice) . CD4 and Foxp3 were stained with antibodies. Then, the lamina propria lymphocytes were analyzed by FACS. <figref idref="f0005">Fig. 8</figref> shows the obtained results. Note that in <figref idref="f0005">Fig. 8</figref> each white circle represents the absolute number of CD4⁺ Foxp3⁺ cells in an individual mouse, ** indicates that "P &lt; 0.001", and * indicates that "P &lt; 0.01."

0114Further, lymphocytes were isolated from the lamina propria of the colon, the lamina propria of the small intestine (SI), Peyer's patches (PPs), and mesenteric lymph nodes (MLNs) of mice (SPF C57BL/6 mice) to which antibiotics were orally administered with water for eight weeks. CD4 and Foxp3 were stained with antibodies. Then, the lymphocytes were analyzed by FACS. Similar results were obtained in two or more independent experiments. <figref idref="f0005">Fig. 9</figref> shows the obtained results (the ratio of the Foxp3⁺ cells in the CD4⁺ cells of an individual mouse) . Note that the following antibiotics were used in combination in accordance with the description in the following document: <ul id="ul0013" list-style="none" compact="compact"><li>ampicillin (A; 500 mg/L, Sigma)</li><li>vancomycin (V; 500 mg/L, NACALAI TESQUE, INC.)</li><li>metronidazole (M; 1g/L, NACALAI TESQUE, INC.)</li><li>neomycin (N; 1g/L, NACALAI TESQUE, INC.)</li></ul>

0115<nplcit id="ncit0026" npl-type="s"><text>Rakoff-Nahoum, J. Paglino, F. Eslami-Varzaneh, S. Edberg, R. Medzhitov, Cell 118, 229 (Jul 23, 2004</text></nplcit>) <nplcit id="ncit0027" npl-type="s"><text>Fagarasan et al., Science 298, 1424 (Nov 15, 2002</text></nplcit>)

0116In <figref idref="f0005">Fig. 9</figref>, each white circle represents the absolute number of the CD4⁺ Foxp3⁺ cells in an individual mouse, each horizontal bar represents the average value of the absolute numbers, * indicates that "P &lt; 0.01," and "AVMN" represents the kinds of the administered antibiotics by using the first letters of the antibiotics.

0117As is apparent from the results shown in <figref idref="f0004 f0005">Figs. 6 to 9</figref>, the frequencies and the absolute numbers of Foxp3⁺ CD4⁺ cells in the small intestine and the peripheral lymph nodes of the GF mice were equal to or greater than those of the SPF mice (refer to <figref idref="f0004 f0005">Figs. 6 to 8</figref>) . In addition, the numbers of the Treg cells in the small intestinal lamina propria, Peyer' s patches, and mesenteric lymph nodes of the SPF mice to which the antibiotics were orally administered for eight weeks were equal to or greater than those of the SPF mice (refer to <figref idref="f0005">Fig. 9</figref>). Meanwhile, the number of the Foxp3⁺ CD4⁺ cells in the colonic lamina propria of the GF mice was decreased significantly in comparison with that of the SPF mice (refer to <figref idref="f0004">Figs. 6 and 7</figref>). This decrease was commonly observed among mice of different genetic backgrounds (Balb/c, IQI, and C57BL/6), as well as among mice bred in different animal facilities (refer to <figref idref="f0004">Fig. 7</figref> for the data regarding the different genetic backgrounds, the data regarding the mice bred in the different animal facilities are not shown in the drawings) . In addition, it was also shown that the number of Treg cells in the colonic lamina propria of the SPF C57BL/6 mice to which the antibiotics were administered was decreased significantly (refer to <figref idref="f0005">Fig. 9</figref>).

(Example 3)

0118Next, it was directly checked whether or not the decrease in the number of the Treg cells in the colonic lamina propria of the GF mice shown in Example 2 was attributed to the absence of microbiota. Specifically, a fecal suspension of B6 SPF mice purchased from The Jackson Laboratory was orally administered to GF-IQI mice (conventionalization). Three weeks after the administration, lymphocytes were isolated from the colonic lamina propria, and the expression of Foxp3 in CD4⁺ lymphocytes was analyzed. <figref idref="f0006">Figs. 10 and 11</figref> show the obtained results. Note that each white circle in <figref idref="f0006">Fig. 11</figref> represents the absolute number of CD4⁺ Foxp3⁺ cells in an individual mouse, the error bars represent standard deviations (SD), * indicates that "P &lt; 0.01" in Student's t-test, and ** indicates that "P &lt; 0.001." As is apparent from the results shown in <figref idref="f0006">Figs. 10 and 11</figref>, the number of Treg cells in the small intestinal lamina propria did not change. However, the number of the Treg cells in the colonic lamina propria increased significantly. Hence, it was shown that host-microbial interaction played an important role in the accumulation of Foxp3⁺ Treg cells in the colonic lamina propria, while the accumulation of the Treg cells in the small intestinal lamina propria had a different mechanism.

(Example 4)

0119Next, the relationship between the gut-associated lymphoid tissues of mice and the number of Foxp3⁺ cells in the colonic lamina propria of the mice was investigated in accordance with the method described in <nplcit id="ncit0028" npl-type="s"><text>M. N. Kweon et al., J Immunol 174, 4365 (Apr 1, 2005</text></nplcit>). Specifically, 100 µg of an extracellular domain recombinant protein (a fusion protein (LTβR-Ig) between a lymphotoxin β receptor (LTβR) and a Fc region of human IgG1, refer to <nplcit id="ncit0029" npl-type="s"><text>Honda et al., J Exp Med 193, 621 (Mar 5, 2001</text></nplcit>)) was injected intraperitoneally into pregnant C57BL/6 mice 14 days after conception. The LTβR-Ig was again injected intraperitoneally into fetuses obtained from such mice, so that mice from which isolated lymphoid follicles (ILFs), Peyer's patches (PPs), and colonic-patches (CPs) were completely removed were produced. Then, the ratios of Foxp3⁺ cells in CD4⁺ cells in the colonic lamina propria of the mice treated with the LTβR-Ig, and mice treated with rat IgG (control) were analyzed by FACS. <figref idref="f0007">Fig. 12</figref> shows the obtained results. Note that in <figref idref="f0007">Fig. 12</figref> each white circle represents the ratio of Foxp3⁺ cells in an individual mouse, and the error bars represent standard deviations. As is apparent from the results shown in <figref idref="f0007">Fig. 12</figref>, it was found that the ratio of the Foxp3⁺ cells in the colonic lamina propria of the mice deficient in isolated lymphoid follicles, Peyer's patches, and the colonic-patches (the mice treated with the LTβR-Ig) rather increased. Accordingly, it was suggested that the decrease in the number of the Treg cells in the colonic lamina propria of the GF mice and the mice treated with the antibiotics was caused because the transmission of specific signals which promotes the accumulation of Treg cells in the colonic lamina propria and which is caused by the intestinal microbes did not occur, rather than simply because of a secondary effect of disorganized gut-associated lymphoid tissues.

(Example 5)

0120To investigate whether or not a specific intestinal flora induced the accumulation of colonic Treg cells, vancomycin as an antibiotic against Gram-positive bacteria or polymyxin B as an antibiotic against Gram-negative bacteria was administered to SPF mice (from 4 weeks of age) for four weeks, and analyzed for the ratio of Foxp3⁺ cells in the CD4⁺ cell group ([%] Foxp3⁺ in CD4). <figref idref="f0018">Fig. 30</figref> shows the obtained results. Note that, in <figref idref="f0018">Fig. 30</figref>, "SPF" indicates the result of SPF mice (control), "poly B" indicates the result of the SPF mice to which polymyxin B was administered, and "Vanco." indicates the result of the SPF mice to which vancomycin was administered. Meanwhile, * indicates that "P &lt; 0.01."

0121As is apparent from the results shown in <figref idref="f0018">Fig. 30</figref>, the number of Treg cells in the colon of the mice to which vancomycin was administered was markedly decreased in comparison with that of the control. In contrast, no influence was observed on the number of Treg cells of the mice to which polymyxin B was administered. Those facts suggested that Gram-positive commensal bacteria played a major role in accumulation of Treg cells.

(Example 6)

0122A recent report has suggested that spore-forming bacteria play an important role in intestinal T cells response (see <nplcit id="ncit0030" npl-type="s"><text>V. Gaboriau-Routhiau et al., Immunity 31, 677 (Oct 16, 2009</text></nplcit>)). In this respect, fecal microorganisms (spore-forming fraction) resistant to 3% chloroform were orally administered to GF mice, which were then analyzed for the ratio of Foxp3⁺ cells in the CD4⁺ cell group ([%] Foxp3⁺ in CD4) . <figref idref="f0018">Fig. 31</figref> shows the obtained results. Note that, in <figref idref="f0018">Fig. 31</figref>, "GF" indicates the result of GF mice, and "+chloro" indicates the result of the GF mice to which the chloroform-treated feces were administered. Meanwhile, ** indicates that "P &lt; 0.001."

0123As is apparent from the results shown in <figref idref="f0018">Fig. 31</figref>, three weeks after the administration of the chloroform-treated feces, the number of Treg cells in the administered mice was markedly increased to the same level as those of the SPF mice and the GF mice to which the untreated feces was forcibly administered (see <figref idref="f0004">Figs. 7</figref> and <figref idref="f0006">11</figref>) .

0124Accordingly, considering the results shown in Example 5 in combination, it was revealed that the specific components of the indigenous microbiota were highly likely to belong to the Gram-positive group, and that the spore-forming fraction played an important role in the induction of Treg cells.

(Example 7)

0125Next, the species of the intestinal microbiota which induced the accumulation of Treg cells in the colon as suggested in Examples 4 to 6 were identified. Specifically, segmented filamentous bacteria (SFB), 16 strains of the Bacteroides spp. (Bactero. (6 strains of <u>B. vulgatus,</u> 7 of the <u>B. acidifaciens group 1,</u> and 3 of the <u>B. acidifaciens</u> group 2)), 3 strains of the Lactobacillus (Lacto. <u>(L. acidophilus,</u><u>L. fermentum,</u> and <u>L. murinum</u>)), and 46 strains of Clostridium spp. (Clost., refer to "<nplcit id="ncit0031" npl-type="s"><text>Itoh, K., and Mitsuoka, T. Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice. Lab. Animals 19: 111-118 (1985</text></nplcit>))"), or microbiota collected from mice (SPF) bred under a conventional environment was orally administered to GF-Balb/c mice or GF-IQI mice. The mice were maintained in vinyl isolators for three weeks. Then, CD4 cells were isolated from the colon and the small intestine of these mice. The numbers of Treg cells in the colon and the small intestine were analyzed by flow cytometry.

0126<figref idref="f0007">Fig. 13</figref> shows FACS dot-plots obtained when a gate was set on CD4⁺ cells of the Balb/c mice. <figref idref="f0008">Fig. 14</figref> shows the ratio of Foxp3⁺ cells in CD4⁺ cells of each mouse.

0127Note that, the bacteria belonging to the genus Clostridium are classified by sequencing of 16S rRNA gene, as follows. Specifically, the 16S rRNA genes of the bacteria were amplified by PCR using 16S rRNA gene-specific primer pairs: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO: 19) and 5'-ATTACCGCGGCKGCTG-3' (SEQ ID NO: 20) (see <nplcit id="ncit0032" npl-type="s"><text>T. Aebischer et al., Vaccination prevents Helicobacter pylori-induced alterations of the gastric flora in mice. FEMS Immunol. Med. Microbiol. 46,221-229(2006</text></nplcit>)). The 1.5-kb PCR product was then introduced into pCR-Blunt Vector. The inserts were sequenced and aligned using the ClustalW software program. The resulting sequences of 16S rRNA genes derived from strain 1-41 of 46 strains of Clostridium spp. were shown in SEQ ID NO: 21-61. Phylogenetic tree which was constructed by the neighbor-joining method with the resulting sequences of the 41 strains of Clostridium and those of known bacteria obtained from Genbank database using Mega software was shown in <figref idref="f0029">Fig.49</figref>.

0128As is apparent from the results shown in <figref idref="f0007">Figs. 13</figref> and <figref idref="f0008">14</figref>, no effect on the number of the Treg cells in the colon was observed in the GF mice in which the segmented filamentous bacteria (SFB) were colonized (refer to <figref idref="f0008">Fig. 14</figref>) . Moreover, mice in which the cocktail of three strains of Lactobacillus was colonized gave similar results (refer to <figref idref="f0008">Fig. 14</figref>). On the other hand, it was shown that the accumulation of Foxp3⁺ cells in the colonic lamina propria was strongly induced in the mice in which 46 strains of Clostridium spp. were colonized. Importantly, such accumulation was promoted irrespective of the genetic backgrounds of the mice, and led to the increase in number similar to that in the SPF mice although intestinal microbiota of only a single genus were colonized. It was also shown that the colonization of the Clostridium did not change the number of Treg cells in the small intestinal lamina propria (refer to <figref idref="f0008">Fig. 14</figref>). Note that, when the 16 strains of Bactericides spp. were colonized, the number of Treg cells in the colon was increased significantly. However, the extent of the increase varied depending on the genetic background of the mice in which the bacteria were colonized (refer to <figref idref="f0007">Figs. 13</figref> and <figref idref="f0008">14</figref>).

(Example 8)

0129Next, CD4 expression, Foxp3 expression, and Helios expression in LP lymphocytes of the thymuses and the colons of SPF mice, GF mice, Lactobacillus-colonized mice, and Clostridium-colonized mice were analyzed by flow cytometry. <figref idref="f0019">Figs. 32</figref> and <figref idref="f0020">33</figref> show the obtained results. Note that, in <figref idref="f0019">Figs. 32</figref> and <figref idref="f0020">33</figref>, "GF" or "Germ Free" indicates the results of the GF mice, "SPF" indicates the results of the SPF mice, "Lacto." indicates the results of the Lactobacillus-colonized mice, and "Clost." indicates the results of the Clostridium-colonized mice. In <figref idref="f0019">Fig. 32</figref>, the vertical axis represents the ratio of Helios^- cells in the Foxp3⁺ cell group ([%] Helios^- in Foxp3⁺), and ** indicates that "P &lt; 0.001."

0130As is apparent from the results shown in <figref idref="f0019">Figs 32</figref> and <figref idref="f0020">33</figref>, most Foxp3⁺ cells found in the SPF mice or the Clostridium-colonized mice did not express Helios. Note that Helios is a transcription factor known to be expressed in thymic-derived natural Treg cells (see <nplcit id="ncit0033" npl-type="s"><text>A. M. Thornton et al., J Immunol 184, 3433 (Apr 1, 2010</text></nplcit>)). Accordingly, it was suggested that most of the Treg cells in the SPF mice and the Clostridium-colonized mice were Treg cells induced in peripheral portions, i.e., so-called iTreg cells.

(Example 9)

0131Next, it was investigated whether or not the colonization of the Clostridium or the like had an influence on other T cells. Specifically, SFB, 16 strains of Bacteroides spp. (Bactero.), 46 strains of Clostridium spp. (Clost.), or microbiota collected from mice bred under a conventional environment (SPF) was colonized in GF IQI mice. Three weeks later, lymphocytes in the colonic lamina propria were isolated from these mice, and stimulated with PMA (50 ng/ml) and ionomycin (1 ug/ml) for four hours in the presence of Golgistop (BD Bioscience) . After the stimulation was given, intracellular cytokines were stained by using an anti-IL-17 PE antibody (TC11-18H10) and an anti-IFN-g FITC antibody (BD Bioscience) in accordance with the manual of a cytofix/cytoperm kit (BD Bioscience) . Then, the ratio of IFN-γ⁺ cells or IL-17⁺ cells in CD4⁺ leucocytes was analyzed by flow cytometry. <figref idref="f0008">Figs. 15</figref> and <figref idref="f0009">16</figref> show the obtained results. Note that, in <figref idref="f0008">Figs. 15</figref> and <figref idref="f0009">16</figref>, each white circle represents the absolute number of CD4⁺ IFN-γ⁺ cells or the absolute number of CD4⁺ IL-17⁺ cells in each individual mouse, and the error bars represent standard deviations (SD) . As is apparent from the results shown in <figref idref="f0008">Figs. 15</figref> and <figref idref="f0009">16</figref>, the colonization of the Clostridium did not have any influence on Th1 cells (CD4⁺ IFN-γ⁺ cells) in the colon, and caused only a slight increase of Th17 cells (CD4⁺ IL-17⁺ cells). Accordingly, it was suggested that the genus Clostridium was a genus of bacteria which specifically induced Treg cells.

(Example 10)

0132It has been reported that 46 strains of Clostridium spp. exert an influence on the accumulation of CD8⁺ intestinal tract intraepithelial lymphocytes (IELs) in the colon. Accordingly, it is conceivable that Clostridium regulates the immune system in various aspects, and that Clostridium exhibits a marked ability to induce and maintain Treg cells especially in the colon, as described above. In addition, a kind of cytokines, transforming growth factor-β (TGF-β), is known to play an important role in regulation of Treg cell generation.

0133In this respect, it was examined whether or not the colonization of Clostridium provided a colonic environment rich in TGF-β. Specifically, first, the whole colons of GF mice, Clostridium-colonized mice, and Lactobacillus-colonized mice were cultured for 24 hours, and the culture supernatants thereof were measured for the concentration of active TGF-β (TGF-β1) by ELISA (the number of mice analyzed was four per group) . <figref idref="f0021">Fig. 34</figref> shows the obtained results. Note that, in <figref idref="f0021">Fig. 34</figref>, "GF" indicates the result of the GF mice, "Clost." indicates the result of the Clostridium-colonized mice, and "Lacto." indicates the result of Lactobacillus-colonized mice. Meanwhile, * indicates that "P &lt; 0.02," and ** indicates that "P &lt; 0.001."

0134As is apparent from the results shown in <figref idref="f0021">Fig. 34</figref>, the amount of TGF-β produced in the colons of the Clostridium-colonized mice was significantly larger than those of the GF mice and the Lactobacillus-colonized mice.

0135Next, intestinal epithelial cells (IECs) of GF mice and Clostridium-colonized mice were cultured for 24 hours, and the culture supernatants thereof were measured for the concentration of active TGF-β (TGF-β1) by ELISA (the number of mice analyzed was four per group) . <figref idref="f0021">Fig. 35</figref> shows the obtained results. Note that, in <figref idref="f0021">Fig. 35</figref>, "GF" indicates the result of the GFmice, and "Clost." indicates the result of the Clostridium-colonized mice. Meanwhile, ** indicates that "P &lt; 0.001."

0136As is apparent from the results shown in <figref idref="f0021">Fig. 35</figref>, TGF-β was detected in the culture supernatant of the IECs isolated from the Clostridium-colonized mice, whereas no TGF-β was detected in the culture supernatant of the IECs isolated from the GF mice.

0137Next, as described above, splenic CD4⁺ T cells were cultured for five days together with a 50% conditioned medium in which IECs isolated from the GF mice or the Clostridium-colonized mice were cultured, and with the anti-CD3 antibody, in the presence or absence of an anti-TGF-β antibody. Then, the T cells were collected, and analyzed for expression of Foxp3 by real-time RT-PCR. <figref idref="f0022">Fig. 36</figref> shows the obtained results. Note that, in <figref idref="f0022">Fig. 36</figref>, "Medium" indicates the result of a medium in which no cells were cultured, "GF" indicates the result of the conditioned medium in which the IECs of the GF mice were cultured, "Clost." indicates the result of the conditioned medium in which the IECs of the Clostridium-colonized mice were cultured, and "Clost. + αTGFβ" indicates the result of the conditioned medium to which the anti-TGF-β antibody was added and in which the IECs of the Clostridium-colonized mice were cultured. Meanwhile, ** indicates that "P &lt; 0.001."

0138As is apparent from the results shown in <figref idref="f0022">Fig. 36</figref>, when the culture supernatant of the IECs derived from the Clostridium-colonized mice was added to the splenic CD4⁺ T cells, the differentiation into Foxp3-expressing cells was accelerated. Meanwhile, the differentiation into the Treg cells was inhibited by the anti-TGF-β antibody.

0139Moreover, the expression of MMP2, MMP9, and MMP13, which are thought to contribute to the activation of latent TGF-β was investigated. The expression of indoleamine 2,3-dioxygenase (IDO), which is thought to be involved in the induction of Treg cells, was also investigated. Specifically, 46 bacterial strains of the genus Clostridium (Clost.), or three bacterial strains of the genus Lactobacillus (Lacto.) were orally administered to C57BL/6 germ-free mice. Three weeks after the administration, IECs were collected, and analyzed for relative mRNA expression levels of MMP2, MMP9, MMP13, and IDO genes by real-time RT-PCR (the number of mice analyzed was three per group). <figref idref="f0022 f0023 f0024">Figs. 37 to 40</figref> show the obtained results. Note that, in <figref idref="f0022 f0023 f0024">Figs. 37 to 40</figref>, "GF#1 to 3" indicate the results of GF mice, "Clost.#1 to 3" indicate the results of the Clostridium-colonized mice, and "Lacto.#1 to 3" indicate the results of the Lactobacillus-colonized mice.

0140For the relationship between the activation of latent TGF-β and the above-describe MMP, see <nplcit id="ncit0034" npl-type="s"><text>D'Angelo et al., J. Biol. Chem. 276, 11347-11353, 2001</text></nplcit>; <nplcit id="ncit0035" npl-type="s"><text>Heidinger et al., Biol. Chem. 387, 69-78, 2006</text></nplcit>; <nplcit id="ncit0036" npl-type="s"><text>Yu et al., Genes Dev. i4, 163-176, 2000</text></nplcit>. For the relationship between IDO and the induction of Treg cells, see <nplcit id="ncit0037" npl-type="s"><text>G. Matteoli et al., Gut 59, 595 (May, 2010</text></nplcit>).

0141As is apparent from the results shown in <figref idref="f0022 f0023">Figs 37 to 39</figref>, in agreement with the production of TGF-β described above, transcription products of the genes encoding MMP2, MMP9, and MMP13 were expressed at higher levels in the IECs derived from the Clostridium-colonized mice than those in the GF mice and in the Lactobacillus-colonized mice.

0142Moreover, as is apparent from the results shown in <figref idref="f0024">Fig. 40</figref>, IDO was expressed only in the Clostridium-colonized mice.

0143Accordingly, it was revealed that the Clostridium activated the IECs, and led to the production of TGF-β and other Treg cell-inducing molecules in the colon.

(Example 11)

0144Next, it was investigated whether or not the Treg cell accumulation induced by the colonization of the Clostridium was dependant on signal transmission by pathogen-associated molecular pattern recognition receptors. Specifically, the numbers of Treg cells in the colonic lamina propria of each of SPF mice of <u>Myd88</u>^-/- (deficient in Myd88 (signaling adaptor for Toll-like receptor)), <u>Rip2</u>^-/- (deficient in Rip2 (NOD receptor adaptor)), and <u>Card9</u>^-/- (deficient in Card9 (essential signal transmission factor for Dectin-1 signal transmission)) were examined. In addition, Clostridium spp. were caused to be colonized in the <u>Myd88</u>^-/-GF mice, and the change in the number of Treg cells was investigated. <figref idref="f0009">Figs. 17</figref> and <figref idref="f0010">18</figref> show the obtained results. As is apparent from the results shown in <figref idref="f0009">Figs. 17</figref> and <figref idref="f0010">18</figref>, the number of Treg cells of each kind of the SPF mice deficient in the associated factors of the pathogen-associated molecular pattern recognition receptors did not change relative to that of wild-type mice of the same litter, which served as a control. In addition, it was found that also when Clostridium spp. were colonized in GF mice deficient in Myd88, the accumulation of Treg cells in the colonic lamina propria was induced. Accordingly, it has been suggested that the mechanism of inducing the accumulation of Treg cells in the colonic lamina propria relies not on activation of recognition pathway for major pathogen-associated molecular patterns as is caused by most of bacterium, but on specific commensal bacterial species.

(Example 12)

0145Intestinal tract Foxp3⁺ Treg cells are known to exert some immunosuppressive functions through IL-10 production (refer to Non-Patent Document 9) . Meanwhile, animals having CD4⁺ Foxp3⁺ cells from which IL-10 is specifically removed are known to develop inflammatory bowel disease (refer to Non-Patent Document 18) . In this respect, first, the expression of IL-10 in lymphocytes of various tissues was examined. Specifically, lymphocytes were isolated from various tissues of SPF <u>Il10</u>^venus mice, and the expression of CD4 and the expression of Venus were analyzed by flow cytometry. <figref idref="f0011">Fig. 19</figref> shows the obtained results. Note that each numeric value in <figref idref="f0011">Fig. 19</figref> represents the ratio of cells within the corresponding one of regions divided into four.

0146Moreover, lymphocytes in the colonic lamina propria were isolated from <u>Il10</u>^venus mice, and the expression of T cell receptor β chain (TCRβ) on the surfaces of the cells was detected by FACS. <figref idref="f0012">Fig. 20</figref> shows the obtained results (FACS dot-plots obtained when a gate was set on CD4⁺ cells) . Note that each numeric value in <figref idref="f0012">Fig. 20</figref> represents the ratio of cells within the corresponding one of regions divided into four.

0147Furthermore, lymphocytes in the colonic lamina propria were isolated from <u>Il10</u>^venus mice. The lymphocytes were stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) for four hours in the presence of Golgistop (BD Bioscience) . Then, after the stimulation was given, intracellular cytokines were stained by using an anti-IL-17 PE antibody, an anti-IL-4 APC antibody (11B11), and an anti-IFN-g FITC antibody (BD Bioscience) in accordance with the manual of a cytofix/cytoperm kit (BD Bioscience) . <figref idref="f0012">Fig. 21</figref> shows the obtained results (FACS dot-plots obtained when a gate was set on CD4⁺ cells). Note that each numeric value in <figref idref="f0012">Fig. 21</figref> represents the ratio of cells within the corresponding one of regions divided into four.

0148In addition, Foxp3⁺ CD4⁺ cells and Foxp3^- CD4⁺ cells were isolated from the spleen (Spl) of Foxp3^eGFP reporter mice, and Venus⁺ cells were isolated from the colonic lamina propria and the small intestine (SI) lamina propria of <u>Il10</u>^venus mice. Then, the obtained cells were analyzed in terms of the expression of predetermined genes. The gene expression was analyzed by real-time RT-PCR using a Power SYBR Green PCR Master Mix (Applied Biosystems) and an ABI 7300 real time PCR system (Applied Biosystems). Here, the value for each cell was normalized for the amount of GAPDH. <figref idref="f0013">Fig. 22</figref> shows the obtained results. Note that in <figref idref="f0013">Fig. 22</figref> the error bars represent standard deviations. As is apparent from the results shown in <figref idref="f0011 f0012 f0013">Figs. 19 to 22</figref>, almost no Venus⁺ cells (IL-10-producing cells) were detected in the cervical lymph nodes (peripheral lymph nodes), thymus, peripheral blood, lung, and liver of mice kept under the SPF conditions. Meanwhile, in the spleen, Peyer's patches, and mesenteric lymph nodes thereof, Venus⁺ cells were slightly detected (refer to <figref idref="f0011">Fig. 19</figref>) . On the other hand, many Venus⁺ cells were found in the lymphocytes in the small intestine lamina propria and colonic lamina propria. In addition, most of the Venus⁺ cells in the intestines were positive for CD4, and also positive for T cell receptor β chain (TCRβ) (refer to <figref idref="f0011">Figs. 19</figref> and <figref idref="f0012">20</figref>). Moreover, it was found that the Venus⁺ CD4⁺ T cells expressed Foxp3 and other Treg cell-associated factors such as a cytotoxic T-Lymphocyte antigen (CTLA-4) and a glucocorticoid-induced TNFR-associated protein (GITR) although the Venus⁺ CD4⁺ T cells showed none of the phenotypes of Th2 (IL-4-producing) and Th17 (IL-17-producing) (refer to <figref idref="f0012">Figs. 21</figref> and <figref idref="f0013">22</figref>) . In addition, it was shown that the expression level of CTLA-4 in the intestinal Venus⁺ cells was higher than that in the splenic GFP⁺ Treg cells isolated from the Foxp3^eGFP reporter mice (refer to <figref idref="f0013">Fig. 22</figref>).

(Example 13)

0149Venus⁺ cells can be classified into at least two subsets, namely, Venus⁺ Foxp3⁺ double positive (DP) Treg cells and Venus⁺ Foxp3^- Treg cells on the basis of intracellular Foxp3 expression. Cells of the latter subset correspond to type 1 regulatory T cells (Tr1) (refer to Non-Patent Documents 8 and 9). In this respect, the Venus⁺ cells (IL-10-producing cells) observed in Example 8 were investigated in terms of the expression of Foxp3. Specifically, the expression of CD4, Foxp3, and Venus in the lamina propria of the colon and the lamina propria of the small intestine of <u>Il10</u>^venus mice kept under GF or SPF conditions was analyzed by FACS, and the numbers of Venus⁺ cells in the intestinal tract lamina propria were compared between SPF and GF <u>Il10</u>^Venus mice. <figref idref="f0013">Fig. 23</figref> shows the obtained results (dot-plots obtained when a gate was set on CD4⁺ cells).

0150In addition, the intracellular expression of Venus and Foxp3 in CD4 cells in various tissues of SPF <u>Il10</u>^venus mice was analyzed by flow cytometry. <figref idref="f0014">Fig. 24</figref> shows the obtained results (dot-plots obtained when a gate was set on CD4⁺ cells). Note that each numeric value in <figref idref="f0014">Fig. 24</figref> represents the ratio of cells within the corresponding one of regions divided into four.

0151Moreover, in order to investigate whether or not the presence of commensal bacteria had any influence on the expression of IL-10 in regulatory cells in the gastrointestinal tracts, germ-free (GF) <u>Il10</u>^Venus mice were prepared. Then, predetermined species of bacteria were caused to be colonized in the obtained GF <u>Il10</u>^venus mice. Three weeks after the species of bacteria were colonized, a CD4⁺ cell group (V⁺F^-, Venus⁺ Foxp3^-cells; V⁺F⁺, Venus⁺ Foxp3⁺ cells; and V-F⁺, Venus^-Foxp3⁺ cells) in which Foxp3 and/or Venus were expressed in the colon and the small intestine was analyzed by flow cytometry. <figref idref="f0015">Fig. 25</figref> shows dot-plots obtained when a gate was set on colonic CD4⁺ cells, and <figref idref="f0015">Figs. 26</figref> and <figref idref="f0016">27</figref> show the ratios in the CD4⁺ cell group of each mouse. Note that each numeric value in <figref idref="f0015">Fig. 25</figref> represents the ratio of cells within the corresponding one of regions divided into four. Meanwhile, the error bars in <figref idref="f0015">Figs. 26</figref> and <figref idref="f0016">27</figref> represent standard deviations, * indicates that "P &lt; 0.02," and ** indicates that "P &lt; 0.001."

0152Moreover, in order to check whether or not the presence of commensal bacteria had any influence on the expression of IL-10 in regulatory cells in the gastrointestinal tracts, antibiotics were orally given with water to five or six <u>Il10</u>^venus mice per group for 10 weeks. The following antibiotics were used in combination. ampicillin (A; 500 mg/L Sigma) vancomycin (V; 500 mg/L NACALAI TESQUE, INC.) metronidazole (M; 1 g/L NACALAI TESQUE, INC.) neomycin (N; 1 g/L NACALAI TESQUE, INC.)

0153Then, CD4 and Foxp3 of lymphocytes in the lamina propria of the colon, the lamina propria of the small intestine (SI) mesenteric lymph nodes (MLN), and Peyer's patches (PPs) were stained with antibodies, and analyzed by FACS. The results were obtained from two or more independent experiments which gave similar results. <figref idref="f0016">Fig. 28</figref> shows the obtained results (the ratio of Venus⁺ cells in CD4⁺ cells in each sample) . Note that each white circle in <figref idref="f0016">Fig. 28</figref> represents an individual sample, each horizontal bar represents an average value, * indicates that "P &lt; 0.02," and "AVMN" represents the kinds of the administered antibiotics by using the first letters of the antibiotics.

0154As is apparent from the results shown in <figref idref="f0013">Figs. 23</figref> and <figref idref="f0014">24</figref>, it was shown that the small intestinal lamina propria was rich in Venus⁺ Foxp3^- cells, namely, Tr1-like cells, and that the Venus⁺ Foxp3⁺ DP Treg cells were present at a high frequency in the colon of the SPF mice (refer to <figref idref="f0013">Figs. 23</figref> and <figref idref="f0014">24</figref>). In contrast, although sufficient numbers of Foxp3⁺ cells were observed also in other tissues, the expression of Venus was not observed in almost all of the cells (refer to <figref idref="f0014">Fig. 24</figref>).

0155In addition, as is apparent from the results shown in <figref idref="f0013">Figs. 23</figref> and <figref idref="f0015 f0016">25 to 28</figref>, it was shown that all regulatory T cell fractions of Venus⁺ Foxp3^-, Venus⁺ Foxp3⁺, and Venus^- Foxp3⁺ in the colon significantly decreased under the GF conditions (<figref idref="f0013">Figs. 23</figref> and <figref idref="f0015 f0016">26 to 27</figref>). Moreover, similar decrease in Venus⁺ cells was observed also in the SPF <u>Il10</u>^Venus mice treated with the antibiotics (refer to <figref idref="f0016">Fig. 28</figref>) .

0156Moreover, as is apparent from the results shown in <figref idref="f0015 f0016">Figs. 25 to 27</figref>, the colonization of Clostridium spp. strongly induced all regulatory T cell fractions of Venus⁺ Foxp3^-, Venus⁺ Foxp3⁺, and Venus^- Foxp3⁺ in the colon, and the degrees of the induction thereof were equal to those in the SPF mice (refer to <figref idref="f0015">Figs. 25</figref> and <figref idref="f0016">27</figref>). In addition, it was found that the colonization of the three strains of Lactobacillus or the colonization of SFB had an extremely small influence on the number of Venus⁺ and/or Foxp3⁺ cells in the colon (refer to <figref idref="f0015">Figs. 25</figref> and <figref idref="f0016">27</figref>). Moreover, the colonization of 16 strains of Bacteroides spp. also induced Venus⁺ cells, but the influence of the colonization was specific to Venus⁺ Foxp3^- Tr1-like cells (refer to <figref idref="f0015">Figs. 25</figref> and <figref idref="f0016">27</figref>). On the other hand, it was found that none of the bacterial species tested exerted any significant influence on the number of IL-10-producing cells in the small intestinal lamina propria (refer to <figref idref="f0015">Fig. 26</figref>).

0157Hence, it was shown that the genus Clostridium colonized in the colon or a physiologically active substance derived from the bacteria provided a signal for inducing the accumulation of IL-10⁺ regulatory T cells in the colonic lamina propria or the expression of IL-10 in T cells. Meanwhile, it was shown that the number of Venus⁺ cells in the small intestine was not significantly influenced by the situation where no commensal bacteria were present or commensal bacteria were decreased (refer to <figref idref="f0013">Figs. 23</figref> and <figref idref="f0015 f0016">26 to 28</figref>) and that IL-10⁺ regulatory cells (Tr1-like cells) accumulated in the small intestinal lamina propria independently of commensal bacteria.

(Example 14)

0158It was investigated whether or not Venus⁺ cells induced by the genus Clostridium had an immunosuppressive function similar to that of Venus⁺ cells in the colon of SPF mice. Specifically, CD4⁺ CD25^- cells (effector T cells, Teff cells) isolated from the spleen were seeded in a flat-bottomed 96-well plate at 2 x 10⁴/well, and cultured for three days together with 2 x 10⁴ splenic CD11c⁺ cells (antigen-representing cells) subjected to 30 Gy radiation irradiation treatment, 0.5 µg/ml of an anti-CD3 antibody, and a lot of Treg cells. In addition, for the last six hours, the CD4⁺ CD25^- cells were cultured, with [³H] -thymidine (1 µCi/well) was added thereto. Note that, Treg cells used in Example 14 were CD4⁺ GFP⁺ T cells isolated from the spleen of Foxp3^eGFP reporter mice, or CD4⁺ Venus⁺ T cells in the colonic lamina propria of GF <u>Il10</u>^venus mice in which Clostridium spp. were colonized or SPF <u>Il10</u>^venus mice. Then, proliferation of the cells was determined based on the uptake amount of [³H]-thymidine, and represented by a count per minute (cpm) value.

0159As is apparent from the results shown in <figref idref="f0017">Fig. 29</figref>, Venus⁺ CD4⁺ cells of the mice in which the genus Clostridium was colonized suppressed in vitro proliferation of CD25^- CD4⁺ activated T cells. The suppression activity was slightly inferior to that of GFP⁺ cells isolated from the Foxp3^eGFP reporter mice, but equal to that of Venus⁺ cells isolated from the SPF <u>Il10</u>^Venus mice. Accordingly, it has been shown that the genus Clostridium induces IL-10-expressing T cells having sufficient immunosuppressive activities, and thereby plays a critical role in maintaining immune homeostasis in the colon.

(Example 15)

0160Next, the influence, on the local immune response, of the colonization of a large number of Clostridium and the resultant proliferation of Treg cells was investigated.

<Dextran Sulfate Sodium (DSS)-Induced Colitis Model>

0161First, the DSS-induced colitis model was prepared as described above, and the influence, on the model mice, of the inoculation of the Clostridium and the proliferation of Treg cells was investigated. Specifically, control mice and Clostridium-inoculated mice were treated with 2% DSS, then observed and measured for six days for the body weight loss, the hardness of stool, and bleeding, and then were evaluated numerically. In addition, on day 6, the colons were collected, dissected, and analyzed histologically by HE staining. <figref idref="f0024 f0025">Figs. 41 to 43</figref> show the obtained results. Note that, in <figref idref="f0024 f0025">Figs. 41 to 43</figref>, "SPF+Clost." or "SPF+Clost.#1 to 3" indicate the results of C57BL/6 mice inoculated with a fecal suspension of Clostridium-colonized mice, and grown in a conventional environment for six weeks, and "SPF" or "SPF#1 to 3" indicate the results of C57BL/6 mice (control mice) grown in a conventional environment for six weeks without being inoculated with the fecal suspension. In addition, in <figref idref="f0024">Fig. 41</figref>, the vertical axis "Disease score" represents the disease activity index (DAI) described above, and the horizontal axis "post 2% DSS (d)" represents the days elapsed after the initial administration of 2% DSS to the mice. Moreover, in <figref idref="f0024">Fig. 41</figref>, * indicates that "P &lt; 0.02," and ** indicates that "P &lt; 0.001." Meanwhile, Treg cells induced by regulatory dendritic cells are known to play a preventive role in a DSS-induced colitis model (see <nplcit id="ncit0038" npl-type="s"><text>S. Manicassamy et al., Science 329, 849 (Aug 13, 2010</text></nplcit>)).

0162As is apparent from the results shown in <figref idref="f0024 f0025">Figs. 41 to 43</figref>, the symptoms of the colitis such as body weight loss and rectal bleeding were significantly suppressed in the mice having a large number of Clostridium (hereinafter also referred to as "Clostridium-abundant mice") in comparison with the control mice (see <figref idref="f0024">Fig. 41</figref>). All the features typical for colonic inflammation, such as shortening of the colon, edema, and hemorrhage, were observed markedly in the control mice in comparison with the Clostridium-abundant mice (see <figref idref="f0025">Fig. 42</figref>). Moreover, histological features such as mucosal erosion, edema, cellular infiltration, and crypt loss were less severe in the DSS-treated Clostridium-abundant mice than in the control mice (see <figref idref="f0025">Fig. 43</figref>).

<Oxazolone-Induced Colitis Model>

0163Next, the oxazolone-induced colitis model was prepared as described above, and the influence, on the model mice, of the inoculation of Clostridium and the proliferation of Treg cells was investigated. Specifically, control mice and Clostridium-inoculated mice were sensitized with oxazolone, and subsequently the inside of the rectums thereof were treated with a 1% oxazolone/50% ethanol solution. Then, the body weight loss was observed and measured. In addition, the colons were dissected, and analyzed histologically by HE staining. <figref idref="f0026">Figs. 44 and 45</figref> show the obtained results. Note that, in <figref idref="f0026">Figs. 44 and 45</figref>, "SPF+Clost." indicates the results of C57BL/6 mice (Clostridium-abundant mice) inoculated with a fecal suspension of Clostridium-colonized mice, and grown in a conventional environment for six weeks, and "SPF" indicates the results of C57BL/6 mice (control mice) grown in a conventional environment for six weeks without being inoculated with the fecal suspension. In addition, in <figref idref="f0026">Fig. 44</figref>, the vertical axis "Weight (% of initial) " represents the body weight after the administration of 1% oxazolone where the body weight before the administration was taken as 100%, and the horizontal axis "post 1% oxazolone (d)" represents the days elapsed after the administration of 1% oxazolone to the mice. Meanwhile, it is known that Th2-type T cells are involved in colitis induced by oxazolone. (see <nplcit id="ncit0039" npl-type="s"><text>M. Boirivant, I. J. Fuss, A. Chu, W. Strober, J Exp Med 188, 1929 (Nov 16, 1998</text></nplcit>)).

0164As is apparent from the results shown in <figref idref="f0026">Figs. 44 and 45</figref>, the colitis proceeded along with persistent body weight loss in the control mice. Meanwhile, the body weight loss of the Clostridium-abundant mice was reduced (see <figref idref="f0026">Fig. 44</figref>). In addition, it was also revealed that portions having histological diseases such as mucosal erosion, edema, cellular infiltration , and hemorrhage were reduced in the colon of the Clostridium-abundant mice (see <figref idref="f0026">Fig. 45</figref>).

(Example 16)

0165Next, the influence, on the systemic immune response (systemic IgE production), of the colonization of a large number of Clostridium and the resultant proliferation of Treg cells was investigated. Specifically, as described above, control mice and Clostridium-inoculated mice were immunized by administering alum-absorbed ovalbumin (OVA) twice at a 2-week interval. Then, sera were collected from these mice, and the OVA-specific IgE level thereof was investigated by ELISA. In addition, splenic cells were collected from the mice in each group, and IL-4 and IL-10 production by in-vitro OVA restimulation was investigated. <figref idref="f0027 f0028">Figs. 46 to 48</figref> show the obtained results. Note that, in <figref idref="f0027 f0028">Figs. 46 to 48</figref>, "SPF+Clost." indicates the results of BALB/c SPF mice (Clostridium-abundant mice) inoculated with a fecal suspension of Clostridium-colonized mice, and grown in a conventional environment, "SPF" indicates the results of BALB/c SPF mice (control mice) grown in a conventional environment without being inoculated with the fecal suspension, and ** indicates that "P &lt; 0.001." Meanwhile, in <figref idref="f0027">Fig. 46</figref>, the vertical axis "OVA-specific IgE (ng/ml)" represents the concentration of OVA-specific IgE in the sera. Moreover, in <figref idref="f0027">Fig. 46</figref>, the horizontal axis represents the days elapsed after the initial administration of the alum-absorbed ovalbumin to the Clostridium-abundant mice or the control mice (4-week old), and "OVA+Alum" indicates the timing of the administration of the alum-absorbed ovalbumin. In addition, in <figref idref="f0027">Figs. 47</figref> and <figref idref="f0028">48</figref>, "OVA" on the horizontal axis indicates the results in the case where the in-vitro OVA restimulation was performed, and "-" indicates the results in the case where no in-vitro OVA restimulation was performed. Moreover, in <figref idref="f0027">Figs. 47</figref> and <figref idref="f0028">48</figref>, the vertical axes "IL-4 (pg/ml)" and "IL-10 (pg/ml)" show the IL-4 concentration and the IL-10 concentration in culture supernatants of splenic cells, respectively.

0166As is apparent from the results shown in <figref idref="f0027 f0028">Figs. 46 to 48</figref>, the IgE level was significantly lower in the Clostridium-abundant mice than in the control mice (see <figref idref="f0027">Fig. 46</figref>). Moreover, the IL-4 production by the OVA restimulation was reduced (see <figref idref="f0027">Fig. 47</figref>) and the IL-10 production thereby was increased (see <figref idref="f0028">Fig. 48</figref>) in the splenic cells of the Clostridium-abundant mice sensitized with OVA and alum, in comparison with those of the control mice.

0167Accordingly, in consideration of the results shown in Example 15 in combination, it has been revealed that the induction of Treg cells by Clostridium in the colon plays an important role in local and systemic immune responses.

(Example 17)

0168Next, GF Balb/c were colonized with three strains of Clostridium belonging to cluster IV(strains 22, 23 and 32 listed in <figref idref="f0029">Fig.49</figref>). Three weeks later, colonic Foxp3⁺ Treg cells were analyzed by FACS. <figref idref="f0030">Fig.50</figref> shows the obtained results. As is apparent from the results shown in <figref idref="f0030">Fig. 50</figref>, gnotobiotic mice colonized with three strains of Clostridium showed an intermediate pattern of Treg induction between GF mice and mice inoculated with all 46 strains.

(Example 18)

0169Next, it was investigated whether or not a spore-forming (for example, a chloroform resistant) fraction of a fecal sample obtained from humans had the effect of inducing proliferation or accumulation of regulatory T cells similar to the spore-forming fraction of the fecal sample obtained from mice.

0170Specifically, human stool from a healthy volunteer (Japanese, male, 29years old) was suspended with phosphate-buffered saline (PBS), mixed with chloroform (final concentration 3%), and then incubated in a shaking water bath for 60 min. After evaporation of chloroform by bubbling with N₂ gas, the aliquots containing chloroform-resistant (for example, spore-forming) fraction of human intestinal bacteria were orally inoculated into germ-free (GF) mice (IQI, 8 weeks old). The treated mice were kept in a vinyl isolator for 3 weeks. The colon was collected and opened longitudinally, washed to remove fecal content, and shaken in Hanks' balanced salt solution (HBSS) containing 5 mM EDTA for 20 min at 37°C. After removing epithelial cells and fat tissue, the colon was cut into small pieces and incubated with RPMI1640 containing 4% fetal bovine serum, 1 mg/ml collagenase D, 0.5 mg/ml dispase and 40µg/ml DNase I (all manufactured by Roche Diagnostics) for 1 hour at 37°C in a shaking water bath. The digested tissue was washed with HBSS containing 5 mMEDTA, resuspended in 5 ml of 40% Percoll (manufactured by GE Healthcare) and overlaid on 2.5 ml of 80% Percoll in a 15-ml Falcon tube. Percoll gradient separation was performed by centrifugation at 780 g for 20 min at 25°C. The interface cells were collected and suspended in staining buffer containing PBS, 2% FBS, 2 mM EDTA and 0.09% NaN₃ and stained for surface CD4 with Phycoerythrin-label ed anti-CD4 Ab (RM4-5, manufactured by BD Biosciences). Intracellular staining of Foxp3 was performed using the Alexa647-labeled anti-Foxp3 Ab (FJK-16s, manufactured by eBioscience) and Foxp3 Staining Buffer Set (manufactured by eBioscience). The percentage of Foxp3positive cells within the CD4 positive lymphocyte population was analyzed by flow cytometry. <figref idref="f0031">Figs. 51 and 52</figref> show the obtained results.

0171In figures, representative histograms (<figref idref="f0031">Fig. 51</figref>) and combined data (<figref idref="f0031">Fig. 52</figref>) for Foxp3 expression by CD4 positive lymphocytes from GFmice (GF) or GF mice gavaged with chloroform-treated human stool (GF+Chloro.) are shown. In addition, numbers in <figref idref="f0031">Fig. 51</figref> indicate the percentages of cells in the gate. Each circle in <figref idref="f0031">Fig. 52</figref> represents a separate animal, error bars indicate the SD, and ** indicates that "P &lt; 0.001."

0172As is apparent from the results shown in <figref idref="f0031">Figs. 51 and 52</figref>, it was found that also when the spore-forming (for example, the chloroform resistant) fraction of human intestinal bacteria was colonized in GF mice, the accumulation of Foxp3⁺ regulatory (Treg) cells in the colonic lamina propria of the mice was induced.

0173Next, it was investigated what species of bacteria grew by gavaging with chloroform-treated human stool.

0174Specifically, using a QIAamp DNA Stool mini kit (manufactured by QIAGEN), bacterial genomic DNA was isolated from the human stool from a healthy volunteer as described above (human stool) or fecal pellets from GFmice gavaged with chloroform-treated human stool (GF+Chloro.) . Quantitative PCR analysis was carried out using a LightCycler 480 (manufactured by Roche). Relative quantity was calculated by the ΔCt method and normalized to the amount of total bacteria, dilution, and weight of the sample. The following primer sets were used: <ul id="ul0014" list-style="none" compact="compact"><li>total bacteria <ul id="ul0015" list-style="none" compact="compact"><li>5'-GGTGAATACGTTCCCGG-3' (SEQ ID NO: 62) and</li><li>5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 63)</li></ul></li><li>Clostridium cluster XIVa (Clostridium coccoides subgroup) <ul id="ul0016" list-style="none" compact="compact"><li>5'-AAATGACGGTACCTGACTAA-3' (SEQ ID NO: 64) and</li><li>5'-CTTTGAGTTTCATTCTTGCGAA-3' (SEQ ID NO: 65)</li></ul></li><li>Clostridium cluster IV (Clostridium leptum) <ul id="ul0017" list-style="none" compact="compact"><li>5'-GCACAAGCAGTGGAGT-3' (SEQ ID NO: 66) and</li><li>5'-CTTCCTCCGTTTTGTCAA-3' (SEQ ID NO: 24)</li></ul></li><li>Bacteroides <ul id="ul0018" list-style="none" compact="compact"><li>5'-GAGAGGAAGGTCCCCCAC-3' (SEQ ID NO: 67) and</li><li>5'-CGCTACTTGGCTGGTTCAG-3' (SEQ ID NO: 68).</li></ul></li></ul>

0175<figref idref="f0032">Fig. 53</figref> shows the obtained results.

0176As is apparent from the results shown in <figref idref="f0032">Fig. 53</figref>, mice gavaged with chloroform-treated human stool exhibited high amounts of spore-forming bacteria, such as Clostridium clusters XIVa and IV, and a severe decrease of non-spore-forming bacteria, such as Bacteroides, compared with the human stool before chloroform treatment.

[Industrial Applicability]

0177As has been described above, the present disclosure makes it possible to provide an excellent composition for inducing proliferation or accumulation of regulatory T cells (Treg cells) by utilizing bacteria belonging to the genus Clostridium or a physiologically active substance or the like derived from the bacteria. Since the composition of the present invention has immunosuppressive effects, the composition can be used as defined in the claims.

0178Healthy individuals can easily and routinely ingest the composition as a food or beverage, such as a health food, to improve their immune functions.

[Sequence Listing]

0179<ul id="ul0019" list-style="none" compact="compact"><li>SEQ ID NO:1 to 20, 62 to 69 <223> Artificially synthesized primer sequence</li><li>SEQ ID NO:21 to 61 <223> 16S rRNA coding gene sequence of each Clostridium strain</li></ul>

SEQUENCE LISTING

0180<ul id="ul0020" list-style="none"><li><110> The University of Tokyo</li><li><120> COMPOSITION FOR INDUCING PROLIFERATION OR ACCUMULATION OF REGULATORY T CELLS</li><li><130> 215208</li><li><150> <patcit id="pcit0003" dnum="JP2010129134A"><text>JP2010/129134</text></patcit> <151> 2010-06-04</li><li><150> <patcit id="pcit0004" dnum="JP2010071746W" dnum-type="L"><text>PCT/JP2010/071746</text></patcit> <151> 2010-12-03</li><li><160> 69</li><li><170> PatentIn version 3.5</li><li><210> 1 <211> 23 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 1 ggcaatagtt ccttcccaga gtt   23</li><li><210> 2 <211> 22 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 2 gggtcgcata ttgtggtact tg   22</li><li><210> 3 <211> 23 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 3 ccttttgtag ccctgctcac tct 23</li><li><210> 4 <211> 21 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 4 gggtcacctg tatggcttca g   21</li><li><210> 5 <211> 21 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 5 tcagtgcaag atctgcaagc a   21</li><li><210> 6 <211> 19 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 6 acaccggaag ccaaacaca   19</li><li><210> 7 <211> 27 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 7 gattttaata agctccaaga ccaaggt   27</li><li><210> 8 <211> 27 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 8 cttctatgca gttgatgaag atgtcaa   27</li><li><210> 9 <211> 21 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 9 cctcgtcccg tagacaaaat g 21</li><li><210> 10 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 10 tctccacttt gccactgcaa   20</li><li><210> 11 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 11 ggacattgtc tttgatggca   20</li><li><210> 12 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 12 cttgtcacgt ggtgtcactg 20</li><li><210> 13 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 13 tctctggacg tcaaatgtgg 20</li><li><210> 14 <211> 19 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 14 gctgaacagc agagccttc   19</li><li><210> 15 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 15 aggtctggat cactccaagg   20</li><li><210> 16 <211> 19 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 16 tcgcctggac cataaagaa   19</li><li><210> 17 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 17 agaggatgcg tgactttgtg   20</li><li><210> 18 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 18 atacagcaga ccttctggca   20</li><li><210> 19 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 19 agagtttgat cmtggctcag   20</li><li><210> 20 <211> 16 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 20 attaccgcgg ckgctg   16</li><li><210> 21 <211> 1460 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1460) <223> 16S rRNA coding gene sequence of Clostridium strain 1</li><li><400> 21 <img file="EP3552613B1_D0001.tif" /><img file="EP3552613B1_D0002.tif" /></li><li><210> 22 <211> 1485 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1485) <223> 16S rRNA coding gene sequence of Clostridium strain 2</li><li><400> 22 <img file="EP3552613B1_D0003.tif" /><img file="EP3552613B1_D0004.tif" /></li><li><210> 23 <211> 1491 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1491) <223> 16S rRNA coding gene sequence of Clostridium strain 3</li><li><400> 23 <img file="EP3552613B1_D0005.tif" /><img file="EP3552613B1_D0006.tif" /></li><li><210> 24 <211> 1491 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1491) <223> 16S rRNA coding gene sequence of Clostridium strain 4</li><li><400> 24 <img file="EP3552613B1_D0007.tif" /><img file="EP3552613B1_D0008.tif" /></li><li><210> 25 <211> 1467 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1467) <223> 16S rRNA coding gene sequence of Clostridium strain 5</li><li><400> 25 <img file="EP3552613B1_D0009.tif" /><img file="EP3552613B1_D0010.tif" /></li><li><210> 26 <211> 1474 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1474) <223> 16S rRNA coding gene sequence of Clostridium strain 6</li><li><400> 26 <img file="EP3552613B1_D0011.tif" /><img file="EP3552613B1_D0012.tif" /></li><li><210> 27 <211> 1484 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1484) <223> 16S rRNA coding gene sequence of Clostridium strain 7</li><li><400> 27 <img file="EP3552613B1_D0013.tif" /><img file="EP3552613B1_D0014.tif" /></li><li><210> 28 <211> 1483 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1483) <223> 16S rRNA coding gene sequence of Clostridium strain 8</li><li><400> 28 <img file="EP3552613B1_D0015.tif" /><img file="EP3552613B1_D0016.tif" /></li><li><210> 29 <211> 1480 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1480) <223> 16S rRNA coding gene sequence of Clostridium strain 9</li><li><400> 29 <img file="EP3552613B1_D0017.tif" /><img file="EP3552613B1_D0018.tif" /></li><li><210> 30 <211> 1489 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1489) <223> 16S rRNA coding gene sequence of Clostridium strain 10</li><li><400> 30 <img file="EP3552613B1_D0019.tif" /><img file="EP3552613B1_D0020.tif" /></li><li><210> 31 <211> 1490 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1490) <223> 16S rRNA coding gene sequence of Clostridium strain 11</li><li><400> 31 <img file="EP3552613B1_D0021.tif" /><img file="EP3552613B1_D0022.tif" /></li><li><210> 32 <211> 1489 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1489) <223> 16S rRNA coding gene sequence of Clostridium strain 12</li><li><400> 32 <img file="EP3552613B1_D0023.tif" /><img file="EP3552613B1_D0024.tif" /></li><li><210> 33 <211> 1456 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1456) <223> 16S rRNA coding gene sequence of Clostridium strain 13</li><li><400> 33 <img file="EP3552613B1_D0025.tif" /><img file="EP3552613B1_D0026.tif" /></li><li><210> 34 <211> 1475 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1475) <223> 16S rRNA coding gene sequence of Clostridium strain 14</li><li><400> 34 <img file="EP3552613B1_D0027.tif" /><img file="EP3552613B1_D0028.tif" /></li><li><210> 35 <211> 1480 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1480) <223> 16S rRNA coding gene sequence of Clostridium strain 15</li><li><400> 35 <img file="EP3552613B1_D0029.tif" /><img file="EP3552613B1_D0030.tif" /></li><li><210> 36 <211> 1486 <212> DNA <213> Clostridium papyrosolvens</li><li><220> <221> rRNA <222> (1)..(1486) <223> 16S rRNA coding gene sequence of Clostridium strain 16</li><li><400> 36 <img file="EP3552613B1_D0031.tif" /><img file="EP3552613B1_D0032.tif" /></li><li><210> 37 <211> 1493 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1493) <223> 16S rRNA coding gene sequence of Clostridium strain 17</li><li><400> 37 <img file="EP3552613B1_D0033.tif" /><img file="EP3552613B1_D0034.tif" /></li><li><210> 38 <211> 1493 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1493) <223> 16S rRNA coding gene sequence of Clostridium strain 18</li><li><400> 38 <img file="EP3552613B1_D0035.tif" /><img file="EP3552613B1_D0036.tif" /></li><li><210> 39 <211> 1483 <212> DNA <213> Clostridium leptum</li><li><220> <221> misc_feature <222> (1)..(1483) <223> 16S rRNA coding gene sequence of Clostridium strain 19</li><li><400> 39 <img file="EP3552613B1_D0037.tif" /><img file="EP3552613B1_D0038.tif" /></li><li><210> 40 <211> 1511 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1511) <223> 16S rRNA coding gene sequence of Clostridium strain 20</li><li><400> 40 <img file="EP3552613B1_D0039.tif" /><img file="EP3552613B1_D0040.tif" /></li><li><210> 41 <211> 1495 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1495) <223> 16S rRNA coding gene sequence of Clostridium strain 21</li><li><400> 41 <img file="EP3552613B1_D0041.tif" /><img file="EP3552613B1_D0042.tif" /></li><li><210> 42 <211> 1491 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1491) <223> 16S rRNA coding gene sequence of Clostridium strain 22</li><li><400> 42 <img file="EP3552613B1_D0043.tif" /><img file="EP3552613B1_D0044.tif" /></li><li><210> 43 <211> 1495 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1495) <223> 16S rRNA coding gene sequence of Clostridium strain 23</li><li><400> 43 <img file="EP3552613B1_D0045.tif" /><img file="EP3552613B1_D0046.tif" /></li><li><210> 44 <211> 1440 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1440) <223> 16S rRNA coding gene sequence of Clostridium strain 24</li><li><400> 44 <img file="EP3552613B1_D0047.tif" /><img file="EP3552613B1_D0048.tif" /></li><li><210> 45 <211> 1495 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1495) <223> 16S rRNA coding gene sequence of Clostridium strain 25</li><li><400> 45 <img file="EP3552613B1_D0049.tif" /><img file="EP3552613B1_D0050.tif" /></li><li><210> 46 <211> 1495 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1495) <223> 16S rRNA coding gene sequence of Clostridium strain 26</li><li><400> 46 <img file="EP3552613B1_D0051.tif" /><img file="EP3552613B1_D0052.tif" /></li><li><210> 47 <211> 1509 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1509) <223> 16S rRNA coding gene sequence of Clostridium strain 27</li><li><400> 47 <img file="EP3552613B1_D0053.tif" /><img file="EP3552613B1_D0054.tif" /></li><li><210> 48 <211> 1583 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1583) <223> 16S rRNA coding gene sequence of Clostridium strain 28</li><li><400> 48 <img file="EP3552613B1_D0055.tif" /><img file="EP3552613B1_D0056.tif" /></li><li><210> 49 <211> 1519 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1519) <223> 16S rRNA coding gene sequence of Clostridium strain 29</li><li><400> 49 <img file="EP3552613B1_D0057.tif" /><img file="EP3552613B1_D0058.tif" /></li><li><210> 50 <211> 1497 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1497) <223> 16S rRNA coding gene sequence of Clostridium strain 30</li><li><400> 50 <img file="EP3552613B1_D0059.tif" /><img file="EP3552613B1_D0060.tif" /></li><li><210> 51 <211> 1475 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1475) <223> 16S rRNA coding gene sequence of Clostridium strain 31</li><li><400> 51 <img file="EP3552613B1_D0061.tif" /><img file="EP3552613B1_D0062.tif" /></li><li><210> 52 <211> 1491 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1491) <223> 16S rRNA coding gene sequence of Clostridium strain 32</li><li><400> 52 <img file="EP3552613B1_D0063.tif" /><img file="EP3552613B1_D0064.tif" /></li><li><210> 53 <211> 1495 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1495) <223> 16S rRNA coding gene sequence of Clostridium strain 33</li><li><400> 53 <img file="EP3552613B1_D0065.tif" /><img file="EP3552613B1_D0066.tif" /></li><li><210> 54 <211> 1493 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1493) <223> 16S rRNA coding gene sequence of Clostridium strain 34</li><li><400> 54 <img file="EP3552613B1_D0067.tif" /><img file="EP3552613B1_D0068.tif" /></li><li><210> 55 <211> 1498 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1498) <223> 16S rRNA coding gene sequence of Clostridium strain 35</li><li><400> 55 <img file="EP3552613B1_D0069.tif" /><img file="EP3552613B1_D0070.tif" /></li><li><210> 56 <211> 1491 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1491) <223> 16S rRNA coding gene sequence of Clostridium strain 36</li><li><400> 56 <img file="EP3552613B1_D0071.tif" /><img file="EP3552613B1_D0072.tif" /></li><li><210> 57 <211> 1493 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1493) <223> 16S rRNA coding gene sequence of Clostridium strain 37</li><li><400> 57 <img file="EP3552613B1_D0073.tif" /><img file="EP3552613B1_D0074.tif" /></li><li><210> 58 <211> 1493 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1493) <223> 16S rRNA coding gene sequence of Clostridium strain 38</li><li><400> 58 <img file="EP3552613B1_D0075.tif" /><img file="EP3552613B1_D0076.tif" /></li><li><210> 59 <211> 1511 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1511) <223> 16S rRNA coding gene sequence of Clostridium strain 39</li><li><400> 59 <img file="EP3552613B1_D0077.tif" /><img file="EP3552613B1_D0078.tif" /></li><li><210> 60 <211> 1499 <212> DNA <213> Clostridium leptum</li><li><220> <221> rRNA <222> (1)..(1499) <223> 16S rRNA coding gene sequence of Clostridium strain 40</li><li><400> 60 <img file="EP3552613B1_D0079.tif" /><img file="EP3552613B1_D0080.tif" /></li><li><210> 61 <211> 1512 <212> DNA <213> Clostridium coccoides</li><li><220> <221> rRNA <222> (1)..(1512) <223> 16S rRNA coding gene sequence of Clostridium strain 41</li><li><400> 61 <img file="EP3552613B1_D0081.tif" /><img file="EP3552613B1_D0082.tif" /></li><li><210> 62 <211> 17 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 62 ggtgaatacg ttcccgg 17</li><li><210> 63 <211> 22 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 63 tacggctacc ttgttacgac tt 22</li><li><210> 64 <211> 20 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 64 aaatgacggt acctgactaa 20</li><li><210> 65 <211> 22 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 65 ctttgagttt cattcttgcg aa 22</li><li><210> 66 <211> 16 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 66 gcacaagcag tggagt 16</li><li><210> 67 <211> 18 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 67 cttcctccgt tttgtcaa 18</li><li><210> 68 <211> 18 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 68 gagaggaagg tcccccac 18</li><li><210> 69 <211> 19 <212> DNA <213> Artificial</li><li><220> <223> Artificially synthesized primer sequence</li><li><400> 69 cgctacttgg ctggttcag 19</li></ul>