Regulation of Th17 cell differentiation by intestinal commensal bacteria.

Interleukin (IL)-17-producing CD4 T cells ('Th17 cells') are most abundant at the intestinal mucosa, and play a critical role in the maintenance of mucosal barrier function. Recent studies indicate that accumulation of intestinal Th17 cells depends on stimulation by intestinal commensal bacteria - particularly by segmented filamentous bacterium. In this review, we summarise recent advances in our understanding of the mechanisms of intestinal Th17 synthesis in mice, and discuss their relevance to infectious and inflammatory diseases.

Altered profiles of intestinal microbiota and organic acids may be the origin of symptoms in irritable bowel syndrome.

BACKGROUND: The profile of intestinal organic acids in irritable bowel syndrome (IBS) and its correlation with gastrointestinal (GI) symptoms are not clear. We hypothesized in this study that altered GI microbiota contribute to IBS symptoms through increased levels of organic acids. METHODS: Subjects were 26 IBS patients and 26 age- and sex-matched controls. Fecal samples were collected for microbiota analysis using quantitative real-time polymerase chain reaction and culture methods, and the determination of organic acid levels using high-performance liquid chromatography. Abdominal gas was quantified by image analyses of abdominal X-ray films. Subjects completed a questionnaire for GI symptoms, quality of life (QOL) and negative emotion. KEY RESULTS: Irritable bowel syndrome patients showed significantly higher counts of Veillonella (P = 0.046) and Lactobacillus (P = 0.031) than controls. They also expressed significantly higher levels of acetic acid (P = 0.049), propionic acid (P = 0.025) and total organic acids (P = 0.014) than controls. The quantity of bowel gas was not significantly different between controls and IBS patients. Finally, IBS patients with high acetic acid or propionic acid levels presented with significantly worse GI symptoms, QOL and negative emotions than those with low acetic acid or propionic acid levels or controls. CONCLUSIONS & INFERENCES: These results support the hypothesis that both fecal microbiota and organic acids are altered in IBS patients. A combination of Veillonella and Lactobacillus is known to produce acetic and propionic acid. High levels of acetic and propionic acid may associate with abdominal symptoms, impaired QOL and negative emotions in IBS.

Randomized placebo-controlled trial assessing the effect of bifidobacteria-fermented milk on active ulcerative colitis.

BACKGROUND: Probiotics are efficacious for treating and maintaining remission of ulcerative colitis. AIM: To conduct a randomized placebo-controlled trial of bifidobacteria-fermented milk supplementation as a dietary adjunct in treating active ulcerative colitis. METHODS: Twenty patients with mild to moderate, active, ulcerative colitis randomly received 100 mL/day of bifidobacteria-fermented milk or placebo for 12 weeks with conventional treatment. RESULTS: Clinical and endoscopic activity indices and histological scores were similar in the two groups before treatment. Although improvements were significant in both groups, the clinical activity index was significantly lower in the bifidobacteria-fermented milk than in the placebo group after treatment. The post-treatment endoscopic activity index and histological score were significantly reduced in the bifidobacteria-fermented milk, but not the placebo group. Increases in faecal butyrate, propionate and short-chain fatty acid concentrations were significant in the bifidobacteria-fermented milk, but not the placebo group. No adverse effects were observed in either group. CONCLUSION: Supplementation with this bifidobacteria-fermented milk product is safe and more effective than conventional treatment alone, suggesting possible beneficial effects in managing active ulcerative colitis. This is a pilot study and further larger studies are required to confirm the result these preliminary results.

Improvement of human faecal flora-associated mouse model for evaluation of the functional foods.

AIMS: Animal models are required for evaluation of the functional foods such as pro/prebiotics exerting effects through the metabolism of the intestinal microflora. The object of this study was to establish new human flora-associated mice reflecting the environment of the human intestinal tract. METHODS AND RESULTS: We inoculated a human faecal suspension into segmented filamentous bacteria (SFB) monoassociated mice as a model system. In both human flora (HF) and SFB-associated mouse (HF-SFB mouse), intestinal characteristics such as the composition of intraepithelial lymphocytes, the expression of major histocompatibility complex (MHC) class II molecules and the number of immunoglobulin A-producing cells in the mucosa was closer to those of conventionally reared mice than was case with human flora-associated mice (HF mice) lacking SFB. Several predominant bacterial groups except lactobacilli in human flora were found in faeces of HF-SFB mice. Lactobacilli established small populations in the gut of HF-SFB mice when administered before inoculation with the human flora. Faecal enzymatic activities and organic acid concentration of HF-SFB mice proportionally reflected those of the donor subject. CONCLUSION: We established a new human flora-associated mouse (HF-SFB mouse), in which intestinal characteristics are normally developed and their major microbial composition reflect the human. SIGNIFICANCE AND IMPACT OF THE STUDY: HF-SFB mice are a valuable model for studying pro/prebiotic effects on the human intestine.

Lactobacillus casei strain Shirota-fermented milk stimulates indigenous Lactobacilli in the pig intestine.

The aim of this study was to determine the effect of a probiotic, i.e. fermented milk prepared with Lactobacillus casei strain Shirota, on indigenous Lactobacilli in the pig large intestine. This fermented milk was given as a probiotic to experimental pigs for 2 weeks. The fecal organic acid concentration increased with the fermented milk; acetate and propionate increased significantly (p<0.05). At the same time, lactate and butyrate tended to increase. The fecal pH was significantly reduced by the fermented milk (p<0.05). Although the number of bacteria of strain Shirota in the intestinal contents was much smaller than those of indigenous Lactobacilli, 10(4) vs 10(8) (cfu/g), the numbers of indigenous Lactobacilli and Bifidobacteria in the pig intestine appeared to increase with the fermented milk. In addition, the phenotypic diversity (phenotypic group numbers) of indigenous Lactobacilli increased from 3 to 8 with the fermented milk supplementation. Thus the fermented milk affected the indigenous Lactobacillus population and constitution.

Strain gauge force transducer and its application in a pig model to evaluate the effect of probiotic on colonic motility.

The aim of this study was to investigate the effect of probiotic, i.e., fermented milk prepared with Lactobacillus casei strain Shirota, on colonic motility by the strain gauge force transducer (SGFT) in a pig model. The contractions of the circular muscle layer of the cecum, upper colon, lower colon, and terminal colon in pigs were directly measured in conscious status by this method. This method was useful for quantitatively evaluating the effects of stimuli on colonic motility. Feeding significantly stimulated the motilities of the upper and lower colon. Defecation significantly stimulated the motilities of the upper and terminal colon. Two weeks' feeding of the fermented milk significantly activated the response to feeding in four portions of the large intestine. It increased motility of the terminal colon that did not promote defecation. The frequency of defecation from 9:00 to 10:00 (the period just after the morning meal) increased significantly, but from 0:00 to 1:00 (the midnight period) it decreased as a result of the ingestion of fermented milk. Such effects of the fermented milk on motility of the terminal colon are discussed in relation to the movement of digesta. The effects may relate to the stimulation of colonic fermentation as shown by a decrease in fecal pH.

Structure of the intestinal flora responsible for development of the gut immune system in a rodent model.

The intestinal flora comprising indigenous, autochthonous bacteria is constantly present in the alimentary tract of host animals, including humans. The indigenous bacteria greatly affect the structure and functions of the intestinal mucosa. Studies involving gnotobiotic mice or rats have shown that the presence of limited kinds of intestinal bacteria is responsible for the development of the gut immune system, such as secretory IgA, major histocompatibility complex molecules and intraepithelial lymphocytes. Understanding of the structure of the intestinal flora or the organization of the microbial population in the intestine, based on evaluation of the immunological responses, may clarify its functions in the host animal.

Physiological roles of gammadelta T-cell receptor intraepithelial lymphocytes in cytoproliferation and differentiation of mouse intestinal epithelial cells.

In this study we aimed to elucidate the physiological role of gammadelta intraepithelial lymphocytes (IEL) in the mouse intestine. For this purpose, we used T-cell receptor (TCR) Vgamma4/Vdelta5 transgenic mice (KN 6 Tg: BALB/c background, H-2d), and compared the immunological and physiological characteristics of the intestinal tracts of KN 6 Tg and non-transgenic (non-Tg) littermates. In KN 6 Tg littermates, 95% of small intestinal (SI) and large intestinal (LI) IEL expressed gammadelta TCR, and their TCR was replaced by Tg gammadelta TCR. In these mice, class II major histocompatibility complex (MHC) expression was up-regulated in the SI epithelium, compared with the non-Tg littermates, under specific pathogen-free (SPF) conditions. Competitive reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the mRNAs of the I-Ealpha chain on the SI epithelial cells was higher in KN 6 Tg than in non-Tg littermates. However, in the LI, class II MHC molecules were not expressed in either KN 6 Tg or non-Tg littermates. The epithelial cell mitotic index in the SI, but not in the LI, was higher in KN 6 Tg than in non-Tg littermates under SPF conditions. However, differentiation markers for SI epithelial cells, such as alkaline phosphatase and disaccharidase (lactase, maltase and sucrase) activities, were similar in KN 6 Tg and non-Tg littermates. MHC class II molecule expression on the SI epithelium was absent in germ-free (GF) Tg mice, but was induced under SPF conditions, coinciding with the increase of interferon-gamma (IFN-gamma) mRNA in gammadelta TCR SI-IEL. These findings suggest that gammadelta TCR IEL regulate epithelial cell regeneration and class II MHC expression, but not cell differentiation in the SI. However, these functions were not observed in the gammadelta TCR IEL in the LI. In addition, the activation step in the gammadelta TCR SI-IEL is dependent on the presence of gut microflora.

Differential roles of segmented filamentous bacteria and clostridia in development of the intestinal immune system.

The presence of microflora in the digestive tract promotes the development of the intestinal immune system. In this study, to evaluate the roles of two types of indigenous microbe, segmented filamentous bacteria (SFB) and clostridia, whose habitats are the small and large intestines, respectively, in this immunological development, we analyzed three kinds of gnotobiotic mice contaminated with SFB, clostridia, and both SFB and clostridia, respectively, in comparison with germfree (GF) or conventionalized (Cvd) mice associated with specific-pathogen-free flora. In the small intestine, the number of alpha beta T-cell receptor-bearing intraepithelial lymphocytes (alpha betaIEL) increased in SFB-associated mice (SFB-mice) but not in clostridium-associated mice (Clost-mice). There was no great difference in Vbeta usage among GF mice, Cvd mice, and these gnotobiotic mice, although the association with SFB decreased the proportion of Vbeta6(+) cells in CD8beta- subsets to some extent, compared to that in GF mice. The expression of major histocompatibility complex class II molecules on the epithelial cells was observed in SFB-mice but not in Clost-mice. On the other hand, in the large intestine, the ratio of the number of CD4(-) CD8(+) cells to that of CD4(+) CD8(-) cells in alpha betaIEL increased in Clost-mice but not in SFB-mice. On association with both SFB and clostridia, the numbers and phenotypes of IEL in the small and large intestines changed to become similar to those in Cvd mice. In particular, the ratio of the number of CD8alpha beta+ cells to that of CD8alpha alpha+ cells in alpha betaIEL, unusually elevated in the small intestines of SFB-mice, decreased to the level in Cvd mice on contamination with both SFB and clostridia. The number of immunoglobulin A (IgA)-producing cells in the lamina propria was more elevated in SFB-mice than in Clost-mice, not only in the ileum but also in the colon. The number of IgA-producing cells in the colons of Clost-mice was a little increased compared to that in GF mice. Taken together, SFB and clostridia promoted the development of both IEL and IgA-producing cells in the small intestine and that of only IEL in the large intestine, respectively, suggesting the occurrence of compartmentalization of the immunological responses to the indigenous bacteria between the small and large intestines.

Inflammatory bowel disease-like enteritis and caecitis in a senescence accelerated mouse P1/Yit strain.

BACKGROUND: A new subline of the senescence accelerated mouse (SAM) P1/Yit strain has been established which shows spontaneous enteric inflammation under specific pathogen free (SPF) conditions. AIMS: To elucidate the pathogenesis of enteric inflammation in this new subline. METHODS: The SPF and germ free (GF) SAMP1/Yit strains were used. Histological, immunological, and microbiological characterisation of the mice with enteric inflammation was performed. RESULTS: Histologically, enteritic inflammation developed as a discontinuous lesion in the terminal ileum and caecum with the infiltration of many inflammatory cells after 10 weeks of age. the activity of myeloperoxidase, and both immunolocalisation and mRNA expression of inducible nitric oxide synthase increased in the lesion. CD3-epsilon positive T cells, neutrophils, and macrophages were more numerous in the inflamed mucosa of the SAMP1/Yit strain. The GF SAMP1/Yit strain did not show any inflammation in the intestinal wall, by the age of 30 weeks, and the enteritis and caecitis developed 10 weeks after the conventionalisation of the GF SAMP1/Yit strain. CONCLUSION: Enteric inflammation in the ileum and caecum developed in the SAMP1/Yit strain. The pathophysiological characteristics of the disease in this mouse have some similarities to those of human inflammatory bowel disease (IBD). This mouse strain should be a useful model system for elucidating the interaction between the pathogenesis of IBD and the gut microflora.

A step-wise expansion of intestinal intraepithelial T lymphocytes in association with microbial colonization is defined by sensitivity to cyclosporin A.

Murine intestinal intraepithelial lymphocytes (IELs) consist of T cells bearing alpha beta-antigen receptor (alpha beta-IELs) and those bearing gamma delta-IELs). Although gamma delta-IELs outnumber alpha beta-IELs in germ-free (GF) mice, oral inoculation of fecal suspension from conventional (CV) mice into GF mice induced the increase in number of alpha beta-IELs, leaving the number of gamma delta-IELs unchanged, and the number of alpha beta-IELs reached the level of CV mice by 3 weeks after conventionalization. Expansion of alpha beta-IELs and increase in their CD44+ subset in conventionalized mice were not affected until 2 weeks after beginning of daily injection of cyclosporin A (CsA). However, further expansion of alpha beta-IELs during 2-3 weeks after conventionalization was blocked by injection of CsA. Although the relative constitution of CD4- 8-, CD4+ 8-, CD4- 8 alpha alpha+, CD4- 8 alpha beta+ and CD4+ 8+ subsets among alpha beta-IELs was comparable between control and CsA-treated groups, CsA injection resulted in the decrease in ratio of high-density fraction cells to low density fraction cells in IELs. CsA completely abrogated the expansion of T cells in peripheral lymph nodes stimulated by alloantigens in vivo, and proliferation of IELs from GF mice induced by immobilized anti-alpha beta-T-cell receptor (TCR) monoclonal antibodies (mAb) in vitro was also eliminated by CsA. These results indicate that microbial colonization-induced expansion of alpha beta-IELs is subdivided into two steps: the early phase of expansion takes place via TCR-non-mediated pathway and the late phase of expansion requires TCR-mediated signal transduction.

Growth retardation and early death of beta-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells.

Carbohydrate chains on a glycoprotein are important not only for protein conformation, transport and stability, but also for cell-cell and cell-matrix interactions. UDP-Gal:N-acetylglucosamine beta-1,4-galactosyltransferase (GalT) (EC 2.4.1.38) is the enzyme which transfers galactose (Gal) to the terminal N-acetylglucosamine (GlcNAc) of complex-type N-glycans in the Golgi apparatus. In addition, it has also been suggested that this enzyme is involved directly in cell-cell interactions during fertilization and early embryogenesis through a subpopulation of this enzyme distributed on the cell surface. In this study, GalT-deficient mice were produced by gene targeting in order to examine the pathological effects of the deficiency. GalT-deficient mice were born normally and were fertile, but they exhibited growth retardation and semi-lethality. Epithelial cell proliferation of the skin and small intestine was enhanced, and cell differentiation in intestinal villi was abnormal. These observations suggest that GalT plays critical roles in the regulation of proliferation and differentiation of epithelial cells after birth, although this enzyme is dispensable during embryonic development.

Proliferative recruitment of intestinal intraepithelial lymphocytes after microbial colonization of germ-free mice.

Intraepithelial lymphocytes (IEL), particularly alpha beta TCR-bearing IEL (alpha beta-IEL), dramatically increase in number after microbial colonization of formerly germ-free (ex-GF) mice (Umesaki et al., Immunology 1993. 79: 32). In this study, the kinetics of expansion of IEL after microbial colonization in ex-GF mice were investigated by the bromodeoxyuridine (BrdUrd) continuous labeling method. In GF mice, gamma delta- and alpha beta-IEL were gradually labeled with BrdUrd, reaching approximately 30% and 15% labeling, respectively, after 10 days of continuous BrdUrd labeling. In conventional (CV) mice, the percentage of BrdUrd-labeled alpha beta-IEL was a little higher than that for gamma delta-IEL. The maximal labeling for alpha beta-IEL and gamma delta-IEL reached 50-60% and 40%, respectively, in 10 days. In the case of conventionalized ex-GF mice, continuous labeling was started 11 days after microbial colonization of GF mice because alpha beta-IEL outnumbered gamma delta-IEL during this period. In this case, 75% of alpha beta-IEL and 67% of gamma delta-IEL were labeled with BrdUrd in 10 days. On the other hand, the apparent half lives of alpha beta-IEL and gamma delta-IEL were 10 and 20 days, respectively in CV mice. These results strongly suggest that the number of IEL, particularly alpha beta-IEL, increases after microbial colonization through recruitment into the cell cycle of a large proportion of IEL or their immediate precursors. The difference in the relative BrdUrd-labeling rate of alpha beta-IEL to that of gamma delta-IEL between germ-free and conventionalized mice indicates a preferential increase in the alpha beta-IEL subset during the course of conventionalization.

Gamma delta TCR-bearing intraepithelial lymphocytes regulate class II major histocompatibility complex molecule expression on the mouse small intestinal epithelium.

Introduction of fecal bacteria into germ-free (GF) Balb/c mice induces class II major histocompatibility complex (MHC) molecules on the small intestinal epithelium. In this study, we elucidated the regulatory mechanisms for the class II MHC molecule induction on the mouse small intestinal epithelium during microbial colonisation of the gut in ex-GF mice. Intraperitoneal injection of interferon-gamma (IFN-gamma) into GF Balb/c mice induced class II MHC expression on the small intestinal epithelial cells. Induction of these molecules was inhibited by peritoneal injection of a monoclonal antibody (mAb) against IFN-gamma on the conventionalisation of GF mice. RNA reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of the small intestinal epithelium indicated that the class II transactivator (CIITA), a regulatory factor for the class II MHC gene, and the I-E alpha chain, but not IFN-gamma receptor mRNA, increased during conventionalisation. The induction of class II MHC on the epithelial cells during the conventionalisation of GF C.B-17 scid mice was much lower than that in GF Balb/c mice. Immunocytochemical and RT-PCR analysis showed that both the number of IFN-gamma producing IEL and the level of the IFN-gamma mRNA in gamma delta TCR IEL were very low in the GF state, and gradually increased after microbial colonisation. After in vivo treatment with a mAb against gamma delta TCR, the number of gamma delta TCR-expressing IEL greatly decreased and the expression of class II MHC molecules on the small intestinal epithelium was repressed during the conventionalisation of GF mice. Taken together, these results suggested that gamma delta TCR-bearing IEL modulate class II MHC molecule expression on the small intestinal epithelium through the production of IFN-gamma during microbial colonisation in ex-GF mice.

Segmented filamentous bacteria are indigenous intestinal bacteria that activate intraepithelial lymphocytes and induce MHC class II molecules and fucosyl asialo GM1 glycolipids on the small intestinal epithelial cells in the ex-germ-free mouse.

In ex-germ-free mice conventionalized by association with fecal microorganisms, the induction fo major histocompatibility complex class II molecules and fucosylation of asialo GM1 glycolipid occur in the small intestinal epithelial cells (IEC). The intestinal intraepithelial lymphocytes (IEL), especially alpha beta T-cell receptor-bearing ones, also remarkably expand and show cytolytic activity. In this study, we investigated the immunological and physiological characteristics of the small intestine induced by a kind of indigenous bacteria of the small intestine, segmental filamentous bacteria (SFB), among chloroform-resistant intestinal bacteria. Monoassociation of SFB with germ-free mice was confirmed by the determination of the base sequences of polymerase chain reaction products of 16S rRNA genes of the fecal bacteria of these mice and in situ hybridization using fluorescein-labeled probes based on them. SFB increased the number of alpha beta TCR-bearing IEL and induced Thy-1 expression and cytolytic activity of IEL. The induction of MHC class II molecules and fucosyl asialo GM1 glycolipids and the increases in the mitotic activity and the ratio of the number of columnar cells to those of goblet cells also occurred in the small intestinal epithelial cells on monoassociation of these bacteria. SFB are important indigenous bacteria for the development of the mucosal architecture and immune system in the small intestine, at least in mice.

Effects of fecal microorganisms and their chloroform-resistant variants derived from mice, rats, and humans on immunological and physiological characteristics of the intestines of ex-germfree mice.

In order to elucidate the nature of intestinal flora affecting the immunological and physiological parameters of the intestine, we produced several kinds of ex-germfree mice associated with fecal organisms and their chloroform-resistant variants derived from mice, rats, and humans. The phenotypes of intraepithelial lymphocytes were changed to those in conventional mice, particularly the increased positive percentage of alpha beta T-cell-receptor and Thy-1-bearing T cells, on association of the microorganisms (MF) and their chloroform resistant variants (MChl) derived from mice, but not rats and humans, with germfree mice. The cytolytic activity of intraepithelial lymphocytes was expressed only in the MF and MChl groups. The induction of the synthesis of fucosyl asialo GM1 glycolipid, the expression of major histocompatibility complex class II molecule, an increase in the mitotic indices of colonic epithelial cells, and a decrease in lactase activity of the small intestinal epithelial cells also occurred only in the two groups. On the other hand, the cecal size (cecal weight/body weight ratio) was reduced in the mice of all groups examined here, there being approximately the same amount and composition of organic acids, such as acetic acid, butyric acid, and propionic acid, in the cecal contents. Taken together, the results suggest that mouse-specific and chloroform-resistant microorganisms, which are difficult to cultivate at present, may contribute to alteration of the immunological and epithelial characteristics of the mouse intestine. Another factor derived from the intestinal flora, for example, bacterial metabolites such as organic acids, may also affect the cecal size.

Cytolytic activity of intestinal intraepithelial lymphocytes in germ-free mice is strain dependent and determined by T cells expressing gamma delta T-cell antigen receptors.

We have compared the cytolytic activities and the cellular compositions of the intestinal intraepithelial lymphocyte (i-IEL) populations in three different combinations of conventional (CV) and germ-free (GF) mice. Cytolytic activity of i-IELs expressing gamma delta T-cell antigen receptors (TCRs) is strain dependent in CV mice (high vs. low), and this strain-dependent variability is unaltered in the GF condition. Although absolute numbers of gamma delta i-IELs are slightly decreased, the composition of CD8 alpha alpha+ and CD4-CD8- subsets and the usage of TCR gamma- and delta-chain variable gene segments by gamma delta i-IELs remain the same in GF mice. By contrast, cytolytic activity of alpha beta TCR-expressing i-IELs is uniformly high in CV mice but attenuated sharply in the GF condition. A conspicuous decrease in the total numbers of alpha beta i-IELs is also noted, and CD8 alpha beta+ and CD4+CD8+ subsets are reduced, whereas the CD8 alpha alpha+ subset is expanded in GF mice. These results indicate that microbial deprivation preferentially influences the alpha beta i-IEL population to decrease and become noncytolytic but has little effect on the pool size or characteristics of gamma delta i-IELs. Consequently, cytolytic activity of freshly isolated i-IELs from GF mice is determined by T cells expressing gamma delta TCRs and is found to be strain dependent.

Expansion of alpha beta T-cell receptor-bearing intestinal intraepithelial lymphocytes after microbial colonization in germ-free mice and its independence from thymus.

A large proportion of intestinal intraepithelial lymphocytes (IEL) comprises alpha beta and gamma delta T-cell receptor (TcR)-bearing T cells. The numbers of alpha beta and gamma delta IEL are reported to be very different between germ-free and conventional microbial conditions. In this study, we investigated the kinetics of both types of TcR-bearing cells after microbial colonization in germ-free mice and the influence of thymus deprivation on IEL populations during the microbial association process. Immediately after association with microbes in germ-free animals, the number of alpha beta TcR-bearing IEL gradually increased. Fourteen days after microbial association the number of alpha beta IEL equalled that of gamma delta TcR-bearing IEL. Approximately 1 month after microbial association, the number of alpha beta IEL was several times greater than that of gamma delta IEL, having almost reached the level in conventional mice reared in a conventional animal room after birth. On the other hand the number of gamma delta IEL hardly changed throughout this microbial association process. Two-colour analysis involving anti-alpha beta TcR and anti-Lyt-2 or Lyt-3 antibodies showed that the major fraction of IEL that increased after microbial association comprised alpha beta TcR-bearing T cells expressing CD8 antigen composed of a homodimer of alpha-chains, which was not detected in other gut associated-lymphoid tissues (GALT) such as Peyer's patch, mesenteric lymph node and lamina propria tissue. The number of alpha beta T cells in these GALT increased within 1 week more quickly than that of IEL. The increase in alpha beta IEL after microbial association was not prevented by thymectomy. These results strongly suggest that the progenitors of alpha beta TcR-bearing IEL expand outside the thymus in response to microbial colonization in germ-free mice.