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The homophilic cell surface receptors CD150 (Slamf1) and CD352 (Slamf6) are known to modulate adaptive immune responses. Although the Th17 response was enhanced in Slamf6−/− C57BL/6 mice upon oral infection with Citrobacter rodentium, the pathologic consequences are indistinguishable from an infection of wild-type C57BL/6 mice. Using a reporter-based binding assay, we show that Slamf6 can engage structures on the outer cell membrane of several Gram− bacteria. Therefore, we examined whether Slamf6, like Slamf1, is also involved in innate responses to bacteria and regulates peripheral inflammation by assessing the outcome of C. rodentium infections in Rag−/− mice. Surprisingly, the pathology and immune responses in the lamina propria of C. rodentium-infected Slamf6−/−Rag−/− mice were markedly reduced as compared with those of Rag−/− mice. Infiltration of inflammatory phagocytes into the lamina propria was consistently lower in Slamf6−/−Rag−/− mice than in Rag−/− animals. Concomitant with the reduced systemic translocation of the bacteria was an enhanced production of IL-22, suggesting that Slamf6 suppresses a mucosal protective program. Furthermore, administering a mAb (330) that inhibits bacterial interactions with Slamf6 to Rag−/− mice ameliorated the infection compared with a control antibody. We conclude that Slamf6-mediated interactions of colonic innate immune cells with specific Gram− bacteria reduce mucosal protection and enhance inflammation, contributing to lethal colitis that is caused by C. rodentium infections in Rag−/− mice.

Several autoimmune disease models depend on an imbalance in the activation of aggressor T(h)1 and CD4(+)CD25(+) regulatory T (T(reg)) cells. Here we compare the requirement for signals through the co-stimulatory molecules CD28 and inducible co-stimulator (ICOS) in chronic murine colitis, a model for inflammatory bowel disease. We used a colitis model in which disease-causing CD45RB(hi) T cells alone or in combination with CD4(+)CD25(+) T cells from either CD28-deficient or wild-type donors were transferred into T cell-deficient animals, half of which were treated with ICOS-blocking reagents. Blocking ICOS on the surface of CD28-deficient T(h)1 cells abrogated development of colitis, whereas blocking CD28 or ICOS alone had little to no effect on disease induction. In contrast to T(h)1 cells, regulatory T cell functioning depended mostly on CD28 signaling with only a minor contribution for ICOS. We conclude that CD28 and ICOS collaborate to development of murine colitis by aggressor T(h)1 cells, and that CD28 is required for T(reg) cells, which should caution against the use of CD28-blocking reagents in inflammatory bowel disease.

Although CD4(+) T cells form a major subset of TCRalphabeta T cells, only a small number of TCRgammadelta T cells express CD4. Factors contributing to the down-regulation of CD4(+) TCRgammadelta T cells have not been identified. The CD5 molecule is expressed on most TCRgammadelta T cells in the spleen, whereas only a few intestinal intraepithelial TCRgammadelta T cells express this molecule in wild-type mice and TCRbeta mutant (beta(-/-)) mice. Unexpectedly, in the present studies, the lack of CD5 led to a remarkable increase of a CD4(+) TCRgammadelta T cell subset in CD5(-/-)beta(-/-) mice. The CD4(+) TCRgammadelta T cells were also detectable in MHC II(-/-)CD5(-/-)beta(-/-) triple-mutant mice. This CD4(+) TCRgammadelta T cell subset provided help in Mycobacterium-induced germinal center (GC) formation and showed a T(h)-like cytokine profile. In contrast, CD5(+) TCRgammadelta T cells suppressed the CD4(+) TCRgammadelta T cell-mediated GC formation, presumably by eliminating this CD4(+) subset. Unlike intraepithelial gammadelta T cells,30% of TCRgammadelta T cells in the colonic lamina propria (LP) expressed CD5. The lack of CD5 also led to increased numbers of CD4(+) TCRgammadelta T cells in the colonic LP and increased susceptibility to development of chronic colitis in beta(-/-) mice. Cell transfer studies suggest that CD5(+) TCRgammadelta T cells are capable of selectively eliminating CD4(+) TCRgammadelta T cells in the intestine. The CD4(+) TCRgammadelta T cells possess immune functions similar to CD4(+) TCRalphabeta T cells.

The spontaneous chronic colitis in TCR alpha mutant (TCRalpha(-/-)) mice mediated by CD4(+) TCRalpha(-)beta(+) T cells is more severe in the absence of mature B cells, suggesting a suppressive role of B cells and Ig in the development of chronic colitis. To investigate the direct role of B cells in the suppression of this colitis, cell transfer studies were performed in TCRalpha(-/-) x Igmu(-/-) (alphamu(-/-)) double-knockout mice. The chronic colitis was markedly attenuated in alphamu(-/-) mice after the adoptive transfer of peripheral B cells from TCRalpha(-/-) mice into 3- to 4-week-old alphamu(-/-) mice prior to the development of colitis. Furthermore, transfer of mature B cells from TCRalpha(-/-) mice markedly decreased the number of pathogenic colonic CD4(+) TCRalpha(-)beta(+) T cells in alphamu(-/-) mice with established colitis. This B cell effect required the presence of functional co-stimulatory molecules CD40 and B7-2 (CD86) but not B7-1 (CD80). These results indicate that mature B cells play an important role in the development of chronic colitis in TCRalpha(-/-) mice by directly regulating the pathogenic T cells (CD4(+) TCRalpha(-)beta(+) T cells).

Since lumenal bacteria have been postulated to play an important role in the pathogenesis of inflammatory bowel disease (IBD), we investigated the humoral response to cecal aerobic bacterial antigens by Western blot analysis in TCR alpha -/- mice which spontaneously develop IBD. The sera from TCR alpha -/- mice revealed an alteration of the recognition pattern against aerobic bacterial antigens from polyclonal to oligoclonal with age. This alteration was not observed in TCR delta -/- and TCR alpha +/- mice. The alteration of the recognition pattern in TCR alpha -/- mice was associated with production of autoantibodies against tropomyosin and the development of IBD. The unique population of CD4+ TCR alpha -beta + cells in TCR alpha -/- mice may be involved in the recognition of these bacterial antigens and the absence of the alpha chain may result in the alteration of immune response.

According to the 'hygiene hypothesis', enhanced microbial exposure due to early childhood infections leads to a reduction of T(h)2-mediated allergic diseases and inflammatory bowel disease. To begin to elucidate the mechanisms underlying this hypothesis, we studied development of T(h)2-mediated colitis of the TCRalpha knockout (KO) mouse in both a specific pathogen-free (SPF) facility and a conventional (CV) facility. After more than five generations in each facility, TCRalpha KO mice kept in the CV facility developed dramatically less colitis than mice that were kept in the SPF facility. Surprisingly, the suppression of colitis in the CV facility correlated with a significant increase in natural IgM production by B-1 B cells. In contrast, B cell-deficient TCRalpha double-knockout (alphamu DKO) mice maintained in the CV facility continued to develop severe colitis, strongly suggesting that B-1 B cells contributed to the suppression of colitis. Indeed, the adoptive transfer of B-1 B cells isolated from the peritoneal cavity of TCRalpha KO mice (SPF) into alphamu DKO mice (CV) suppressed the development of colitis in the recipient mice. We conclude that B-1 cells play a regulatory role in T(h)2-mediated colitis under non-hygienic conditions, possibly by generating natural antibodies in response to microbial flora.

Thymocytes bearing autoreactive TCR are eliminated from the organism by a process termed negative selection. The molecular basis of this deletion has been recently shown to be a consequence of TCR-triggered activation of a caspase by certain peptide-MHC ligands in the immature CD4+CD8+ double-positive (DP) thymocyte subpopulation. Of note, the numerically minor TCRhigh DP thymocyte subpopulation, unlike the major TCRlow DP subset, is resistant to negative selection. Despite exposure to cognate peptide, TCRhigh DP thymocytes mature into single-positive thymocytes and are exported into the periphery. Here we investigated the mechanism by which these thymocytes escape negative selection. Using a cytochemical assay in conjunction with a caspase-specific affinity ligand, we demonstrate that the resistance of the TCRhigh DP thymocytes to negative selection correlates with the disappearance of TCR-triggered caspase activity in these cells. Thus thymocytes which have presumably begun the positive selection process inactivate the thymic caspase pathway and are no longer susceptible to negative selection.

Mutations in Bruton's tyrosine kinase (Btk) gene, in mice, result in reduced numbers and responses of peripheral B cells. Surface Ig-mediated signaling is defective in Btk mutant B cells as they do not proliferate upon slg cross-linking and lack thymus-independent (TI) type II responses. Signals through sIg and CD40 play a critical role in B cell maturation. To investigate the consequences of the lack of both Btk and CD40 on B cell development and function, mice were generated that were homozygous for targeted mutations in the Btk and the CD40 genes (BtkMCD40M). The CD40 mutation (CD40M) had a synergistic effect on the BtkM defects. In BtkMCD40M mice the number of B cells was reduced 3- to 4-fold compared to BtkM mice and mature B cells (IgMlow/IgDhigh) were virtually absent; serum levels of all Ig isotypes were diminished; and antibody responses to TI-I TI-II and thymus-dependent antigens were impaired. Furthermore, although wild-type BtkM and CD40M mice produced germinal centers in response to TI-I antigen, the BtkMCD40M mice did not. Maturational and functional B cell defects in BtkMCD40M mice may result from a combination of intrinsic B cell defects, lack of CD40L-dependent T cell help and microenvironmental defects. These data suggest that signals through Btk and CD40 are necessary for the production and maintenance of the mature B cell.

We describe the development and function of the peripheral lymphoid system of mutant mice rendered deficient in either alpha beta or gamma delta T cells via targeting of TCR genes in embryonic stem cells. In the spleen of alpha beta T cell-deficient mice, gamma delta T cells do not compensate in numbers for the lack of alpha beta T cells, but B cells do. alpha beta T cell-deficient mice are unable to mount an antibody response to ovalbumin and do not reject skin allografts. Natural killer cell function is not impaired in any of the mutant mice. TCR mutant mice will prove useful in dissecting differential functions of alpha beta and gamma delta T cells in vivo.