SUMMARY The intestinal microbiota is important for induction of inflammatory bowel disease (IBD). IBD is associated with complex shifts in microbiota composition, but it is unclear whether specific bacterial subsets induce IBD and, if so, whether their proportions in the microbiota are altered during disease. Here we fulfilled Koch’s postulates in host-genotype-specific fashion using a mouse model of IBD with human-relevant disease-susceptibility mutations. From screening experiments we isolated common commensal Bacteroides species, introduced them into antibiotic-pretreated mice, and quantitatively re-isolated them in culture. The bacteria colonized IBD-susceptible and non-susceptible mice equivalently, but induced disease exclusively in susceptible animals. Conversely, commensal Enterobacteriaceae were >100-fold enriched during spontaneous disease but an Enterobacteriaceae isolate failed to induce disease in antibiotic-pretreated mice despite robust colonization. We thus demonstrate that IBD-associated microbiota alterations do not necessarily reflect underlying disease etiology. These findings establish important experimental criteria and a conceptual framework for understanding microbial contributions to IBD.

We generated the DUC18 T cell receptor transgenic mouse expressing an H-2Kd -restricted transgenic T cell receptor specific for the syngeneic CMS5 fibrosarcoma rejection antigen mutated ERK2(136-144). DUC18 mice were capable of specifically eliminating lethal CMS5 tumor challenges, and transfer of DUC18 splenocytes to naive nontransgenic recipients conferred protection from subsequent and established CMS5 tumor burdens. Eradication of established tumor burdens by adoptive transfer of DUC18 splenocytes was dose and time dependent. Transferred tumor-specific T cells remained functional in vivo and capable of rejecting small tumors even in the presence of large, established tumor burdens. These findings highlight the kinetic battle between tumor growth and the production of a tumor-specific response and have critical implications for effective adoptive immunotherapy.

Summary Microbes interact with the host immune system via several potential mechanisms. One essential step for each mechanism is the method by which intestinal microbes or their antigens access specific host immune cells. Using genetically-susceptible mice (dnKO) that develops spontaneous, fulminant colitis, triggered by Bacteroides thetaiotaomicron (B. theta), we investigated the mechanism of intestinal microbial access under conditions that stimulate colonic inflammation. B. theta antigens localized to host immune cells through outer membrane vesicles (OMVs) that harbor bacterial sulfatase activity. We deleted the anaerobic sulfatase maturating enzyme (anSME) from B. theta, which is required for post-translational activation of all B. theta sulfatase enzymes. This bacterial mutant strain did not stimulate colitis in dnKO mice. Lastly, access of B. theta OMVs to host immune cells was sulfatase-dependent. These data demonstrate that bacterial OMVs and associated enzymes promote inflammatory immune stimulation in genetically susceptible hosts.

The molecular basis underlying the specificity of alloreactive T cells for peptide-major histocompatibility complex ligands has been elusive. Here we describe a screen of 60 I-E(k)-alloreactive T cells and 83 naturally processed peptides that identified 9 reactive T cells. Three of the T cells responded to multiple, distinct peptides that shared no sequence homology. These T cells recognized each peptide-major histocompatibility complex ligand specifically and used a distinct constellation of I-E(k) contact residues for each interaction. Our studies show that alloreactive T cells have a 'germline-encoded' capacity to recognize multiple, distinct ligands and thus show 'polyspecificity', not degeneracy. Our findings help to explain the high frequency of alloreactive T cells and provide insight into the nature of T cell specificity.

The T cell receptor (TCR) ␣␤ heterodimer determines the peptide and MHC specificity of a T cell. It has been proposed that in vivo selection processes maintain low TCR affinities because T cells with higher-affinity TCRs would (i) have reduced functional capacity or (ii) cross-react with self-peptides resulting in clonal deletion. We used the class II-restricted T cell clone 3.L2, specific for murine hemoglobin (Hb͞I-E k ), to explore these possibilities by engineering higher-affinity TCR mutants. A 3.L2 single-chain TCR (V␤-linker-V␣) was mutagenized and selected for thermal stability and surface expression in a yeast display system. Stabilized mutants were used to generate a library with CDR3 mutations that were selected with Hb͞I-E k to isolate a panel of affinity mutants with KD values as low as 25 nM. Kinetic analysis of soluble single-chain TCRs showed that increased affinities were the result of both faster on-rates and slower off-rates. T cells transfected with the mutant TCRs and wild-type TCR responded to similar concentrations of peptide, indicating that the increased affinity was not detrimental to T cell activation. T cell transfectants maintained exquisite hemoglobin peptide specificity, but an altered peptide ligand that acted as an antagonist for the wild-type TCR was converted to a strong agonist with higher-affinity TCRs. These results show that T cells with high-affinity class II reactive TCRs are functional, but there is an affinity threshold above which an increase in affinity does not result in significant enhancement of T cell activation.antigen specificity ͉ major histocompatibility complex ͉ T cell activity