SummaryThe instruction of the immune system to be tolerant of self, thereby preventing autoimmunity, is facilitated by the education of T cells in a specialized organ, the thymus, where self-reactive cells are either eliminated or differentiated into tolerogenic Foxp3+ regulatory T(Treg) cells1. However, it is unknown whether T cells are also educated to be tolerant of foreign antigens, such as those from commensal bacteria, in order to prevent immunopathology such as inflammatory bowel disease2–4. Here, we show that encounter with commensal microbiota results in the peripheral generation of Treg cells, rather than pathogenic effectors. We observed that colonic Treg cells utilized T cell antigen receptors (TCRs)different from those used by Treg cells in other locations, implying an important role for local antigens in shaping the colonic Treg cell population. Many of the local antigens appeared to be derived from commensal bacteria based on the in vitro reactivity of common colon Treg TCRs. Interestingly, these TCRs did not facilitate thymic Treg cell development, implying that manycolonic Treg cells arise instead via antigen-driven peripheral Treg cell development. Further analysis of two of these TCRs by the creation of retroviral bone marrow chimeras and a TCR transgenic linerevealed that microbiota indigenous to our mouse colony was required for the generation of colonic Treg cells from otherwise naive T cells. If T cells expressing these TCRs fail to undergo Treg cell development and instead become effector cells, they have the potential to induce colitis, as evidenced by adoptive transfer studies. These results suggest that the efficient peripheral generation of antigen-specific populations of Treg cells in response to an individual’s microbiota provides important post-thymic education of the immune system to foreign antigens, thereby providing tolerance to commensal microbiota.

Because the deletion of self-reactive T cells is incomplete, thymic development of natural Foxp3+CD4+ regulatory T (Treg) cells is required for preventing autoimmunity. However, the role of T cell receptor (TCR) specificity in thymic Treg cell development remains controversial. To address this issue, we generated a transgenic line expressing a naturally occurring Treg cell-derived TCR. Surprisingly, efficient thymic Treg cell development occurred only when the antigen-specific Treg cell precursors were present at low clonal frequency (<1%) within a normal thymus. Using retroviral vectors and bone marrow chimeras, we observed similar behavior with two other Treg cell-derived TCRs. These data demonstrate that thymic Treg cell development is a TCR-instructive process involving a niche which can be saturable at much lower clonal frequencies than the niche for positive selection.

Although regulatory T (T reg) cells are thought to develop primarily in the thymus, the peripheral events that shape the protective T reg cell population are unclear. We analyzed the peripheral CD4 + T cell receptor (TCR) repertoire by cellular phenotype and location in mice with a fi xed TCR ␤ chain. We found that T reg (Foxp3 + ) cells showed a marked skewing of TCR usage by anatomical location in a manner similar to antigen-experienced (CD44 hi Foxp3 -) but not naive (CD44 lo Foxp3 -) cells, even though CD44 hi and T reg cells used mostly dissimilar TCRs. This was likely unrelated to peripheral conversion, which we estimate generates only a small percentage of peripheral T reg cells in adults. Conversion was readily observed, however, during the immune response induced by Foxp3 -cells in lymphopenic hosts. Interestingly, the converted Foxp3 + and expanded Foxp3 -TCR repertoires were different, suggesting that generation of Foxp3 + cells is not an automatic process upon antigen activation of Foxp3 -T cells. Retroviral expression of these TCRs in primary monoclonal T cells confi rmed that conversion did not require prior cellular conditioning. Thus, these data demonstrate that TCR specifi city plays a crucial role in the process of peripheral conversion and in shaping the peripheral T reg cell population to the local antigenic landscape.

To study mechanisms of peripheral self-tolerance, we injected small numbers of naive CD4+ TCR-transgenic T cells into mice expressing the MHC/peptide ligand under the control of an MHC class II promoter. The donor T cells expand rapidly to very large numbers, acquire memory markers, and go out into tissues, but the animals remain healthy, and the accumulated T cells are profoundly anergic to restimulation with Ag in vitro. Provision of a costimulatory signal by coinjection of an agonist Ab to OX40 (CD134), a TNFR family member expressed on activated CD4 T cells, results in death of the mice within 12 days. TCR-transgenic T cells recovered at 5 days from anti-OX40-treated mice have a unique phenotype: they remain unresponsive to Ag in vitro, but they are larger, more granular, and strongly IL-2R positive. Some spontaneously secrete IFN-γ directly ex vivo, and the majority make IFN-γ in response to PMA and ionomycin. Although they are anergic by conventional tests requiring Ag recognition, they respond vigorously to cytokines, proliferating in response to IL-2, and secreting IFN-γ when TCR signaling is bypassed with IL-12 and IL-18. We conclude that the costimulatory signal through OX40 allows otherwise harmless, proliferating, autoreactive T cells to acquire effector cell functions. The ability of these T cells to respond to cytokines by synthesizing additional inflammatory cytokines without a TCR signal may drive the fatal pathogenic process in vivo.

The Gram-negative bacterium Salmonella enterica has developed an array of sophisticated tools to manipulate the host cell and establish an intracellular niche, for successful propagation as a facultative intracellular pathogen. While Salmonella exerts diverse effects on its host cell, only the cell biology of the classic “trigger”-mediated invasion process and the subsequent development of the Salmonella-containing vacuole have been investigated extensively. These processes are dependent on cohorts of effector proteins translocated into host cells by two type III secretion systems (T3SS), although T3SS-independent mechanisms of entry may be important for invasion of certain host cell types. Recent studies into the intracellular lifestyle of Salmonella have provided new insights into the mechanisms used by this pathogen to modulate its intracellular environment. Here we discuss current knowledge of Salmonella-host interactions including invasion and establishment of an intracellular niche within the host.