To ensure immune tolerance, regulatory T cell (Treg) numbers must be maintained by cell division. This process has been thought to be strictly dependent on the Treg TCR interacting with MHC class II (MHCII). Here, we report that Treg division does not absolutely require cell-autonomous TCR signaling in vivo, depending on the degree of IL-2-mediated stimulation provided. At steady state IL-2 levels, Tregs require cell-autonomous TCR signaling to divide. However, when given exogenous IL-2 or when STAT5 is selectively activated in Tregs, Treg division can occur independently of MHCII and TCR signaling. Thus, depending on the amount of IL-2 receptor stimulation, a wide range of TCR signals support Treg division, which may contribute to preservation of a diverse repertoire of Treg TCR specificities. These findings also have therapeutic implications, as TCR signaling by Tregs may not be required when using IL-2 to increase Treg numbers for treatment of inflammatory disorders.

Transcription factors regulate T cell fates at every stage of development and differentiation. Members of the Foxp family of forkhead transcription factors are essential for normal T lineage development; Foxp3 is required for T regulatory cell generation and function, and Foxp1 is necessary for generation and maintenance of naïve T cells. Foxp4, an additional member of the Foxp family, is highly homologous to Foxp1 and has been shown to dimerize with other Foxp proteins. We report the initial characterization of Foxp4 in T lymphocytes. Foxp4 is expressed in both thymocytes and peripheral CD4+ and CD8+ T cells. We used a CD4Cre mediated approach to evaluate the cell autonomous role for Foxp4 in murine T lymphocytes. T cell development, peripheral cellularity and cell surface phenotype are normal in the absence of Foxp4. Furthermore, Foxp3+ T regulatory cells develop normally in Foxp4 deficient animals and naïve Foxp4 deficient CD4 T cells can differentiate to inducible T regulatory cells in vitro. In wild-type T cells, expression of Foxp4 increases following activation, but deletion of Foxp4 does not affect T cell proliferative responses or in vitro effector T cell differentiation. In vivo, despite effective control of Toxoplasma gondii and acute lymphocytic choriomeningitis virus infections, effector cytokine production during antigen specific recall responses are reduced in the absence of Foxp4. We conclude that Foxp4 is dispensable for T cell development, but necessary for normal T cell cytokine recall responses to antigen following pathogenic infection.

Summary CD4+Foxp3+ regulatory T cells (Tregs) are required for maintenance of self-tolerance, as demonstrated by profound autoimmunity in mice and humans with inactivating Foxp3 mutations. Recent studies demonstrate that Tregs are anatomically compartmentalized within secondary lymphoid organs based on their TCR repertoire and specific organ-protective function; however, whether this reflects differential homing or in situ selection is not known. Here, using Foxp3-GFP reporter mice, we have examined the ability of polyclonal Tregs from cervical LN to return to their site-of-origin following adoptive transfer to non-lymphopenic congenic recipients. We find that bulk cervical LN Tregs do not home directly to cervical LN but, rather, accumulate site-specifically over time following transfer. Site-specific enrichment is both more rapid and more pronounced among a population of recently activated (CD69+) Tregs. These data suggest that compartmentalization of Tregs within secondary lymphoid organs may be governed by antigen recognition and implicate CD69 as a potential marker of recently activated Tregs recognizing local-expressed antigens.

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CD4+ memory T cells are generated in response to infection or vaccination, provide protection to the host against re-infection, and persist through a combination of enhanced survival and slow homeostatic turnover. We used timed deletion of the TCR-signaling adaptor molecule SH2-domain-containing phosphoprotein of 76 kDa (SLP-76) with MHC:peptide tetramers to study the requirements for tonic TCR signals in the maintenance of polyclonal antigen-specific CD4+ memory T cells. SLP-76 deficient I-Ab:GP61 cells are unable to rapidly generate effector cytokine or proliferate in response to secondary infection. In mice infected with lymphocytic choriomeningitis virus (LCMV) or Listeria monocytogenes expressing the LCMV GP61-80 peptide, SLP-76 deficient I-Ab:GP61+ cells exhibit reduced division, similar to that seen in in vitro generated CD44hi and endogenous CD4+CD44hi cells. Competitive bone marrow chimera experiments demonstrated that the decrease in homeostatic turnover in the absence of SLP-76 is a cell intrinsic process. Surprisingly, despite the reduction in turnover, I-Ab:GP61+ antigen-specific memory cells persist in normal numbers for more than 30 weeks post LCMV infection in the absence of SLP-76. These data suggest the independent maintenance of a population of antigen-specific CD4+ memory T cells in the absence of SLP-76 and normal levels of homeostatic division.