Basis of Treg development in the thymus.

HHS Public Access Author manuscript Author Manuscript Cell Cycle. Author manuscript; available in PMC 2019 August 12. Published in final edited form as: Cell Cycle. 2014 ; 13(4): 501–502. doi:10.4161/cc.27787. Basis of Treg development in the thymus Xuguang Tai, Alfred Singer* Experimental Immunology Branch; National Cancer Institute; National Institutes of Health; Bethesda, MD USA Author Manuscript Developing T-cells express antigen receptors (TCR) with diverse recognition specificities that are subjected to positive and negative selection pressures before emigrating out of the thymus.1,2 Although negative selection deletes T-cells bearing autoreactive TCR, thymic selection is imperfect, so that some T-cells with auto-reactive TCR escape from the thymus into the periphery, where their autoreactive potential must be suppressed by regulatory Tcells (Tregs). Tregs are a specialized subpopulation of CD4 T-cells with suppressive capability that differ from conventional CD4 T-cells in their expression of the forkhead family transcription factor Foxp3 and in their dependence on γc-dependent cytokines such as IL-2.3 Curiously, thymic selection of Tregs closely resembles thymic deletion of conventional autoreactive T-cells, in that both events require high-affinity TCR signals, and both events require engagement of CD28 costimulatory ligands.1,4 Consequently, it has not been understood why Tregs survive CD28 costimulatory signals in the thymus1 and why Tregs, unlike all other MHC class II selected T-cells in the thymus, require γc cytokine signaling.3 Author Manuscript To address these issues, we recently examined the effect of Foxp3 protein expression on cell viability.5 We found that Foxp3 induced developing CD4 T-cells to express a distinctive proapoptotic protein signature (abbreviated as Puma2+p-Bim2+p-JNK2+DUSP6−) and to repress expression of the prosurvival protein Bcl-2, with the result that Foxp3 was potentially lethal to cells that expressed it. However, we also found that Foxp3 lethality was prevented by γc cytokine signals, which upregulated Bcl-2 expression to levels that counterbalanced the lethal effects of the proapoptotic protein signature. Thus, Foxp3+Tregs require γc cytokines such as IL-2 to upregulate Bcl-2 expression to levels that protect Tregs from Foxp3-induced apoptosis. Author Manuscript The requirement for IL-2 survival signals could be circumvented by a Bcl-2 transgene, which protected Tregs from Foxp3-induced cell death in the absence of signaling by γc cytokines.5 This finding revealed that IL-2 was not needed to induce Foxp3 gene expression and contradicts the conventional perspective that IL-2 is required to induce Foxp3 gene expression in developing CD4 thymocytes.6,7 In fact, expression of the Bcl-2 transgene not only obviated the requirement for IL-2, it resulted in approximately 3-fold more Foxp3+ Tcells than normally arise in the thymus, indicating that the thymus contained insufficient IL-2 to rescue all newly arising Tregs. This observation was not unique to Bcl-2 transgenic mice, as mice deficient in the proapoptotic proteins Puma and Bim also contained 3-fold * Correspondence to: Alfred Singer; . Tai and Singer Page 2 Author Manuscript more Foxp3+ T-cells in the thymus.5 The Foxp3+ T-cells that were increased in number in both Bcl-2 transgenic and Puma/Bim-deficient mice were Foxp3+CD25− CD4 T-cells that were phenotypically distinct from functionally mature Tregs that are Foxp3+CD25+. Notably, such Foxp3+CD25− T-cells possessed the potential to differentiate into functionally mature Foxp3+CD25+ Tregs when signaled by IL-2.5 Thus, Foxp3+CD25− T-cells are precursors of Foxp3+CD25+ mature Tregs. Author Manuscript Author Manuscript It has conventionally been thought that all Foxp3+ cells in the thymus have been IL-2 signaled, because IL-2 signaling is necessary to induce Foxp3 gene expression.6,7 In the conventional perspective, mature Foxp3+CD25+ Tregs must arise in the thymus from IL-2signaled Foxp3−CD25+ precursors. However, the existence of Foxp3+CD25− Treg precursor cells requires that the conventional view of Treg differentiation be replaced with a more expanded view. We think 2 distinct developmental pathways actually exist, by which conventional CD4 T-cells are signaled to differentiate in the thymus into mature Tregs (Fig. 1). We think that most mature Tregs arise from Foxp3+CD25− precursors, while a minority are the progeny of Foxp3−CD25+ precursors. Interestingly, both major and minor developmental pathways are initiated by CD28 costimulation of conventional CD4 T-cells and are completed by IL-2-signaled differentiation of precursors into functionally mature Tregs (Fig. 1). Importantly, the 2 pathways differ in the prominence of Foxp3-mediated apoptosis and their dependence on IL-2 survival signals. In the major pathway, Foxp3+CD25− precursors are highly susceptible to Foxp3-induced apoptosis, with most precursors dying in the thymus, because IL-2 is present in insufficient amounts in the thymus. In the minor pathway, Foxp3−CD25+ precursors express Foxp3 only after receiving IL-2 signals, so they are not susceptible to Foxp3-induced apoptosis. It remains to be determined if important functional differences exist in the mature Foxp3+CD25+ Tregs derived from the 2 different precursor populations. Finally, it is interesting to consider why developing Tregs are the only cells to survive costimulation in the thymus. It is known that Foxp3 targets Zap70 and Itk, 2 kinases involved in TCR signal transduction, so that TCR/CD28 signaling might be dampened sufficiently to allow Foxp3+ cells to avoid clonal deletion.8 If so, Foxp3 expression would be a double-edged sword that first protects Treg precursors from clonal deletion, only to then make them susceptible to Foxp3-mediated apoptosis if they fail to be signaled by prosurvival γc cytokines. References Author Manuscript 1. Pobezinsky LA, et al. Nat Immunol 2012; 13:569–78; 10.1038/ni.2292 [PubMed: 22544394] 2. Singer A, et al. Nat Rev Immunol 2008; 8:788–801; 10.1038/nri2416 [PubMed: 18802443] 3. Josefowicz SZ, et al. Annu Rev Immunol 2012; 30:531–64; 10.1146/annurev.immunol. 25.022106.141623 [PubMed: 22224781] 4. Tai X, et al. Nat Immunol 2005; 6:152–62; 10.1038/ni1160 [PubMed: 15640801] 5. Tai X, et al. Immunity 2013; 38:1116–28; 10.1016/j.immuni.2013.02.022 [PubMed: 23746651] 6. Burchill MA, et al. Immunity 2008; 28:112–21; 10.1016/j.immuni.2007.11.022 [PubMed: 18199418] 7. Lio CW, et al. Immunity 2008; 28:100–11; 10.1016/j.immuni.2007.11.021 [PubMed: 18199417] 8. Marson A, et al. Nature 2007; 445:931–5; 10.1038/nature05478 [PubMed: 17237765] Cell Cycle. Author manuscript; available in PMC 2019 August 12. Tai and Singer Page 3 Author Manuscript Author Manuscript Figure 1. Author Manuscript Two distinct developmental pathways for Treg cell differentiation in the thymus. Developing (Foxp3−CD25−) CD4 thymocytes that receive TCR/CD28 costimulatory signals diff (...truncated)