CTLA-4 proteins contribute to the suppressor function of regulatory T cells (Tregs), but the mechanism by which they do so remains incompletely understood. In the present study, we assessed CTLA-4 ...protein function in both Tregs and conventional (Tconv) CD4+ T cells. We report that CTLA-4 proteins are responsible for all 3 characteristic Treg functions of suppression, TCR hyposignaling, and anergy. However, Treg suppression and anergy only required the external domain of CTLA-4, whereas TCR hyposignaling required its internal domain. Surprisingly, TCR hyposignaling was neither required for Treg suppression nor anergy because costimulatory blockade by the external domain of CTLA-4 was sufficient for both functions. We also report that CTLA-4 proteins were localized in Tregs in submembrane vesicles that rapidly recycled to/from the cell surface, whereas CTLA-4 proteins in naive Tconv cells were retained in Golgi vesicles away from the cell membrane and had no effect on Tconv cell function. However, TCR signaling of Tconv cells released CTLA-4 proteins from Golgi retention and caused activated Tconv cells to acquire suppressor function. Therefore, the results of this study demonstrate the importance of intracellular localization for CTLA-4 protein function and reveal that CTLA-4 protein externalization imparts suppressor function to both regulatory and conventional CD4+ T cells.
...to Treg differentiation, which requires agonist signalling disruption, we found that CD4+CD25+ precursors require persistent agonist signalling to activate the IL-2 gene and to become IL-2+Teffs.5 ...Thus, the fate of surviving CD4 thymocytes depends on the duration of late agonist signalling, with disrupted agonist signalling inducing Foxp3+Tregs and persistent agonist signalling inducing IL-2+Teffs (Figure 1). Because transforming growth factor beta (TGFβ) has previously been shown to induce Tregs through unknown mechanisms,8,9 our finding that late agonist signalling disruption was required to induce Foxp3 gene expression prompted us to examine whether TGFβ functions by disrupting agonist signalling in autoreactive CD4 thymocytes. ...excessive IL-2 is produced in the TGFβR1-deficient mice.5 Our discovery that CD4+CD25+ precursors first become Foxp3+CD25− preTregs and then Foxp3+CD25+ mature Tregs defines a precursor–progeny relationship that reveals a new Treg developmental pathway, which we have called the ‘primary pathway’ (Figure 1). Since most Foxp3+CD25− preTregs die by Foxp3-induced apoptosis in normal mice,11 their existence as immediate Treg precursors has been largely overlooked. ...we propose that thymic Treg differentiation goes through the primary pathway because this pathway is TGFβ dependent and occurs in normal mice. Overall, our analysis of autoreactive CD4 thymocyte differentiation revealed an unexpected similarity with normal thymocyte development described by the kinetic signalling model,2,13,14 as signalling disruption versus signalling persistence is the underlying mechanism of lineage fate determination in both.
Major histocompatibility complex class I (MHC I) positive selection of CD8
T cells in the thymus requires that T cell antigen receptor (TCR) signaling end in time for cytokines to induce Runx3d, the ...CD8-lineage transcription factor. We examined the time required for these events and found that the overall duration of positive selection was similar for all CD8
thymocytes in mice, despite markedly different TCR signaling times. Notably, prolonged TCR signaling times were counter-balanced by accelerated Runx3d induction by cytokines and accelerated differentiation into CD8
T cells. Consequently, lineage errors did not occur except when MHC I-TCR signaling was so prolonged that the CD4-lineage-specifying transcription factor ThPOK was expressed, preventing Runx3d induction. Thus, our results identify a compensatory signaling mechanism that prevents lineage-fate errors by dynamically modulating Runx3d induction rates during MHC I positive selection.
T cell differentiation in the thymus proceeds in an ordered sequence of developmental events characterized by variable expression of CD4 and CD8 coreceptors. Here, we report that immature ...single-positive (ISP) thymocytes are molecularly distinct from all other T cell populations in the thymus in their expression of a gene profile that is dependent on the transcription factor BRD4. Conditional deletion of BRD4 at various stages of thymic differentiation reveals that BRD4 selectively regulates the further differentiation of ISPs by targeting cell cycle and metabolic pathways, but it does not affect the extensive proliferation that results in the generation of ISPs. These studies lead to the conclusion that the ISP subpopulation is not a hybrid transitional state but a molecularly distinct subpopulation that is selectively dependent on BRD4.
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•ISP thymocyte differentiation is selectively dependent on BRD4•Deletion of BRD4 in ISPs downregulates cell cycle and metabolic pathways•BRD4 is not necessary for proliferation of the DN thymocytes•Treg and iNKT cell differentiation is BRD4 dependent
Thymocytes differentiate from immature DN to ISP, DP, and single-positive thymocytes. Gegonne et al. report the finding that BRD4 is required at the transition from immature ISP to DP thymocytes but not for the differentiation of DN thymocytes or the maturation of conventional single-positive thymocytes from the DP stage.
Foxp3+ Tregs are potent immunosuppressive CD4+ T cells that are critical to maintain immune quiescence and prevent autoimmunity. Both the generation and maintenance of Foxp3+ Tregs depend on the ...cytokine IL-2. Hence, the expression of the IL-2 receptor α-chain (CD25) is not only considered a specific marker, but also a nonredundant requirement for Tregs. Here, we report that Foxp3+ Tregs in the small intestine (SI) epithelium, a critical barrier tissue, are exempt from such an IL-2 requirement, since they had dramatically downregulated CD25 expression, showed minimal STAT5 phosphorylation ex vivo, and were unable to respond to IL-2 in vitro. Nonetheless, SI epithelial Tregs survived and were present at the same frequency as in other lymphoid organs, and they retained potent suppressor function that was associated with high levels of CTLA-4 expression and the production of copious amounts of IL-10. Moreover, adoptive transfer experiments of Foxp3+ Tregs revealed that such IL-2-independent survival and effector functions were imposed by the SI epithelial tissue, suggesting that tissue adaptation is a mechanism that tailors the effector function and survival requirements of Foxp3+ Tregs specific to the tissue environment.
CTLA-4-deficient mice develop a lethal autoimmune lymphoproliferative disorder that is strictly dependent on in vivo CD28 costimulation. Nevertheless, it is not known whether there is a specific site ...on the CD28 molecule that is required for induction of autoimmunity. Using CTLA-4-deficient mice expressing CD28 molecules with various point mutations in the CD28 cytosolic tail, the present study documents that in vivo costimulation for induction of autoimmune disease strictly requires an intact C-terminal proline motif that promotes lymphocyte-specific protein tyrosine kinase Lck binding to the CD28 cytosolic tail, because point mutations in C-terminal proline residues (Pro-187 and Pro-190) completely prevented disease induction. In contrast, in vivo costimulation for disease induction did not require either an intact YMNM motif or an intact N-terminal proline motif, which, respectively, promote phosphoinositide 3-kinase and IL2-inducible T cell kinase binding to the CD28 cytosolic tail. Thus, in vivo CD28 costimulation for induction of autoimmune disease is strictly and specifically dependent on an intact C-terminal proline motif that serves as a lymphocyte-specific protein tyrosine Lck kinase binding site in the CD28 cytosolic tail.
MHC-independent αβTCRs (TCRs) recognize conformational epitopes on native self-proteins and arise in mice lacking both MHC and CD4/CD8 coreceptor proteins. Although naturally generated in the thymus, ...these TCRs resemble re-engineered therapeutic chimeric antigen receptor (CAR) T cells in their specificity for MHC-independent ligands. Here we identify naturally arising MHC-independent TCRs reactive to three native self-proteins (CD48, CD102, and CD155) involved in cell adhesion. We report that naturally arising MHC-independent TCRs require high affinity TCR-ligand engagements in the thymus to signal positive selection and that high affinity positive selection generates a peripheral TCR repertoire with limited diversity and increased self-reactivity. We conclude that the affinity of TCR-ligand engagements required to signal positive selection in the thymus inversely determines the diversity and self-tolerance of the mature TCR repertoire that is selected.
In addition to TCR signaling, the activation and proliferation of naive T cells require CD28-mediated co-stimulation. Once engaged, CD28 is phosphorylated and can then activate signaling pathways by ...recruiting molecules to its YMNM motif and two PxxP motifs. In this study, we analyzed the relationship between tyrosine phosphorylation and the co-stimulatory function of CD28 in murine primary CD4(+) T cells. Tyrosine phosphorylation is decreased in CD28 where the N-terminal PxxP motif is mutated (nPA). In cells expressing nPA, activation of Akt and functional co-stimulation were decreased. In contrast, where the C-terminal PxxP motif is mutated, tyrosine phosphorylation and activation of the ERK, Akt and NF-κB were intact, but proliferation and IL-2 production were decreased. Using the Y(189) to F mutant, we also demonstrated that in naive CD4(+) T cells, tyrosine at position 189 in the YMNM motif is critical for both tyrosine phosphorylation and the functional co-stimulatory effects of CD28. This mutation did not affect unfractionated T-cell populations. Overall, our data suggest that CD28 signaling uses tyrosine phosphorylation-dependent and phosphorylation-independent pathways.
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