How autoreactive thymocytes differentiate into regulatory versus effector CD4+ T cells after avoiding clonal deletion.

Thymocytes bearing autoreactive T cell receptors (TCRs) are agonist-signaled by TCR/co-stimulatory molecules to either undergo clonal deletion or to differentiate into specialized regulatory T (Treg) or effector T (Teff) CD4+ cells. How these different fates are achieved during development remains poorly understood. We now document that deletion and differentiation are agonist-signaled at different times during thymic selection and that Treg and Teff cells both arise after clonal deletion as alternative lineage fates of agonist-signaled CD4+CD25+ precursors. Disruption of agonist signaling induces CD4+CD25+ precursors to initiate Foxp3 expression and become Treg cells, whereas persistent agonist signaling induces CD4+CD25+ precursors to become IL-2+ Teff cells. Notably, we discovered that transforming growth factor-ß induces Foxp3 expression and promotes Treg cell development by disrupting weaker agonist signals and that Foxp3 expression is not induced by IL-2 except under non-physiological in vivo conditions. Thus, TCR signaling disruption versus persistence is a general mechanism of lineage fate determination in the thymus that directs development of agonist-signaled autoreactive thymocytes.

Reversal of the T cell immune system reveals the molecular basis for T cell lineage fate determination in the thymus.

T cell specificity and function are linked during development, as MHC-II-specific TCR signals generate CD4 helper T cells and MHC-I-specific TCR signals generate CD8 cytotoxic T cells, but the basis remains uncertain. We now report that switching coreceptor proteins encoded by Cd4 and Cd8 gene loci functionally reverses the T cell immune system, generating CD4 cytotoxic and CD8 helper T cells. Such functional reversal reveals that coreceptor proteins promote the helper-lineage fate when encoded by Cd4, but promote the cytotoxic-lineage fate when encoded in Cd8-regardless of the coreceptor proteins each locus encodes. Thus, T cell lineage fate is determined by cis-regulatory elements in coreceptor gene loci and is not determined by the coreceptor proteins they encode, invalidating coreceptor signal strength as the basis of lineage fate determination. Moreover, we consider that evolution selected the particular coreceptor proteins that Cd4 and Cd8 gene loci encode to avoid generating functionally reversed T cells because they fail to promote protective immunity against environmental pathogens.

A TCR mechanotransduction signaling loop induces negative selection in the thymus.

The T cell antigen receptor (TCR) expressed on thymocytes interacts with self-peptide major histocompatibility complex (pMHC) ligands to signal apoptosis or survival. Here, we found that negative-selection ligands induced thymocytes to exert forces on the TCR and the co-receptor CD8 and formed cooperative TCR-pMHC-CD8 trimolecular 'catch bonds', whereas positive-selection ligands induced less sustained thymocyte forces on TCR and CD8 and formed shorter-lived, independent TCR-pMHC and pMHC-CD8 bimolecular 'slip bonds'. Catch bonds were not intrinsic to either the TCR-pMHC or the pMHC-CD8 arm of the trans (cross-junctional) heterodimer but resulted from coupling of the extracellular pMHC-CD8 interaction to the intracellular interaction of CD8 with TCR-CD3 via associated kinases to form a cis (lateral) heterodimer capable of inside-out signaling. We suggest that the coupled trans-cis heterodimeric interactions form a mechanotransduction loop that reinforces negative-selection signaling that is distinct from positive-selection signaling in the thymus.

Identification of lineage-specifying cytokines that signal all CD8+-cytotoxic-lineage-fate 'decisions' in the thymus.

T cell antigen receptor (TCR) signaling in the thymus initiates positive selection, but the CD8+-lineage fate is thought to be induced by cytokines after TCR signaling has ceased, although this remains controversial and unproven. We have identified four cytokines (IL-6, IFN-γ, TSLP and TGF-ß) that did not signal via the common γ-chain (γc) receptor but that, like IL-7 and IL-15, induced expression of the lineage-specifying transcription factor Runx3d and signaled the generation of CD8+ T cells. Elimination of in vivo signaling by all six of these 'lineage-specifying cytokines' during positive selection eliminated Runx3d expression and completely abolished the generation of CD8+ single-positive thymocytes. Thus, this study proves that signaling during positive selection by lineage-specifying cytokines is responsible for all CD8+-lineage-fate 'decisions' in the thymus.

Timing and duration of MHC I positive selection signals are adjusted in the thymus to prevent lineage errors.

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.

Lck availability during thymic selection determines the recognition specificity of the T cell repertoire.

Thymic selection requires signaling by the protein tyrosine kinase Lck to generate T cells expressing αß T cell antigen receptors (TCR). For reasons not understood, the thymus selects only αßTCR that are restricted by major histocompatibility complex (MHC)-encoded determinants. Here, we report that Lck proteins that were coreceptor associated promoted thymic selection of conventionally MHC-restricted TCR, but Lck proteins that were coreceptor free promoted thymic selection of MHC-independent TCR. Transgenic TCR with MHC-independent specificity for CD155 utilized coreceptor-free Lck to signal thymic selection in the absence of MHC, unlike any transgenic TCR previously described. Thus, the thymus can select either MHC-restricted or MHC-independent αßTCR depending on whether Lck is coreceptor associated or coreceptor free. We conclude that the intracellular state of Lck determines the specificity of thymic selection and that Lck association with coreceptor proteins during thymic selection is the mechanism by which MHC restriction is imposed on a randomly generated αßTCR repertoire.

Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function.

Lethal-7 (let-7) microRNAs (miRNAs) are the most abundant miRNAs in the genome, but their role in developing thymocytes is unclear. We found that let-7 miRNAs targeted Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and thereby the effector functions of natural killer T cells (NKT cells). Dynamic upregulation of let-7 miRNAs during the development of NKT thymocytes downregulated PLZF expression and directed their terminal differentiation into interferon-γ (IFN-γ)-producing NKT1 cells. Without upregulation of let-7 miRNAs, NKT thymocytes maintained high PLZF expression and terminally differentiated into interleukin 4 (IL-4)-producing NKT2 cells or IL-17-producing NKT17 cells. Upregulation of let-7 miRNAs in developing NKT thymocytes was signaled by IL-15, vitamin D and retinoic acid. Such targeting of a lineage-specific transcription factor by miRNA represents a previously unknown level of developmental regulation in the thymus.

TCR affinity for thymoproteasome-dependent positively selecting peptides conditions antigen responsiveness in CD8(+) T cells.

In the thymus, low-affinity T cell antigen receptor (TCR) engagement facilitates positive selection of a useful T cell repertoire. Here we report that TCR responsiveness of mature CD8(+) T cells is fine tuned by their affinity for positively selecting peptides in the thymus and that optimal TCR responsiveness requires positive selection on major histocompatibility complex class I-associated peptides produced by the thymoproteasome, which is specifically expressed in the thymic cortical epithelium. Thymoproteasome-independent positive selection of monoclonal CD8(+) T cells results in aberrant TCR responsiveness, homeostatic maintenance and immune responses to infection. These results demonstrate a novel aspect of positive selection, in which TCR affinity for positively selecting peptides produced by thymic epithelium determines the subsequent antigen responsiveness of mature CD8(+) T cells in the periphery.

The transcription factor ThPOK suppresses Runx3 and imposes CD4(+) lineage fate by inducing the SOCS suppressors of cytokine signaling.

Lineage fate in the thymus is determined by mutually exclusive expression of the transcription factors ThPOK and Runx3, with ThPOK imposing the CD4(+) lineage fate and Runx3 promoting the CD8(+) lineage fate. While it is known that cytokine signals induce thymocytes to express Runx3, it is not known how ThPOK prevents thymocytes from expressing Runx3 and adopting the CD8(+) lineage fate, nor is it understood why ThPOK itself imposes the CD4(+) lineage fate on thymocytes. We now report that genes encoding members of the SOCS (suppressor of cytokine signaling) family are critical targets of ThPOK and that their induction by ThPOK represses Runx3 expression and promotes the CD4(+) lineage fate. Thus, induction of SOCS-encoding genes is the main mechanism by which ThPOK imposes the CD4(+) lineage fate in the thymus.

IL-7 signaling must be intermittent, not continuous, during CD8⁺ T cell homeostasis to promote cell survival instead of cell death.

The maintenance of naive CD8(+) T cells is necessary for lifelong immunocompetence but for unknown reasons requires signaling via both interleukin 7 (IL-7) and the T cell antigen receptor (TCR). We now report that naive CD8(+) T cells required IL-7 signaling to be intermittent, not continuous, because prolonged IL-7 signaling induced naive CD8(+) T cells to proliferate, produce interferon-γ (IFN-γ) and undergo IFN-γ-triggered cell death. Homeostatic engagement of the TCR interrupted IL-7 signaling and thereby supported the survival and quiescence of CD8(+) T cells. However, CD8(+) T cells with insufficient TCR affinity for self ligands received prolonged IL-7 signaling and died during homeostasis. In this study we identified regulation of the duration of IL-7 signaling by homeostatic engagement of the TCR as the basis for in vivo CD8(+) T cell homeostasis.

Clonal deletion and the fate of autoreactive thymocytes that survive negative selection.

Clonal deletion of autoreactive thymocytes is important for self-tolerance, but the intrathymic signals that induce clonal deletion have not been clearly identified. We now report that clonal deletion during negative selection required CD28-mediated costimulation of autoreactive thymocytes at the CD4(+)CD8(lo) intermediate stage of differentiation. Autoreactive thymocytes were prevented from undergoing clonal deletion by either a lack of CD28 costimulation or transgenic overexpression of the antiapoptotic factors Bcl-2 or Mcl-1, with surviving thymocytes differentiating into anergic CD4(-)CD8(-) double-negative thymocytes positive for the T cell antigen receptor αß subtype (TCRαß) that 'preferentially' migrated to the intestine, where they re-expressed CD8α and were sequestered as CD8αα(+) intraepithelial lymphocytes (IELs). Our study identifies costimulation by CD28 as the intrathymic signal required for clonal deletion and identifies CD8αα(+) IELs as the developmental fate of autoreactive thymocytes that survive negative selection.

Signaling by intrathymic cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes the differentiation of cytotoxic-lineage T cells.

Immature CD4(+)CD8(+) (double-positive (DP)) thymocytes are signaled via T cell antigen receptors (TCRs) to undergo positive selection and become responsive to intrathymic cytokines such as interleukin 7 (IL-7). We report here that cytokine signaling is required for positively selected thymocytes to express the transcription factor Runx3, specify CD8 lineage choice and differentiate into cytotoxic-lineage T cells. In DP thymocytes genetically engineered to be cytokine responsive, IL-7 signaling induced TCR-unsignaled DP thymocytes to express Runx3 and to differentiate into mature CD8(+) T cells, completely circumventing positive selection. We conclude that TCR-mediated positive selection converts DP cells into cytokine-responsive thymocytes, but it is subsequent signaling by intrathymic cytokines that specifies CD8 lineage choice and promotes differentiation into cytotoxic-lineage T cells.

Foxp3 transcription factor is proapoptotic and lethal to developing regulatory T cells unless counterbalanced by cytokine survival signals.

Immune tolerance requires regulatory T (Treg) cells to prevent autoimmune disease, with the transcription factor Foxp3 functioning as the critical regulator of Treg cell development and function. We report here that Foxp3 was lethal to developing Treg cells in the thymus because it induced a unique proapoptotic protein signature (Puma⁺⁺⁺p-Bim⁺⁺p-JNK⁺⁺DUSP6⁻) and repressed expression of prosurvival Bcl-2 molecules. However, Foxp3 lethality was prevented by common gamma chain (γc)-dependent cytokine signals that were present in the thymus in limiting amounts sufficient to support only ∼1 million Treg cells. Consequently, most newly arising Treg cells in the thymus were deprived of this signal and underwent Foxp3-induced death, with Foxp3⁺CD25⁻ Treg precursor cells being the most susceptible. Thus, we identify Foxp3 as a proapoptotic protein that requires developing Treg cells to compete with one another for limiting amounts of γc-dependent survival signals in the thymus.

Structure of MHC-Independent TCRs and Their Recognition of Native Antigen CD155.

During normal T cell development in the thymus, αß TCRs signal immature thymocytes to differentiate into mature T cells by binding to peptide-MHC ligands together with CD4/CD8 coreceptors. Conversely, in MHC and CD4/CD8 coreceptor-deficient mice, the thymus generates mature T cells expressing MHC-independent TCRs that recognize native conformational epitopes rather than linear antigenic-peptides presented by MHC. To date, no structural information of MHC-independent TCRs is available, and their structural recognition of non-MHC ligand remains unknown. To our knowledge in this study, we determined the first structures of two murine MHC-independent TCRs (A11 and B12A) that bind with high nanomolar affinities to mouse adhesion receptor CD155. Solution binding demonstrated the Vαß-domain is responsible for MHC-independent B12A recognition of its ligand. Analysis of A11 and B12A sequences against various MHC-restricted and -independent TCR sequence repertoires showed that individual V-genes of A11 and B12A did not exhibit preference against MHC-restriction. Likewise, CDR3 alone did not discriminate against MHC binding, suggesting VDJ recombination together with Vα/Vß pairing determine their MHC-independent specificity for CD155. The structures of A11 and B12A TCR are nearly identical to those of MHC-restricted TCR, including the conformations of CDR1 and 2. Mutational analysis, together with negative-staining electron microscopy images, showed that the CDR regions of A11 and B12A recognized epitopes on D1 domain of CD155, a region also involved in CD155 binding to poliovirus and Tactile in human. Taken together, MHC-independent TCRs adopt canonical TCR structures to recognize native Ags, highlighting the importance of thymic selection in determining TCR ligand specificity.

αß T cell receptors that do not undergo major histocompatibility complex-specific thymic selection possess antibody-like recognition specificities.

Major histocompatibility complex (MHC) restriction is the cardinal feature of T cell antigen recognition and is thought to be intrinsic to αß T cell receptor (TCR) structure because of germline-encoded residues that impose MHC specificity. Here, we analyzed αßTCRs from T cells that had not undergone MHC-specific thymic selection. Instead of recognizing peptide-MHC complexes, the two αßTCRs studied here resembled antibodies in recognizing glycosylation-dependent conformational epitopes on a native self-protein, CD155, and they did so with high affinity independently of MHC molecules. Ligand recognition was via the αßTCR combining site and involved the identical germline-encoded residues that have been thought to uniquely impose MHC specificity, demonstrating that these residues do not only promote MHC binding. This study demonstrates that, without MHC-specific thymic selection, αßTCRs can resemble antibodies in recognizing conformational epitopes on MHC-independent ligands.

Chromatin remodeling complex NURF regulates thymocyte maturation.

The maturation of T cells requires signaling from both cytokine and T-cell receptors to gene targets in chromatin, but how chromatin architecture influences this process is largely unknown. Here we show that thymocyte maturation post-positive selection is dependent on the nucleosome remodeling factor (NURF). Depletion of Bptf (bromodomain PHD finger transcription factor), the largest NURF subunit, in conditional mouse mutants results in developmental arrest beyond the CD4(+) CD8(int) stage without affecting cellular proliferation, cellular apoptosis, or coreceptor gene expression. In the Bptf mutant, specific subsets of genes important for thymocyte development show aberrant expression. We also observed defects in DNase I-hypersensitive chromatin structures at Egr1, a prototypical Bptf-dependent gene that is required for efficient thymocyte development. Moreover, chromatin binding of the sequence-specific factor Srf (serum response factor) to Egr1 regulatory sites is dependent on Bptf function. Physical interactions between NURF and Srf suggest a model in which Srf recruits NURF to facilitate transcription factor binding at Bptf-dependent genes. These findings provide evidence for causal connections between NURF, transcription factor occupancy, and gene regulation during thymocyte development.

Upregulation of CD4 expression during MHC class II-specific positive selection is essential for error-free lineage choice.

The lineage fate of developing thymocytes is determined by the persistence or cessation of T cell receptor (TCR) signaling during positive selection, with persistent TCR signaling required for CD4 lineage choice. We show here that transcriptional upregulation of CD4 expression is essential for error-free lineage choice during major histocompatibility complex class II (MHC II)-specific positive selection and is critical for error-free lineage choice in TCR-transgenic mice whose thymocytes compete for the identical selecting ligand. CD4 upregulation occurred for endogenously encoded CD4 coreceptors, but CD4 transgenes were downregulated during positive selection, disrupting MHC II-specific TCR signaling and causing lineage errors regardless of the absolute number or signaling strength of transgenic CD4 proteins. Thus, the kinetics of CD4 coreceptor expression during MHC II-specific positive selection determines the integrity of CD4 lineage choice, revealing an elegant symmetry between coreceptor kinetics and lineage choice.

Coreceptor gene imprinting governs thymocyte lineage fate.

Immature thymocytes are bipotential cells that are signalled during positive selection to become either helper- or cytotoxic-lineage T cells. By tracking expression of lineage determining transcription factors during positive selection, we now report that the Cd8 coreceptor gene locus co-opts any coreceptor protein encoded within it to induce thymocytes to express the cytotoxic-lineage factor Runx3 and to adopt the cytotoxic-lineage fate, findings we refer to as 'coreceptor gene imprinting'. Specifically, encoding CD4 proteins in the endogenous Cd8 gene locus caused major histocompatibility complex class II-specific thymocytes to express Runx3 during positive selection and to differentiate into CD4(+) cytotoxic-lineage T cells. Our findings further indicate that coreceptor gene imprinting derives from the dynamic regulation of specific cis Cd8 gene enhancer elements by positive selection signals in the thymus. Thus, for coreceptor-dependent thymocytes, lineage fate is determined by Cd4 and Cd8 coreceptor gene loci and not by the specificity of T-cell antigen receptor/coreceptor signalling. This study identifies coreceptor gene imprinting as a critical determinant of lineage fate determination in the thymus.

Regulation of MHC class I expression by Foxp3 and its effect on regulatory T cell function.

Expression of MHC class I molecules, which provide immune surveillance against intracellular pathogens, is higher on lymphoid cells than on any other cell types. In T cells, this is a result of activation of class I transcription by the T cell enhanceosome consisting of Runx1, CBFß, and LEF1. We now report that MHC class I transcription in T cells also is enhanced by Foxp3, resulting in higher levels of class I in CD4(+)CD25(+) T regulatory cells than in conventional CD4(+)CD25(-) T cells. Interestingly, the effect of Foxp3 regulation of MHC class I transcription is cell type specific: Foxp3 increases MHC class I expression in T cells but represses it in epithelial tumor cells. In both cell types, Foxp3 targets the upstream IFN response element and downstream core promoter of the class I gene. Importantly, expression of MHC class I contributes to the function of CD4(+)CD25(+) T regulatory cells by enhancing immune suppression, both in in vitro and in vivo. These findings identify MHC class I genes as direct targets of Foxp3 whose expression augments regulatory T cell function.

MHC restriction is imposed on a diverse T cell receptor repertoire by CD4 and CD8 co-receptors during thymic selection.

Mature αß T cells recognize foreign antigenic peptides presented by MHC molecules but do not recognize native antigenic proteins; features known as MHC restriction. How MHC restriction is imposed on αß T cells has intrigued immunologists for several decades. One model proposes that germline-encoded elements in the T cell receptor (TCR) variable regions are evolutionarily conserved to only recognize MHC. However, we propose an alternative model that posits that MHC restriction is imposed by CD4 and CD8 co-receptors during thymic selection. Thus, we think that TCRs are structurally able to recognize a huge diversity of ligands but only TCRs with MHC specificity survive thymic selection.