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.

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.

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.

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.