To investigate the temporal regulation of the commitment of immature thymocytes to either the CD4(+) or the CD8(+) lineage in the thymus, we developed a transgenic mouse that expressed a tetracycline-inducible gene encoding the tyrosine kinase zeta chain-associated protein kinase of 70 kD (Zap70), which restored development in Zap70(-/-) thymocytes arrested at the preselection, CD4(+)CD8(+) double-positive (DP) stage. After induction of the expression of Zap70 and the production of Zap70 protein, CD4(+) single-positive (SP) cells that expressed Zbtb7b (which encodes the CD4(+) T cell-associated transcription factor ThPOK) became abundant within 30 hours, whereas CD8(+) SP cells were not detectable until day 4. We found that mature CD4(+) and CD8(+) cells arose from phenotypically distinct subsets of DP thymocytes that developed with different kinetics and contrasting sensitivities to stimulation of the T cell antigen receptor (TCR). In wild-type mice, expression of endogenous Zap70 progressively increased during maturation of the DP subsets, and the abundance of Zap70 protein determined the sensitivity of the cells to stimulation of the TCR. This temporal gradient in the amount of Zap70 protein enabled the selection of CD4(+) and CD8(+) repertoires in separate temporal windows and at different TCR signaling thresholds, thereby facilitating discrimination of distinct positive selection signals in these lineages.

It has long been recognized that the T-cell compartment has more CD4 helper than CD8 cytotoxic T cells, and this is most evident looking at T-cell development in the thymus. However, it remains unknown how thymocyte development so favors CD4 lineage development. To identify the basis of this asymmetry, we analyzed development of synchronized cohorts of thymocytes in vivo and estimated rates of thymocyte death and differentiation throughout development, inferring lineage-specific efficiencies of selection. Our analysis suggested that roughly equal numbers of cells of each lineage enter selection and found that, overall, a remarkable ∼75% of cells that start selection fail to complete the process. Importantly it revealed that class I-restricted thymocytes are specifically susceptible to apoptosis at the earliest stage of selection. The importance of differential apoptosis was confirmed by placing thymocytes under apoptotic stress, resulting in preferential death of class I-restricted thymocytes. Thus, asymmetric death during selection is the key determinant of the CD4:CD8 ratio in which T cells are generated by thymopoiesis. CD4 T cells | CD8 T cellsD evelopment of CD4 and CD8 lineage cells from common thymic precursors is one of the most fundamental developmental processes in the adaptive immune system. The predominance of CD4 over CD8 T-cell populations in the periphery has been apparent since helper and cytotoxic T cells were first delineated more than 30 y ago (1), but the cause of this signature bias has remained obscure. A key contribution arises from the ratio in which CD4 and CD8 lineage T cells are generated by the thymus. Single-positive (SP) thymocytes exist in the thymus at ∼4:1, a ratio that is highly conserved across mouse strains and other species, suggesting that the developmental mechanisms involved are fundamental to the processes that give rise to mature T cells in the thymus. During thymocyte development, T-cell antigen receptor (TCR) genes undergo somatic rearrangements to generate a broad repertoire of TCR structures. Negative and positive selection of thymocytes results in the deletion of autoreactive thymocytes and ensures that class I and class II MHC reactivity is correlated with CD8 and CD4 lineage specification. The molecular mechanisms underpinning these processes are increasingly well understood. Although survival of thymocytes is regulated by expression of Bcl2 family members, up-regulation of the BH3-only family member Bim has been specifically implicated as a key event in negative selection of thymocytes (2). During positive selection, class II recognition is thought to induce strong persistent signaling that results in a cascade of transcriptional regulation by factors such as GATA3 and T-helper-inducing POZ/Krüppel-like factor, which results in fixation of cells to the CD4 lineage (3, 4). In contrast, weaker or transient signaling by class I MHC results in the cytokine-dependent induction of a Runx3-mediated transcriptional program that induces CD8 lineage commitment (5-8).Despit...

The thymic medulla represents a key site for the induction of T cell tolerance. In particular, autoimmune regulator (Aire)-expressing medullary thymic epithelial cells (mTECs) provide a spectrum of tissue-restricted Ags that, through both direct presentation and cross-presentation by dendritic cells, purge the developing T cell repertoire of autoimmune specificities. Despite this role, the mechanisms of Aire+ mTEC development remain unclear, particularly those stages that occur post-Aire expression and represent mTEC terminal differentiation. In this study, in mouse thymus, we analyze late-stage mTEC development in relation to the timing and requirements for Aire and involucrin expression, the latter a marker of terminally differentiated epithelium including Hassall’s corpuscles. We show that Aire expression and terminal differentiation within the mTEC lineage are temporally separable events that are controlled by distinct mechanisms. We find that whereas mature thymocytes are not essential for Aire+ mTEC development, use of an inducible ZAP70 transgenic mouse line—in which positive selection can be temporally controlled—demonstrates that the emergence of involucrin+ mTECs critically depends upon the presence of mature single positive thymocytes. Finally, although initial formation of Aire+ mTECs depends upon RANK signaling, continued mTEC development to the involucrin+ stage maps to activation of the LTα–LTβR axis by mature thymocytes. Collectively, our results reveal further complexity in the mechanisms regulating thymus medulla development and highlight the role of distinct TNFRs in initial and terminal differentiation stages in mTECs.

Antigen-presenting cells (APCs) occupy diverse anatomical tissues, but their tissue-restricted homeostasis remains poorly understood. Here, working in mouse models of inflammation, we found that mTOR-dependent metabolic adaptation was required at discrete locations. mTOR was dispensable for DC homeostasis in secondary lymphoid tissues but necessary to regulate cellular metabolism and accumulation of CD103+ DCs and alveolar macrophages in lung. Moreover, while numbers of mTOR-deficient lung CD11b+ DCs were not changed, they were metabolically reprogrammed to skew allergic inflammation from eosinophilic Th2 to neutrophilic Th17 polarity. The mechanism for this change was independent of translational control but dependent on inflammatory DC, which produced IL-23 and increased fatty acid oxidation. mTOR therefore mediates metabolic adaptation of APCs in distinct tissues, influencing the immunological character of allergic inflammation.

The developmental pathways of regulatory T cells (Treg) generation in the thymus are not fully understood. Here, we reconstituted thymic development of Zap70 deficient thymocytes with a tetracycline inducible Zap70 transgene to allow temporal dissection of Treg development. We find that Treg develop with distinctive kinetics, first appearing by day 4 amongst CD4 single positive (SP) thymocytes. Accepted models of CD25+FoxP3+ Treg selection suggest development via CD25+FoxP3− CD4 SP precursors. In contrast, our kinetic analysis revealed the presence of abundant CD25− FoxP3+ cells that are highly efficient at maturing to CD25+FoxP3+ cells in response to IL-2. CD25− FoxP3+ cells more closely resembled mature Treg both with respect to kinetics of development and avidity for self peptide MHC. These population also exhibited distinct requirements for cytokines during their development. CD25−FoxP3+ cells were IL-15 dependent while generation of CD25+FoxP3+ specifically required IL-2. Finally, we found that IL-2 and IL-15 arose from distinct sources in vivo. IL-15 was of stromal origin, while IL-2 was of exclusively from haemopoetic cells that depended on intact CD4 lineage development but not either antigen experienced or NKT cells.

PI3K inhibitors with differential selectivity to distinct PI3K isoforms have been tested extensively in clinical trials, largely to target tumor epithelial cells. PI3K signaling also regulates the immune system and inhibition of PI3Kδ modulate the tumor immune microenvironment of pre-clinical mouse tumor models by relieving T-regs-mediated immunosuppression. PI3K inhibitors as a class and PI3Kδ specifically are associated with immune-related side effects. However, the impact of mixed PI3K inhibitors in tumor immunology is under-explored. Here we examine the differential effects of AZD8835, a dual PI3Kα/δ inhibitor, specifically on the tumor immune microenvironment using syngeneic models. Continuous suppression of PI3Kα/δ was not required for anti-tumor activity, as tumor growth inhibition was potentiated by an intermittent dosing/schedule in vivo. Moreover, PI3Kα/δ inhibition delivered strong single agent anti-tumor activity, which was associated with dynamic suppression of T-regs, improved CD8+ T-cell activation and memory in mouse syngeneic tumor models. Strikingly, AZD8835 promoted robust CD8+ T-cell activation dissociated from its effect on T-regs. This was associated with enhancing effector cell viability/function. Together these data reveal novel mechanisms by which PI3Kα/δ inhibitors interact with the immune system and validate the clinical compound AZD8835 as a novel immunoncology drug, independent of effects on tumor cells. These data support further clinical investigation of PI3K pathway inhibitors as immuno-oncology agents.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0457-0) contains supplementary material, which is available to authorized users.

The homeostatic maintenance of normal numbers of mature T lymphocytes in the immune system depends on signaling from the T cell antigen receptor (TCR) and the interleukin-7 receptor (IL-7R); however, it is unclear whether there is crosstalk between these two receptors. Here, we have identified a central role for TCR signaling during the development of T lymphocytes in the thymus in the determination of IL-7 function in mature T lymphocytes. We showed that Il7r expression in mature T cells was modulated by developmental TCR-dependent signals elicited during the process of positive selection in the thymus and that this mechanism was common to both CD4(+) and CD8(+) T cells. Control of Il7r expression by the TCR was limited to thymocytes because neither the abundance nor the function of IL-7Rα was affected by TCR signaling in peripheral T cells. Finally, we showed that thymocytes without optimal IL-7Rα abundance failed to form part of the pool of mature T lymphocytes that patrol the periphery of normal hosts, highlighting the importance of this mechanism in shaping the repertoire of lymphocytes that make up this population.

T cell receptor (TCR) signaling plays a central role in directing developmental fates of thymocytes. Current models suggest TCR signal duration directs CD4 vs CD8 lineage development. To investigate the role of TCR signaling during positive selection directly, we switched signaling off in a cohort of selecting thymocytes and followed, in time, their subsequent fate. We did this using an inducible Zap70 transgenic mouse model that allowed Zap70 dependent signaling to be turned on and then off again. Surprisingly, loss of TCR signaling in CD4+CD8lo thymocytes did not prevent their development to committed CD4 single positives (SP), nor their continued maturation to HSAloSPs. However, numbers of CD4SPs underwent a substantial decline following loss of Zap70 expression suggesting an essential survival role for the kinase. Termination of TCR signaling is considered an essential step in CD8 lineage development. Loss of Zap70 expression, however, resulted in the rapid death of CD8 lineage precursor thymocytes and a failure to generate CD8 SPs. Significantly, extending the window Zap70 expression was sufficient for generation and export of both CD4 and CD8 T cells. These data reveal a parallel requirement for TCR-mediated survival signaling, but an asymmetric requirement for TCR-mediated maturation signals.