Manoj Saini

Why is PTPN22 a good candidate susceptibility gene for autoimmune disease

The PTPN22 locus is one of the strongest risk factors outside of the major histocompatability complex that associates with autoimmune diseases. PTPN22 encodes lymphoid protein tyrosine phosphatase (Lyp) which is expressed exclusively in immune cells. A single base change in the coding region of this gene resulting in an arginine to tryptophan amino acid substitution within a polyproline binding motif associates with type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosis, Hashimotos thyroiditis, Graves disease, Addisons disease, Myasthenia Gravis, vitiligo, systemic sclerosis juvenile idiopathic arthritis and psoriatic arthritis. Here, we review the current understanding of the PTPN22 locus from a genetic, geographical, biochemical and functional perspective.

Lymphotoxin Signals from Positively Selected Thymocytes Regulate the Terminal Differentiation of Medullary Thymic Epithelial Cells

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.

Regulation of Zap70 expression during thymocyte development enables temporal separation of CD4 and CD8 repertoire selection at different signaling thresholds.

Regulated expression of Zap70 determines whether thymocytes develop into CD4+ or CD8+ T cells. Timing Is Everything As they develop into mature CD4+ or CD8+ single-positive (SP) T cells in the thymus, immature thymocytes undergo positive selection, during which they have both CD4 and CD8 [double-positive (DP) cells] and their T cell antigen receptor (TCR) is stimulated by self-peptide–major histocompatibility complexes. It remains unclear whether it is the strength or the duration of TCR signaling that determines how a cell “chooses” to become a CD4+ or CD8+ SP cell (see the Perspective by Alarcón and van Santen). Saini et al. generated transgenic mice that expressed an inducible gene encoding the tyrosine kinase Zap70, a critical mediator of TCR signaling, in a Zap70-deficient background (TetZap70 mice). Thymocytes in Zap70-deficient mice are arrested at the DP stage because of defective TCR signaling, but this block was removed by induction of Zap70 expression in the TetZap70 mice, after which CD4+ SP cells developed more quickly than did CD8+ SP cells. The temporal difference in SP cell development, also seen in wild-type mice, occurred because CD4+ SP cells required less Zap70 protein to develop than did CD8+ SP cells. In addition, this study showed that DP thymocytes were heterogeneous, with CD4+ and CD8+ SP cells arising from different DP subpopulations. Together, these data suggest that regulation of the abundance of Zap70 enables the temporal differential development of CD4+ and CD8+ cells, which is based on signal strength through the TCR, thus incorporating both the “strength” and the “duration” models of positive selection. 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 ζ 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.

Abrogation of CD30 and OX40 signals prevents autoimmune disease in FoxP3-deficient mice

FoxP3-deficient mice are rescued from tissue and organ destruction and subsequent lethal autoimmune disease by the combined absence of OX40 and CD30 signals.

Regulation of T cell-dendritic cell interactions by IL-7 governs T-cell activation and homeostasis.

Interleukin-7 (IL-7) plays a central role in the homeostasis of the T-cell compartment by regulating T-cell survival and proliferation. Whether IL-7 can influence T-cell receptor (TCR) signaling in T cells remains controversial. Here, using IL-7-deficient hosts and TCR-transgenic T cells that conditionally express IL-7R, we examined antigen-specific T-cell responses in vitro and in vivo to viral infection and lymphopenia to determine whether IL-7 signaling influences TCR-triggered cell division events. In vitro, we could find no evidence that IL-7 signaling could costimulate T-cell activation over a broad range of conditions, suggesting that IL-7 does not directly tune TCR signaling. In vivo, however, we found an acute requirement for IL-7 signaling for efficiently triggering T-cell responses to influenza A virus challenge. Furthermore, we found that IL-7 was required for the enhanced homeostatic TCR signaling that drives lymphopenia-induced proliferation by a mechanism involving efficient contacts of T cells with dendritic cells. Consistent with this, saturating antigen-presenting capacity in vivo overcame the triggering defect in response to cognate peptide. Thus, we demonstrate a novel role for IL-7 in regulating T cell-dendritic cell interactions that is essential for both T-cell homeostasis and activation in vivo.

Lymphoid tissue inducer cells: bridges between the ancient innate and the modern adaptive immune systems

Phylogeny indicates that adaptive immunity evolved first in diffusely distributed lymphoid tissues found in the lamina propria (LP) of the gut. B follicular structures appeared later, probably initially in isolated lymphoid follicles in the LP and then in organized lymphoid tissues such as lymph nodes and Peyers patches. The development of these new lymphoid structures was enabled by gene duplication and evolution of new tumor necrosis family members. Here, we argue that lymphoid tissue inducer cells (LTis) had a pivotal role, not only in the development of organized lymphoid structures, but also in the subsequent genesis of the CD4-dependent class-switched memory antibody responses. In this review, we concentrate on the latter function: the sustenance by LTis of CD4 T-cell responses for protective immunity.

The Long-Term Survival Potential of Mature T Lymphocytes Is Programmed During Development in the Thymus

T cell receptor signaling in thymocytes determines their responsiveness to a survival cytokine later in life. Programming T Cell Survival The development of thymocytes in the thymus critically depends on signals through the T cell antigen receptor (TCR) and the receptor for the homeostatic cytokine interleukin-7 (IL-7). During development, thymocytes lose IL-7Rα but regain the receptor after they receive signals through the TCR in a process known as positive selection. When thymocytes leave the thymus as mature T lymphocytes and move to peripheral lymphoid organs, they still depend on TCR and IL-7R signaling for their survival. Sinclair et al. found that TCR signaling in developing thymocytes in the mouse activated the reexpression of Il7r and the reappearance of IL-7Rα on the cell surface. The abundance of surface IL-7Rα correlated with the strength of positive selection, and those T cells with the highest abundance of IL-7Rα had the greatest chance of survival in the periphery. In contrast, TCR signaling in peripheral T cells did not alter the abundance or function of IL-7Rα. Together, these data suggest that TCR-dependent regulation of IL-7Rα abundance on thymocytes determines the long-term survivability of the resulting T cells. 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.

Mathematical Modeling Reveals the Biological Program Regulating Lymphopenia-Induced Proliferation

Recognition of peptide-MHC by the TCR induces T lymphocytes to undergo cell division. Although recognition of foreign peptide induces a program of cellular division and differentiation by responding T cells, stimulation by self-peptide MHC complexes in lymphopenic conditions induces a slower burst of divisions that may or may not be accompanied by effector differentiation. Although both responses are triggered by signals from the TCR, it is not known whether they represent distinct programs of cell cycle control. In this study, we use a mathematical modeling approach to analyze the proliferative response of TCR transgenic F5 T cells to lymphopenia. We tested two fundamentally different models of cell division: one in which T cells are triggered into an “autopilot” deterministic burst of divisions, a model successfully used elsewhere to describe T cell responses to cognate Ag, and a second contrasting model in which cells undergo independent single stochastic divisions. Whereas the autopilot model provided a very poor description of the F5 T cell responses to lymphopenia, the model of single stochastic divisions fitted the experimental data remarkably closely. Furthermore, this model proved robust because specific predictions of cellular behavior made by this model concerning the onset, rate, and nature of division were successfully validated experimentally. Our results suggest cell division induced by lymphopenia involves a process of single stochastic divisions, which is best suited to a homeostatic rather than differentiation role.

IL-7 determines the homeostatic fitness of T cells by distinct mechanisms at different signalling thresholds in vivo.

The cytokine interleukin (IL)‐7 is essential for Treg‐cell homeostasis. It remains unclear, however, whether IL‐7 regulates the homeostatic fitness of T cells quantitatively and, if so, by what mechanisms. We addressed this question by analysing T cells exposed to different levels of IL‐7 signalling in vivo. Using TCR transgenic mice that conditionally express IL‐7Rα, we show that T‐cell longevity in the absence of survival cues is not a cell‐intrinsic property but rather a dynamic process of which IL‐7 signalling is a key regulator. Naïve T cells deficient in IL‐7Rα expression underwent rapid cell death within hours of in vitro culture. In contrast, the same T cells from lymphopenic hosts, in which IL‐7 is non‐limiting, were able to survive in culture independently of growth factors for many days. Surprisingly, different levels of IL‐7 signalling in vivo evoked distinct molecular mechanisms to regulate homeostatic fitness. When IL‐7 was non‐limiting, increased survival was associated with up‐regulation of anti‐apoptotic Bcl2 family members. In contrast, in T‐cell replete conditions i.e. when IL‐7 is limiting, we found evidence that IL‐7 regulated T‐cell fitness by distinct non‐transcriptional mechanisms. Together, these data demonstrate a quantitative aspect to IL‐7 signalling dependent on distinct molecular mechanisms.

Lymphoid tissue inducer cells and the evolution of CD4 dependent high-affinity antibody responses.

Phylogeny indicates that in mammals memory CD4-dependent antibody responses evolved after monotremes split from the common ancestor of marsupial and eutherian mammals. This was strongly associated with the development of segregated B and T cell areas and the development of a linked lymph node network. The evolution of the lymphotoxin beta receptor in these higher mammals was key to the development of these new functions. Here, we argue that lymphoid tissue inducer cells played a pivotal role not only in the development of organized lymphoid structures but also in the subsequent genesis of the CD4-dependent class-switched memory antibody responses that depend on an organized infrastructure to work. In this review, we concentrate on the role of this cell type in the making of a tolerant CD4 T cell repertoire and in the sustenance of CD4 T cell responses for protective immunity.