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De novo synthesis of the pyrimidine, cytidine triphosphate (CTP), is crucial for DNA/RNA metabolism and depends on the CTP synthetases, CTPS1 and −2. Partial CTPS1 deficiency in humans has previously been shown to lead to immunodeficiency, with impaired expansion of T and B cells. Here, we examine the effects of conditional and inducible inactivation of Ctps1 and/or Ctps2 on mouse embryonic development and immunity. We report that deletion of Ctps1, but not Ctps2, is embryonic-lethal. Tissue and cells with high proliferation and renewal rates, such as intestinal epithelium, erythroid and thymic lineages, activated B and T lymphocytes, and memory T cells strongly rely on CTPS1 for their maintenance and growth. However, both CTPS1 and CTPS2 are required for T cell proliferation following TCR stimulation. Deletion of Ctps1 in T cells or treatment with a CTPS1 inhibitor rescued Foxp3-deficient mice from fatal systemic autoimmunity and reduced the severity of experimental autoimmune encephalomyelitis. These findings support that CTPS1 may represent a target for immune suppression. Cytidine nucleotide triphosphate (CTP) is a key precursor involved in the metabolism of DNA, RNA and phospholipids. In this study, the authors examine the physiological consequences of CTP synthase (Ctps) 1 and 2 deletion in vivo and demonstrate that Ctps1 protects mice from fatal autoimmunity. [ABSTRACT FROM AUTHOR]

Lymphocyte-specific protein tyrosine kinase (LCK) is an SRC-family kinase critical for initiation and propagation of T-cell antigen receptor (TCR) signaling through phosphorylation of TCR-associated CD3 chains and recruited downstream molecules. Until now, only one case of profound T-cell immune deficiency with complete LCK deficiency [1] caused by a biallelic missense mutation (c.1022T>C, p.L341P) and three cases of incomplete LCK deficiency [2] caused by a biallelic splice site mutation (c.188-2A>G) have been described. Additionally, deregulated LCK expression has been associated with genetically undefined immune deficiencies and hematological malignancies. Here, we describe the second case of complete LCK deficiency in a 6-month-old girl born to consanguineous parents presenting with profound T-cell immune deficiency. Whole exome sequencing (WES) revealed a novel pathogenic biallelic missense mutation in LCK (c.1393T>C, p.C465R), which led to the absence of LCK protein expression and phosphorylation, and a consecutive decrease in proximal TCR signaling. Loss of conventional CD4+ and CD8+ αβT-cells and homeostatic T-cell expansion was accompanied by increased γδT-cell and Treg percentages. Surface CD4 and CD8 co-receptor expression was reduced in the patient T-cells, while the heterozygous mother had impaired CD4 and CD8 surface expression to a lesser extent. We conclude that complete LCK deficiency is characterized by profound T-cell immune deficiency, reduced CD4 and CD8 surface expression, and a characteristic TCR signaling disorder. CD4 and CD8 surface expression may be of value for early detection of mono- and/or biallelic LCK deficiency. [ABSTRACT FROM AUTHOR]

The use of chimeric antigen receptor (CAR)-engineered regulatory T cells (Tregs) has emerged as a promising strategy to promote immune tolerance. However, in conventional T cells (Tconvs), CAR expression is often associated with tonic signaling, which can induce CAR-T cell dysfunction. The extent and effects of CAR tonic signaling vary greatly according to the expression intensity and intrinsic properties of the CAR. Here, we show that the 4-1BB CSD-associated tonic signal yields a more dramatic effect in CAR-Tregs than in CAR-Tconvs with respect to activation and proliferation. Compared to CD28 CAR-Tregs, 4-1BB CAR-Tregs exhibit decreased lineage stability and reduced in vivo suppressive capacities. Transient exposure of 4-1BB CAR-Tregs to a Treg stabilizing cocktail, including an mTOR inhibitor and vitamin C, during ex vivo expansion sharply improves their in vivo function and expansion after adoptive transfer. This study demonstrates that the negative effects of 4-1BB tonic signaling in Tregs can be mitigated by transient mTOR inhibition. Chimeric antigen receptor engineering in T cells has been shown to be of great potential therapeutic benefit in a range of immune pathologies, although the functionality of such cell therapies can be limited due to tonic signalling and the induction of dysfunction. Here the authors show transient inhibition of mTOR can rescue their 41-BB-CAR-Tregs from tonic signalling-induced dysfunction. [ABSTRACT FROM AUTHOR]

Primary immunodeficiencies (PIDs) provide researchers with unique models to understand in vivo immune responses in general and immunity to infections in particular. In humans, impaired immune control of Epstein‐Barr virus (EBV) infection is associated with the occurrence of several different immunopathologic conditions; these include non‐malignant and malignant B‐cell lymphoproliferative disorders, hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory condition, and a chronic acute EBV infection of T cells. Studies of PIDs associated with a predisposition to develop severe, chronic EBV infections have led to the identification of key components of immunity to EBV – notably the central role of T‐cell expansion and its regulation in the pathophysiology of EBV‐associated diseases. On one hand, the defective expansion of EBV‐specific CD8 T cells results from mutations in genes involved in T‐cell activation (such as RASGRP1, MAGT1, and ITK), DNA metabolism (CTPS1) or co‐stimulatory pathways (CD70, CD27, and TNFSFR9 (also known as CD137/4‐1BB)) leads to impaired elimination of proliferating EBV‐infected B cells and the occurrence of lymphoma. On the other hand, protracted T‐cell expansion and activation after the defective killing of EBV‐infected B cells is caused by genetic defects in the components of the lytic granule exocytosis pathway or in the small adapter protein SH2D1A (also known as SAP), a key activator of T‐ and NK cell‐cytotoxicity. In this setting, the persistence of EBV‐infected cells results in HLH, a condition characterized by unleashed T‐cell and macrophage activation. Moreover, genetic defects causing selective vulnerability to EBV infection have highlighted the role of co‐receptor molecules (CD27, CD137, and SLAM‐R) selectively involved in immune responses against infected B cells via specific T‐B cell interactions. [ABSTRACT FROM AUTHOR]

Cernunnos is a DNA repair factor of the nonhomologous end-joining machinery. Its deficiency in humans causes radiosensitive severe combined immune deficiency (SCID) with microcephaly, characterized in part by a profound lymphopenia. In contrast to the human condition, the immune system of Cernunnos knockout (KO) mice is not overwhelmingly affected. In particular, Cernunnos is dispensable during V(D)J recombination in lymphoid cells. Nevertheless, the viability of thymocytes is reduced in Cernunnos KO mice, owing to the chronic activation of a P53-dependent DNA damage response. This translates into a qualitative alteration of the T cell repertoire to one in which the most distal Vα and Jα segments are missing. This results in the contraction of discrete T cell populations, such as invariant natural killer T (iNKT) and mucosa-associated invariant T (MAIT) cells, in both humans and mice. [ABSTRACT FROM AUTHOR]

Hemophagocytic lymphohistiocytosis (HLH) manifests as the uncontrolled activation of T lymphocytes and macrophages infiltrating multiple organs. Molecular studies of individuals with HLH have demonstrated in most of these conditions a critical role of granule-dependent cytotoxic activity in the regulation of lymphocyte homeostasis, and have allowed the characterization of key effectors regulating cytotoxic granule release. The cytolytic process may now be considered a multistep process, including cell activation; the polarization of cytotoxic granules toward the conjugated target cell; the tethering, priming, and fusion of the cytotoxic granules with the plasma membrane; and the release of their contents (perforin and granzymes) into the intercellular cleft, leading to target cell death. Cytolytic cells have a second effector function involving the production of cytokines, principally γ-interferon, which is secreted independently of the exocytosis cytotoxic granule pathway. An analysis of the mechanisms underlying HLH has identified γ-interferon as a key cytokine inducing uncontrolled macrophage activation, and thus represents a potential therapeutic target. [ABSTRACT FROM AUTHOR]

Mucosal-associated invariant T cells, the most abundant invariant T cell subset in humans, arise via a distinct developmental pathway that represents a hybrid of that seen for NKT and γδ T cells, two other unconventional T cell subsets. [ABSTRACT FROM AUTHOR]

Cytolytic lymphocytes kill virus-infected cells as well as tumor cells by the exocytosis of the content of specialized secretory lysosomes at the immunological synapse (IS). Perforin and granzymes are the molecular effectors that induce rapid target cell death. Cytolytic T cells are activated by specific antigen recognition whereas the cytolytic activity of natural killer cells is initiated by specific activating receptors or combinations thereof and is inhibited by self MHC class I recognition. The cytolytic process has received considerable attention and can now be described as a multi-step process including cell activation, polarization of specialized lysosomes — lytic granules — toward the IS, tethering of the lytic granules to the plasma membrane, priming for fusion with the plasma membrane, effective fusion and release of granule content in the IS cleft, and death of the target. This is a highly flexible system that could enable a cytolytic cell to subsequently kill target cells bound at different sites around the effector cell. Cytolytic cells exert a second effector function consisting of the secretion of cytokines, notably interferon γ. The latter secretory process functions independently from the exocytic pathway of the lytic granules. [Copyright &y& Elsevier]

SAP (or SH2D1A), an adaptor-like molecule expressed in immune cells, is composed almost exclusively of a Src homology 2 (SH2) domain. In humans, SAP is mutated and either absent or non-functional in X-linked lymphoproliferative (XLP) syndrome, a disease characterized by an inappropriate response to Epstein-Barr virus (EBV) infection. Through its SH2 domain, SAP associates with tyrosines in the cytoplasmic domain of the SLAM family of immune cell receptors, and is absolutely required for the function of these receptors. This property results from the ability of SAP to promote the selective recruitment and activation of FynT, a cytoplasmic Src-related protein tyrosine kinase (PTK). Here, we demonstrate that SAP operates in this pathway by binding to the SH3 domain of FynT, through a second region in the SAP SH2 domain distinct from the phosphotyrosine-binding motif. We demonstrate that this interaction is essential for SAPmediated signalling in T cells, and for the capacity of SAP to modulate immune cell function. These observations characterize a biologically important signalling mechanism in which an adaptor molecule composed only of an SH2 domain links a receptor devoid of intrinsic catalytic activity to the kinase required for its function. [ABSTRACT FROM AUTHOR]

Signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a short intracellular molecule that is mutated in humans with X-linked lymphoproliferative (XLP) disease. Although the exact role and mechanism of action of SAP are not known, it has the capacity to interact with the cytoplasmic region of SLAM and other related immune cell receptors. As SAP is composed almost exclusively of a Src homology 2 (SH2) domain, it has been proposed that it functions as a natural blocker of SH2 domain?mediated interactions. We report here that the SLAM receptor is capable of triggering a protein tyrosine phosphorylation signal in T cells via a mechanism that is strictly dependent on SAP expression. This signal involves the SH2 domain?containing inositol phosphatase (SHIP); the adaptor molecules Dok2, Dok1 and Shc; and Ras GTPase?activating protein RasGAP. SAP is essential for this pathway because it facilitates the selective recruitment and activation of the Src-related protein tyrosine kinase FynT. We also show that signaling via the SLAM-SAP pathway in an established T cell line can alter the profile of cytokine production during T cell activation. These findings identify a mechanism by which a putative adaptor molecule is required for receptor-mediated signaling events in the immune system. They also provide insights into the pathophysiology of a severe human lymphoproliferative disease. [ABSTRACT FROM AUTHOR]

Background: Signals emanating from the antigen T-cell receptor (TCR) are required for T-cell development and function. The T lymphocyte–specific protein tyrosine kinase (Lck) is a key component of the TCR signaling machinery. On the basis of its function, we considered LCK a candidate gene in patients with combined immunodeficiency. Objective: We identify and describe a child with a T-cell immunodeficiency caused by a homozygous missense mutation of the LCK gene (c.1022T>C) resulting from uniparental disomy. Methods: Genetic, molecular, and functional analyses were performed to characterize the Lck deficiency, and the associated clinical and immunologic phenotypes are reported. Results: The mutant LCK protein (p.L341P) was weakly expressed with no kinase activity and failed to reconstitute TCR signaling in LCK-deficient T cells. The patient presented with recurrent respiratory tract infections together with predominant early-onset inflammatory and autoimmune manifestations. The patient displayed CD4+ T-cell lymphopenia and low levels of CD4 and CD8 expression on the T-cell surface. The residual T lymphocytes had an oligoclonal T-cell repertoire and exhibited a profound TCR signaling defect, with only weak tyrosine phosphorylation signals and no Ca2+ mobilization in response to TCR stimulation. Conclusion: We report a new form of T-cell immunodeficiency caused by a LCK gene defect, highlighting the essential role of Lck in human T-cell development and responses. Our results also point out that defects in the TCR signaling cascade often result in abnormal T-cell differentiation and functions, leading to an important risk factor for inflammation and autoimmunity. [ABSTRACT FROM AUTHOR]

Signaling lymphocytic activation molecule (SLAM) family receptors mediate important regulatory signals in immune cells, as a result of their exquisite ability to associate with members of the SLAM-associated protein (SAP) family of adaptors. As discussed herein, recent findings show that the SLAM and SAP families carry out pivotal functions in innate-like and conventional lymphocytes. They are critically needed for the development of innate-like lymphocytes such as NKT cells. In addition, they influence several of the functions of conventional lymphocytes, including the ability of CD4+ T cells to secrete certain cytokines and mediate B cell help; CD8+ T cell proliferation and cytokine production; NK cell-mediated cytotoxicity; and B cell antibody production. These unique functional properties appear to be facilitated by the ability of SLAM-related receptors to serve as self-ligands during homotypic interactions between immune cells. The importance of the SLAM-SAP pathway in normal immunity is highlighted by the finding that SAP is mutated in humans suffering from the immunodeficiency X-linked lymphoproliferative disease. [Copyright &y& Elsevier]

Naive CD4 lymphocytes undergo a polarization process in the periphery to become induced Th17 (iTh17) cells. Using retinoic acid-related orphan receptor γt (RORγt)-gfp mice, we found that RORγt and the transcription factor promyelocytic leukemia zinc finger (PLZF) are valuable new markers to identify the recently described natural Th17 (nTh17) cell population. nTh17 cells are thymically committed to promptly produce large amounts of IL-17 and IL-22. In this study, we show that, in addition to responding to TCR cross-linking, nTh17 cells secrete IL-17 and IL-22 when stimulated with IL-23 plus IL-1β, either in recombinant form or in supernatants from TLR4-activated dendritic cells. This innate-like ability of RORγt+ nTh17 cells to respond to TLR4-induced cytokines was not shared by iTh17 cells. The other distinct properties of RORγt+ nTh17 cells are their high expression of PLZF and their absence from lamina propria; iTh17 cells are found therein. RORγt+ nTh17 cells are present in the thymus of germ-free RORγt-gfp and IL-6-/- RORγt-gfp mice, indicating that these cells do not require symbiotic microbiota or IL-6 for their generation. Finally, we found that PLZF+RORγt+ nTh17 cells represent one of the primary IL-17-producing innate-like T cell populations in a TLR7 imiquimod model of psoriasis-like disorder, indicating their involvement in this kind of lesion. Collectively, our results reveal RORγt and PLZF as characteristic markers for identifying nTh17 cells and demonstrate one of their novel properties: the ability to respond promptly to TLR-dependent proinflammatory stimuli without TCR engagement, placing them as members of the innate-like T cell family. [ABSTRACT FROM AUTHOR]

Invariant natural killer (iNKT) T cells and mucosal-associated invariant T (MAIT) cells represent peculiar T-lymphocyte subpopulations with innate-like properties that differ from conventional T cells. iNKT are reduced in the primary immunodeficiency caused by mutations in the X-linked inhibitor of apoptosis (XIAP). By studying the mechanism of this depletion, we herein report that iNKT cells exhibit a high susceptibility to apoptosis that is not observed with conventional T cells. Elevated expression of caspases 3 and 7 accounts for the proapoptotic phenotype of iNKT cells, which is inhibited by XIAP although it exerts a moderate effect in conventional T cells. Similarly, MAIT cells exhibit a proapoptotic propensity with elevated expression of activated caspases and are decreased in XIAP-deficient individuals. Knockdown of the transcription factor PLZF/ZBTB-16, which is involved in the effector program of iNKT cells, diminishes their proapoptotic phenotype. Conversely, overexpression of PLZF/ZBTB-16 in conventional T cells leads to a proapoptotic phenotype. Our findings identify a previously unknown pathway of regulation of innate-like T-cell homeostasis depending on XIAP and PLZF. The proapoptotic feature of iNKT cells also gives a reliable explanation of their exhaustion observed in different human conditions including the XIAP immunodeficiency. [ABSTRACT FROM AUTHOR]

Background: The outcomes of gene therapy to correct congenital immunodeficiencies are unknown. We reviewed long-term outcomes after gene therapy in nine patients with X-linked severe combined immunodeficiency (SCID-X1), which is characterized by the absence of the cytokine receptor common γ chain. Methods: The nine patients, who lacked an HLA-identical donor, underwent ex vivo retrovirus-mediated transfer of γ chain to autologous CD34+ bone marrow cells between 1999 and 2002. We assessed clinical events and immune function on long-term follow-up. Results: Eight patients were alive after a median follow-up period of 9 years (range, 8 to 11). Gene therapy was initially successful at correcting immune dysfunction in eight of the nine patients. However, acute leukemia developed in four patients, and one died. Transduced T cells were detected for up to 10.7 years after gene therapy. Seven patients, including the three survivors of leukemia, had sustained immune reconstitution; three patients required immunoglobulin-replacement therapy. Sustained thymopoiesis was established by the persistent presence of naive T cells, even after chemotherapy in three patients. The T-cell−receptor repertoire was diverse in all patients. Transduced B cells were not detected. Correction of the immunodeficiency improved the patients' health. Conclusions: After nearly 10 years of follow-up, gene therapy was shown to have corrected the immunodeficiency associated with SCID-X1. Gene therapy may be an option for patients who do not have an HLA-identical donor for hematopoietic stem-cell transplantation and for whom the risks are deemed acceptable. This treatment is associated with a risk of acute leukemia. (Funded by INSERM and others.) N Engl J Med 2010;363:355-64. [ABSTRACT FROM AUTHOR]