The protein tyrosine kinase Tec regulates mast cell function

Mast cells play crucial roles in a variety of normal and pathophysiological processes and their activation has to be tightly controlled. Here, we demonstrate that the protein tyrosine kinase Tec is a crucial regulator of murine mast cell function. Tec was activated upon FcεRI stimulation of BM‐derived mast cells (BMMC). The release of histamine in the absence of Tec was normal in vitro and in vivo; however, leukotriene C₄ levels were reduced in Tec^?/? BMMC. Furthermore, the production of IL‐4 was severely impaired, and GM‐CSF, TNF‐α and IL‐13 levels were also diminished. Finally, a comparison of WT, Tec^?/?, Btk^?/? and Tec^?/?Btk^?/? BMMC revealed a negative role for Btk in the regulation of IL‐4 production, while for the efficient production of TNF‐α, IL‐13 and GM‐CSF, both Tec and Btk were required. Our results demonstrate a crucial role for Tec in mast cells, which is partially different to the function of the well‐characterized family member Btk.     

Conditional deletion of SLP-76 in mature T cells abrogates peripheral immune responses

The adaptor protein Src homology 2 domain‐containing leukocyte‐specific protein of 76 kDa (SLP‐76) is central to the organization of intracellular signaling downstream of the T‐cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP‐76 protein in thymocytes. Here, we show that following tamoxifen‐regulated conditional deletion of SLP‐76, mature, antigen‐inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR‐generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP‐76 are resistant to induction of the CD4⁺ T‐cell‐mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP‐76‐mediated signaling in initiating T‐cell‐directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.     

Perturbed thymopoiesis in vitro in the absence of suppressor of cytokine signalling 1 and 3

Cytokine signals are central to the differentiation of thymocytes and their stepwise progression through defined developmental stages. The intensity and duration of cytokine signals are regulated by the suppressor of cytokine signalling (SOCS) proteins. A clear role for SOCS1 during the later stages of thymopoiesis has been established, but little is known about its role during early thymopoiesis, nor the function of its closest relative, SOCS3. Here, we find that both SOCS1 and SOCS3 are expressed during early thymopoiesis, with expression coincident during the double negative (DN)2 and DN3 stages. We examined thymocyte differentiation in vitro by co-culture of SOCS-deficient bone marrow cells with OP9 cells expressing the Notch ligand Delta-like1 (OP9-DL1). Cells lacking SOCS1 were retarded at the DN3:DN4 transition and appeared unable to differentiate into double positive (DP) thymocytes. Cells lacking both SOCS1 and SOCS3 were more severely affected, and displayed an earlier block in T cell differentiation at DN2, the stage at which expression of SOCS1 and SOCS3 coincides. This indicates that, in addition to their specific roles, SOCS1 and SOCS3 share overlapping roles during thymopoiesis. This is the first demonstration of functional redundancy within the SOCS family, and has uncovered a vital role for SOCS1 and SOCS3 during two important checkpoints in early T cell development.     

Differential T‐cell receptor signals for T helper cell programming

Upon encounter with their cognate antigen, naive CD4 T cells become activated and are induced to differentiate into several possible T helper (Th) cell subsets. This differentiation depends on a number of factors including antigen‐presenting cells, cytokines and co‐stimulatory molecules. The strength of the T‐cell receptor (TCR) signal, related to the affinity of TCR for antigen and antigen dose, has emerged as a dominant factor in determining Th cell fate. Recent studies have revealed that TCR signals of high or low strength do not simply induce quantitatively different signals in the T cells, but rather qualitatively distinct pathways can be induced based on TCR signal strength. This review examines the recent literature in this area and highlights important new developments in our understanding of Th cell differentiation and TCR signal strength.     

Tyrosine phosphorylation of scavenger receptor cysteine‐rich WC1 is required for the WC1‐mediated potentiation of TCR‐induced T‐cell proliferation

Workshop cluster 1 (WC1) molecules are transmembrane glycoproteins uniquely expressed by γδ T cells. They belong to the scavenger receptor cysteine‐rich superfamily and are encoded by a multi‐gene family, which is divided on the basis of antibody reactivity, into three groups, WC1.1, WC1.2, and WC1.3. The potential role of WC1 as a co‐stimulatory molecule for the γδ TCR is suggested by the presence of several tyrosine‐based motifs in their intracellular domains. In this study, we found that WC1 was constitutively phosphorylated in ex vivo bovine γδ T cells and associated with src family tyrosine kinases. Crosslinking of WC1 molecules resulted in an increase in WC1 phosphorylation and co‐crosslinking of WC1 and γδ TCR together prolonged WC1 phosphorylation. We identified the second tyrosine residue as the primary phosphorylation target in WC1.1 and WC1.2 intracellular sequences in both in vitro and in vivo assays. The cytoplasmic tails of WC1.1 and WC1.2 were phosphorylated on serine and PKC activity was required for PMA‐induced endocytosis of WC1.1 or WC1.2. We found that phosphorylation of the second tyrosine in the WC1 cytoplasmic domain was required for the WC1‐mediated potentiation of TCR‐induced T‐cell proliferation, suggesting that WC1 acts as a co‐stimulatory molecule for γδ TCR.     

Vav1 transduces TCR signals required for LFA-1 function and cell polarization at the immunological synapse

Activation of T lineage cells through the TCR by peptide-MHC complexes on APC is critically dependent on rearrangement of the actin cytoskeleton. Vav1 is a guanine nucleotide exchange factor for members of the Rho/Rac family of GTPases which is activated following TCR stimulation, suggesting that it may transduce TCR signals to the activation of some or all actin-controlled processes. We show that Vav1-deficient double-positive thymocytes are less efficient at forming conjugates with APC presenting agonist peptide than wild-type cells are. Furthermore we demonstrate that Vav1 is required for TCR-induced activation of the integrin LFA-1, which is likely to explain the defect in conjugate formation. However, once Vav1-deficient cells form a conjugate, the assembly of proteins into an immunological synapse at the conjugate interface is normal. In contrast, thymocyte polarization is defective in the absence of Vav1, as judged by the relocalization of the microtubule-organizing center. These data demonstrate that Vav1 transduces signals to only a subset of cytoskeleton-dependent events at the immunological synapse.     

T cell stimulation in the absence of exogenous antigen: a T cell signal is induced by both MHC-dependent and -independent mechanisms

Mature T cells residing in peripheral lymphoid organs have frequent contact with antigen presenting cells (APC). Such contact may be required for T cell survival, but the degree to which signals in mature T cells are induced by TCR recognition of self peptide/MHC complexes is unclear. We have used induction of the early growth response gene 1 (Egr1) as an indicator of signal transduction in 3.L2 (I-Ek-restricted) T cells interacting with APC in the absence of exogenous antigen. The data show that Egr1 can be induced in 3.L2 T cells by TCR recognition of self peptides presented by I-Ek. However, a more transient induction of Egr1 can be induced in 3.L2 T cells interacting with dendritic cells derived from class II/beta2m double-deficient mice. Egr1 induction after T cell-APC contact was also observed in a freshly isolated polyclonal CD4 T cell population. The data suggest that self peptide/MHC recognition by the TCR induces a signal in T cells and that dendritic cells can also induce a more transient T cell signal by an MHC-independent mechanism.     

Pathways of memory CD8+ T‐cell development

CD8⁺ T‐cell responses must have at least two components, a replicative cell type that proliferates in the secondary lymphoid tissue and that is responsible for clonal expansion, and cytotoxic cells with effector functions that mediate the resolution of the infection in the peripheral tissues. To confer memory, the response must also generate replication‐competent T cells that persist in the absence of antigen after the primary infection is cleared. The current models of memory differentiation differ in regards to whether or not memory CD8⁺ T cells acquire effector functions during their development. In this review we discuss the existing models for memory development and the consequences that the recent finding that memory CD8⁺ T cells may express granzyme B during their development has for them. We propose that memory CD8⁺ T cells represent a self‐renewing population of T cells that may acquire effector functions but that do not lose the naïve‐like attributes of lymphoid homing, antigen‐independent persistence or the capacity for self‐renewal.     

c‐IAP ubiquitin protein ligase activity is required for 4‐1BB signaling and CD8+ memory T‐cell survival

Cellular inhibitor of apoptosis proteins (c‐IAP) 1 and 2 are widely expressed ubiquitin protein ligases that regulate a variety of cellular functions, including the sensitivity of T cells to costimulation. 4‐1BB is a TNF receptor family member that signals via a complex that includes TRAF family members and the c‐IAPs to upregulate NF‐κB and ERK, and has been implicated in memory T‐cell survival. Here, we show that effector and memory T cells from mice expressing a dominant negative E3‐inactive c‐IAP2 (c‐IAP2^H570A) have impaired signaling downstream of 4‐1BB. When infected with lymphocytic choriomeningitis virus, unlike mice in which c‐IAPs were acutely downregulated by c‐IAP antagonists, the primary response of c‐IAP2^H570A mice was normal. However, the number of antigen‐specific CD8⁺ but not CD4⁺ T cells declined more rapidly and to a greater extent in c‐IAP2^H570A mice than in WT controls. Studies with T‐cell adoptive transfer demonstrated that the enhanced decay of memory cells was T‐cell intrinsic. Thus, c‐IAP E3 activity is required for 4‐1BB coreceptor signaling and maintenance of CD8⁺ T‐cell memory.     

T‐cell modulation by cyclophosphamide for tumour therapy

The power of T cells for cancer treatment has been demonstrated by the success of co‐inhibitory receptor blockade and adoptive T‐cell immunotherapies. These treatments are highly successful for certain cancers, but are often personalized, expensive and associated with harmful side effects. Other T‐cell‐modulating drugs may provide additional means of improving immune responses to tumours without these disadvantages. Conventional chemotherapeutic drugs are traditionally used to target cancers directly; however, it is clear that some also have significant immune‐modulating effects that can be harnessed to target tumours. Cyclophosphamide is one such drug; used at lower doses than in mainstream chemotherapy, it can perturb immune homeostasis, tipping the balance towards generation of anti‐tumour T‐cell responses and control of cancer growth. This review discusses its growing reputation as an immune‐modulator whose multiple effects synergize with the microbiota to tip the balance towards tumour immunity offering widespread benefits as a safe, and relatively inexpensive component of cancer immunotherapy.     

The who's who of T‐cell differentiation: Human memory T‐cell subsets

Following antigen encounter and subsequent resolution of the immune response, a single naïve T cell is able to generate multiple subsets of memory T cells with different phenotypic and functional properties and gene expression profiles. Single‐cell technologies, first and foremost flow cytometry, have revealed the complex heterogeneity of the memory T‐cell compartment and its organization into subsets. However, a consensus has still to be reached, both at the semantic (nomenclature) and phenotypic level, regarding the identification of these subsets. Here, we review recent developments in the characterization of the heterogeneity of the memory T‐cell compartment, and propose a unified classification of both human and nonhuman primate T cells on the basis of phenotypic traits and in vivo properties. Given that vaccine studies and adoptive cell transfer immunotherapy protocols are influenced by these recent findings, it is important to use uniform methods for identifying and discussing functionally distinct subsets of T cells.     

N‐Ethyl‐N‐nitrosourea mutagenesis in the mouse provides strong genetic and in vivo evidence for the role of the Caspase Recruitment Domain (CARD) of CARD‐MAGUK1 in T regulatory cell development

Natural regulatory T (nTreg) cells generated in the thymus are essential throughout life for the maintenance of T‐cell homeostasis and the prevention of autoimmunity. T‐cell receptor (TCR)/CD28‐mediated activation of nuclear factor‐κB and (J)un (N)‐terminal kinase pathways is known to play a key role in nTreg cell development but many of the predicted molecular interactions are based on extrapolations from non‐Treg cell TCR stimulation with non‐physiological ligands. For the first time, we provide strong genetic evidence of a scaffold function for the Caspase Recruitment Domain (CARD) of the TCR signalling protein CARD‐MAGUK1 (CARMA1) in nTreg cell development in vivo. We report two, new, N‐ethyl‐N‐nitrosourea‐derived mutant mice, Vulpo and Zerda, with a profound block in the development of nTreg cells in the thymus as well as impaired inducible Treg cell differentiation in the periphery. Despite independent heritage, both mutants harbour different point mutations in the CARD of the CARMA1 protein. Mutations in vulpo and zerda do not affect expression levels of CARMA1 but still impair signalling through the TCR due to defective downstream Bcl‐10 recruitment by the mutated CARD of CARMA1. Phenotypic differences observed between Vulpo and Zerda mutants suggest a role for the CARD of CARMA1 independent of Bcl‐10 activation of downstream pathways. We conclude that our forward genetic approach demonstrates a critical role for the CARD function of CARMA1 in Treg cell development in vivo.     

Mitochondrial and cytosolic roles of PINK1 shape induced regulatory T‐cell development and function

Mutations in PTEN‐induced kinase 1 (PINK1), a serine/threonine kinase linked to familial early‐onset Parkinsonism, compromise mitochondrial integrity and metabolism and impair AKT signaling. As the activation of a naïve T cell requires an AKT‐dependent reorganization of a cell's metabolic machinery, we sought to determine if PINK1‐deficient T cells lack the ability to undergo activation and differentiation. We show that CD4⁺ T cells from PINK1 knockout mice fail to properly phosphorylate AKT upon activation, resulting in reduced expression of the IL‐2 receptor subunit CD25. Following, deficient IL‐2 signaling mutes the activation‐induced increase in respiratory capacity and mitochondrial membrane potential. Under polarization conditions favoring the development of induced regulatory T cells, PINK1^?/? T cells exhibit a reduced ability to suppress bystander T‐cell proliferation despite normal FoxP3 expression kinetics. Our results describe a critical role for PINK1 in integrating extracellular signals with metabolic state during T‐cell fate determination, and may have implications for the understanding of altered T‐cell populations and immunity during the progression of active Parkinson's disease or other immunopathologies.     

Altered T‐cell entry and egress in the absence of Coronin 1A attenuates murine acute graft versus host disease

Acute graft‐versus‐host disease (aGvHD) is a major limitation to the use of allogeneic stem cell transplantation for the treatment of patients with relapsed malignant disease. Previous work using animals lacking secondary lymphoid tissue (SLT) suggested that activation of donor T cells in SLT is critically important for the pathogenesis of aGvHD. However, these studies did not determine if impaired migration into, and more importantly, out of SLT, would ameliorate aGvHD. Here, we show that T cells from mice lacking Coronin 1A (Coro 1A^?/?), an actin‐associated protein shown to be important for thymocyte egress, do not mediate acute GvHD. The attenuation of aGvHD was associated with decreased expression of the critical trafficking proteins C‐C chemokines receptor type 7 (CCR7) and sphingosine 1 phosphate receptor on donor T cells. This was mediated in part by impaired activation of the canonical NF‐κB pathway in the absence of Coro 1A. As a result of these alterations, donor T cells from Coro 1A^?/? mice were not able to initially traffic to SLT or exit SLT after BM transplantation. However, this alteration did not abrogate the graft‐versus‐leukemia response. Our data suggest that blocking T‐cell migration into and out of SLT is a valid approach to prevent aGvHD.     

Invariant natural killer T‐cell development and function with loss of microRNA‐155

Invariant natural killer T (iNKT) cells are adaptive T cells with innate‐like characteristics including rapid cytokine production and a proliferative response to stimulation. Development of these cells in the thymus is dependent on expression of the microRNA (miRNA) processing enzyme Dicer, indicating that iNKT cells probably have distinct miRNA requirements for gene regulation during development. The miRNA miR‐155 has previously been shown to have numerous roles in T cells, including regulation of proliferation and differentiation, and positive modulation of interferon‐γ expression. We examined the role of miR‐155 in the development and function of iNKT cells. Using germline‐deficient miR‐155 mice, we showed that loss of miR‐155 resulted in unchanged iNKT cell frequency and cell number. Although miR‐155 was up‐regulated in iNKT cells upon activation with α‐galactosylceramide, loss of miR‐155 did not affect cytokine production or proliferation by iNKT cells. Hence, cytokine production occurs in iNKT cells independently of miR‐155 expression.     

CD8‐β ADP‐ribosylation affects CD8+ T‐cell function

The CD8αβ coreceptor is crucial for effective peptide: MHC‐I recognition by the TCR of CD8⁺ T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD⁺ to transfer ADP‐ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD⁺, ART2.2 caused ADP‐ribosylation of CD8‐β on murine CD8⁺ T cells in vitro and in vivo. Treatment with NAD⁺ prevented binding of anti‐CD8‐β mAb YTS156.7.7 but not of mAb H35–17.2, indicating that NAD⁺ caused modification of certain epitopes and not a general loss of CD8‐β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2‐deficient T cells or in the presence of inhibitory anti‐ART2.2 single‐domain antibodies. ADP‐ribosylation of CD8‐β occurred during cell isolation, particularly when cells were isolated from CD38‐deficient mice. Incubation of ART2‐expressing, but not of ART2‐deficient, OVA‐specific CD8⁺ T cells with NAD⁺ interfered with binding of OVA_257–264:MHC‐I tetramers. In line with this result, treatment of WT mice with NAD⁺ resulted in reduced CD8⁺ T‐cell mediated cytotoxicity in vivo. We propose that ADP‐ribosylation of CD8‐β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD⁺.