A novel human autoimmune syndrome caused by combined hypomorphic and activating mutations in ZAP-70

A brother and sister developed a previously undescribed constellation of autoimmune manifestations within their first year of life, with uncontrollable bullous pemphigoid, colitis, and proteinuria. The boy had hemophilia due to a factor VIII autoantibody and nephrotic syndrome. Both children required allogeneic hematopoietic cell transplantation (HCT), which resolved their autoimmunity. The early onset, severity, and distinctive findings suggested a single gene disorder underlying the phenotype. Whole-exome sequencing performed on five family members revealed the affected siblings to be compound heterozygous for two unique missense mutations in the 70-kD T cell receptor ζ-chain associated protein (ZAP-70). Healthy relatives were heterozygous mutation carriers. Although pre-HCT patient T cells were not available, mutation effects were determined using transfected cell lines and peripheral blood from carriers and controls. Mutation R192W in the C-SH2 domain exhibited reduced binding to phosphorylated ζ-chain, whereas mutation R360P in the N lobe of the catalytic domain disrupted an autoinhibitory mechanism, producing a weakly hyperactive ZAP-70 protein. Although human ZAP-70 deficiency can have dysregulated T cells, and autoreactive mouse thymocytes with weak Zap-70 signaling can escape tolerance, our patients’ combination of hypomorphic and activating mutations suggested a new disease mechanism and produced previously undescribed human ZAP-70–associated autoimmune disease.The adaptive immune system is tightly regulated to allow responses against invading pathogens while avoiding injurious hyperactivity and misdirected responses to self-proteins. Impairment of lymphocyte pathways by genetic defects in mediators of immune signaling and activation can lead to immunodeficiency, but also to immune dysregulation, autoimmunity, and malignancy (Notarangelo, 2014). Essential steps in T cell activation and signaling include antigen recognition by the TCR–CD3 complex; tyrosine phosphorylation of immunoreceptor activation motifs (ITAMs) of the CD3 and ζ-chains by the tyrosine kinase Lck; interaction between phosphorylated ITAMs and the cytoplasmic tyrosine kinase ZAP-70; phosphorylation of ZAP-70 by Lck to relieve its autoinhibition and promote its activation; and ZAP-70–mediated phosphorylation of its adaptor substrates, leading to downstream events, including activation of the Ras–MAPK pathway and increased intracellular calcium.ZAP-70, a critical T cell signaling molecule, is expressed predominantly in T and NK cells. It exists in an autoinhibited state, which is relieved by a two-step process. The first step, binding of the ZAP-70 tandem SH2 domains to doubly phosphorylated ITAMs of the ζ-chain, requires dissociation of the SH2 linker from the back of the kinase domain and repositioning of the SH2 domains to align with ζ-chain ITAMs. This change in structure facilitates a second conformational change whereby ZAP-70 tyrosines Y315 and Y319 in interdomain B are exposed and phosphorylated by Lck, leading to stabilization of the active conformation of the ZAP-70 catalytic domain to permit phosphorylation of downstream signaling molecules (Au-Yeung et al., 2009; Yan et al., 2013; Klammt et al., 2015). The phosphorylation of Y319 is particularly important because, in the nonphosphorylated state, it interacts with the N-lobe of the catalytic domain to maintain its inactive conformation.Deficiency of ZAP-70 in humans causes a profound combined immunodeficiency (CID) in which CD8 T cells are absent and CD4 T cells are defective (Arpaia et al., 1994; Elder et al., 1994; Roifman, 1995). Affected individuals are susceptible to life-threatening infections and require hematopoietic cell transplantation (HCT) to survive (Arpaia et al., 1994; Chan et al., 1994; Katamura et al., 1999; Elder et al., 2001; Turul et al., 2009; Fischer et al., 2010; Roifman et al., 2010). Some ZAP-70–deficient patients also have skin infiltration with dysfunctional CD4 T cells, elevated serum IgE, and eosinophilia (Katamura et al., 1999; Turul et al., 2009).In contrast to humans, mice with complete Zap-70 deficiency manifest developmental arrest of both CD4 and CD8 T lineages. A hypomorphic murine Zap-70 mutation with reduced ζ-chain binding caused attenuated TCR signaling that permitted survival of autoreactive T cells normally deleted in the thymus (Tanaka et al., 2010). In response to innate stimuli, these self-reactive murine T cells contributed to the development of non–tissue-specific autoantibodies (such as rheumatoid factor and antibody to cyclic citrullinated peptide) and autoimmune arthritis (Sakaguchi et al., 2012). Other hypomorphic alleles of Zap-70 in the mouse have also been associated with nonspecific autoantibodies (e.g., antinuclear antibodies; Siggs et al., 2007). In contrast, antibody-mediated autoimmune disease due to hypomorphic ZAP-70 alleles in human patients has not been reported.We present two siblings with unique mutations of ZAP70 who lacked clinical immunodeficiency, but instead had a novel constellation of early onset, severe autoimmune manifestations, including bullous pemphigoid. Compound heterozygosity for hypomorphic and hyperactive mutant ZAP70 alleles in these patients represents a new genetic mechanism underlying inappropriate T cell activation.     

Dual-reactive B cells are autoreactive and highly enriched in the plasmablast and memory B cell subsets of autoimmune mice

Rare dual-reactive B cells expressing two types of Ig light or heavy chains have been shown to participate in immune responses and differentiate into IgG⁺ cells in healthy mice. These cells are generated more often in autoreactive mice, leading us to hypothesize they might be relevant in autoimmunity. Using mice bearing Igk allotypic markers and a wild-type Ig repertoire, we demonstrate that the generation of dual-κ B cells increases with age and disease progression in autoimmune-prone MRL and MRL/lpr mice. These dual-reactive cells express markers of activation and are more frequently autoreactive than single-reactive B cells. Moreover, dual-κ B cells represent up to half of plasmablasts and memory B cells in autoimmune mice, whereas they remain infrequent in healthy mice. Differentiation of dual-κ B cells into plasmablasts is driven by MRL genes, whereas the maintenance of IgG⁺ cells is partly dependent on Fas inactivation. Furthermore, dual-κ B cells that differentiate into plasmablasts retain the capacity to secrete autoantibodies. Overall, our study indicates that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in autoimmunity.While developing in the BM, B cells undergo stochastic rearrangement of Ig heavy (IgH) and Ig light (IgL) chain V(D)J gene segments resulting in the random expression of Ig H and L (κ and λ) chains in the emerging B cell population (Schlissel, 2003; Nemazee, 2006). During V(D)J recombination, allelic and isotypic exclusion at the Ig loci are also established, leading to the expression of a unique H and L chain pair and, therefore, of BCRs with unique specificity in each B cell (Langman and Cohn, 2002; Nemazee, 2006; Vettermann and Schlissel, 2010). These mechanisms ensure that developing B cells expressing BCRs reactive with self-antigens (i.e., autoreactive B cells) undergo tolerance induction, whereas those expressing BCRs specific for a foreign antigen or a peripheral self-antigen proceed in differentiation and selection into the periphery (Burnet, 1959). Autoreactive B cells are silenced by central tolerance in the BM via receptor editing and, less frequently, clonal deletion (Halverson et al., 2004; Ait-Azzouzene et al., 2005), whereas peripheral B cell tolerance proceeds via anergy and clonal deletion (Goodnow et al., 2005; Pelanda and Torres, 2006, 2012; Shlomchik, 2008). Despite these tolerance mechanisms, small numbers of autoreactive B cells are detected in peripheral tissues of healthy mice and humans (Grandien et al., 1994; Wardemann et al., 2003) and their numbers are increased in autoimmunity (Andrews et al., 1978; Izui et al., 1984; Warren et al., 1984; Samuels et al., 2005; Yurasov et al., 2005, 2006; Liang et al., 2009).A small population of dual-reactive B cells expressing two types of L chains (or more rarely H chains) has been observed both in mice and humans (Nossal and Makela, 1962; Pauza et al., 1993; Giachino et al., 1995; Gerdes and Wabl, 2004; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007; Kalinina et al., 2011). These allelically and isotypically (overall haplotype) included B cells are <5% of all peripheral B cells in normal mice (Barreto and Cumano, 2000; Rezanka et al., 2005; Casellas et al., 2007; Velez et al., 2007), but they are more frequent in Ig knockin mice in which newly generated B cells are autoreactive and actively undergo receptor editing (Li et al., 2002a,b; Liu et al., 2005; Huang et al., 2006; Casellas et al., 2007). B cells that coexpress autoreactive and nonautoreactive antibodies can escape at least some of the mechanisms of central and peripheral B cell tolerance and be selected into the mature peripheral B cell population (Kenny et al., 2000; Li et al., 2002a,b; Gerdes and Wabl, 2004; Liu et al., 2005; Huang et al., 2006), sometimes with a preference for the marginal zone (MZ) B cell subset (Li et al., 2002b).Furthermore, dual-reactive B cells observed within a normal polyclonal Ig repertoire exhibit characteristics of cells that develop through the receptor editing process, including delayed kinetics of differentiation and more frequent binding to self-antigens (Casellas et al., 2007). Hence, dual-reactive B cells might play a role in autoantibody generation and autoimmunity. However, the contribution of these B cells to autoimmunity has not yet been established. Our hypothesis is that haplotype-included autoreactive B cells are positively selected within the context of genetic backgrounds that manifest defects in immunological tolerance and contribute to the development of autoimmunity.Until recently, the analysis of dual-reactive B cells was impaired by the inability to detect dual-κ cells, which are the most frequent among haplotype-included B cells (Casellas et al., 2007; Velez et al., 2007). To overcome this issue, we took advantage of Igk^h mice that bear a gene-targeted human Ig Ck allele in the context of a wild-type Ig repertoire (Casellas et al., 2001) and crossed these to MRL-Fas^lpr/lpr (MRL/lpr) and MRL mice that develop an autoimmune pathology with characteristics similar to human lupus (Izui et al., 1984; Rordorf-Adam et al., 1985; Theofilopoulos and Dixon, 1985; Cohen and Eisenberg, 1991; Watanabe-Fukunaga et al., 1992). MRL/lpr mice, moreover, display defects in receptor editing (Li et al., 2002a; Lamoureux et al., 2007; Panigrahi et al., 2008) and reduced tolerance induction (Li et al., 2002a), which could potentially contribute to higher frequency of haplotype-included autoreactive B cells.We found that the frequency of dual-κ cells increased with age and progression of disease in autoimmune-prone mice and independent of the expression of Fas. Dual-κ B cells exhibited higher prevalence of autoreactivity than single-κ B cells and were frequently selected into the antigen-activated cell subsets in MRL/lpr and MRL mice where up to half of the plasmablasts and memory B cells were dual-κ B cells. Moreover, disruption of Fas expression appeared to mediate increased survival of dual-reactive memory B cells. Overall, these data indicate that dual-reactive B cells significantly contribute to the plasmablast and memory B cell populations of autoimmune-prone mice suggesting a role in the development of autoimmunity.     

Complement drives Th17 cell differentiation and triggers autoimmune arthritis

Activation of serum complement triggers Th17 cell–dependent spontaneous autoimmune disease in an animal model. In genetically autoimmune-prone SKG mice, administration of mannan or β-glucan, both of which activate serum complement, evoked Th17 cell–mediated chronic autoimmune arthritis. C5a, a chief component of complement activation produced via all three complement pathways (i.e., lectin, classical, and alternative), stimulated tissue-resident macrophages, but not dendritic cells, to produce inflammatory cytokines including IL-6, in synergy with Toll-like receptor signaling or, notably, granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF secreted by activated T cells indeed enhanced in vitro IL-6 production by C5a-stimulated macrophages. In vivo, C5a receptor (C5aR) deficiency in SKG mice inhibited the differentiation/expansion of Th17 cells after mannan or β-glucan treatment, and consequently suppressed the development of arthritis. Transfer of SKG T cells induced Th17 cell differentiation/expansion and produced arthritis in C5aR-sufficient recombination activating gene (RAG)^−/− mice but not in C5aR-deficient RAG^−/− recipients. In vivo macrophage depletion also inhibited disease development in SKG mice. Collectively, the data suggest that complement activation by exogenous or endogenous stimulation can initiate Th17 cell differentiation and expansion in certain autoimmune diseases and presumably in microbial infections. Blockade of C5aR may thus be beneficial for controlling Th17-mediated inflammation and autoimmune disease.There is recent evidence that IL-17–secreting CD4⁺ T cells (Th17 cells) play a key role in autoimmune diseases, such as rheumatoid arthritis (RA) and multiple sclerosis (Harrington et al., 2005; Veldhoen et al., 2006; Korn et al., 2009). It remains unclear, however, how pathogenic self-reactive Th17 cells are generated from naive T cells, and are activated by external or internal stimuli in autoimmune disease.SKG mice, a mutant of the gene encoding ZAP-70 on the BALB/c background, spontaneously develop CD4⁺ T cell–mediated autoimmune arthritis clinically and immunologically resembling human RA (Sakaguchi et al., 2003). The mutation alters the sensitivity of developing T cells to positive and negative selection in the thymus, leading to thymic production of potentially arthritogenic autoimmune T cells (Sakaguchi et al., 2003; Hirota et al., 2007). The SKG arthritis is critically dependent on Th17 cells, as deficiency of either IL-17 or IL-6 completely inhibits the disease (Hirota et al., 2007). Importantly, they spontaneously develop severe arthritis in a microbially conventional environment but not under a specific pathogen–free (SPF) condition, suggesting that environmental stimuli such as microbial infection may expand or trigger the differentiation of arthritogenic Th17 cells (Yoshitomi et al., 2005). Indeed, injection of zymosan, a crude extract of yeast cell wall containing β-glucans or purified β-glucans, such as laminarin, activates innate immunity via Toll-like receptor (TLR) and Dectin-1, and drives preferential differentiation and expansion of Th17 cells, thereby triggering arthritis in SKG mice under a SPF condition (Yoshitomi et al., 2005; LeibundGut-Landmann et al., 2007). Because zymosan is also an activator of the alternative pathway of complement (Mullaly and Kubes, 2007) and β-glucan structure can be recognized by ficolin-L, an initiator of the lectin pathway (Garlatti et al., 2007), it is also likely that complement activation may contribute to triggering Th17-mediated autoimmune disease.In this report, we show that complement activation via all three pathways (i.e., the lectin, classical, and alternative pathways) and the resulting generation of the common product C5a potently promote the differentiation/expansion of self-reactive T cells to Th17 cells that mediate autoimmune arthritis in SKG mice. The results indicate that exogenous or endogenous stimuli that activate complement can be a triggering cause of Th17-mediated autoimmune disease and that C5a is a key molecular target in controlling Th17-mediated autoimmunity as well as microbial immunity.     

The Src family kinases Hck,Fgr, and Lyn are critical for the generation of the in vivo inflammatory environment without a direct role in leukocyte recruitment

Although Src family kinases participate in leukocyte function in vitro, such as integrin signal transduction, their role in inflammation in vivo is poorly understood. We show that Src family kinases play a critical role in myeloid cell–mediated in vivo inflammatory reactions. Mice lacking the Src family kinases Hck, Fgr, and Lyn in the hematopoietic compartment were completely protected from autoantibody-induced arthritis and skin blistering disease, as well as from the reverse passive Arthus reaction, with functional overlap between the three kinases. Though the overall phenotype resembled the leukocyte recruitment defect observed in β₂ integrin–deficient (CD18^−/−) mice, Hck^−/−Fgr^−/−Lyn^−/− neutrophils and monocytes/macrophages had no cell-autonomous in vivo or in vitro migration defect. Instead, Src family kinases were required for the generation of the inflammatory environment in vivo and for the release of proinflammatory mediators from neutrophils and macrophages in vitro, likely due to their role in Fcγ receptor signal transduction. Our results suggest that infiltrating myeloid cells release proinflammatory chemokine, cytokine, and lipid mediators that attract further neutrophils and monocytes from the circulation in a CD18-dependent manner. Src family kinases are required for the generation of the inflammatory environment but not for the intrinsic migratory ability of myeloid cells.Src family kinases are best known for their role in malignant transformation and tumor progression, as well as signaling through cell surface integrins (Parsons and Parsons, 2004; Playford and Schaller, 2004). Due to their role in cancer development and progression, Src family kinases have become major targets of cancer therapy (Kim et al., 2009; Zhang and Yu, 2012). Src family kinases are also present in immune cells with dominant expression of Lck and Fyn in T cells and NK cells; Lyn, Fyn, and Blk in B cells and mast cells; and Hck, Fgr, and Lyn in myeloid cells such as neutrophils and macrophages (Lowell, 2004).The best known function of Src family kinases in the immune system is their role in integrin signal transduction. Indeed, Hck, Fgr, and Lyn mediate outside-in signaling by β₁ and β₂ integrins in neutrophils and macrophages (Lowell et al., 1996; Meng and Lowell, 1998; Mócsai et al., 1999; Suen et al., 1999; Pereira et al., 2001; Giagulli et al., 2006; Hirahashi et al., 2006), Lck participates in LFA-1–mediated T cell responses (Morgan et al., 2001; Fagerholm et al., 2002; Feigelson et al., 2001; Suzuki et al., 2007), and Src family kinases are required for LFA-1–mediated signal transduction and target cell killing by NK cells (Riteau et al., 2003; Perez et al., 2004).Src family kinases also mediate TCR signal transduction by phosphorylating the TCR-associated immunoreceptor tyrosine-based activation motifs (ITAMs), leading to recruitment and activation of ZAP-70 (van Oers et al., 1996; Zamoyska et al., 2003; Palacios and Weiss, 2004). However, their role in receptor-proximal signaling by the BCR and Fc receptors is rather controversial. Although the combined deficiency of Lyn, Fyn, and Blk results in defective BCR-induced NF-κB activation, receptor-proximal BCR signaling (ITAM phosphorylation) is not affected (Saijo et al., 2003). Genetic deficiency of Lyn, the predominant Src family kinase in B cells, even leads to enhanced BCR signaling and B cell–mediated autoimmunity (Hibbs et al., 1995; Nishizumi et al., 1995; Chan et al., 1997). Similarly, both positive (Hibbs et al., 1995; Nishizumi and Yamamoto, 1997; Parravicini et al., 2002; Gomez et al., 2005; Falanga et al., 2012) and negative (Kawakami et al., 2000; Hernandez-Hansen et al., 2004; Odom et al., 2004; Gomez et al., 2005; Falanga et al., 2012) functions for Fyn and Lyn during Fc receptor signaling in mast cells have been reported. In addition, Hck^−/−Fgr^−/− neutrophils respond normally to IgG immune complex–induced activation (Lowell et al., 1996) and Fc receptor–mediated phagocytosis of IgG-coated red blood cells is delayed but not blocked in Hck^−/−Fgr^−/−Lyn^−/− macrophages (Fitzer-Attas et al., 2000; Lowell, 2004). The differential requirement for Src family kinases in TCR, BCR, and Fc receptor signaling is thought to derive from the fact that Syk, but not ZAP-70, is itself able to phosphorylate ITAM tyrosines (Rolli et al., 2002), making Src family kinases indispensable for signaling by the ZAP-70–coupled TCR but not by the Syk-coupled BCR and Fc receptors.Autoantibody production and immune complex formation is one of the major mechanisms of autoimmunity-induced tissue damage. In vivo models of those processes include the K/B×N serum transfer arthritis (Korganow et al., 1999) and autoantibody-induced blistering skin diseases (Liu et al., 1993; Sitaru et al., 2002, 2005), which mimic important aspects of human rheumatoid arthritis, bullous pemphigoid, and epidermolysis bullosa acquisita. Activation of neutrophils or macrophages (Liu et al., 2000; Wipke and Allen, 2001; Sitaru et al., 2002, 2005; Solomon et al., 2005), recognition of immune complexes by Fcγ receptors (Ji et al., 2002; Sitaru et al., 2002, 2005), and β₂ integrin–mediated leukocyte recruitment (Watts et al., 2005; Liu et al., 2006; Chiriac et al., 2007; Monach et al., 2010; Németh et al., 2010) are indispensable for the development of those in vivo animal models.The role of Src family kinases in β₂ integrin signaling and the requirement for β₂ integrins during autoantibody-induced in vivo inflammation prompted us to test the role of Src family kinases in autoantibody-induced inflammatory disease models. We found that Hck^−/−Fgr^−/−Lyn^−/− mice were completely protected from autoantibody-induced arthritis and inflammatory blistering skin disease. Surprisingly, this was not due to a cell-autonomous defect in β₂ integrin–mediated leukocyte migration but to defective generation of an inflammatory microenvironment, likely due to the role of Src family kinases in immune complex–induced neutrophil and macrophage activation.     

Annular PIP3 accumulation controls actin architecture and modulates cytotoxicity at the immunological synapse

The immunological synapse formed by a T lymphocyte on the surface of a target cell contains a peripheral ring of filamentous actin (F-actin) that promotes adhesion and facilitates the directional secretion of cytokines and cytolytic factors. We show that growth and maintenance of this F-actin ring is dictated by the annular accumulation of phosphatidylinositol trisphosphate (PIP₃) in the synaptic membrane. PIP₃ functions in this context by recruiting the exchange factor Dock2 to the periphery of the synapse, where it drives actin polymerization through the Rho-family GTPase Rac. We also show that synaptic PIP₃ is generated by class IA phosphoinositide 3-kinases that associate with T cell receptor microclusters and are activated by the GTPase Ras. Perturbations that inhibit or promote PIP₃-dependent F-actin remodeling dramatically affect T cell cytotoxicity, demonstrating the functional importance of this pathway. These results reveal how T cells use lipid-based signaling to control synaptic architecture and modulate effector responses.Stimulation of the TCR induces dramatic cytoskeletal remodeling that reshapes the interface between the T cell and the APC into an immunological synapse (IS; Gomez and Billadeau, 2008; Dustin et al., 2010). First, an intense burst of actin polymerization drives radially symmetric spreading over the APC. Subsequently, the filamentous actin (F-actin) within this circular lamellipodium resolves into an annular configuration (Bunnell et al., 2001; Stinchcombe et al., 2006; Sims et al., 2007). The resulting F-actin ring regulates the trafficking and clustering of signaling complexes and integrins (Varma et al., 2006; Nguyen et al., 2008; Babich et al., 2012; Yi et al., 2012). It also provides a structural framework for specifying effector function. Clearance of F-actin from the central synaptic membrane is coupled to the polarization of the microtubule-organizing center (MTOC) toward the APC (Huse, 2012). These events together facilitate the directional release of soluble factors into the IS. This is particularly important for CD8⁺ CTLs, which kill APCs by directional secretion of cytolytic perforins and granzymes (Stinchcombe and Griffiths, 2007).The pathways regulating synaptic F-actin architecture are not well understood. Studies suggest that TCR-induced actin polymerization and cell spreading require the Rho family GTPase Rac (Ku et al., 2001; Sanui et al., 2003; Nolz et al., 2006; Zipfel et al., 2006). Like all small GTPases, Rac is activated by specific guanine nucleotide exchange factors (GEFs) that catalyze its transition from an inactive, GDP-bound form into an active, GTP-bound form that recruits downstream effectors (Jaffe and Hall, 2005). T cells express several GEFs that could potentially regulate Rac. The most prominent is Vav, which functions as a core component of the TCR signaling complex (Tybulewicz, 2005). Recent work, however, has suggested that GEFs other than Vav might control Rac-dependent F-actin remodeling at the IS (Miletic et al., 2009). T cells also express Dock2, a Rac-specific CDM family GEF that catalyzes nucleotide exchange via its conserved DHR-2 domain (Côté and Vuori, 2007). T cells lacking Dock2 display marked defects in Rac activation and TCR trafficking (Sanui et al., 2003), implying that Dock2 might be involved in shaping synaptic F-actin. The N-terminal region of Dock2 binds constitutively to the scaffolding protein Elmo, which confers stabilization and enhances GEF activity toward Rac. Dock2 also contains a so-called DHR-1 domain, which binds specifically to phosphatidylinositol 3,4,5-trisphosphate (PIP₃; Côté and Vuori, 2007). In that regard, it is intriguing that TCR signaling induces robust PIP₃ accumulation at the IS (Costello et al., 2002; Harriague and Bismuth, 2002; Huppa et al., 2003; Garçon et al., 2008). The possibility that this pool of PIP₃ might regulate F-actin organization via recruitment of Dock2, however, has not been explored.It is generally thought that synaptic PIP₃ is produced by class I phosphoinositide 3-kinases (PI3Ks). It remains controversial, however, precisely which isoforms contribute to this process (Alcázar et al., 2007; Garçon et al., 2008; Sauer et al., 2008), and it is also unclear how these proteins might be recruited and activated by TCR signaling. Previous studies in T cells have focused on the role of phosphotyrosine (pTyr)-containing signaling motifs that can bind and allosterically activate certain PI3K isoforms (Carpenter et al., 1993; Holt et al., 1994; Pagès et al., 1994; Zhang et al., 1998; Shim et al., 2004, 2011). However, class I PI3Ks also interact with the small GTPase Ras (Rodriguez-Viciana et al., 1994, 1996), which functions synergistically with pTyr peptides to induce full PI3K activity (Jimenez et al., 2002). Ras is strongly activated by TCR signaling (Genot and Cantrell, 2000), but whether it promotes synaptic PIP₃ accumulation through PI3K is not known.In the present study, we demonstrate that the size and shape of the synaptic F-actin ring is dictated by an annular accumulation of PIP₃ in the overlying plasma membrane. This PIP₃ is generated by class IA PI3K isoforms downstream of Ras, and coordinates F-actin architecture by recruiting the Dock2/Elmo complex to the periphery of the IS. Specific perturbations in PIP₃-Dock2 signaling either reduce or enhance CTL-mediated killing, indicative of an important role in T cell effector responses. These results identify a previously uncharacterized mechanism for controlling synaptic F-actin, and provide insight into how lipid second messenger signaling shapes lymphocyte structure and function.     

Coreceptor affinity for MHC defines peptide specificity requirements for TCR interaction with coagonist peptide–MHC

Recent work has demonstrated that nonstimulatory endogenous peptides can enhance T cell recognition of antigen, but MHCI- and MHCII-restricted systems have generated very different results. MHCII-restricted TCRs need to interact with the nonstimulatory peptide–MHC (pMHC), showing peptide specificity for activation enhancers or coagonists. In contrast, the MHCI-restricted cells studied to date show no such peptide specificity for coagonists, suggesting that CD8 binding to noncognate MHCI is more important. Here we show how this dichotomy can be resolved by varying CD8 and TCR binding to agonist and coagonists coupled with computer simulations, and we identify two distinct mechanisms by which CD8 influences the peptide specificity of coagonism. Mechanism 1 identifies the requirement of CD8 binding to noncognate ligand and suggests a direct relationship between the magnitude of coagonism and CD8 affinity for coagonist pMHCI. Mechanism 2 describes how the affinity of CD8 for agonist pMHCI changes the requirement for specific coagonist peptides. MHCs that bind CD8 strongly were tolerant of all or most peptides as coagonists, but weaker CD8-binding MHCs required stronger TCR binding to coagonist, limiting the potential coagonist peptides. These findings in MHCI systems also explain peptide-specific coagonism in MHCII-restricted cells, as CD4–MHCII interaction is generally weaker than CD8–MHCI.The vast majority of the peptides presented by MHC molecules are derived from self-proteins and do not activate mature T cells. Antigen recognition and T cell activation must thus be tuned to allow for recognition of the small minority of disease-associated peptide–MHC (pMHC) “needles in the haystack” of nonstimulatory endogenous pMHC (Davis et al., 2007; Gascoigne, 2008; Gascoigne et al., 2010). Several experiments have shown that T cell activation by small amounts of antigen is enhanced by the presence of endogenous peptides (Irvine et al., 2002; Yachi et al., 2005). Although this activation enhancement or coagonist phenomenon has been reported for both MHC class I (MHCI)–restricted T cells and thymocytes (Yachi et al., 2005, 2007; Anikeeva et al., 2006; Juang et al., 2010) and for MHCII-restricted T cells (Irvine et al., 2002; Li et al., 2004; Krogsgaard et al., 2005), the relative importance of TCR recognition of the endogenous pMHC appears to be very different for CD4 and CD8 T cells (Davis et al., 2007; Gascoigne, 2008; Gascoigne et al., 2010).The number of potential coagonist peptides for a given CD4 T cell are very limited (Krogsgaard et al., 2005; Ebert et al., 2009; Lo et al., 2009), whereas coagonism for CD8 T cells or thymocytes occurs with a wide range of different nonstimulatory peptides (Yachi et al., 2005, 2007; Juang et al., 2010). This evidence thus suggests that MHCII-restricted TCRs discriminate between endogenous peptides, whereas MHCI-restricted TCRs do not. However, recent data indicate that nonstimulatory pMHCI ligands show a very weak but possibly biologically significant interaction with TCR (Juang et al., 2010). This suggested that TCRs might play a role in coagonism in MHCI-restricted cells but that its specificity is only evident for very weakly stimulatory TCR ligands such as those involved in positive selection.The CD8 coreceptor’s interaction with nonstimulatory MHCI has been suggested to be important for coagonism in MHCI-restricted cells (Yachi et al., 2005; Gascoigne, 2008; Gascoigne et al., 2010). Nonstimulatory pMHC alone can recruit CD8 to the T cell–APC interface (Yachi et al., 2005; Rybakin et al., 2011). Also, coagonist pMHCs became antagonists in CD8-negative cells (Stone et al., 2011). These results, along with the lack of peptide specificity for coagonists, suggest that non-cognate CD8 coreceptor binding to nonstimulatory pMHC is the dominant mechanism of activation enhancement for MHCI-restricted T cells. In addition, CD8 affinity for the MHC presenting the antigenic peptide (agonist) plays a direct role in signaling through the TCR, where increasing the affinity of CD8 can increase ligand potency and even bypass peptide specificity requirements altogether (Laugel et al., 2007; Wooldridge et al., 2007, 2010). Because there is a range of affinities for CD8 binding to different MHCI molecules (Cole et al., 2012), the relative requirements for CD8, or for TCR interaction with the nonstimulatory ligand, might be expected to vary with the strength of CD8–MHC binding. Interestingly, the two mouse MHCI-restricted TCR models that have been analyzed in coagonism experiments (OT-I [Yachi et al., 2005, 2007; Juang et al., 2010] and 2C [Stone et al., 2011]) recognize H-2K^b or L^d, which show relatively high-affinity CD8 binding (Cole et al., 2012).A stochastic, computational model has been used to investigate the role of coreceptors in TCR triggering, and results suggest that CD8 plays a dual role of stabilizing the TCR–pMHC interaction and of delivering the CD8-associated kinase Lck to the TCR to initiate signaling, with the latter effect being the more important (Artyomov et al., 2010). This model explicitly combined two key features, membrane-protein mobility and protein–protein interactions (Lis et al., 2009), which allowed incorporation of many biophysical measurements for MHC, TCR, and coreceptor interactions. Here, an extension of this model allows us to describe coagonism enabled by self-peptides, taking into account the distinct activation states of Lck (Nika et al., 2010; Stirnweiss et al., 2013).Because of the glaring discrepancies in the requirements for TCR discrimination between coagonist peptides in MHCI- and MHCII-restricted systems, there is a need for a unifying concept to explain activation enhancement for both T cell lineages. In this paper, we used H-2K^b and H-2D^b single chain (sc)–pMHCs (Yu et al., 2002; Choudhuri et al., 2005; Palmowski et al., 2009), which allowed us to dissect the distinct contributions of CD8 affinity and of TCR affinity for both antigenic and nonstimulatory pMHCs. Using H-2K^b– and H-2D^b–restricted TCRs and stochastic computer simulations of the kinetics of T cell activation, we describe two distinct mechanisms by which CD8 affinity for pMHC can influence the requirements for coagonists. Mechanism 1 describes CD8 binding to nonstimulatory pMHC as an absolute requirement for coagonism and shows that higher-affinity CD8–pMHC interactions can mitigate peptide specificity requirements for coagonists and increase the magnitude of enhancement. Mechanism 2 describes how the affinity of CD8 for agonist pMHC influences the requirements for TCR interaction with coagonist pMHC. A relatively simple kinetic model of T cell activation is sufficient to account for all coagonist phenomena, thus unifying disparate observations from CD4 and CD8 T cells.     

Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain

The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide–MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.TCR signaling controls many different aspects of T cell function. The ligand-binding TCR heterodimer assembles with three dimeric signaling modules (CD3γε, CD3δε, and ζζ) that contain cytoplasmic immunoreceptor tyrosine-based activation motifs (ITAMs), characterized by two tyrosines and two aliphatic residues (YxxL/I_6-12YxxL/I; Reth, 1989; Kuhns et al., 2006). After phosphorylation of both ITAM tyrosines by the Src kinase Lck, ZAP-70 is bound through its tandem SH2 domains. Engagement of both ZAP-70 SH2 domains destabilizes its inactive state, and ZAP-70 then phosphorylates downstream substrates, including LAT and SLP-76, which function as scaffolds for recruitment of many signaling molecules (Balagopalan et al., 2010; Jordan and Koretzky, 2010; Wang et al., 2010).TCR recognition is highly sensitive: transient calcium flux can be induced by one or a few peptide–MHC (pMHC) ligands at the interface between T cells and APCs, and ∼10 ligands are sufficient for sustained calcium flux (Irvine et al., 2002). Several mechanisms prevent ligand-independent TCR activation, including phosphatases that can dephosphorylate TCR ITAMs or downstream signaling molecules (Kuhns et al., 2006). After recognition of agonist pMHC ligands, TCRs form microclusters that physically exclude the phosphatases CD45 and CD148, which have substantially larger extracellular domains than the TCR (Choudhuri et al., 2005; Varma et al., 2006). Interestingly, TCR microclusters even form when the most proximal kinase in the cascade, Lck, is pharmacologically inhibited with PP2 (Campi et al., 2005). TCR binding to pMHC molecules occurs with rapid kinetics, due to the alignment of the interacting molecules on opposing membranes. Off-rates are very fast (typical t_1/2 of less than one second), and productive signaling in microclusters results from many sequential binding events (serial triggering; Valitutti et al., 1995; Huang et al., 2010; Huppa et al., 2010).Studies by several groups have shown that the cytoplasmic domains of the CD3ε and ζ chains are not suspended in the cytosol, but rather bound to the inner leaflet of the plasma membrane (PM). Membrane binding of CD3ε and ζ cytoplasmic domains is primarily mediated by clusters of basic residues that interact with anionic lipids, including phosphatidylserine (PS) and phosphatidylinositol species (such as phosphatidylinositol 4,5-bisphosphate; PIP₂; Aivazian and Stern, 2000; Sigalov et al., 2006; Xu et al., 2008; DeFord-Watts et al., 2009, 2011; Zhang et al., 2011). Structural studies of the CD3ε cytoplasmic domain in a lipid-bound state by nuclear magnetic resonance (NMR) spectroscopy showed that the two tyrosines of the ITAM can partition into the hydrophobic core of the lipid bilayer (Xu et al., 2008). The NMR data also indicated that this binding mode is dynamic, and a shift in the equilibrium of lipid-bound versus unbound state may thus render the tyrosines accessible during TCR triggering. In vitro studies showed that lipid binding can prevent phosphorylation of the ITAM tyrosine residues by recombinant Lck (Xu et al., 2008).Here, we investigated mechanisms responsible for dissociation of the CD3ε cytoplasmic domain from the plasma membrane during TCR signaling. We identified early local changes in the lipid composition of TCR microclusters that reduced the density of PS as well as the negative charge of the inner leaflet of the PM. This was accompanied by a decrease in lateral diffusion of PS in the immunological synapse. These results identify a mechanism for release of the CD3ε cytoplasmic domain from the PM during T cell activation.     

Shp1 regulates T cell homeostasis by limiting IL-4 signals

The protein-tyrosine phosphatase Shp1 is expressed ubiquitously in hematopoietic cells and is generally viewed as a negative regulatory molecule. Mutations in Ptpn6, which encodes Shp1, result in widespread inflammation and premature death, known as the motheaten (me) phenotype. Previous studies identified Shp1 as a negative regulator of TCR signaling, but the severe systemic inflammation in me mice may have confounded our understanding of Shp1 function in T cell biology. To define the T cell–intrinsic role of Shp1, we characterized mice with a T cell–specific Shp1 deletion (Shp1^fl/fl CD4-cre). Surprisingly, thymocyte selection and peripheral TCR sensitivity were unaltered in the absence of Shp1. Instead, Shp1^fl/fl CD4-cre mice had increased frequencies of memory phenotype T cells that expressed elevated levels of CD44. Activation of Shp1-deficient CD4⁺ T cells also resulted in skewing to the Th2 lineage and increased IL-4 production. After IL-4 stimulation of Shp1-deficient T cells, Stat 6 activation was sustained, leading to enhanced Th2 skewing. Accordingly, we observed elevated serum IgE in the steady state. Blocking or genetic deletion of IL-4 in the absence of Shp1 resulted in a marked reduction of the CD44^hi population. Therefore, Shp1 is an essential negative regulator of IL-4 signaling in T lymphocytes.T cells are characterized by their ability to expand dramatically in an antigen-specific manner during an immune challenge. After an initial immune response, a small proportion of responding T cells survive and give rise to memory cells (Bruno et al., 1996). Memory T cells express elevated levels of CD44 and can be divided further into central-memory (CD62L^hi CCR7^hi) and effector-memory (CD62L^lo CCR7^lo) compartments. However, not all T cells that display the phenotype of memory cells are the product of a classical antigen-specific immune response (Sprent and Surh, 2011). For example, such cells are found in unimmunized mice, including those raised in germ-free and antigen-free conditions (Dobber et al., 1992; Vos et al., 1992). The precise ontogeny of such cells remains elusive, although several mechanisms by which naive cells can adopt a memory phenotype have been characterized. Naive T cells introduced into lymphopenic environments adopt a memory phenotype through a process of homeostatic proliferation in response to IL-7 and MHC (Goldrath et al., 2000; Murali-Krishna and Ahmed, 2000). Additionally, increased production of IL-4 has been linked to the development of memory phenotype–innate T cell populations in studies of several knockout mouse models (Lee et al., 2011).The T cell response is tightly regulated by the balance of phosphorylation and dephosphorylation of intracellular signaling molecules. Shp1 (encoded by Ptpn6) is a protein tyrosine phosphatase expressed ubiquitously in hematopoietic cells and has been broadly characterized as a negative regulator of immune cell activation (Pao et al., 2007a; Lorenz, 2009). The physiological relevance of Shp1 as a key negative regulator of the immune response is illustrated by the motheaten (me) and motheaten viable (me^v) mutations, which ablate Shp1 expression or greatly reduce Shp1 activity, respectively (Shultz et al., 1993; Tsui et al., 1993). Homozygous me/me or me^v/me^v mice (hereafter, referred to collectively as me mice) suffer from severe systemic inflammation and autoimmunity, which result in retarded growth, myeloid hyperplasia, hypergammaglobulinemia, skin lesions, interstitial pneumonia, and premature death. More recently, a study has identified a third allele of Ptpn6, named spin, which encodes a hypomorphic form of Shp1 (Croker et al., 2008). Mice homozygous for spin develop a milder autoimmune/inflammatory disease that is ablated in germ-free conditions.Shp1 has been implicated in signaling from many immune cell surface receptors (Zhang et al., 2000; Neel et al., 2003), including the TCR (Plas et al., 1996; Lorenz, 2009), BCR (Cyster and Goodnow, 1995; Pani et al., 1995), NK cell receptors (Burshtyn et al., 1996; Nakamura et al., 1997), chemokine receptors (Kim et al., 1999), FAS (Su et al., 1995; Takayama et al., 1996; Koncz et al., 2008), and integrins (Roach et al., 1998; Burshtyn et al., 2000). Shp1 also has been demonstrated to regulate signaling from multiple cytokine receptors by dephosphorylating various Jak (Klingmüller et al., 1995; Jiao et al., 1996; Minoo et al., 2004) and/or Stat (Kashiwada et al., 2001; Xiao et al., 2009) molecules. Several of these cytokines are pertinent to T cell biology. For example, Stat 5 is an essential mediator of signals from IL-2 and IL-7 (Rochman et al., 2009). IL-4 signaling results in Stat 6 phosphorylation and has potent Th2 skewing effects. Additionally, IL-4 has mitogenic effects on CD8⁺ T cells (Rochman et al., 2009). Notably, mutation of the immunoreceptor tyrosine-based inhibitory motif (ITIM) in IL-4Rα results in ablation of Shp1 binding and hypersensitivity to IL-4 stimulation (Kashiwada et al., 2001), implicating Shp1 as a regulator of this cytokine receptor.Although development of the me phenotype does not require T cells (Shultz, 1988; Yu et al., 1996), several aspects of T cell biology reportedly are controlled by Shp1 (Lorenz, 2009). Most previous studies that examined the role of Shp1 in T cells used cells derived from me/me or me^v/me^v mice (Carter et al., 1999; Johnson et al., 1999; Zhang et al., 1999; Su et al., 2001) or cells expressing a dominant-negative allele of Shp1 (Plas et al., 1996, 1999; Zhang et al., 1999). Several such reports have concluded that Shp1 negatively regulates the strength of TCR signaling during thymocyte development and/or peripheral activation (Carter et al., 1999; Johnson et al., 1999; Plas et al., 1999; Zhang et al., 1999; Su et al., 2001). Despite the large number of studies that implicate Shp1 in control of TCR signaling, there is no consensus on which component of the TCR signaling cascade is targeted by the catalytic activity of Shp1. Suggested Shp1 targets downstream of T cell activation include TCR-ζ (Chen et al., 2008), Lck (Lorenz et al., 1996; Stefanová et al., 2003), Fyn (Lorenz et al., 1996), ZAP-70 (Plas et al., 1996; Chen et al., 2008), and SLP-76 (Mizuno et al., 2005). Shp1 also is implicated in signal transduction downstream of several immune inhibitory receptors that negatively regulate T cell activity, such as PD-1 (Chemnitz et al., 2004), IL-10R (Taylor et al., 2007), CEACAM1 (Lee et al., 2008), and CD5 (Perez-Villar et al., 1999).The severe inflammation characteristic of the me phenotype might have confounded studies examining the cell-intrinsic role of Shp1 in various hematopoietic cell types. We previously generated a floxed Shp1 allele that facilitates analysis of the role of Shp1 in various lineages (Pao et al., 2007b). Previous studies have used this approach to study the role of Shp1 in T cells during antiviral and antitumor immune responses, respectively (Fowler et al., 2010; Stromnes et al., 2012). However, a more fundamental analysis of the cell-intrinsic role of Shp1 during T cell development, homeostasis, and activation has not been reported. Here, we provide evidence that a major role for Shp1 in T cells is to maintain normal T cell homeostasis through negative regulation of IL-4 signaling.     

Inherited IL-17RC deficiency in patients with chronic mucocutaneous candidiasis

Chronic mucocutaneous candidiasis (CMC) is characterized by recurrent or persistent infections of the skin, nail, oral, and genital mucosae with Candida species, mainly C. albicans. Autosomal-recessive (AR) IL-17RA and ACT1 deficiencies and autosomal-dominant IL-17F deficiency, each reported in a single kindred, underlie CMC in otherwise healthy patients. We report three patients from unrelated kindreds, aged 8, 12, and 37 yr with isolated CMC, who display AR IL-17RC deficiency. The patients are homozygous for different nonsense alleles that prevent the expression of IL-17RC on the cell surface. The defect is complete, abolishing cellular responses to IL-17A and IL-17F homo- and heterodimers. However, in contrast to what is observed for the IL-17RA– and ACT1-deficient patients tested, the response to IL-17E (IL-25) is maintained in these IL-17RC–deficient patients. These experiments of nature indicate that human IL-17RC is essential for mucocutaneous immunity to C. albicans but is otherwise largely redundant.In humans, chronic mucocutaneous candidiasis (CMC) is characterized by infections of the skin, nail, digestive, and genital mucosae with Candida species, mainly C. albicans, a commensal of the gastrointestinal tract in healthy individuals (Puel et al., 2012). CMC is frequent in acquired or inherited disorders involving profound T cell defects (Puel et al., 2010b; Vinh, 2011; Lionakis, 2012). Human IL-17 immunity has recently been shown to be essential for mucocutaneous protection against C. albicans (Puel et al., 2010b, 2012; Cypowyj et al., 2012; Engelhardt and Grimbacher, 2012; Huppler et al., 2012; Ling and Puel, 2014). Indeed, patients with primary immunodeficiencies and syndromic CMC have been shown to display impaired IL-17 immunity (Puel et al., 2010b). Most patients with autosomal-dominant (AD) hyper-IgE syndrome (AD-HIES) and STAT3 deficiency (de Beaucoudrey et al., 2008; Ma et al., 2008; Milner et al., 2008; Renner et al., 2008; Chandesris et al., 2012) and some patients with invasive fungal infection and autosomal-recessive (AR) CARD9 deficiency (Glocker et al., 2009; Lanternier et al., 2013) or Mendelian susceptibility to mycobacterial diseases (MSMD) and AR IL-12p40 or IL-12Rβ1 deficiency (de Beaucoudrey et al., 2008, 2010; Prando et al., 2013; Ouederni et al., 2014) have low proportions of IL-17A–producing T cells and CMC (Cypowyj et al., 2012; Puel et al., 2012). Patients with AR autoimmune polyendocrine syndrome type 1 (APS-1) and AIRE deficiency display CMC and high levels of neutralizing autoantibodies against IL-17A, IL-17F, and/or IL-22 (Browne and Holland, 2010; Husebye and Anderson, 2010; Kisand et al., 2010, 2011; Puel et al., 2010a).These findings paved the way for the discovery of the first genetic etiologies of CMC disease (CMCD), an inherited condition affecting individuals with none of the aforementioned primary immunodeficiencies (Puel et al., 2011; Casanova and Abel, 2013; Casanova et al., 2013, 2014). AR IL-17RA deficiency, AR ACT1 deficiency, and AD IL-17F deficiency were described, each in a single kindred (Puel et al., 2011; Boisson et al., 2013). A fourth genetic etiology of CMCD, which currently appears to be the most frequent, has also been reported: heterozygous gain-of-function (GOF) mutations of STAT1 impairing the development of IL-17–producing T cells (Liu et al., 2011; Smeekens et al., 2011; van de Veerdonk et al., 2011; Hori et al., 2012; Takezaki et al., 2012; Tóth et al., 2012; Al Rushood et al., 2013; Aldave et al., 2013; Romberg et al., 2013; Sampaio et al., 2013; Soltész et al., 2013; Uzel et al., 2013; Wildbaum et al., 2013; Frans et al., 2014; Kilic et al., 2014; Lee et al., 2014; Mekki et al., 2014; Mizoguchi et al., 2014; Sharfe et al., 2014; Yamazaki et al., 2014). We studied three unrelated patients with CMCD without mutations of IL17F, IL17RA, ACT1, or STAT1. We used a genome-wide approach based on whole-exome sequencing (WES). We found AR complete IL-17RC deficiency in all three patients.     

Activating receptors promote NK cell expansion for maintenance,IL-10 production,and CD8 T cell regulation during viral infection

Natural killer (NK) cells have the potential to deliver both direct antimicrobial effects and regulate adaptive immune responses, but NK cell yields have been reported to vary greatly during different viral infections. Activating receptors, including the Ly49H molecule recognizing mouse cytomegalovirus (MCMV), can stimulate NK cell expansion. To define Ly49H''s role in supporting NK cell proliferation and maintenance under conditions of uncontrolled viral infection, experiments were performed in Ly49h^−/−, perforin 1 (Prf1)^−/−, and wild-type (wt) B6 mice. NK cell numbers were similar in uninfected mice, but relative to responses in MCMV-infected wt mice, NK cell yields declined in the absence of Ly49h and increased in the absence of Prf1, with high rates of proliferation and Ly49H expression on nearly all cells. The expansion was abolished in mice deficient for both Ly49h and Prf1 (Ly49h^−/−Prf1^−/−), and negative consequences for survival were revealed. The Ly49H-dependent protection mechanism delivered in the absence of Prf1 was a result of interleukin 10 production, by the sustained NK cells, to regulate the magnitude of CD8 T cell responses. Thus, the studies demonstrate a previously unappreciated critical role for activating receptors in keeping NK cells present during viral infection to regulate adaptive immune responses.Classical (non–T) NK cells are generally found at low frequencies in leukocyte populations (Biron et al., 1999). They have the potential to mediate antiviral and immunoregulatory functions through a variety of mechanisms (Orange et al., 1995; Su et al., 2001; Lee et al., 2007; Robbins et al., 2007; Strowig et al., 2008). By altering cell availability, in vivo conditions changing NK cell numbers may indirectly influence all of their effects. Activating receptors on NK cells are linked to stimulatory pathways overlapping with those used by TCRs to drive cell expansions (Murali-Krishna et al., 1998; Pitcher et al., 2003; French et al., 2006; MacFarlane and Campbell, 2006; Biron and Sen, 2007; Lee et al., 2007) and can induce NK cell proliferation (Dokun et al., 2001; French et al., 2006). Although particular activating receptors have been reported to recognize microbial products (Lanier, 1998; Vidal and Lanier, 2006; Jonjic et al., 2008), dramatic NK cell expansion has not been observed during infections. Except under rare experimental conditions (Caligiuri et al., 1991; Yamada et al., 1996; Fehniger et al., 2001; Huntington et al., 2007a; Sun et al., 2009), NK cell division is generally induced for limited periods of time as a consequence of transient innate cytokine exposure (Biron et al., 1984; Biron et al., 1999; Dokun et al., 2001; Nguyen et al., 2002; Yokoyama et al., 2004). Increasing proportions of NK cell subsets with activating receptors recognizing particular viral ligands can be detected during certain infections (Dokun et al., 2001; Gumá et al., 2006), but this is observed without dramatic increases in overall NK cell numbers, and many viral infections induce striking reductions in NK cell functions, frequencies, and yields (Biron et al., 1999; Tarazona et al., 2002; Lehoux et al., 2004; Reed et al., 2004; Azzoni et al., 2005; Vossen et al., 2005; Morishima et al., 2006). Thus, particular conditions of viral challenges must result in differential regulation of NK cell proportions and numbers, with consequences for the delivery of NK cell functions.An NK cell activating receptor in the mouse is Ly49H (Lanier, 1998; Gosselin et al., 1999; Smith et al., 2000; Vidal and Lanier, 2006). This molecule recognizes a mouse CMV (MCMV) ligand (Arase et al., 2002; Smith et al., 2002), is expressed on NK cell subsets in strains of particular genetic backgrounds, including C57BL/6 (B6) mice, and is reported to be an exclusive marker for the classical NK cell subset (Smith et al., 2000). Through an associated molecule, Ly49H stimulates using signaling pathways overlapping with those used by the TCR (MacFarlane and Campbell, 2006; Biron and Sen, 2007). Additional markers for all NK cells include CD49b, expressed on other activated cell types (Arase et al., 2001); NKp46, selectively expressed on classical NK cells (Gazit et al., 2006; Walzer et al., 2007a; Walzer et al., 2007b); CD122, the IL-2Rβ chain, expressed on all NK cells and activated T cells (Huntington et al., 2007b); and NK1.1, exclusively expressed on C57BL6 (B6) NK and NKT cells (Lian and Kumar, 2002; MacDonald, 2002; Yokoyama et al., 2004; Huntington et al., 2007b). The TCR with associated CD3 molecules is not expressed on their cell surfaces (Biron et al., 1999). The mechanisms for NK cell–enhanced resistance to MCMV infection are incompletely characterized (Lee et al., 2007), but Ly49H contributes to their protective effects (Scalzo et al., 1990; Brown et al., 2001; Daniels et al., 2001; Lee et al., 2001; Lee et al., 2003). Engagement of the Ly49H receptor can lead to killing of target cells (Arase et al., 2002; Smith et al., 2002), and the correlation of increases in viral burdens resulting from the absence of Ly49H (Scalzo et al., 1990) as compared with those resulting from defects in cytotoxicity functions, such as mutation of the membrane pore-forming protein perforin 1 (Prf1; Tay and Welsh, 1997; Loh et al., 2005; van Dommelen et al., 2006), supports a role for Ly49H-dependent killing of virus-infected cells in the delivery of NK cell antiviral effects. The receptor may have other functions associated with its ability to stimulate proliferation, however, as the proportions of NK cells expressing Ly49H are increased during MCMV infection (Dokun et al., 2001).The studies presented in this paper were undertaken to dissect the proliferative from the cytotoxic functions accessed through Ly49H, and to define the contribution of the regulation of NK cell numbers to protection during infection. To carry out the work, responses were evaluated, during MCMV infections, in wild-type (wt) B6 mice and mice deficient in Ly49h (Fodil-Cornu et al., 2008), Prf1 (Kägi et al., 1994), or both. As expected, single Ly49h^−/− and Prf1^−/− mice had profoundly increased viral burdens, but significant differences in NK cell expansion were discovered. The NK cell populations were decreasing in infected Ly49h^−/− mice, whereas infected Prf1^−/− mice had an unexpected dramatic proliferation of NK cells uniformly expressing Ly49H. The expansion was proven to be dependent on Ly49H. During uncontrolled infection in the absence of Prf1, Ly49H beneficially promoted effects for survival, because the sustained NK cells produced IL-10 to control the magnitude of the CD8 T cell response and limit immunopathology. The data suggest that Ly49H-dependent cytotoxicity acts to control viral infection and NK cell expansion, but that in the absence of the killing function, Ly49H promotes a continued NK cell expansion critical for supporting life over death because the NK cells are available to regulate adaptive immune responses.     

Allelic polymorphism in the T cell receptor and its impact on immune responses

In comparison to human leukocyte antigen (HLA) polymorphism, the impact of allelic sequence variation within T cell receptor (TCR) loci is much less understood. Particular TCR loci have been associated with autoimmunity, but the molecular basis for this phenomenon is undefined. We examined the T cell response to an HLA-B*3501–restricted epitope (HPVGEADYFEY) from Epstein-Barr virus (EBV), which is frequently dominated by a TRBV9*01⁺ public TCR (TK3). However, the common allelic variant TRBV9*02, which differs by a single amino acid near the CDR2β loop (Gln55→His55), was never used in this response. The structure of the TK3 TCR, its allelic variant, and a nonnaturally occurring mutant (Gln55→Ala55) in complex with HLA-B*3501^HPVGEADYFEY revealed that the Gln55→His55 polymorphism affected the charge complementarity at the TCR–peptide-MHC interface, resulting in reduced functional recognition of the cognate and naturally occurring variants of this EBV peptide. Thus, polymorphism in the TCR loci may contribute toward variability in immune responses and the outcome of infection.MHC molecules play a critical role in protective immunity by presenting antigenic peptide fragments for T cell recognition (Davis and Bjorkman, 1988). MHC polymorphism enhances immune defense across the population by ensuring wide variation in the T cell response to infecting pathogens through presentation of a broad array of target epitopes (Lawlor et al., 1990; Germain and Margulies, 1993). There are ∼4,000 different variants of HLA (Robinson et al., 2003), with polymorphism generally concentrated in the antigen-binding cleft, controlling the size and diversity of the peptide repertoire presented by each HLA molecule. Although HLA molecules can differ from each other by >30 amino acids, differences of only a few amino acids (micropolymorphism) can have a major impact on immune responses (Archbold et al., 2009). Namely, HLA micropolymorphism can influence the repertoire of peptides presented on the surface of APCs (Macdonald et al., 2003; Burrows et al., 2007), the conformation of HLA-bound peptide (Hülsmeyer et al., 2004; Tynan et al., 2005c), the dependence on chaperones for antigen loading (Zernich et al., 2004), αβ TCR recognition (Tynan et al., 2005a,b, 2007), and susceptibility to infectious disease (Limou et al., 2009).To engage the vast repertoire of MHC-bound antigenic peptides, TCRs are diversified through the random rearrangement of V and J genes at the TCRα locus, and V, D, and J genes at the TCRβ locus of developing thymic T cells. Further potential diversity is created through untemplated addition or deletion of a variable number of nucleotides at the V-(D)-J junctional sites, called N regions. The residual repertoire of unique TCRs after thymic selection is between 10 and 100 million in humans (Arstila et al., 1999). Despite this vast potential repertoire, immune responses often show strong unexplained biases in TCR selection, resulting in immunodominance of certain “public” TCRs that are widely used in individuals with shared MHC types (Acha-Orbea et al., 1988; Argaet et al., 1994; Burrows et al., 1995; Turner et al., 2006; Gras et al., 2008). The first and second complementarity-determining regions (CDRs) of the TCR are germline encoded within the TRAV and TRBV gene segments, whereas the CDR3 regions are derived from the V-(D)-J and N regions. From the growing number of unique TCR–peptide-MHC (pMHC)–I structures determined, it is apparent that a rough docking mode is preserved in which the Vα domain is positioned over the MHC-I α2 helix and the N-terminal end of the peptide, whereas the Vβ domain is more often positioned over the MHC-I α1 helix and the C-terminal end of the peptide, although the precise interatomic interactions vary considerably between TCR–pMHC complexes (Rudolph et al., 2006; Godfrey et al., 2008).As with the MHC genes, allelic sequence variation is also a feature of the TCR loci; however, the full extent of TCR polymorphism and its functional significance in influencing protective immunity is unknown. Nevertheless, several single nucleotide polymorphism (SNP) studies have revealed considerable polymorphism within the TRAV and TRBV gene segments (Subrahmanyan et al., 2001; Mackelprang et al., 2006). In one study, the TCR loci from 40 individuals across four ethnic groups were fully sequenced, and >550 SNPs were found, with many being situated in coding/regulatory regions of functional TCR genes and several causing null and nonfunctional mutations. On average, the coding region of each TCR variable gene contained two SNPs, with many more found in the 5′, 3′, and intronic sequences of these segments. Furthermore, a total of 51 SNPs in the TRA locus and 72 SNPs in the TRB locus resulted in amino acid changes (Subrahmanyan et al., 2001; Mackelprang et al., 2006), although the structural and functional consequences of this sequence variation have not been investigated. Importantly, particular TCR loci have been associated with increased susceptibility to common immune diseases such as multiple sclerosis (Seboun et al., 1989; Hibberd et al., 1992; Hockertz et al., 1998), asthma (Moffatt et al., 1994, 1997; Cho et al., 2001), and narcolepsy (Hallmayer et al., 2009).In this study, we have investigated the functional and structural impact of natural micropolymorphism within genes encoding a public TCR that recognizes an 11–amino acid epitope, ⁴⁰⁷HPVGEADYFEY⁴¹⁷ (referred to as HPVG), derived from the EBNA-1 protein of EBV. This epitope is highly immunogenic in EBV-exposed healthy individuals expressing HLA-B*3501 (Blake et al., 1997; Lee et al., 2004; Tellam et al., 2004; Miles et al., 2006). Although EBV is a genetically stable DNA virus, sequence variation within the HPVG epitope has been previously described (Snudden et al., 1995; Wang et al., 2002; Zhang et al., 2004; Dolan et al., 2006). Unrelated EBV⁺, HLA-B*3501⁺ individuals frequently generate CTLs against the HPVG epitope that express immunodominant public TCR α and β chains characterized by TRAV20, TRAJ58, TRBV9, and TRBJ2-2 usage (Miles et al., 2006). We now show that allelic variation within this TRBV9 gene, which led to a Gln (TRBV9*01) to His (TRBV9*02) substitution at position 55 (ImMunoGeneTics unique numbering; Lefranc, 2003), resulted in a reduction in TCR binding affinity and diminished functional recognition of the cognate viral epitope as well as the naturally occurring variants of this epitope. These factors dictate the preferential selection of the TRBV9*01 TCR β chain gene and the exclusion of TRBV9*02 in this antiviral immune response. Our data therefore illustrate both the sensitivity and significance of allelic polymorphism within the TCR loci in protective immunity.