Semin Immunopathol (2010) 32:107–116 DOI 10.1007/s00281-010-0196-x REVIEW ZAP70: a master regulator of adaptive immunity Alain Fischer & Capucine Picard & Karine Chemin & Stéphanie Dogniaux & Françoise le Deist & Claire Hivroz Received: 16 October 2009 / Accepted: 29 December 2009 / Published online: 5 February 2010 # Springer-Verlag 2010 Abstract The protein tyrosine kinase ZAP70 became the subject of intense scrutiny in the early nineties, when ZAP70 mutations were characterized in several young patients presenting with severe T cell immunodeficiencies. The association of a lack of expression of ZAP70 with an immunodeficiency consisting in a markedly reduced T lymphocyte-mediated immunity highlighted the crucial role of this tyrosine kinase in T cell development and function. This discovery was soon accompanied by the characterization of the substrates of ZAP70 and the signalling cascades that depend on ZAP70 activity. These studies demonstrated that ZAP70 was indeed at the crossroad of several signalling pathways that control T lymphocyte development and function. Recently, a revival of interest for this protein came again from studies associating abnormal ZAP70 expression with pathological conditions. Some chronic lymphocytic leukemia B cells were shown to express ZAP70, and this expression was correlated with bad prognosis. Mouse models also revealed that partial defects in ZAP70 activity can be associated with autoimmunity. These last results suggested that ZAP70 is involved in the fine balance between immunity and tolerance. In this review, we will discuss the role of ZAP70 in T cell activation and focus on what we learnt from pathological conditions associated with defective expression or activity of the ZAP70 kinase. A. Fischer INSERM, U768, Hôpital Necker-Enfants Malades, Paris, France C. Picard Génétique Humaine des Maladies Infectieuses, Faculté Necker, INSERM, Unité 550, Paris, France A. Fischer Unité d’Immuno-Hématologie Pédiatrique, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Paris, France K. Chemin : S. Dogniaux : C. Hivroz (*) Centre de Recherche, Institut Curie, Pavillon Pasteur, 26 Rue d’ULM, Paris 75248, France e-mail: claire.hivroz@curie.fr A. Fischer : C. Picard Faculté Necker, Université Paris Descartes, Paris, France K. Chemin : S. Dogniaux : C. Hivroz Immunité et Cancer, INSERM, Unité 932, Paris 75248, France C. Picard Centre d’Étude des Déficits Immunitaires, Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Paris, France F. le Deist Department of Microbiology and Immunology, and Centre of Research, CHU Sainte-Justine, Université de Montréal, Montreal, QC, Canada Keywords Immunodeficiency . Autoimmunity . Tyrosine kinase . Immunoreceptor The T cell antigenic receptor (TCR) complex is an octameric receptor composed of two chains, αβ or γδ, which bind to specific ligands, i.e., antigenic peptide presented on major histocompatibility complex (MHC) molecules. These chains are noncovalently associated with the CD3 subunits γε and δε and the ζ homodimer that 108 mediate signal transduction. Binding of the antigenic receptors to their ligands induces tyrosine phosphorylation of numerous proteins that will ultimately lead to functional response. Yet, none of the chains of the TCR-CD3-ζ complex possesses intrinsic tyrosine kinase activity; instead, they recruit intracellular kinase activities that bind to the CD3-ζ complex after recognition by the TCR of the peptide-MHC complexes. Four families of tyrosine kinases are involved in TCR signal transduction: Csk kinases, Src kinases, Tec kinases, and the ZAP70 kinase. The key role of this kinase in human T cell function has been highlighted in the nineties by the studies of immunodeficient patients carrying mutations in the ZAP70 gene. Since then, ZAP70 defects have been associated with autoimmunity and cancer in human and mice and have thus been the subject of much attention from the immunologists. Structure of the ZAP70 protein Recognition of specific MHC-peptide by the TCR at the surface of antigen-presenting cells (APC) leads to the phosphorylation of tyrosine residues present in the intracellular tail of the CD3 and ζ subunits. These chains bear specific motives called immunoreceptor tyrosine-based activation motif (ITAM, [(D/E)xxYxxI/Lx(6–8)YxxI/L]) containing two tyrosines, which are phosphorylated by Src kinases. In 1992, A Weiss' group described a tyrosine kinase activity of 70 kDa that was associated with the ζ chain, the ζ-associated protein of 70 kDa or ZAP70, which was shown to exhibit unique characteristics [13]. ZAP70 protein expression is essentially restricted to thymocytes and peripheral T cells. However, leukemic and primary B cells also express ZAP70. There are only two members in the ZAP70 kinase family, i.e., ZAP70 and Syk. In quiescent T lymphocytes, ZAP70 is a cytosolic protein, and it is recruited at the plasma membrane of T cells following TCR stimulation. This recruitment is mediated by binding of the two SH2 domains of ZAP70 to the doubly phosphorylated ITAMs of ζ, which serve to dock the kinase at the stimulated TCR complex [10, 36] (Fig. 1). Docking of ZAP70 at the plasma membrane is required for its activation. The binding of ZAP70 to the doubly phosphorylated ITAM not only targets ZAP70 to the plasma membrane, where it is activated by Lck-mediated phosphorylation [12, 25, 73, 84], but also contributes to relieve an autoinhibited conformation of the kinase that was revealed by the crystal structure of the full length ZAP70 [21]. Thus, the SH2 domains are critical for both ZAP70 recruitment at the plasma membrane and ZAP70 activation. The interdomain B is another critical region of ZAP70 that separates the C-terminal SH2 domain from the kinase domain. This region contains three tyrosine residues (Y292, Semin Immunopathol (2010) 32:107–116 Y315, and Y319) that are phosphorylated by Lck upon TCR triggering and interact with several signalling proteins. The tyrosine Y292 has been shown to bind the ubiquitin ligase c-Cbl [44, 45] and to control both ζ ubiquitination [44, 81] and TCR downmodulation [20, 94]. These results suggest that Y292 phosphorylation regulates the duration of residence of the activated TCR at the T cell surface. Concerning Y315, it has been shown that once phosphorylated by Lck, Y315 interacts with the CT10 regulator of kinase II (CrkII) adapter protein [33]. This interaction is supposed to play a role in TCR-induced actin remodeling [33]. Indeed, disruption of complex formation by a mutation in the CrkII SH3.1 domain resulted in impaired actin polymerization and IL-2 production upon TCR stimulation. Thus, ZAP70 could influence T cell activation indirectly through interactions with actin network-remodeling proteins [65]. The Y315 has also been involved in TCR-induced activation of the integrin LFA-1 [35] and, hence, in T cell adhesion [29, 35] Mutational analysis of Y319 showed that it is a binding site for the SH2 domain of Lck and for the C-terminal SH2 domain of PLCγ [22, 55, 87]. Mutation of ZAP70 Y319 induces a decrease in PLCγ phosphorylation that is accompanied by a defect in Ca2+ mobilization and IL-2 secretion [22, 87]. These results indicate that Y319 is a critical positive APC Peptide/MHC α β TCR/CD3/ ε ε ζζ γ δ T CD4 Lck 3 1 Interdomain A 10 102 N-SH2 162 Interdomain B 254 C-SH2 2 ZAP70 292 315 319 337 Y Y Y 493 492Y Y Kinase 592 : Phosphorylation on Y residues Fig. 1 Recruitment and activation of ZAP70. Recognition by the T cell antigenic receptor of its specific peptide-major histocompatibility complex induces (1) activation of the p56lck kinase, which phosphorylates tyrosine residues present in the ITAM motives of the CD3-ζ complexes; (2) recruitment of the cytosolic ZAP70 kinase on ITAM motives of the CD3-ζ complexes via its two SH2 domains; and (3) activation of ZAP70 by a p56lck-dependent phosphorylation of the interdomain B and kinase domain of ZAP70. A schematical representation of ZAP70 is shown Semin Immunopathol (2010) 32:107–116 109 regulator of ZAP70 kinase-dependent signals. Finally, deletion of the entire interdomain B induces a reduction in ZAP70 kinase activity [95] showing that this domain regulates ZAP70 function. Indeed, recent studies suggest that Y315 and Y319 control the autoinhibitory activity of ZAP70 [9, 21]. The kinase domain itself contains two Y residues that are also phosphorylated. A ZAP70 Y492F mutant exhibited increased kinase activity, while kinase activity of the Y493F mutant was impaired [12]. Phosphorylation of Y493 by PTKs belonging to the src family has been shown to upregulate ZAP70 activity and to be required for TCR-induced induction of IL-2 secretion by T lymphocytes [12]. In contrast, phosphorylation of Y492 seems to negatively regulate ZAP70 kinase activity [83]. These data demonstrate that the activity of ZAP70 is strictly controlled both in quiescent and in TCR-activated T lymphocytes. Signalling pathways controlled by ZAP70 in T cells One of the most salient defect described in T lymphocytes or thymocytes lacking ZAP70 expression is the absence of increased intracellular free Ca2+ concentration upon TCR Pat. P-SLP76 Pat. Anti-CD3 (Ca2 +)i SLP76 e Ctl 0 P-LAT LAT t im (Ca2 +)i tim e Pat. An ti-C D3 PM A PLC-γ Ctl Anti-CD3 Nb of cells P-PLC-γ 0 - + - + An ti-C PM D3 A Ctl Anti-CD3: P-MAPK Fig. 2 Defective T cell antigenic receptor (TCR)-induced signalling in CD4+ T cells from a ZAP70-deficient patient [49]. Purified CD4+ T cells were left unactivated or stimulated for 5 min with a crosslinked anti-CD3. PLCγ, SLP76, and LAT were immunoprecipitated. Phosphorylation of the protein was revealed by Western blot analysis with an anti-phosphotyrosine mAb (P-protein); total expression of the protein is shown. Results show an absence of CD3-induced phosphorylation of PLCγ, SLP76, and LAT. TCR-induced increase of the free intracellular Ca2+ was measured in CD4+ T cells from the patient or a control donor labeled with indo-1 as described in [49]. No mobilization of the intracellular Ca2+ was observed in the patient's T cells. Phosphorylation of the mitogen-activated protein kinases (MAPK) Erk1 and Erk2 induced after 5 min of CD3 or PMA stimulation was revealed by Western blot with a mAb specific for the phosphorylated forms of Erk1,2. Western blot revealed an absence of CD3-induced phosphorylation of the MAPKs in the patient's cells. PMA stimulation used as a control showed a normal phosphorylation triggering [3, 14, 49, 52, 88] (Fig. 2). This defective Ca2+ signalling observed in ZAP70-deficient T cells may in turn explain many of their functional defects as demonstrated by the dysfunction of T lymphocytes defective in the ORAI1 Ca2+ channel or in the STIM1 protein that is involved in the control of this channel [31, 42, 57]. Yet, other defects such as defective activation of the mitogen-activated protein kinases (MAPK) Erk1 and Erk2 have been reported in thymocytes of ZAP70-deficient mice and CD4+ T lymphocytes of ZAP70-deficient patients [48] (Fig. 2). A systematic analysis of the proteins that lack tyrosine phosphorylation upon TCR triggering of ZAP70-deficient cells revealed the defective phosphorylation of two key proteins of T cell activation, i.e., LAT and SLP-76 (Fig. 2). It has also been shown that ZAP70-dependent tyrosine phosphorylation of LAT controls its localization at the immunological synapse [8]. LAT and SLP76 are both devoid of intrinsic enzymatic activity but contain numerous tyrosine residues, which, once phosphorylated, can bind to other proteins thus driving the spatial organization of signalling complexes involved in the downstream activating cascades. The pleiotropic effect of ZAP70 on TCR-induced signalling may thus be the consequence of the ZAP70dependent phosphorylation of the two scaffolding proteins LAT and SLP-76. ZAP70 also controls cytoskeleton modifications [6, 7, 11], adhesion, and mobility [68, 74] of T lymphocytes. It is required for a correct interaction with the APC by controlling the formation of a functional immunological synapse. Indeed, ZAP70 controls the polarization of the microtubule organizing center “en face” of the APC, thus ensuring the targeted delivery of effectors such as cytokines to the APC [7]. It is worth noting that ZAP70, which plays a key role in T cell activation, has also been demonstrated to exert inhibitory effects on T cell activation. Like many other cell surface receptors, TCR-CD3-ζ complexes are constitutively internalized and recycled back to cell surface [41, 43, 50]. It was shown in the early 1980s that the activation of T cells by Ag-loaded APCs, or mAbs directed against the TCR/CD3 complex, results in the downmodulation of TCR-CD3-ζ expression at the cell surface [59, 92] and reviewed in [2]. This downmodulation by reducing the number of receptors at the cell surface prevents sustained signalling in T-APC conjugates and modulates the responsiveness of T cells to further antigenic stimulation [80, 92]. It may also facilitate the serial engagement of many TCRs by a small number of TCR/peptide-MHC complexes [79]. We have shown that ZAP70 activity is indeed required for the TCR-induced downmodulation of the TCR-CD3-ζ complexes and their degradation [24]. It was later shown that the Y292 tyrosine residue, which is present in the interdomain of ZAP70 that binds to the ubiquitin ligase c-Cbl, is involved 110 in dynamics, internalization, and degradation of the TCR/ CD3 complexes in response to TCR triggering [20]. Thus, ZAP70, which switches on T cell activation, is also involved in its switching off by regulating TCR expression at the T cell surface. ZAP70 is not only involved in activation of mature T cells but also in the control of mouse and human thymocyte development. During thymic development, T cells that bind self peptide-MHC complexes with an intermediate avidity are selected for survival (positive selection), whereas T cells that bind with high avidity (autoreactive T cells) are eliminated (negative selection). Mice lacking ZAP70 have been shown to develop neither CD4 nor CD8 singlepositive T cells [52]. Moreover, ZAP70-deficient thymocytes were not deleted by peptide antigens showing that negative selection required ZAP70 activity [52]. This is very different in ZAP70-deficient patients, wherein only the CD8+ thymocyte development is affected [3, 60]. The role ZAP70 and Syk have in the different steps of mouse and human thymocytes development has been recently reviewed by A Weiss [4]. Signalling pathways controlled by ZAP70 in B cells Unlike Syk, the other member of the ZAP70-Syk family of tyrosine kinase, which expression among hematopoietic cells is ubiquitous, ZAP70 was first thought to be uniquely expressed in T lymphocytes, thymocytes, and NK cells. Hence, microarray analyses revealed that some chronic lymphocytic leukemia B (B-CLL) cells expressed ZAP70 [62]. This expression was then correlated with poor prognosis, this “marker” being a more accurate predictor of the disease outcome than the unmutated IgVH status that was used before [54]. Of note, the level of ZAP70 expression in B-CLL does not change over time [58]. This prognostic value of B-CLL ZAP70 expression is particularly interesting since it is technically easy and of low cost to analyze the presence of ZAP70 in B-CLL patients. This discovery suggested that the BCR might, like the TCR, use ZAP70 to induce signalling in B cells. ZAP70 expression in B-CLL was indeed associated with enhanced BCR-induced phosphorylation of several signalling molecules including Syk [17]. In addition, the introduction of ZAP70 in B-CLL cells resulted in enhanced BCR-induced signalling, while ZAP70 was shown to associate with the surface BCR complex after anti-IgM treatment of ZAP70+ B-CLL [15]. However, a recent study [16] showed that the ability of ZAP70 to enhance BCR signalling in B-CLL was independent of its kinase activity, as both WT ZAP70 and a catalytically inactive ZAP70 mutant induced similar increases in intracellular free Ca2+ concentration upon BCR triggering. This finding suggests that ZAP70 may Semin Immunopathol (2010) 32:107–116 facilitate BCR signalling through its adaptor function and/ or associate with a regulator of Syk. Thus, ZAP70 expression in B-CLL might result in a more efficient BCR signalling that would account for inappropriate activation of the cells and hence disease progression. Yet, further studies are needed to understand the precise mechanisms linking ZAP70 to poor prognosis B-CLL. An increase in ZAP70 expression was recently reported in other abnormal B cells. CD38-, CD5-, and CD23-positive B cells found in inflamed synovial from rheumatoid arthritis patients were shown to be enriched in ZAP70 protein. These ZAP70+ B cells exhibited an increased survival in vitro compared to ZAP70 negative B cells. These observations link once more ZAP70 abnormal expression with inflammatory and autoimmune phenotype [75]. ZAP70-dependent signalling also plays a role in primary B cells. The role of Syk in primary B lymphocyte development and activation was described 15 years ago [18, 77]. More recently, B cell progenitors and splenic B cells were shown to express ZAP70 suggesting that this kinase may play a role in the development or activation of B cells. Indeed, several authors noticed that B cell development was not completely blocked in Syk-deficient mice and that mice deficient in both Syk and ZAP70 had a more complete block in B cell development [66]. These results demonstrate that there is partial redundancy between Syk and ZAP70 that is unique to B cell development, since as described earlier, ZAP70-deficient mice lack both CD4+ and CD8+ mature T lymphocytes [52] indicating that Syk cannot compensate for ZAP70 deficiency in T cell development at least in mice. ZAP70 and autoimmunity in mice and humans Some years ago, Sakaguchi et al. characterized a punctual mutation in the SKG mouse strain. SKG mice spontaneously develop inflammatory arthritis. These mice were used as model of rheumatoid arthritis since they exhibit many clinical similarities with rheumatoid arthritis patients. Indeed, these mice have T cell infiltrates into the synovia; they develop hypergammaglobulinemia, anti-self antibodies, and high titers of rheumatoid factor (reviewed in [63]). These SKG mice were shown to carry a missense mutation in the ZAP70 gene responsible for a tryptophan to cysteine (W163C) exchange within the C-terminal SH2 domain of ZAP70 [64]. In these mice, the onset of arthritis did not occur spontaneously but was induced when mice were submitted to fungal infection [91]. Adoptive transfer of CD4+ T of affected SKG mice led to the disease in immunodeficient mice, indicating that T cells were responsible for the disease. Biochemical studies showed that the W163C mutation precludes binding of ZAP70 to the ζ chain, Semin Immunopathol (2010) 32:107–116 thus leading to a reduction of Ca2+ signalling and phosphorylation of LAT [64]. This decreased TCR signalling impaired both positive and negative selection of thymocytes, which led to the survival of otherwise deleted autoreactive T cells. It was also proposed that the decrease in TCR signalling might alter the development and function of CD4+CD25+ regulatory T cells (Tregs) in SKG mice [63] and that this could participate in the onset of autoimmune arthritis. More recently, another hypomorphic ZAP70 allelic series was described in mice. It consists of two mutant strains each with partial defects in TCR signalling because of amino acid substitutions within the catalytic site of ZAP70. One Zap70 variant (I367F), murdock (mrd), moderately decreased TCR signalling and thymic selection without compromising immunological tolerance, while the other (W504R), mrtless (mrt), abolished thymic-positive selection and led to an immunodeficiency. Combination of the two mutations in ZAP70mrd/mrt mice revealed that intermediate ZAP70 activity was associated with abnormal Treg development, production of anti-DNA autoantibodies and hyper-IgE, whereas neither allele resulted in autoimmunity or hyper-IgE in a homozygous state [67]. This study suggests that inherited quantitative variation in TCR signalling may lead to paradoxical autoimmune and immunodeficient states because the threshold of TCR signalling to induce thymic selection, Tregs function, and T effector function are different. Thus, reduced TCR signalling creates a cellular imbalance between immunogenic and tolerogenic functions of T cells, leading to paradoxical autoimmunity associated with immunodeficiency. Autoimmunity was also reported in another mouse model, wherein only partial TCR signalling was observed. Indeed, a single amino acid substitution in LAT (LATY136F), one of the substrate of ZAP70, which diminishes TCR signalling without abolishing it, is also associated with severe inflammatory disease and autoimmunity [1, 37, 64, 69]. LatY136F/Y136F mice exhibit a profound yet incomplete block of αβ T cell development [1, 71]. Paradoxically, the few CD4+ T cells that escape this developmental block expand, leading to a fivefold increased number of CD4+ T cells in secondary lymphoid organs of 6-week-old LatY136F/Y136F mice as compared to wild-type controls. These expanding LatY136F/Y136F CD4+ T cells have a stable T helper 2 (Th2) effector phenotype that induce massive polyclonal B cell activation leading to hypergammaglobulinemia IgG1 and IgE and autoimmune nephritis [34]. These LatY136F/Y136F CD4+ T cells become refractory to TCR signals in the course of the disease [82]. The pathology developed by LatY136F/Y136F mice seems to be mediated by conventional (Foxp3−) CD4+ T cells and not to the absence of function of LatY136F/Y136F Treg, since 111 transfer of wild-type Treg (Foxp3+) cells in neonatal LatY136F/Y136F mice was unable to prevent the occurrence of the disorder [82]. The polyclonal CD4+ T cells found in LatY136/Y136F mice were shown to have higher affinity for self-peptides bound to self-MHCII molecules than in LAT wild-type mice [1]. It was thus proposed that triggering of the self-reactive TCRs expressed by LatY136/Y136F mice, combined with the decreased function of LAT Y136 creates, like in the ZAP70mrd/mrt model, an imbalance among the positive and negative signals normally delivered by the TCR, ultimately leading to lymphoproliferation, inflammation, and autoimmunity [70]. This explanation has recently been challenged by a study from B. Malissen's group, which shows that LAT-Y136F expression in postthymic CD4+ T cells also triggers a lymphoproliferative disorder with high amount of Th2 cytokine production and hypergammaglobulinemia. These results show that even when normal thymic selection occurs, defects in TCR signalling can result in autoimmunity [51]. What remains unclear are the mechanisms by which destruction of a major hub of TCR signalling pathways results in expansion and differentiation of Th2 effectors leading to lymphoproliferative and inflammatory disorders. In Jurkat T cells and post-thymic CD4+ T cells deprived of LAT molecules [32, 38, 93], kinases such as Fyn, Lck, and ZAP70 are fully functional. Indeed, in LAT-Y136F expressing post-thymic CD4+ T cells, the TCR triggering induces a spectrum of protein tyrosine phosphorylation events that are very similar to the one observed in wt LAT CD4+ T cells [51]. The cause for the lymphoproliferative disease observed in LatY136/Y136F mice may be related to the lack of a negative regulatory loop that normally controls the proximal TCR-triggered LAT-independent signalling pathways. This negative regulatory loop may be due to the recruitment by LAT of signalling molecules such as the tyrosine phosphatase SHP2 [89] or the lipid phosphatase SHIP-1 that negatively regulate early TCR signals [23, 90]. ZAP70 and primary immunodeficiencies Characterization of ZAP70 mutations in patients presenting with T cell deficiencies was instrumental in demonstrating the key role of ZAP70 in development and activation of T lymphocytes. Indeed, the first mutations of ZAP70deficient patients came out before the ZAP70-deficient mouse model was generated. Although rare, ZAP70 mutations have been described in about 20 patients from different families, leading to a distinct phenotype of human severe T cell immunodeficiency. In most cases, the mutations are located in the kinase domain and result in the absence of the ZAP70 protein [3, 14, 27, 49, 53, 78]. In one patient, compound heterozygous for two missense 112 Semin Immunopathol (2010) 32:107–116 Table 1 ZAP70 mutations reported in the literature for patients with T cell immunodeficiencies Mutation Mutated domain P80Q/M572L N-SH2 domain Kinase domain 836+121G>A/836+121G>A (hypomorphic) 279 first aa analogous to wild-type 21aa encoded in intron 7 and premature termination in interdomain B L337R/L337R Interdomain B R465C/R465C Kinase domain R465H/R465H Kinase domain K504-P508delfsX35/K504-P508delfsX35 Premature termination residue 538 of kinase domain A507V/A507V Kinase domain S518R/K541-K542insLEQ K541-K542insLEQ/K541-K542insLEQ Kinase domain Insertion LEQ kinase domain Insertion LEQ kinase domain C564R/C564R Kinase domain mutations in the ZAP70 gene were characterized. One mutation (P80Q) affected a residue in the N-SH2 domain and the other (M572L), the kinase domain. Both mutations caused a temperature-sensitive degradation of ZAP70 [40, 46]. In another case, the patient inherited a homozygous missense mutation (R465C) within the kinase domain, which only modestly affects ZAP70 stability but completely inhibited its catalytic activity [28]. This same mutation was also described in a mouse model (Table 1). All patients reported with complete deficiency in ZAP70 activity presented with severe clinical phenotype characterized by an onset in the first months of life with recurrent infections similar to those observed in severe combined immunodeficiency. They have severe viral infection, Pneumocystis jirovicii pneumonia, candidiasis, bacterial infection, and chronic diarrhea. Patients exhibit low number or a total absence of CD8+ T cells in the periphery and normal or elevated numbers of circulating nonfunctional CD4+ T cells. CD4+ T cells do not proliferate in response to PHA, anti-CD3 stimulation, or antigenic stimulation. All patients have normal numbers of B cells, but some patients had normal or elevated serum immunoglobulins (Ig) levels [78] and defective antibody production [26, 47]. The only available treatment so far is allogeneic hematopoietic stem cell transplantation that has been successfully used in several cases [5, 30]. In vitro studies have shown that retrovirus-mediated transduction of T cells from two ZAP70-deficient patients restored the TCR-induced signalling and resulted in the selective growth advantage of gene-corrected T cells, suggesting that gene therapy could be used in the future to correct such defects [72]. Patient and kindred Reference Onset of the disease One patient Early [46] One patient Late [56] One One One One Early Early Early Early [78] [28] [76] [27, 49] Early [53, 78] Early [14] Early [3] Early [78] patient patient patient patient Four patients from two kindreds Three patients from 1 kindred Three patients from 2 kindreds One patient Recent studies, revealed a clinical heterogeneity in the ZAP70-deficient patients that was initially not observed. This is likely related to a better examination of patients presenting with milder immunodeficiencies sharing some characteristics with complete ZAP70 defects. Thus, hypomorphic ZAP70 mutations that led to partial ZAP70 deficiencies were discovered. We have reported the case of a 9-year-old child with a combined immunodeficiency characterized by a low number of CD8+ and CD4+ T cells and poorly functional T cells [56]. This immunodeficiency was due to an inherited homozygous hypomorphic ZAP70 mutation. This mutation 836+121G>A introduced a new splicing acceptor site and created an in-frame major splice product containing a stop codon predicted to encode a truncated ZAP70 lacking the kinase domain. However, this splice product was not expressed in the patient's T cells. In this patient, low level of the wild-type splice form was however detected that resulted in the expression of wildtype ZAP70 protein by both CD4+ and CD8+ T cells at 20% of the level observed in control T cells. This residual expression of wild-type ZAP70 was accompanied by a reduced but detectable TCR-induced signalling. The clinical consequences of the reduction of ZAP70 activity found in this patient were attenuated as compared to patients presenting with an absence of ZAP70 activity. The patient had a normal growth; he had a severe chicken pox infection at the age of 4 and developed bronchiectasia with recurrent infections. In contrast to patients with complete ZAP70 deficiencies, he had no other severe infections [5, 26, 61, 78]. His antibody response to immunization was affected but not entirely absent, since low to normal response to Haemophilus influenzae and tetanus toxoid were detected, but there was no Semin Immunopathol (2010) 32:107–116 antibodies production to polysaccharides antigens. Unlike the hypomorphic ZAP70 deficiencies mouse models described above, this patient had no evidence of autoimmunity or lymphoproliferative disease and presented with normal numbers of CD4+ Tregs in the periphery. This absence of autoimmunity may reflect the differences in mouse and human T cell development cited above and, in particular, in Treg development. Alternatively, it may be the consequence of a quantitative difference in the residual TCR signalling in the different models and/or a role of the genetic background in determining susceptibility to autoimmunity. Analysis of more patients with partial ZAP70 deficiencies may help discriminate between the different hypotheses. The only common feature with the mouse model was the hyper-IgE that was found in the hypomorphic ZAP70 patient we described [56] as well as in two other cases. One of these patients had two missense mutations in the ZAP70 gene that both resulted in a temperature-dependent degradation of ZAP70 [46], skin lesions infiltrated by CD4+ T cells, and elevated IgE [76]. The other patient was homozygous for the C564R mutation located in the kinase domain; he also developed eczematous skin lesions simulating atopic dermatitis with eosinophilia and elevated IgE [78]. Although the cause of these hyper-IgE is not known, it may be related to the fact that mutant T cells had the capacity to induce antigen-specific IgE production from B cells in response to TCR stimulation [76]. It may also be secondary to potentially abnormal function of ZAP70deficient CD4+ Treg cells. ZAP70-deficient patients have a common clinical phenotype: a nearly complete lack of peripheral CD8+ T cells and normal to elevated numbers of peripheral CD4+ T cells that do not respond to TCR triggering. This phenotype is distinct from ZAP70-deficient mice, in which a complete absence of both CD4+ and CD8+ mature T cells was characterized [39, 52]. This discordance in phenotype was observed even in a patient and a mouse mutant with a common mutation in the DLAARN motif of the kinase domain [28, 85]. This is a good example that phenocopy between human and mouse models is not the rule. This difference between mice and human may be explained by the fact that Syk is expressed at higher levels in human thymocytes than in mouse thymocytes [19]. Thus, in human T lymphocytes, high levels of Syk expression in the thymus may compensate for ZAP70 for the development of human CD4+ thymocytes. In support of this, thymic sections performed on patients showed that CD4+CD8+ T cells are present in the thymic cortex; however, only CD4+, but not CD8+, thymocytes are detected in the medulla [3], but then, why would Syk not compensate for ZAP70 in CD8+ T cell development? CD8 T cell maturation would fail because a primary signal mediated by the tyrosine kinase lck-bound to CD8 would not be powerful enough to allow sufficient Tyr-(P) of CD3 subunits in order to allow Syk to bind and be activated thereafter. Indeed, lck binding to CD8 is much 113 reduced compared to CD4/lck interaction [86]. This explanation yet remains hypothetical. Although ZAP70-deficient CD4+ mature thymocytes make it out in the periphery, they do not function normally, thus revealing that Syk can no longer compensate for ZAP70 in mature CD4+ T cells. Actually, Syk is barely expressed in this T cell population. ZAP70-deficient CD4+ T cells neither proliferate nor produce cytokines in response to mitogens or TCR triggering. Biochemical analysis of the signalling pathways downstream of the TCR revealed a reduced or absent increase in free intracellular Ca2+ concentration as well as a reduced tyrosine phosphorylation of many protein including LAT, PLCγ1, but also of the TCR-associated ζ chain [49]. In this last case, binding of ZAP70 to the ζ chain may account for the protection of ζ from phosphatase activities. Conclusions Since its first description as a ζ-associated kinase activity, ZAP70 has been intensively studied. Its physiological role has been remarkably illustrated by the diseases that are associated with a defect in ZAP70 expression or activity. ZAP70 controls the signalling through the two immunoreceptors, BCR and TCR. This kinase is upstream of many signalling pathways that control key events of a lymphocyte life. It regulates thymocyte development as well as motility, adhesion, and cytokine secretion of mature T cells. Recent findings show that ZAP70 may be important in both switching on and off some immunoreceptor-induced signalling events. It is thus especially interesting to study ZAP70 regulation and to characterize ZAP70 substrates since it will help understanding the fine-tuning of both normal and pathological immune responses. Moreover, ZAP70 is surely an interesting target for pharmacological research since it controls T cell immunity, autoimmunity, and B cell neoplasia. Acknowledgements We thank Françoise Selz, Alexandra Arnold, Corinne Jacques, Stéphanie Ndaga, and Chantal Harré for technical assistance. This work was supported by grants from Institut Curie, INSERM, FRM, and ARC. References 1. 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