Reconstitution of T cell receptor signaling in ZAP-70-deficient cells by retroviral transduction of the ZAP-70 gene

A variant of severe combined immunodeficiency syndrome (SCID) with a selective inability to produce CD8 single positive T cells and a signal transduction defect in peripheral CD4+ cells has recently been shown to be the result of mutations in the ZAP-70 gene. T cell receptor (TCR) signaling requires the association of the ZAP-70 protein tyrosine kinase with the TCR complex. Human T cell leukemia virus type I- transformed CD4+ T cell lines were established from ZAP-70-deficient patients and normal controls. ZAP-70 was expressed and appropriately phosphorylated in normal T cell lines after TCR engagement, but was not detected in T cell lines from ZAP-70-deficient patients. To determine whether signaling could be reconstituted, wild-type ZAP-70 was introduced into deficient cells with a ZAP-70 retroviral vector. High titer producer clones expressing ZAP-70 were generated in the Gibbon ape leukemia virus packaging line PG13. After transduction, ZAP-70 was detected at levels equivalent to those observed in normal cells, and was appropriately phosphorylated on tyrosine after receptor engagement. The kinase activity of ZAP-70 in the reconstituted cells was also appropriately upregulated by receptor aggregation. Moreover, normal and transduced cells, but not ZAP-70-deficient cells, were able to mobilize calcium after receptor ligation, indicating that proximal TCR signaling was reconstituted. These results indicate that this form of SCID may be corrected by gene therapy.     

The Vav Binding Site (Y315) in ZAP-70 Is Critical for Antigen Receptor–mediated Signal Transduction

Stimulation of antigen receptors in T and B cells leads to the activation of the Src and Syk families of protein tyrosine kinases (PTK). These PTKs subsequently phosphorylate numerous intracellular substrates, including the 95-kD protooncogene product Vav. Vav is essential for both T and B cell development and T and B cell antigen receptor–mediated signal transduction. After receptor ligation, Vav associates with phosphorylated Syk and ZAP-70 PTKs, an interaction that depends upon its SH2 domain. Here we demonstrate that a point mutation of tyrosine 315 (Y315F) in ZAP-70, a putative Vav SH2 domain binding site, eliminated the Vav– ZAP-70 interaction. Moreover, the Y315 mutation impaired the function of ZAP-70 in antigen receptor signaling. Surprisingly, this mutation also resulted in marked reduction in the tyrosine phosphorylation of ZAP-70, Vav, SLP-76, and Shc. These data demonstrate that the Vav binding site in ZAP-70 plays a critical role in antigen receptor–mediated signal transduction.     

T cell development and T cell responses in mice with mutations affecting tyrosines 292 or 315 of the ZAP-70 protein tyrosine kinase

After stimulation of the T cell receptor (TCR), the tyrosine residues 292 and 315 in interdomain B of the protein tyrosine kinase ZAP-70 become phosphorylated and plausibly function as docking sites for Cbl and Vav1, respectively. The two latter proteins have been suggested to serve as substrates for ZAP-70 and to fine-tune its function. To address the role of these residues in T cell development and in the function of primary T cells, we have generated mice that express ZAP-70 molecules with Tyr to Phe substitution at position 292 (Y292F) or 315 (Y315F). When analyzed in a sensitized TCR transgenic background, the ZAP-70 Y315F mutation reduced the rate of positive selection and delayed the occurrence of negative selection. Furthermore, this mutation unexpectedly affected the constitutive levels of the CD3-zeta p21 phosphoisoform. Conversely, the ZAP-70 Y292F mutation upregulated proximal events in TCR signaling and allowed more T cells to produce interleukin 2 and interferon gamma in response to a given dose of antigen. The observation that ZAP-70 Y292F T cells have a slower rate of ligand-induced TCR downmodulation suggests that Y292 is likely involved in regulating the duration activated TCR reside at the cell surface. Furthermore, we showed that Y292 and Y315 are dispensable for the TCR-induced tyrosine phosphorylation of Cbl and Vav1, respectively. Therefore, other molecules present in the TCR signaling cassette act as additional adaptors for Cbl and Vav1. The present in vivo analyses extend previous data based on transformed T cell lines and suggest that residue Y292 plays a role in attenuation of TCR signaling, whereas residue Y315 enhances ZAP-70 function.     

In vivo correction of ZAP-70 immunodeficiency by intrathymic gene transfer

SCID patients have been successfully treated by administration of ex vivo gene-corrected stem cells. However, despite its proven efficacy, such treatment carries specific risks and difficulties. We hypothesized that some of these drawbacks may be overcome by in situ gene correction of T lymphoid progenitors in the thymus. Indeed, in vivo intrathymic transfer of a gene that provides a selective advantage for transduced prothymocytes should result in the generation of functional T lymphocyte progeny, allowing long-term immune reconstitution. We assessed the feasibility of this approach in a murine model of ZAP-70-deficient SCID. A T cell-specific ZAP-70-expressing lentiviral vector was injected into thymi of adult ZAP-70-/- mice without prior conditioning. This resulted in the long-term differentiation of mature TCR-alphabeta+ thymocytes, indicating that the vector had integrated into progenitor cells. Moreover, peripheral ZAP-70-expressing T cells demonstrated a partially diversified receptor repertoire and were responsive to alloantigens in vitro and in vivo. Improved treatment efficacy was achieved in infant ZAP-70-/- mice, in which the thymus is proportionately larger and a higher percentage of prothymocytes are in cycle. Thus, intrathymic injection of a lentiviral vector could represent a simplified and potentially safer alternative to ex vivo gene-modified hematopoietic stem cell transplantation for gene therapy of T cell immunodeficiencies.     

ZAP-70 protein promotes tyrosine phosphorylation of T cell receptor signaling motifs (ITAMs) in immature CD4(+)8(+) thymocytes with limiting p56(lck)

As a result of interaction with epithelial cells in the thymic cortex, immature CD4(+)8(+) (double positive, DP) thymocytes express relatively few T cell receptors (TCRs) and contain diminished numbers of coreceptor-associated p56(lck) (lck) PTK molecules. As a result, TCR signal transduction in DP thymocytes is significantly impaired, despite its importance for repertoire selection. We report here that, in DP thymocytes, tyrosine phosphorylation of TCR signaling motifs (ITAMs) by lck, an early event in TCR signal transduction, is dependent upon ZAP-70 protein independent of ZAP-70's kinase activity. Furthermore, the dependence on ZAP-70 protein for ITAM phosphorylation diminishes as available lck increases. Importantly, ZAP-70's role in ITAM phosphorylation in DP thymocytes is not limited to protecting phosphorylated ITAMs from dephosphorylation. Rather, this study indicates that ZAP-70 protein augments ITAM phosphorylation in DP thymocytes and so compensates in part for the relative deficiency of coreceptor-associated lck.     

ZAP-70 expression in B-cell chronic lymphocytic leukemia: evaluation by external (isotypic) or internal (T/NK cells) controls and correlation with IgV(H) mutations

BACKGROUND: Expression of T cell specific zeta-associated protein 70 (ZAP-70) by B-cell chronic lymphocytic leukemia (B-CLL) cells, as investigated by flow cytometry, has both prognostic relevance and predictive power as surrogate for immunoglobulin heavy chain variable region (IgV(H)) mutations, although a standardization of the cytometric protocol is still lacking. METHODS: Flow cytometric analyses for ZAP-70 were performed in peripheral blood samples from 145 B-CLL (124 with IgV(H) mutations) by a standard three-color protocol. Identification of ZAP-70(+) cell population was based on an external negative control, i.e., the isotypic control (ISO method) or an internal positive control, i.e., the population of residual normal T/NK cells (TNK method). A comparison between these two approaches was performed. RESULTS: While 86/145 cases were concordant as for ZAP-70 expression according to the two methods (ISO(+)TNK(+) or ISO(-)TNK(-)), 59/145 cases had discordant ZAP-70 expression, mainly (56/59) showing a ISO(+)TNK(-) profile. These latter cases express higher levels of ZAP-70 in their normal T cell component. Moreover, discordant ISO(+)TNK(-) cases had a IgV(H) gene mutation profile similar to that of concordantly positive cases and different from ZAP-70 concordantly negative B-CLL. CONCLUSION: Analysis of ZAP-70 expression by B-CLL cells by using the ISO method allows to overcome the variability in the expression of ZAP-70 by residual T cells and yields a better correlation with IgV(H) gene mutations. A receiver operating characteristic analysis suggests to employ a higher cut-off than the commonly used 20%. A parallel evaluation of the prognostic value of ZAP-70 expression, as determined according to the ISO and TNK methods, is still needed.     

Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLL

BACKGROUND: In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity. METHODS: B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.2]. Zap-70-PE was then added to this tube. ZAP-70-PE was added to a second tube without unlabeled antibody to ZAP-70. The mean fluorescence intensity of the ZAP-70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP-70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non-specific ZAP-70 fluorescence in the B-CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP-70 fluorescence intensity in the tube with unlabeled antibody to ZAP-70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP-70 cells in the tube without unlabeled antibody to ZAP-70. The brighter the ZAP-70 fluorescence above the non-specific background, the higher the %POS. RESULTS: Due to the varying amount of non-specific staining between patient B-CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP-70 in B-CLL cells than other methods, which use the ratio of B-CLL fluorescence to normal B or T-cell fluorescence. Using this improved method, ZAP-70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP-70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut-off value lower than previous values published, due to exclusion of non-specific staining. Both cut-offs were based upon patient specimen distribution profiling. CONCLUSIONS: Use of a blocking antibody resulted in a robust, reproducible clinical B-CLL assay that is not influenced by the need to measure the amount of ZAP-70 in other cells. ZAP-70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes.     

HIV infection--induced posttranslational modification of T cell signaling molecules associated with disease progression.

In attempt to elucidate the mechanism of the HIV infection induced T cell unresponsiveness, we studied signal-transducing molecules proximal to the T cell receptor (TCR) in T lymphocytes of HIV-infected individuals. Total amounts of protein tyrosine kinases (PTKs) Lck, Fyn, and ZAP-70 and the zeta chain of the TCR were found significantly decreased in T cells of symptomatic/AIDS patients as well as in T cells of individuals in acute and early asymptomatic stages of HIV infection. Unexpectedly, the detection of Lck, Fyn, and ZAP-70 was reversed after the treatment of cell lysates with dithiothreitol. This suggests that PTKs Lck, Fyn, and ZAP-70 were modified by a mechanism altering the status of sulfhydryl groups. Moreover, this mechanism seems to affect selectively T cells of HIV infected patients since B cell PTKs Syk and Lyn were detected structurally and functionally intact. Interestingly, similar alterations of signaling molecules were not detected in T cells of HIV-infected long-term asymptomatic individuals. Modification of T cell PTKs may thus underlie the HIV-induced impairment of lymphocyte function and may potentially predict disease progression.     

Retrovirus-mediated transduction of primary ZAP-70-deficient human T cells results in the selective growth advantage of gene-corrected cells: implications for gene therapy

Humans lacking the ZAP-70 protein tyrosine kinase present with an absence of CD8+ T cells and defective CD4+ T cells in the periphery. This severe combined immunodeficiency is fatal unless treated by allogeneic bone marrow transplantation. However, in the absence of suitable marrow donors, the development of alternative forms of therapy is desirable. Because lymphocytes are long-lived, it is possible that introduction of the wild-type ZAP-70 gene into CD4+ ZAP-70-deficient T cells will restore their immune function in vivo. Initial investigations evaluating the feasibility of gene therapy for ZAP-70 deficiency were performed using HTL V-I-transformed lymphocytes. Although transformation was useful in circumventing problems associated with the maintenance of ZAP-70-deficient T cells and low gene transfer levels, the presence of HTL V-I precluded any biological studies. Here, we investigated a retrovirus-mediated approach for the correction of primary T cells derived from two ZAP-70-deficient patients. Upon introduction of the wild-type ZAP-70 gene, TCR-induced MAPK activation, IL-2 secretion and proliferation were restored to approximately normal levels. Importantly, this gain-of-function was associated with a selective growth advantage of gene-corrected cells, thereby indicating the feasibility of a gene therapy-based strategy.     

Requirement for tyrosine residues 315 and 319 within zeta chain-associated protein 70 for T cell development

Engagement of the T cell antigen receptor (TCR) induces the transphosphorylation of the zeta chain-associated protein of 70,000 Mr (ZAP-70) protein tyrosine kinase (PTK) by the CD4/8 coreceptor associated Lck PTK. Phosphorylation of Tyr 493 within ZAP-70's activation loop results in the enzymatic activation of ZAP-70. Additional tyrosines (Tyrs) within ZAP-70 are phosphorylated that play both positive and negative regulatory roles in TCR function. Phosphorylation of Tyr residues (Tyrs 315 and 319) within the Interdomain B region of the ZAP-70 PTK plays important roles in the generation of second messengers after TCR engagement. Here, we demonstrate that phosphorylation of these two Tyr residues also play important roles in mediating the positive and negative selection of T cells in the thymus.     

Absence of ZAP-70 prevents signaling through the antigen receptor on peripheral blood T cells but not on thymocytes

Recently, a severe combined immunodeficiency syndrome with a deficiency of CD8+ peripheral T cells and a TCR signal transduction defect in peripheral CD4+ T cells was associated with mutations in ZAP-70. Since TCR signaling is required in developmental decisions resulting in mature CD4 (and CD8) T cells, the presence of peripheral CD4+ T cells expressing TCRs incapable of signaling in these patients is paradoxical. Here, we show that the TCRs on thymocytes, but not peripheral T cells, from a ZAP-70-deficient patient are capable of signaling. Moreover, the TCR on a thymocyte line derived from this patient can signal, and the homologous kinase Syk is present at high levels and is tyrosine phosphorylated after TCR stimulation. Thus, Syk may compensate for the loss of ZAP-70 and account for the thymic selection of at least a subset of T cells (CD4+) in ZAP-70-deficient patients.     

p56lck interacts via its src homology 2 domain with the ZAP-70 kinase

p56lck, a member of the src family of protein tyrosine kinases, is an essential component in T cell receptor (TCR) signal transduction. p56lck contains a src homology 2 (SH2) domain found in a number of proteins involved in intracellular signaling. SH2 domains have been implicated in protein-protein interactions by binding to sequences in target proteins containing phosphorylated tyrosine. Using an in vitro assay, we have studied specific binding of tyrosine-phosphorylated proteins to a recombinant p56lck SH2 domain. In nonactivated Jurkat cells, two tyrosine-phosphorylated proteins were detected. Stimulation with anti-CD3 monoclonal antibodies induced the binding of seven additional tyrosine-phosphorylated proteins to the SH2 domain of p56lck. We have identified the zeta-associated tyrosine kinase, ZAP-70, as one of these proteins. Evidence suggests that binding of ZAP-70 to p56lck SH2 is direct and not mediated by zeta. The significance of this interaction was further investigated in vivo. p56lck could be coprecipitated with the zeta/ZAP-70 complex and conversely, ZAP-70 was detected in p56lck immunoprecipitates of activated Jurkat cells. The physical association of p56lck and ZAP-70 during activation supports the recently proposed functional cooperation of these two tyrosine kinases in TCR signaling.     

The pre-T cell receptor (TCR) complex is functionally coupled to the TCR-zeta subunit

The pre-T cell receptor (TCR) complex regulates early T cell development and consists of a heterodimer of the TCR-beta subunit in association with the pre-TCR-alpha chain. Notably, in contrast to alpha/beta-expressing T cells, several studies suggested that the TCR- zeta chain is not stably associated with this pre-TCR complex. To examine the proximal signaling processes mediated by the pre-TCR complex and the role of the TCR-zeta chain in these processes, we stimulated pre-TCR-expressing cells and analyzed the interactions of the TCR/CD3 invariant chains with the Syk/ZAP-70 family of protein tyrosine kinases. Stimulation of the pre-TCR complex led to the tyrosine phosphorylation of the CD3 epsilon and TCR-zeta chains, as well as the phosphorylation and association of ZAP-70 and Syk with phosphorylated CD3 epsilon and TCR-zeta. These results demonstrate that the pre-TCR complex is functionally coupled to the TCR-zeta subunit and to the ZAP-70 and Syk protein tyrosine kinases.     

A hypomorphic allele of ZAP-70 reveals a distinct thymic threshold for autoimmune disease versus autoimmune reactivity

ZAP-70 is critical for T cell receptor (TCR) signaling. Tyrosine to phenylalanine mutations of Y315 and Y319 in ZAP-70 suggest these residues function to recruit downstream effector molecules, but mutagenesis and crystallization studies reveal that these residues also play an important role in autoinhibition ZAP-70. To address the importance of the scaffolding function, we generated a zap70 mutant mouse (YYAA mouse) with Y315 and Y319 both mutated to alanines. These YYAA mice reveal that the scaffolding function is important for normal development and function. Moreover, the YYAA mice have many similarities to a previously identified ZAP-70 mutant mouse, SKG, which harbors a distinct hypomorphic mutation. Both YYAA and SKG mice have impaired T cell development and hyporesponsiveness to TCR stimulation, markedly reduced numbers of thymic T regulatory cells and defective positive and negative selection. YYAA mice, like SKG mice, develop rheumatoid factor antibodies, but fail to develop autoimmune arthritis. Signaling differences that result from ZAP-70 mutations appear to skew the TCR repertoire in ways that differentially influence propensity to autoimmunity versus autoimmune disease susceptibility. By uncoupling the relative contribution from T regulatory cells and TCR repertoire during thymic selection, our data help to identify events that may be important, but alone are insufficient, for the development of autoimmune disease.Signal transduction by the TCR plays a critical role in T cell development and in the protective and pathological responses mediated by mature T cells. The repertoire of mature T cells and the discrimination of self from nonself are largely determined within the thymus through TCR-dependent processes known as positive and negative selection. It is generally thought that quantitative or qualitative differences in TCR signaling determine the binary decision between positive or negative selection (Starr et al., 2003). Likewise, whether a productive mature T cell response will be made is dictated by signaling events induced by the TCR.ZAP-70, a Syk family tyrosine kinase that associates with the TCR CD3 and ζ subunits, plays a critical role in TCR signaling in immature thymocyte selection and in mature T cell responses (Chan et al., 1992). ZAP-70 contains two N-terminal SH2 domains that mediate its association with doubly phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) after their phosphorylation by Lck. The ZAP-70 C-terminal catalytic domain phosphorylates the downstream adaptor molecules LAT and SLP-76 that play critical roles in T cell development and in T cell responses (Horejsí et al., 2004). Interdomain B bridges the C-terminal SH2 and the kinase domains and contains three tyrosine residues (Y292, Y315, and Y319) that are inducibly phosphorylated. Based on tyrosine to phenylalanine mutations, we and others have previously shown that phosphorylation of Y292 may exert a negative regulatory effect on ZAP-70, perhaps by functioning as a docking site for c-Cbl (Lupher et al., 1997; Meng et al., 1999; Rao et al., 2000). In contrast, phenylalanine mutations of Y315 and Y319 suggested that these sites positively regulate ZAP-70 function by recruiting Vav1 and Lck, respectively (Straus et al., 1996; Wu et al., 1997; Pelosi et al., 1999). These sites have also been reported to bind c-Crk (Gelkop and Isakov, 1999) and phospholipase Cγ1 (Williams et al., 1999). Thus, in addition to its catalytic activity, ZAP-70 may have an important scaffolding role in recruiting downstream effector molecules.Surprisingly, in addition to this scaffolding role, we recently discovered that Y315 and Y319 play an important role in regulating ZAP-70 kinase activity. Whereas mutation of both residues to phenylalanine potently inhibits ZAP-70 function, deletion of interdomain B or mutation of these sites to alanine relieves an autoinhibitory conformation and renders the kinase relatively Lck-independent (Brdicka et al., 2005). Stabilizing the autoinhibited ZAP-70 conformation with the YYFF mutations (Y315F and Y319F) allowed the crystallization of full-length ZAP-70. The crystal structure, together with targeted mutagenesis studies, suggest that the Y315 and Y319 stabilize the autoinhibited conformation of the ZAP-70 kinase through hydrophobic interactions involving the aromatic rings of the Y315 and Y319 residues with residues in the inter-SH2 domain (Interdomain A) and the C-terminal lobe of the catalytic domain (Deindl et al., 2007). Comparison of the alignment of the SH2 domains in the autoinhibited conformation to the ITAM-bound SH2 domains (Hatada et al., 1995) further suggests that ZAP-70 undergoes a conformational change upon binding to a doubly phosphorylated ITAM, which allows Y315 and Y319 to be more accessible for phosphorylation. Y315 and Y319 appear to be Lck phosphorylation sites and their phosphorylation stabilizes the active “open” ZAP-70 conformation and prevents the kinase from returning to the autoinhibited conformation (Brdicka et al., 2005; Deindl et al., 2007; Levin et al., 2008). What remains unclear is the relative importance of their scaffolding function in effector molecule recruitment.Two independent groups have generated knockin mice (Magnan et al., 2001) and transgenic mice (Gong et al., 2001) to study the in vivo individual contributions of Y315 and Y319 of ZAP-70 to signal transduction and to T cell development. However, those studies involved mutation of either Y315 or Y319 to phenylalanine, which would have helped stabilize the autoinhibited conformation and could have resulted in attenuated T cell signaling and consequent alteration of positive and negative selection. TCR-mediated calcium increase is greatly diminished in ZAP-70^−/− mice expressing the Y319F transgenic mice whereas the Y315F mutation has a modest effect on calcium responses. Consistent with results from transgenic mice, phosphorylation of phospholipase C γ1, but not Vav1, is reduced in the Y315F knockin mice. These results suggest that Y315 and Y319 contribute to efficient generation of second messengers involved in thymic selection. However, these studies did not take into account the notion that the tyrosine to phenylalanine mutations might also stabilize the autoinhibitory conformation of ZAP-70 rather than only affecting the recruitment of downstream effectors.To address the relative importance of the scaffolding function of these residues, we generated a knockin ZAP-70 mutant strain with Y315 and Y319 simultaneously mutated to alanine residues. By creating a YYAA ZAP-70 knockin mouse we were able to study the contribution of the scaffolding function of these sites without the confounding issue of autoinhibition in the context of phenylalanine mutants. These YYAA mice showed many of the features of a previously identified spontaneous mutant mouse called SKG. The SKG mouse has a syndrome that shares features with human rheumatoid arthritis, including autoimmune arthritis and rheumatoid factor production (Sakaguchi et al., 2003). SKG mice have a single point mutation in the C-terminal SH2 domain of ZAP-70 that results in attenuated TCR signaling and aberrant positive and negative selection. Results suggested that the autoimmune arthritis could be caused by expansion of autoreactive CD4⁺ T cells that have escaped negative selection. Interestingly, unlike SKG mice, although the YYAA mice have similar defects in positive and negative selection in TCR transgenic systems and can be induced to develop rheumatoid factor antibodies, they do not develop autoimmune arthritis. Quantitative assessment of negative selection in response to endogenous superantigens suggests that YYAA and SKG mice have distinct TCR repertoires that could account for the increased susceptibility of both lines of mice to develop rheumatoid factor antibodies, but interestingly, only SKG mice develop arthritis. Our findings demonstrate the importance of phosphorylation of Y315 and Y319 in ZAP-70 as binding sites for key effector molecules. As well, these findings stress the importance of these sites for proper regulation of TCR signaling and for normal T cell repertoire selection and peripheral T cell function in resistance to autoimmune disease susceptibility.     

T cells from late tumor-bearing mice express normal levels of p56lck, p59fyn, ZAP-70, and CD3 zeta despite suppressed cytolytic activity

Loss of T cell-associated signal transduction molecules has recently been implicated in immune suppression in tumor-bearing hosts. In the present study, we have examined this and related phenomenon extensively in a large number of tumor-bearing mice, analyzed individually. Splenic T cells from tumor-bearing mice were isolated and characterized with respect to the following: (a) levels of three tyrosine kinases, p56lck, p59fyn, and ZAP-70; (b) expression of CD3-zeta; (c) alloreactive responses; and (d) antigen-specific responses. Contrary to recent reports, T cells from tumor-bearing mice were observed to express normal levels of lck, fyn, ZAP-70, and CD3-zeta. Further, T cells showed healthy alloreactive and antigen-specific responses until approximately 3 wk after post tumor challenge, when the tumors constituted approximately 20% of the body weight. Alterations with respect to some parameters were observed only in mice that had been bearing larger tumors for a considerably longer period. As human tumors are unlikely to grow to such large sizes (e.g., > 20% of the total body weight), the significance of the alterations in T cell expression of lck, fyn, ZAP-70, or CD3-zeta in the immune status of cancer patients is unclear. Altogether, these results indicate that alterations in T cell signal transduction molecules do not account for the profound tumor-specific suppression observed during tumor growth.     

Lck regulates the tyrosine phosphorylation of the T cell receptor subunits and ZAP-70 in murine thymocytes

The Src-family and Syk/ZAP-70 family of protein tyrosine kinases (PTK) are required for T cell receptor (TCR) functions. We provide evidence that the Src-family PTK Lck is responsible for regulating the constitutive tyrosine phosphorylation of the TCR zeta subunit in murine thymocytes. Moreover, ligation of the TCR expressed on thymocytes from Lck-deficient mice largely failed to induce the phosphorylation of TCR- zeta, CD3 epsilon, or ZAP-70. In contrast, we find that the TCR-zeta subunit is weakly constitutively tyrosine phosphorylated in peripheral T cells isolated from Lck-null mice. These data suggest that Lck has a functional role in regulation of TCR signal transduction in thymocytes. In peripheral T cells, other Src-family PTKs such as Fyn may partially compensate for the absence of Lck.     

Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process

The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold-standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta-associated protein (ZAP-70) expression was associated with unmutated IgVH genes and ZAP-70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP-70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP-70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti-ZAP-70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti-ZAP-70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP-70 levels were expressed as percentage positive (POS) relative to isotype control or normal B-cells or normal T-cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T-cells. The reported level of ZAP-70 expression varied greatly depending on the antibody and the method used to express the results. The CLL/T-cell ZAP-70 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP-70 expression. We assessed the variation of ZAP-70 expression levels in fresh cells according to storage time, which demonstrated that ZAP-70 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures.     

Physical dissociation of the TCR-CD3 complex accompanies receptor ligation

During antigen recognition by T cells, CD4 and the T-cell receptor (TCR)/CD3/zeta complex are thought to interact with the same major histocompatibility complex II molecule in a stable ternary complex. Evidence has suggested that the association of CD4 with TCR/CD3/zeta requires the interaction of the protein tyrosine kinase p56lck with CD4. We have taken a biochemical approach to understand the mechanism by which p56lck and, in particular, its src homology (SH) 2 domain contributes to the association of CD4 with TCR/CD3/zeta during activation. We have previously shown that the p56lck SH2 domain binds directly to tyrosine-phosphorylated ZAP-70. Here we formally demonstrate the in vivo association of p56lck with the homologous protein tyrosine kinases Syk and ZAP-70 after CD3 stimulation of Jurkat cells. A tyrosine-phosphorylated peptide containing the sequence predicted to be optimal for binding to the SH2 domain of src family kinases specifically competes for this association, indicating that tyrosine-phosphorylated ZAP-70 and Syk bind to p56lck by an SH2- mediated interaction. We also show that the same peptide is able to compete for the activation-dependent TCR/CD4 association in Jurkat cells. Moreover, ZAP-70 and CD4 cocap only after CD3 stimulation in human T lymphoblasts. We propose that the interaction of the p56lck SH2 domain with zeta-associated tyrosine-phosphorylated ZAP-70 and/or Syk enables CD4 to associate with antigen-stimulated TCR/CD3/zeta complexes.     

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.     

Comparison of bone marrow and peripheral blood ZAP-70 status examined by flow cytometric immunophenotyping in patients with chronic lymphocytic leukemia

BACKGROUND:: The mutational status of the immunoglobulin heavy chain variable gene in patients with chronic lymphocytic leukemia correlates with prognosis. Patients with mutated IgVH genes fare better than those with unmutated genes. Gene expression profiling studies identified the tyrosine kinase ZAP-70 to be expressed in unmutated CLL samples. Flow cytometric examination of ZAP-70 expression in tumor cells has been proposed to be a convenient surrogate marker for IgVH mutational status. However, a few studies have shown a small number of discordant results between ZAP-70 positivity, IgVH mutational status, and clinical outcome. There have been no reported studies comparing bone marrow samples with peripheral blood for ZAP-70 expression in CLL patients. METHODS:: We searched our flow cytometry files from October 2004 through April 2006 and identified CLL in 311 bone marrow and peripheral blood specimens from 256 patients. We defined ZAP-70 positivity as greater than 30% of the CD19(+) B-cells above the isotype control value that coexpress ZAP-70. Statistical analyses were performed using the Fisher exact test and student t-test. RESULTS:: A significantly greater number of bone marrow specimens were positive for ZAP-70 when compared with the number of peripheral blood specimens. Of all the ZAP-70 negative specimens, CLL cells from bone marrow had a greater mean percentage of ZAP-70 positive cells when compared with the CLL cells from peripheral blood. Finally, six patients were identified who were ZAP-70 positive in the bone marrow but ZAP-70 negative in the peripheral blood. CONCLUSIONS:: These results may be due to either an increase in the false positive rate in bone marrow specimens or to an intrinsic feature of CLL cells in the compartment that is biologically distinct from peripheral tumor cells. As prognosis and treatment decisions may be based on ZAP-70 results from either specimen type, it is prudent to further examine this observation. (c) 2006 International Society for Analytical Cytology.