ZAP-70 mutation: a case with familial autoimmune haemolytic anaemia and immune deficiency.

Zeta-chain associated protein kinase 70 kDa (ZAP-70) deficiency is one of the rare immunodeficiency disorders due to autosomal recessive homozygous or compound heterozygous loss-of-function mutations in the ZAP-70 GENE In the literature, patients with ZAP-70 deficiency have been reported with a broad spectrum of clinical manifestations including recurrent respiratory infections (81.8%), cutaneous involvement (57.9%), lymphoproliferation (32.4%), autoimmunity (19.4%), enteropathy (18.4%) and increased risk of malignancies (8.1%). The most common immunological phenotype in those patients was low CD8+ T cell counts (97.9%) and normal non-functioning CD4+ T cell. Haematopoietic stem cell transplantation was applied as a curative treatment for this disorder.

The Ubiquitin-Associated and SH3 Domain-Containing Proteins (UBASH3) Family in Mammalian Development and Immune Response.

UBASH3A and UBASH3B are protein families of atypical protein tyrosine phosphatases that function as regulators of various cellular processes during mammalian development. As UBASH3A has only mild phosphatase activity, its regulatory effects are based on the phosphatase-independent mechanisms. On the contrary, UBASH3B has strong phosphatase activity, and the suppression of its receptor signalling is mediated by Syk and Zap-70 kinases. The regulatory functions of UBASH3A and UBASH3B are particularly evident in the lymphoid tissues and kidney development. These tyrosine phosphatases are also known to play key roles in autoimmunity and neoplasms. However, their involvement in mammalian development and its regulatory functions are largely unknown and are discussed in this review.

Normal B cells express ZAP70 in chronic lymphocytic leukemia: A link between autoimmunity and lymphoproliferation?

ZAP70 has a prognostic value in chronic lymphocytic leukemia (CLL), through altered B-cell receptor signaling, which is important in CLL pathogenesis. A good correlation between ZAP70 expression in CLL cells and the occurrence of autoimmune phenomena has been reported. Yet, the great majority of CLL-associated autoimmune cytopenia is due to polyclonal immunoglobulin (Ig) G synthesized by nonmalignant B cells, and this phenomenon is poorly understood. Here, we show, using flow cytometry, that a substantial percentage of CD5- nonmalignant B cells from CLL patients expresses ZAP70 compared with CD5- B cells from healthy subjects. This ZAP70 expression in normal B cells from CLL patients was also evidenced by the detection of ZAP70 mRNA at single-cell level with polyclonal Ig heavy- and light-chain gene transcripts. ZAP70+ normal B cells belong to various B-cell subsets and their presence in the naïve B-cell subset suggests that ZAP70 expression may occur during early B-cell development in CLL patients and potentially before malignant transformation. The presence of ZAP70+ normal B cells is associated with autoimmune cytopenia in CLL patients in our cohort of patients, and recombinant antibodies produced from these ZAP70+ nonmalignant B cells were frequently autoreactive including anti-platelet reactivity. These results provide a better understanding of the implication of ZAP70 in CLL leukemogenesis and the mechanisms of autoimmune complications of CLL.

Novel Compound Heterozygous ZAP70 R37G A507T Mutations in Infant with Severe Immunodeficiency.

Zeta-chain associated protein kinase 70 kDa (ZAP70) combined immunodeficiency (CID) is an autosomal recessive severe immunodeficiency that is characterized by abnormal T-cell receptor signaling. Children with the disorder typically present during the first year of life with diarrhea, failure to thrive, and recurrent bacterial, viral, or opportunistic infections. To date, the only potential cure is hematopoietic stem cell transplant (HSCT). The majority of described mutations causing disease occur in the homozygous state, though heterozygotes are reported without a clear understanding as to how the individual mutations interact to cause disease. This case describes an infant with novel ZAP-70 deficiency mutations involving the SH2 and kinase domains cured with allogeneic HSCT utilizing a reduced-intensity conditioning regimen and graft manipulation. We then were able to further elucidate the molecular signaling alterations imparted by these mutations that lead to altered immune function. In order to examine the effect of these novel compound ZAP70 heterozygous mutations on T cells, Jurkat CD4+ T cells were transfected with either wild type, or with individual ZAP70 R37G and A507T mutant constructs. Downstream TCR signaling events and protein localization results link these novel mutations to the expected immunological outcome as seen in the patient's primary cells. This study further characterizes mutations in the ZAP70 gene as combined immunodeficiency and the clinical phenotype.

Decoding the deactivation mechanism of R192W mutation of ZAP-70 using molecular dynamics simulations and binding free energy calculations.

CONTEXT: ZAP-70 (zeta-chain-associated protein of 70 kDa), serving as a critical regulator for T cell antigen receptor signaling, represents an attractive therapeutic target for autoimmunity disease. How the mechanistical mechanism of ZAP-70 to a human autoimmune syndrome-associated R192W mutation remains unclear. The results indicated that the R192W mutation of ZAP-70 clearly affected the conformational flexibility of the N-terminal ITAM-Y2P. Structural analysis unveiled that the R192W mutation of ZAP-70 caused the exposure of the N-terminal ITAM-Y2P to the solvent. MM-GBSA binding free energy calculations exhibited that the R192W mutation decreased the binding affinity of ITAM-Y2P to the ZAP-70 mutant. Residue-based free energy decomposition further revealed that the protein-peptide interaction networks involving electrostatic interactions provide significant contributions for complex formation. The energy unfavorable residues include Arg43, Arg192, Tyr240, and Lys244 from ZAP-70 and Asn301, Leu303, pY304, and pY315 from ITAM-Y2P in the R192W mutant. Our obtained results may help the understanding of the deactivation mechanism of ZAP-70 induced by the R192W mutation. METHODS: In the work, multiple replica molecular dynamics simulations and molecular mechanics-generalized Born surface area (MM-GBSA) method were performed to reveal the doubly phosphorylated ITAMs (ITAM-Y2P)-mediated deactivation mechanism of ZAP-70 induced by the R192W mutation.

Natural isoaspartyl protein modification of ZAP70 alters T cell responses in lupus.

Protein posttranslational modifications (PTMs) arise in a number of normal cellular biological pathways and in response to pathology caused by inflammation and/or infection. Indeed, a number of PTMs have been identified and linked to specific autoimmune responses and metabolic pathways. One particular PTM, termed isoaspartyl (isoAsp or isoD) modification, is among the most common spontaneous PTM occurring at physiological pH and temperature. Herein, we demonstrate that isoAsp modifications arise within the ZAP70 protein tyrosine kinase upon T-cell antigen receptor (TCR) engagement. The enzyme protein L-isoaspartate O-methyltransferase (PCMT1, or PIMT, EC 2.1.1.77) evolved to repair isoaspartyl modifications in cells. In this regard, we observe that increased levels of isoAsp modification that arise under oxidative stress are correlated with reduced PIMT activity in patients with systemic lupus erythematosus (SLE). PIMT deficiency leads to T cell hyper-proliferation and hyper-phosphorylation through ZAP70 signaling. We demonstrate that inducing the overexpression of PIMT can correct the hyper-responsive phenotype in lupus T cells. Our studies reveal a phenotypic role of isoAsp modification and phosphorylation of ZAP70 in lupus T cell autoimmunity and provide a potential therapeutic target through the repair of isoAsp modification.

Combined immunodeficiency caused by pathogenic variants in the ZAP70 C-terminal SH2 domain.

Introduction: ZAP-70, a protein tyrosine kinase recruited to the T cell receptor (TCR), initiates a TCR signaling cascade upon antigen stimulation. Mutations in the ZAP70 gene cause a combined immunodeficiency characterized by low or absent CD8+ T cells and nonfunctional CD4+ T cells. Most deleterious missense ZAP70 mutations in patients are located in the kinase domain but the impact of mutations in the SH2 domains, regulating ZAP-70 recruitment to the TCR, are not well understood. Methods: Genetic analyses were performed on four patients with CD8 lymphopenia and a high resolution melting screening for ZAP70 mutations was developed. The impact of SH2 domain mutations was evaluated by biochemical and functional analyses as well as by protein modeling. Results and discussion: Genetic characterization of an infant who presented with pneumocystis pneumonia, mycobacterial infection, and an absence of CD8 T cells revealed a novel homozygous mutation in the C-terminal SH2 domain (SH2-C) of the ZAP70 gene (c.C343T, p.R170C). A distantly related second patient was found to be compound heterozygous for the R170C variant and a 13bp deletion in the ZAP70 kinase domain. While the R170C mutant was highly expressed, there was an absence of TCR-induced proliferation, associated with significantly attenuated TCR-induced ZAP-70 phosphorylation and a lack of binding of ZAP-70 to TCR-ζ. Moreover, a homozygous ZAP-70 R192W variant was identified in 2 siblings with combined immunodeficiency and CD8 lymphopenia, confirming the pathogenicity of this mutation. Structural modeling of this region revealed the critical nature of the arginines at positions 170 and 192, in concert with R190, forming a binding pocket for the phosphorylated TCR-ζ chain. Deleterious mutations in the SH2-C domain result in attenuated ZAP-70 function and clinical manifestations of immunodeficiency.

Zeta-associated protein 70 expression in chronic lymphocytic leukemia: Relevance in Indian context.

Background: Chronic lymphocytic leukemia (CLL) is prognosticated using the Rai and the Binet's staging. In the past few years, new parameters have been considered for prognostication. One such marker that has been a subject of speculation and found useful by some western studies is zeta-associated protein 70 (ZAP-70). Aim: To investigate the prevalence of ZAP-70 and find out its association with other prognostic markers like Rai and Binet's stage and CD38 in Indian CLL patients. Materials and Methods: Twenty-nine newly diagnosed cases of CLL were selected over 1 year. Immunophenotyping was done and expression of CD38 and ZAP-70 was evaluated on gated CLL cells. Statistical Analysis: Qualitative data were expressed as frequency and percentage. Differences between groups were evaluated using Student's t-test for quantitative data and Chi-square test/Fisher's exact t-test for qualitative variables. A P value less than 0.05 was considered significant. Results and Conclusion: We found a lower prevalence rate of ZAP-70 (2/29, 6.89%) with no association with any of the conventional poor prognostic factors. A large number of our CLL patients fall into the good prognostic group (22/29, ZAP 70-/CD38-) with a least number in the poor prognostic group (2/29, ZAP-70 + CD38+). Also, no association was found between ZAP-70 and CD38. The findings of the present study suggest that the majority of CLL patients in India have a good prognosis, may not require treatment, and have good overall survival. Geographical variations, genetic makeup, and natural history of the CLL could be the cause of such differences from western literature.

Lymphocyte disturbance and functional assessment of the [Asp521Asn] ZAP70 mutation.

Activated zeta-chain-associated protein kinase 70 (ZAP70) phosphorylates the TCRαß:CD3:zeta complex to diversify and amplify TCR signaling. Patients with ZAP70 mutations can present with phenotypes of immune dysregulation as well as infection. We identified the first Taiwanese boy with the [Asp521Asn] ZAP70 mutation who presented with recurrent pneumonia, inflammatory bowel disease-like diarrhea, transient hematuria and autoimmune hepatitis. He had isolated CD8 lymphopenia, eosinophilia, hypogammaglobulinemia, and impaired lymphocyte proliferation. Downstream CD3/CD28 signaling, phosphorylation of AKT, ZAP70 and Ca2+ influx were decreased in [Asp521Asn] ZAP70 lymphocytes. Immunophenotyping analysis revealed expansion of transitional B and CD21-low B cells, Th2-skewing T follicular helper cells, but lower Treg cells. The Asp521Asn-ZAP70 hindered TCR-CD3 downstream phosphorylation and disturbed lymphocyte subgroup "profiles" leading to autoimmunity/autoinflammation. Further large-scale studies are warranted to clarify this lymphocyte disturbance. The prognosis significantly depends on hematopoietic stem cell transplantation, but not the genotype, the presence of opportunistic infections or immune dysregulation.

RNA-Sequencing Analysis and Expression of Lymphocyte-Specific Protein Tyrosine Kinase, Linker for Activation of T Cells, and 70-kDa T-Cell Receptor Zeta-Chain Associated Protein Kinase in Rat Liver Transplant Rejection.

OBJECTIVES: The prevention and treatment of liver transplant rejection remain challenging. We investigated the pathophysiological mechanisms of liver transplant rejection in rats and screened candidate genes to determine their degree of rejection response for possible development of potential therapeutic targets. MATERIALS AND METHODS: Brown Norway-Brown Norway transplant tolerant models and Lewis-Brown Norway transplant rejection models were established. We collected liver tissue and venous blood at 7 days posttransplant for hematoxylin and eosin staining and RNA sequencing analysis, respectively. We conducted differential expression gene analysis, KEGG and GO enrichment analysis. We performed immunohistochemistry to detect highly expressed immunerelated proteins, including lymphocyte-specific protein tyrosine kinase, linker for activation of T cells, and 70-kDa T-cell receptor zeta-chain-associated protein kinase. RESULTS: Significant differences were found in liver function and Banff scores between rejection and tolerant groups, indicating the successful establishment of liver transplant models. RNA-sequencing screened 7521 differentially expressed genes, with 3355 upregulated and 3058 downregulated. KEGG analysis of upregulated genes showed that 8 of the top 20 enrichment pathways were associated with immune system processes and 5 were related to immune system diseases. Among these immune pathways, 289 genes were upregulated; of these, 147 genes were removed after comparison with the IMMPORT database, of which 97 genes were significantly changed. Our GO analysis showed upregulated genes mainly participating in immune response processes, with downregulated genes mainly participating in metabolic processes. Real-time polymerase chain reaction and immunohistochemistry verified expression of the immune-related proteins, consistent with RNAsequencing results, which were mainly expressed in inflammatory cells in sinus and portal vein. CONCLUSIONS: Immune-related genes were found to be associated with liver transplant rejection. The 3 immune-related genes that we analyzed may play a role in liver transplant rejection and can possibly serve as candidate markers for monitoring the degree of liver transplant rejection.

Cyclophilin A associates with and regulates the activity of ZAP70 in TCR/CD3-stimulated T cells.

The ZAP70 protein tyrosine kinase (PTK) couples stimulated T cell antigen receptors (TCRs) to their downstream signal transduction pathways and is sine qua non for T cell activation and differentiation. TCR engagement leads to activation-induced post-translational modifications of ZAP70, predominantly by kinases, which modulate its conformation, leading to activation of its catalytic domain. Here, we demonstrate that ZAP70 in TCR/CD3-activated mouse spleen and thymus cells, as well as human Jurkat T cells, is regulated by the peptidyl-prolyl cis-trans isomerase (PPIase), cyclophilin A (CypA) and that this regulation is abrogated by cyclosporin A (CsA), a CypA inhibitor. We found that TCR crosslinking promoted a rapid and transient, Lck-dependent association of CypA with the interdomain B region, at the ZAP70 regulatory domain. CsA inhibited CypA binding to ZAP70 and prevented the colocalization of CypA and ZAP70 at the cell membrane. In addition, imaging analyses of antigen-specific T cells stimulated by MHC-restricted antigen-fed antigen-presenting cells revealed the recruitment of ZAP70-bound CypA to the immunological synapse. Enzymatically active CypA downregulated the catalytic activity of ZAP70 in vitro, an effect that was reversed by CsA in TCR/CD3-activated normal T cells but not in CypA-deficient T cells, and further confirmed in vivo by FRET-based studies. We suggest that CypA plays a role in determining the activity of ZAP70 in TCR-engaged T cells and impact on T cell activation by intervening with the activity of multiple downstream effector molecules.

AQP9 and ZAP70 as immune-related prognostic biomarkers suppress proliferation, migration and invasion of laryngeal cancer cells.

BACKGROUND: Laryngeal cancer represents a common malignancy that originates from the larynx, with unfavorable prognosis. Herein, this study systematically analyzed the immune signatures of laryngeal cancer and to evaluate their roles on tumor progression. METHODS: Differentially expressed immune-related genes (IRGs) were screened between laryngeal cancer and normal tissues from TCGA dataset. Then, two prognosis-related IRGs AQP9 and ZAP70 were analyzed by a series of survival analysis. Based on them, molecular subtypes were constructed by unsupervised cluster analysis. Differences in survival outcomes, HLA expression and immune cell infiltrations were assessed between subtypes. Expression of AQP9 and ZAP70 was validated in laryngeal cancer tissues and cells by RT-qPCR and immunohistochemistry. After silencing and overexpressing AQP9 and ZAP70, CCK-8, EdU, wound healing and transwell assays were performed in TU212 and LCC cells. RESULTS: Totally, 315 IRGs were abnormally expressed in laryngeal cancer. Among them, AQP9 and ZAP70 were distinctly correlated to patients' prognosis. Two subtypes were developed with distinct survival outcomes, HLA expression and immune microenvironment. Low expression of AQP9 and ZAP70 was confirmed in laryngeal cancer. AQP9 and ZAP70 up-regulation distinctly suppressed proliferation, migration, and invasion of laryngeal cancer cells. The opposite results were investigated when their knockdown. CONCLUSIONS: Our findings revealed the roles of AQP9 and ZAP70 in progression of laryngeal cancer, and suggested that AQP9 and ZAP70 could potentially act as candidate immunotherapeutic targets for laryngeal cancer.

T and NK cell lymphoma cell lines do not rely on ZAP-70 for survival.

B-cell receptor (BCR) signalling is critical for the survival of B-cell lymphomas and is a therapeutic target of drugs such as Ibrutinib. However, the role of T-cell receptor (TCR) signalling in the survival of T/Natural Killer (NK) lymphomas is not clear. ZAP-70 (zeta associated protein-70) is a cytoplasmic tyrosine kinase with a critical role in T-cell receptor (TCR) signalling. It has also been shown to play a role in normal NK cell signalling and activation. High ZAP-70 expression has been detected by immunohistochemistry in peripheral T cell lymphoma (PTCL) and NK cell lymphomas (NKTCL). We therefore, studied the role of TCR pathways in mediating the proliferation and survival of these malignancies through ZAP-70 signalling. ZAP-70 protein was highly expressed in T cell lymphoma cell lines (JURKAT and KARPAS-299) and NKTCL cell lines (KHYG-1, HANK-1, NK-YS, SNK-1 and SNK-6), but not in multiple B-cell lymphoma cell lines. siRNA depletion of ZAP-70 suppressed the phosphorylation of ZAP-70 substrates, SLP76, LAT and p38MAPK, but did not affect cell viability or induce apoptosis in these cell lines. Similarly, while stable overexpression of ZAP-70 mediates increased phosphorylation of target substrates in the TCR pathway, it does not promote increased survival or growth of NKTCL cell lines. The epidermal growth factor receptor (EGFR) inhibitor Gefitinib, which has off-target activity against ZAP-70, also did not show any differential cell kill between ZAP-70 overexpressing (OE) or knockdown (KD) cell lines. Whole transcriptome RNA sequencing highlighted that there was very minimal differential gene expression in three different T/NK cell lines induced by ZAP-70 KD. Importantly, ZAP-70 KD did not significantly enrich for any downstream TCR related genes and pathways. Altogether, this suggests that high expression and constitutive signalling of ZAP-70 in T/NK lymphoma is not critical for cell survival or downstream TCR-mediated signalling and gene expression. ZAP-70 therefore may not be a suitable therapeutic target in T/NK cell malignancies.

Differences in the dynamics of the tandem-SH2 modules of the Syk and ZAP-70 tyrosine kinases.

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine-based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.

Ssu72 phosphatase directly binds to ZAP-70, thereby providing fine-tuning of TCR signaling and preventing spontaneous inflammation.

ZAP-70 is required for the initiation of T cell receptor (TCR) signaling, and Ssu72 is a phosphatase that regulates RNA polymerase II activity in the nucleus. However, the mechanism by which ZAP-70 regulates the fine-tuning of TCR signaling remains elusive. Here, we found that Ssu72 contributed to the fine-tuning of TCR signaling by acting as tyrosine phosphatase for ZAP-70. Affinity purification-mass spectrometry and an in vitro assay demonstrated specific interaction between Ssu72 and ZAP-70 in T cells. Upon TCR stimulation, Ssu72-deficient T cells increased the phosphorylation of ZAP-70 and downstream molecules and exhibited hyperresponsiveness, which was restored by reducing ZAP-70 phosphorylation. In vitro assay demonstrated that recombinant Ssu72 reduced tyrosine phosphorylation of ZAP-70 via phosphatase activity. Cd4-CreSsu72fl/fl mice showed a defect in the thymic development of invariant natural killer T cells and reductions in CD4+ and CD8+ T cell numbers in the periphery but more CD44hiCD62Llo memory T cells and fewer CD44loCD62Lhi naïve T cells, compared with wild-type mice. Furthermore, Cd4-CreSsu72fl/fl mice developed spontaneous inflammation at 6 mo. In conclusion, Ssu72 phosphatase regulates the fine-tuning of TCR signaling by binding to ZAP-70 and regulating its tyrosine phosphorylation, thereby preventing spontaneous inflammation.

ZAP70 activation is an early event of T cell immunity that involved in the anti-bacterial adaptive immune response of Nile tilapia.

ZAP70 is essential for initiating the early events of T-cell antigen receptor (TCR) signaling cascade to ensure proper T cell activation and function. However, whether this molecule takes part in the T cell immune response of early vertebrates remains unclear. In the present study, using a teleost model Nile tilapia (Oreochromis niloticus), we investigated the potential involvement of ZAP70 in the T cell activation and adaptive immunity of fish species. Both primary and tertiary structures of O. niloticus ZAP70 (On-ZAP70) are highly conserved with those from other vertebrates. On-ZAP70 protein was widely expressed in lymphoid tissues, and with the highest level in thymus. Once Nile tilapia was infected by Aeromonas hydrophila, mRNA of On-ZAP70 in spleen lymphocytes was induced on day 5 and 8 after infection; meanwhile, phosphorylation of On-ZAP70 was also enhanced, suggesting that On-ZAP70 potentially participated in primary adaptive immune response of Nile tilapia. Furthermore, the frequency of ZAP70 positive lymphocytes was increased during the anti-bacterial adaptive immune response. More importantly, when spleen lymphocytes were activated by T cell specific mitogen PHA, a dramatical augment of On-ZAP70 could be observed at transcription, phosphorylation and cellular level, indicating the involvement of this molecule in T cells activation of Nile tilapia. Altogether, our results demonstrated that ZAP70 activation is an early event of T cell immunity that involved in the anti-bacterial adaptive immune response of Nile tilapia, and thus provided a new evidence to understand the evolution of the lymphocyte-mediated adaptive immunity.

ZAP-70 constitutively regulates gene expression and protein synthesis in chronic lymphocytic leukemia.

The expression of ZAP-70 in a subset of chronic lymphocytic leukemia (CLL) patients strongly correlates with a more aggressive clinical course, although the exact underlying mechanisms remain elusive. The ability of ZAP-70 to enhance B-cell receptor (BCR) signaling, independently of its kinase function, is considered to contribute. We used RNA-sequencing and proteomic analyses of primary cells differing only in their expression of ZAP-70 to further define how ZAP-70 increases the aggressiveness of CLL. We identified that ZAP-70 is directly required for cell survival in the absence of an overt BCR signal, which can compensate for ZAP-70 deficiency as an antiapoptotic signal. In addition, the expression of ZAP-70 regulates the transcription of factors regulating the recruitment and activation of T cells, such as CCL3, CCL4, and IL4I1. Quantitative mass spectrometry of double-cross-linked ZAP-70 complexes further demonstrated constitutive and direct protein-protein interactions between ZAP-70 and BCR-signaling components. Unexpectedly, ZAP-70 also binds to ribosomal proteins, which is not dependent on, but is further increased by, BCR stimulation. Importantly, decreased expression of ZAP-70 significantly reduced MYC expression and global protein synthesis, providing evidence that ZAP-70 contributes to translational dysregulation in CLL. In conclusion, ZAP-70 constitutively promotes cell survival, microenvironment interactions, and protein synthesis in CLL cells, likely to improve cellular fitness and to further drive disease progression.