Calcineurin is an adaptor required for assembly of the TCR signaling complex.

The serine/threonine phosphatase calcineurin is a component of the T cell receptor (TCR) signalosome, where it promotes T cell activation by dephosphorylating LckS59. Using small interfering RNA (siRNA)-mediated knockdown and CRISPR-Cas9-targeted genetic disruption of the calcineurin A chain α and ß isoforms, we find that calcineurin also functions as an adaptor in TCR-signaled human T cells. Unlike inhibition of its phosphatase activity, in the absence of calcineurin A, TCR signaling results in attenuated actin rearrangement, markedly reduced TCR-Lck microcluster formation and recruitment of the adaptor RhoH, and diminished phosphorylation of critical targets downstream of Lck such as TCRζ and ZAP-70. Reconstitution of deficient T cells with either calcineurin Aα or Aß restores TCR microcluster formation and signaling, as does reconstitution with a phosphatase-inactive Aα chain. These results assign a non-enzymatic adaptor function to calcineurin in the formation and stabilization of a functional TCR signaling complex.

Identification of hub modules and therapeutic targets associated with CD8+T-cells in HF and their pan-cancer analysis.

Heart failure (HF) is a terminal condition of multiple cardiovascular disorders. Cancer is a deadly disease worldwide. The relationship between HF and cancer remains poorly understood. The Gene Expression Omnibus database was used to download the RNA sequencing data of 356 patients with hypertrophic cardiomyopathy-induced HF and non-HF. A co-expression network was established through the weighted correlation network analysis (WGCNA) to identify hub genes of HF and cancer. Cox risk analysis was performed to predict the prognostic risks of HF hub genes in pan-cancer. HF was linked to immune response pathway by the analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). A positive correlation was observed between the expression levels of 4 hub genes and the infiltration of CD8+T-cells in pan-cancer. 4 hub genes were identified as beneficial prognostic factors in several cancers. Western blotting and real-time polymerase chain reaction validated the high expression of GZMM, NKG7, and ZAP70 in both mice and patients with HF compared to control groups. Our study highlights the shared immune pathogenesis of HF and cancer and provides valuable insights for developing novel therapeutic strategies, offering new opportunities for improving the management and treatment outcomes of both HF and cancer.

Genetic evidence for predisposition to acute leukemias due to a missense mutation (p.Ser518Arg) in ZAP70 kinase: a case-control study.

BACKGROUND: The apparent lack of additional missense mutations data on mixed-phenotype leukemia is noteworthy. Single amino acid substitution by these non-synonymous single nucleotide variations can be related to many pathological conditions and may influence susceptibility to disease. This case-control study aimed to unravel whether the ZAP70 missense variant (rs104893674 (C > A)) underpinning mixed-phenotype leukemia. METHODS: The rs104893674 was genotyped in clients who were mixed-phenotype acute leukemia-, acute lymphoblastic leukemia- and acute myeloid leukemia-positive and matched healthy controls, which have been referred to all major urban hospitals from multiple provinces of country- wide, IRAN, from February 11' 2019 to June 10' 2023, by amplification refractory mutation system-polymerase chain reaction method. Direct sequencing for rs104893674 of the ZAP70 gene was performed in a 3130 Genetic Analyzer. RESULTS: We found that the AC genotype of individuals with A allele at this polymorphic site (heterozygous variant-type) contribute to the genetic susceptibility to acute leukemia of both forms, acute myeloid leukemia and acute lymphoblastic leukemia as well as with a mixed phenotype. In other words, the ZAP70 missense variant (rs104893674 (C > A)) increases susceptibility of distinct cell populations of different (myeloid and lymphoid) lineages to exhibiting cancer phenotype. The results were all consistent with genotype data obtained using a direct DNA sequencing technique. CONCLUSION: Of special interest are pathogenic missense mutations, since they generate variants that cause specific molecular phenotypes through protein destabilization. Overall, we discovered that the rs104893674 (C > A) variant chance in causing mixed-phenotype leukemia is relatively high.

Cbl-b inhibition promotes less differentiated phenotypes of T cells with enhanced cytokine production.

For adoptive therapy with T cell receptor engineered T (TCR-T) cells, the quantity and quality of the final cell product directly affect their anti-tumor efficacy. The post-transfer efficacy window of TCR-T cells is keen to optimizing attempts during the manufacturing process. Cbl-b is a E3 ubiquitin ligase previously shown with critical negative impact in T cell functions. This study investigated whether strategic inclusion of a commercially available small inhibitor targeting Cbl-b (Cbl-b-IN-1) prior to T cell activation could enhance the quality of the final TCR-T cell product. Examination with both PBMCs and TCR-T cells revealed that Cbl-b-IN-1 treatment promoted TCR expression efficiency, T cell proliferation potential and, specifically, cell survival capability post antigenic stimulation. Cbl-b-IN-1 exposure facilitated T cells in maintaining less differentiated states with enhanced cytokine production. Further, we found that Cbl-b-IN-1 effectively augmented the activation of TCR signaling, shown by increased phosphorylation levels of Zeta-chain-associated protein kinase 70 (ZAP70) and phospholipase c-γ1 (PLCγ1). In conclusion, our results evidence that the inclusion of Cbl-b inhibitor immediately prior to TCR-T cell activation may enhance their proliferation, survival, and function potentials, presenting an applicable optimization strategy for immunotherapy with adoptive cell transfer.

Generation of antitumor chimeric antigen receptors incorporating T cell signaling motifs.

Chimeric antigen receptor (CAR) T cells have been used to successfully treat various blood cancers, but adverse effects have limited their potential. Here, we developed chimeric adaptor proteins (CAPs) and CAR tyrosine kinases (CAR-TKs) in which the intracellular ζ T cell receptor (TCRζ) chain was replaced with intracellular protein domains to stimulate signaling downstream of the TCRζ chain. CAPs contain adaptor domains and the kinase domain of ZAP70, whereas CAR-TKs contain only ZAP70 domains. We hypothesized that CAPs and CAR-TKs would be more potent than CARs because they would bypass both the steps that define the signaling threshold of TCRζ and the inhibitory regulation of upstream molecules. CAPs were too potent and exhibited high tonic signaling in vitro. In contrast, CAR-TKs exhibited high antitumor efficacy and significantly enhanced long-term tumor clearance in leukemia-bearing NSG mice as compared with the conventional CD19-28ζ-CAR-T cells. CAR-TKs were activated in a manner independent of the kinase Lck and displayed slower phosphorylation kinetics and prolonged signaling compared with the 28ζ-CAR. Lck inhibition attenuated CAR-TK cell exhaustion and improved long-term function. The distinct signaling properties of CAR-TKs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an antitumor chimeric receptor.

Phospho-mimetic CD3ε variants prevent TCR and CAR signaling.

Introduction: Antigen binding to the T cell antigen receptor (TCR) leads to the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex, and thereby to T cell activation. The CD3ε subunit plays a unique role in TCR activation by recruiting the kinase LCK and the adaptor protein NCK prior to ITAM phosphorylation. Here, we aimed to investigate how phosphorylation of the individual CD3ε ITAM tyrosines impacts the CD3ε signalosome. Methods: We mimicked irreversible tyrosine phosphorylation by substituting glutamic acid for the tyrosine residues in the CD3ε ITAM. Results: Integrating CD3ε phospho-mimetic variants into the complete TCR-CD3 complex resulted in reduced TCR signal transduction, which was partially compensated by the involvement of the other TCR-CD3 ITAMs. By using novel CD3ε phospho-mimetic Chimeric Antigen Receptor (CAR) variants, we avoided any compensatory effects of other ITAMs in the TCR-CD3 complex. We demonstrated that irreversible CD3ε phosphorylation prevented signal transduction upon CAR engagement. Mechanistically, we demonstrated that glutamic acid substitution at the N-terminal tyrosine residue of the CD3ε ITAM (Y39E) significantly reduces NCK binding to the TCR. In contrast, mutation at the C-terminal tyrosine of the CD3ε ITAM (Y50E) abolished LCK recruitment to the TCR, while increasing NCK binding. Double mutation at the C- and N-terminal tyrosines (Y39/50E) allowed ZAP70 to bind, but reduced the interaction with LCK and NCK. Conclusions: The data demonstrate that the dynamic phosphorylation of the CD3ε ITAM tyrosines is essential for CD3ε to orchestrate optimal TCR and CAR signaling and highlights the key role of CD3ε signalosome to tune signal transduction.

[Understanding New Regulatory Mechanism of TCR Signal Transduction].

Signal-transducing adaptor protein-2 (STAP-2) is a unique scaffold protein that regulates several immunological signaling pathways, including LIF/LIF receptor and LPS/TLR4 signals. STAP-2 is required for Fas/FasL-dependent T cell apoptosis and SDF-1α-induced T cell migration. Conversely, STAP-2 modulates integrin-mediated T cell adhesion, suggesting that STAP-2 is essential for several negative and positive T cell functions. However, whether STAP-2 is involved in T cell-antigen receptor (TCR)-mediated T cell activation is unknown. STAP-2 deficiency was recently reported to suppress TCR-mediated T cell activation by inhibiting LCK-mediated CD3ζ and ZAP-70 activation. Using STAP-2 deficient mice, it was demonstrated that STAP-2 is required for the pathogenesis of Propionibacterium acnes-induced granuloma formation and experimental autoimmune encephalomyelitis. Here, detailed functions of STAP-2 in TCR-mediated T cell activation, and how STAP-2 affects the pathogenesis of T cell-mediated inflammation and immune diseases, are reviewed.

A Novel Homozygous RHOH Variant Associated with T Cell Dysfunction and Recurrent Opportunistic Infections.

RHOH, an atypical small GTPase predominantly expressed in hematopoietic cells, plays a vital role in immune function. A deficiency in RHOH has been linked to epidermodysplasia verruciformis, lung disease, Burkitt lymphoma and T cell defects. Here, we report a novel germline homozygous RHOH c.245G > A (p.Cys82Tyr) variant in a 21-year-old male suffering from recurrent, invasive, opportunistic infections affecting the lungs, eyes, and brain. His sister also succumbed to a lung infection during early adulthood. The patient exhibited a persistent decrease in CD4+ T, B, and NK cell counts, and hypoimmunoglobulinemia. The patient's T cell showed impaired activation upon in vitro TCR stimulation. In Jurkat T cells transduced with RHOHC82Y, a similar reduction in activation marker CD69 up-regulation was observed. Furthermore, the C82Y variant showed reduced RHOH protein expression and impaired interaction with the TCR signaling molecule ZAP70. Together, these data suggest that the newly identified autosomal-recessive RHOH variant is associated with T cell dysfunction and recurrent opportunistic infections, functioning as a hypomorph by disrupting ZAP70-mediated TCR signaling.

ITPRIPL1 binds CD3ε to impede T cell activation and enable tumor immune evasion.

Cancer immunotherapy has transformed treatment possibilities, but its effectiveness differs significantly among patients, indicating the presence of alternative pathways for immune evasion. Here, we show that ITPRIPL1 functions as an inhibitory ligand of CD3ε, and its expression inhibits T cells in the tumor microenvironment. The binding of ITPRIPL1 extracellular domain to CD3ε on T cells significantly decreased calcium influx and ZAP70 phosphorylation, impeding initial T cell activation. Treatment with a neutralizing antibody against ITPRIPL1 restrained tumor growth and promoted T cell infiltration in mouse models across various solid tumor types. The antibody targeting canine ITPRIPL1 exhibited notable therapeutic efficacy against naturally occurring tumors in pet clinics. These findings highlight the role of ITPRIPL1 (or CD3L1, CD3ε ligand 1) in impeding T cell activation during the critical "signal one" phase. This discovery positions ITPRIPL1 as a promising therapeutic target against multiple tumor types.

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.

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.

Identification of LAT/ZAP70 characterized immune subtypes of prostate cancer.

BACKGROUND: While immunotherapy has shown potent efficacy in clinical practices, patient selection to receive checkpoint blockade is still challenging in prostate cancer (PCa). LAT and ZAP70 functions in lymphocyte activation and plays a critical role in T cell receptor (TCR) signal transduction. However, PCa genomic and clinical data regarding the role of LAT and ZAP70 are limited. We aim to identify and characterize LAT/ZAP70 defined subtypes of PCa. METHODS: We elaborated the TCGA PCa data and metastatic castration-resistant prostate cancer (mCRPC) RNA-seq data bioinformatic analysis and systematically elucidated the role of intra-tumoral expressed LAT and ZAP70 in the progression-free survival and immunotherapeutic-related signals. LAT/ZAP70-associated immune infiltration was evaluated using bioinformatic tools. Immunohistochemical staining of serial sections was used to confirm the expression and distribution of LAT, ZAP70 and androgen receptor (AR) in PCa tissues. RESULTS: Specifically, LAT and ZAP70 revealed increased expressions in PCa when compared to normal tissues and positively associated with intra-tumoral immune cells infiltration. LAT/ZAP70 defined immune-high early-stage PCa revealed higher TP53 mutation frequency and poor prognosis. Transcriptome analysis indicated immune-related signals and CTLA4 expression were highly enhanced in immune-high PCa parallel with higher protein level of MYC and lower AR expression. In mCRPC, LAT/ZAP70 defined immune-high patients also revealed upregulated immune related signals, higher CTLA4 expression and DNA repair deficiency. CONCLUSION: LAT/ZAP70 defined immune-high PCa linked to immune infiltration and predicts poor prognosis. Immune-high PCa may receive effective response from immune checkpoint inhibitor parallel with systemic treatment.

A Cysteine Residue within the Kinase Domain of Zap70 Regulates Lck Activity and Proximal TCR Signaling.

Alterations in both the expression and function of the non-receptor tyrosine kinase Zap70 are associated with numerous human diseases including immunodeficiency, autoimmunity, and leukemia. Zap70 propagates the TCR signal by phosphorylating two important adaptor molecules, LAT and SLP76, which orchestrate the assembly of the signaling complex, leading to the activation of PLCγ1 and further downstream pathways. These events are crucial to drive T-cell development and T-cell activation. Recently, it has been proposed that C564, located in the kinase domain of Zap70, is palmitoylated. A non-palmitoylable C564R Zap70 mutant, which has been reported in a patient suffering from immunodeficiency, is incapable of propagating TCR signaling and activating T cells. The lack of palmitoylation was suggested as the cause of this human disease. Here, we confirm that Zap70C564R is signaling defective, but surprisingly, the defective Zap70 function does not appear to be due to a loss in palmitoylation. We engineered a C564A mutant of Zap70 which, similarly to Zap70C564R, is non-palmitoylatable. However, this mutant was capable of propagating TCR signaling. Moreover, Zap70C564A enhanced the activity of Lck and increased its proximity to the TCR. Accordingly, Zap70-deficient P116 T cells expressing Zap70C564A displayed the hyperphosphorylation of TCR-ζ and Zap70 (Y319), two well-known Lck substrates. Collectively, these data indicate that C564 is important for the regulation of Lck activity and proximal TCR signaling, but not for the palmitoylation of Zap70.

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.

SYK and ZAP70 kinases in autoimmunity and lymphoid malignancies.

SYK and ZAP70 nonreceptor tyrosine kinases serve essential roles in initiating B-cell receptor (BCR) and T-cell receptor (TCR) signaling in B- and T-lymphocytes, respectively. Despite their structural and functional similarity, expression of SYK and ZAP70 is strictly separated during B- and T-lymphocyte development, the reason for which was not known. Aberrant co-expression of ZAP70 with SYK was first identified in B-cell chronic lymphocytic leukemia (CLL) and is considered a biomarker of aggressive disease and poor clinical outcomes. We recently found that aberrant ZAP70 co-expression not only functions as an oncogenic driver in CLL but also in various other B-cell malignancies, including acute lymphoblastic leukemia (B-ALL) and mantle cell lymphoma. Thereby, aberrantly expressed ZAP70 redirects SYK and BCR-downstream signaling from NFAT towards activation of the PI3K-pathway. In the sole presence of SYK, pathological BCR-signaling in autoreactive or premalignant cells induces NFAT-activation and NFAT-dependent anergy and negative selection. In contrast, negative selection of pathological B-cells is subverted when ZAP70 diverts SYK from activation of NFAT towards tonic PI3K-signaling, which promotes survival instead of cell death. We discuss here how both B-cell malignancies and autoimmune diseases frequently evolve to harness this mechanism, highlighting the importance of developmental separation of the two kinases as an essential safeguard.