Tyrosine Kinase ZAP-70 Research Articles

Degron-Based bioPROTACs for Controlling Signaling in CAR T Cells.

Chimeric antigen receptor (CAR) T cells have made a tremendous impact in the clinic, but potent signaling through the CAR can be detrimental to treatment safety and efficacy. The use of protein degradation to control CAR signaling can address these issues in preclinical models. Existing strategies for regulating CAR stability rely on small molecules to induce systemic degradation. In contrast to small molecule regulation, genetic circuits offer a more precise method to control CAR signaling in an autonomous cell-by-cell fashion. Here, we describe a programmable protein degradation tool that adopts the framework of bioPROTACs, heterobifunctional proteins that are composed of a target recognition domain fused to a domain that recruits the endogenous ubiquitin proteasome system. We develop novel bioPROTACs that utilize a compact four-residue degron and demonstrate degradation of cytosolic and membrane protein targets using either a nanobody or synthetic leucine zipper as a protein binder. Our bioPROTACs exhibit potent degradation of CARs and can inhibit CAR signaling in primary human T cells. We demonstrate the utility of our bioPROTACs by constructing a genetic circuit to degrade the tyrosine kinase ZAP70 in response to recognition of a specific membrane-bound antigen. This circuit can disrupt CAR T cell signaling only in the presence of a specific cell population. These results suggest that bioPROTACs are powerful tools for expanding the CAR T cell engineering toolbox.

CD28 Shapes T Cell Receptor Signaling by Regulating ZAP70 Activation and Lck Dynamics.

T cell activation requires T cell receptor (TCR) engagement, which initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation. TCR microclusters (MC) are submicron-sized molecular condensates and basic signaling units that form immediately after TCR ligation. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in MCs and increased ZAP70 activation. This CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.

Hyperactive STAT5 hijacks T cell receptor signaling and drives immature T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive immature T cell cancer. Mutations in IL7R have been analyzed genetically, but downstream effector functions such as STAT5A and STAT5B hyperactivation are poorly understood. Here, we studied the most frequent and clinically challenging STAT5BN642H driver in T cell development and immature T cell cancer onset and compared it with STAT5A hyperactive variants in transgenic mice. Enhanced STAT5 activity caused disrupted T cell development and promoted an early T cell progenitor-ALL phenotype, with upregulation of genes involved in T cell receptor (TCR) signaling, even in absence of surface TCR. Importantly, TCR pathway genes were overexpressed in human T-ALL and mature T cell cancers and activation of TCR pathway kinases was STAT5 dependent. We confirmed STAT5 binding to these genes using ChIP-Seq analysis in human T-ALL cells, which were sensitive to pharmacologic inhibition by dual STAT3/5 degraders or ZAP70 tyrosine kinase blockers in vitro and in vivo. We provide genetic and biochemical proof that STAT5A and STAT5B hyperactivation can initiate T-ALL through TCR pathway hijacking and suggest similar mechanisms for other T cell cancers. Thus, STAT5 or TCR component blockade are targeted therapy options, particularly in patients with chemoresistant clones carrying STAT5BN642H.

ZAP-70 Integrates Tonic B Cell Receptor and Chemokine Signaling in IGHV unmutated Chronic Lymphocytic Leukemia

The importance of B cell receptor (BCR) signaling for the progression of Chronic Lymphocytic Leukemia (CLL) has impressively been demonstrated through the clinical success of small molecule inhibitors interfering with this pathway, providing effective treatment options for a vast majority of patients. The scientific evidence which supported the development of drugs like BTK and PI3K inhibitors largely originate from studies of antigen-induced BCR-signaling. Notably, the presence of a constitutive, so-called tonic BCR-signal was long postulated to co-exist besides antigen-driven activation and is based on the identification of constitutively activated signaling molecules down-stream of the BCR. We here provide direct evidence for the presence of a tonic BCR-signal, which critically relies on the expression of the tyrosine kinase ZAP-70. We have previously established a method to inhibit ZAP-70 expression in primary CLL cells usingRNA-interference in combination with an extended co-culture system. Our previous work demonstrated that ZAP-70 is required for the constitutive gene expression of T cell chemokines CCL3 and CCL4 and contributes to the dysregulation of global protein synthesis and MYC expression in CLL ( Blood, 2021). Applying this method, we here provide evidence for the presence of a constitutive tonic BCR signal, which is dependent on ZAP-70 expression and exclusively found in IGHV unmutated CLL, but not in IGHV mutated cases discordantly expressing ZAP-70. We demonstrate that ZAP-70 is directly involved in the constitutive activation of AKT and GSK-3beta. Furthermore, ZAP-70 expression increases steady state activation of CD19 and the downstream kinase SRC, providing direct evidence for the existence of a ZAP-70 dependent tonic BCR signal in I GHV unmutated CLL only. To identify the underlying signaling events driving this signal, we performed mass spectrometry in combination with proximity ligation assays on CLL cells in the absence of an induced BCR-signal. Unexpectedly, we identified that the majority of ZAP-70 binding proteins do not related to BCR-signaling components, but to cytoskeleton organizing proteins involved in cell migration. Indeed, migration assays demonstrated that the loss of ZAP-70 directly and significantly impaired cell migration induced by CCL19 and CCL21, in keeping with previous data indication an association between ZAP-70 and cell migration. These results were confirmed using a switchable model of ZAP-70, which allowed us to toggle ZAP-70 expression on and off at any given time. To further define the mechanisms of ZAP-70 mediated cell migration, we studied CCL19 and CCL21 signaling in ZAP-70 proficient and deficient cells. Unexpectedly, we identified that ZAP-70 is required for chemokine-mediated activation of AKT. In summary, we here demonstrate that a tonic BCR-signal is present only in IGHV-unmutated CLL and dependent on the expression of ZAP-70. Furthermore, ZAP-70 converges two seemingly independent signaling events, namely chemokine- and tonic BCR signaling. Our data provide further explanations for the more aggressive clinical course of ZAP-70 positive CLL.

A Story of Kinases and Adaptors: The Role of Lck, ZAP-70 and LAT in Switch Panel Governing T-Cell Development and Activation.

Specific antigen recognition is one of the immune system's features that allows it to mount intense yet controlled responses to an infinity of potential threats. T cells play a relevant role in the host defense and the clearance of pathogens by means of the specific recognition of peptide antigens presented by antigen-presenting cells (APCs), and, to do so, they are equipped with a clonally distributed antigen receptor called the T-cell receptor (TCR). Upon the specific engagement of the TCR, multiple intracellular signals are triggered, which lead to the activation, proliferation and differentiation of T lymphocytes into effector cells. In addition, this signaling cascade also operates during T-cell development, allowing for the generation of cells that can be helpful in the defense against threats, as well as preventing the generation of autoreactive cells. Early TCR signals include phosphorylation events in which the tyrosine kinases Lck and ZAP70 are involved. The sequential activation of these kinases leads to the phosphorylation of the transmembrane adaptor LAT, which constitutes a signaling hub for the generation of a signalosome, finally resulting in T-cell activation. These early signals play a relevant role in triggering the development, activation, proliferation and apoptosis of T cells, and the negative regulation of these signals is key to avoid aberrant processes that could generate inappropriate cellular responses and disease. In this review, we will examine and discuss the roles of the tyrosine kinases Lck and ZAP70 and the membrane adaptor LAT in these cellular processes.

Spleen tyrosine kinase mediates the γδTCR signaling required for γδT cell commitment and γδT17 differentiation.

The γδT cells that produce IL-17 (γδT17 cells) play a key role in various pathophysiologic processes in host defense and homeostasis. The development of γδT cells in the thymus requires γδT cell receptor (γδTCR) signaling mediated by the spleen tyrosine kinase (Syk) family proteins, Syk and Zap70. Here, we show a critical role of Syk in the early phase of γδT cell development using mice deficient for Syk specifically in lymphoid lineage cells (Syk-conditional knockout (cKO) mice). The development of γδT cells in the Syk-cKO mice was arrested at the precursor stage where the expression of Rag genes and αβT-lineage-associated genes were retained, indicating that Syk is required for γδT-cell lineage commitment. Loss of Syk in γδT cells weakened TCR signal-induced phosphorylation of Erk and Akt, which is mandatory for the thymic development of γδT17 cells. Syk-cKO mice exhibited a loss of γδT17 cells in the thymus as well as throughout the body, and thereby are protected from γδT17-dependent psoriasis-like skin inflammation. Collectively, our results indicate that Syk is a key player in the lineage commitment of γδT cells and the priming of γδT17 cell differentiation.

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.

An evolutionary divergent thermodynamic brake in ZAP-70 fine-tunes the kinetic proofreading in T cells

T cell signaling starts with assembling several tyrosine kinases and adapter proteins to the T cell receptor (TCR), following the antigen binding to the TCR. The stability of the TCR–antigen complex and the delay between the recruitment and activation of each kinase determines the T cell response. Integration of such delays constitutes a kinetic proofreading mechanism to regulate T cell response to the antigen binding. However, the mechanism of these delays is not fully understood. Combining biochemical experiments and kinetic modeling, here we report a thermodynamic brake in the regulatory module of the tyrosine kinase ZAP-70, which determines the ligand selectivity, and may delay the ZAP-70 activation upon antigen binding to TCR. The regulatory module of ZAP-70 comprises of a tandem SH2 domain that binds to its ligand, doubly-phosphorylated ITAM peptide (ITAM-Y2P), in two kinetic steps: a fast step and a slow step. We show the initial encounter complex formation between the ITAM-Y2P and tandem SH2 domain follows a fast-kinetic step, whereas the conformational transition to the holo-state follows a slow-kinetic step. We further observed a thermodynamic penalty imposed during the second phosphate-binding event reduces the rate of structural transition to the holo-state. Phylogenetic analysis revealed the evolution of the thermodynamic brake coincides with the divergence of the adaptive immune system to the cell-mediated and humoral responses. In addition, the paralogous kinase Syk expressed in B cells does not possess such a functional thermodynamic brake, which may explain the higher basal activation and lack of ligand selectivity in Syk.

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.

Deletion of Rhoh Accelerates Development of Diffuse Large B Cell Lymphoma in Bcl-6 Transgenic Mouse Model

Diffuse large B cell lymphoma (DLBCL) is an aggressive malignancy of mature B lymphocytes, accounting for 30%-40% of cases of non-Hodgkin's lymphoma. DLBCL has been classified by gene expression profiling into three subtypes GC-B-cell like (GCB)-DLBCL, activated B-cell like (ABC)-DLBCL and primary mediastinal B cell lymphoma (PMBL) or by immunohistological analysis into two subtypes, GCB-DLBCL and non-GCB- DLBCL. Rho H, a hematopoietic-specific Rho GTPase is required for CD3-zeta phosphorylation and recruitment of the protein tyrosine kinases Zap70 and Lck to the cellular membrane and immunologic synapse (Gu et al. Nat Immunol. 2006). Therefore, Rho H deficient (Rhoh-/-) mice exhibit T-cell deficiency due to impaired T-cell receptor-mediated selection and maturation of thymocytes. RHOH was first identified as a hypermutable gene and translocation partner in human B cell malignancies (Dallery E, et al . Oncogene. 1995) and RHOH mutations have been reported in 35 of 100 DLBCL cases (Hiraga J, et al. Leukemia. 2007). However, the effect of RHOH mutations and translocations on RHOH expression and the biology of lymphoma are completely unknown.We crossed Rhoh-/- and the DLBCL mouse model Iµ-HABcl-6 transgenic mouse (Cattoretti G, et al. Cancer Cell. 2005) to generate Rhoh-/- ;Bcl-6Tg and RhohWT;Bcl-6Tg mouse, and compared outcomes of these groups. Rhoh-/-; Bcl-6Tg mice developed severe splenomegaly and succumbed to disease more quickly compared with RhohWT; Bcl-6Tg mice (Figure1, Log-rank test p<0.01). Histological examination showed loss of splenic architecture and invasion of large, heterogenous lymphoid cells resembling human DLBCL. In contrast to RhohWT; Bcl-6Tg mice immunohistochemistry showed that RhohKO lymphoma cells were uniformly and strongly positive for IRF4 suggesting that loss of Rho H alters the phenotype of lymphoma at the cellular level to a ABC-DLBCL-like phenotype. Moreover, the number of Ki-67 positive cells showed a 2.5-fold increase in the spleens of Rhoh-/- lymphoma mice compared to RhohWT lymphoma mice (p<0.05). Cleaved Caspase-3, an indicator of apoptosis, was not significantly different between Rhoh-/- and RhohWT lymphoma cells. Western blot analysis of lymphoma cells showed that expression of BCL-6 was consistently increased in Rhoh-/- lymphoma cells compared with RhohWT lymphoma cells while expression of RelA/p65, NF-κB subunit, was upregulated in nuclear fraction of both Rhoh-/- and RhohWT lymphoma cells. Finally, expression of BLIMP-1, a key transcription factor associated with differentiation of B cells into plasma cells, was downregulated in Rhoh-/- lymphoma cells. We conclude that deletion of Rhoh in BCL6-driven DLBCL leads to ABC-DLBCL-like phenotype in this model of lymphoma associated with increased proliferation and more rapid progression of disease. These results support a role for Rho H mutations in the development of diffuse large B cell lymphoma. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

CD4+ T cells are more sensitive to inhibition of TCR signaling under Th17 polarizing conditions

Abstract Following TCR stimulation, in the presence of polarizing cytokines, naïve CD4+ T cells differentiate into effector cells (e.g. Th1, Th2, and Th17). To study how proximal TCR signaling molecules influence T cell differentiation, we used small molecule inhibitors to reduce signaling through the cytoplasmic tyrosine kinases Zap-70 or Lck. We used PP2 to inhibit Lck signaling and a chemical-genetic system to inhibit Zap-70. In this model system, mouse cells expressing an analog-sensitive mutant of Zap-70 are sensitive to inhibitor while wild-type cells are not. Surprisingly, we found that the generation of IL-17 producing Th17 cells was more sensitive to inhibition than the generation of either IFNg producing Th1 cells or IL-4 producing Th2 cells. These results raised the possibility that cells undergoing Th17 differentiation require TCR signaling of a higher intensity. To test this possibility, we analyzed the TCR-induced expression of IRF4 and Nur77-GFP, which are both inducibly expressed in relative proportion to the strength of TCR signaling. For both IRF4 and Nur77-GFP, the magnitude of expression was decreased under Th17 conditions. Additionally, increasing downstream signaling through NFAT rescued the Th17 sensitivity to inhibition. These results suggest that factors in Th17 cultures suppress TCR signal strength, thus cells stimulated under Th17 conditions experience weaker TCR signals and become more sensitive to further inhibition of TCR signaling.

Malignant ascite-derived extracellular vesicles inhibit T cell activity by upregulating Siglec-10 expression.

Background and purpose: To evade immune defense, cancer cells can employ extracellular vesicles (EVs) to inhibit the anti-tumor activity of lymphocytes in the tumor microenvironment. However, the mechanisms and key molecules that mediate the effects of EVs on lymphocytes are unclear.Patients and methods: We used Quantibody® Human Cytokine Antibody Array 440 to determine the tumor immunity-related cytokine profile of peripheral blood lymphocytes (PBLs) stimulated with EVs derived from peritoneal washes or malignant ascites. We detected 21 upregulated and 27 downregulated proteins, including the immunosuppressive receptors Siglec-10, SLAM, PD-1, and TIM-3.Results: Flow cytometry analysis of PBLs or ovarian cancer ascites suggested that Siglec-10 expression on CD3+ T cells was higher in ovarian cancer patients than in healthy controls and in the malignant ascites of ovarian cancer patients than in their blood. Moreover, the expression of CD24, the Siglec-10 ligand, was associated with tumor stage and cancer cell metastasis. Finally, compared to the benign peritoneal wash-derived EVs, the malignant EVs significantly upregulated Siglec-10 expression on Jurkat T cells, inhibited the protein kinase C activity induced by phorbol 12-myristate 13-acetate and ionomycin, and impaired the phosphorylation of the tyrosine kinase ZAP-70 activated by crosslinking with an anti-CD3 antibody.Conclusion: The EVs secreted by malignant ovarian cells upregulated Siglec-10 expression on T cells and impaired T cell activation in the tumor microenvironment. We believe that a comprehensive understanding of the regulation of Siglec-10 and CD24 by malignant EVs has clinical importance, as it will aid in the development of better immunotherapeutic strategies for ovarian cancer.

Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome.

Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins.

Inhibition of T Cell Receptor Activation by Semi-Synthetic Sesquiterpene Lactone Derivatives and Molecular Modeling of Their Interaction with Glutathione and Tyrosine Kinase ZAP-70.

A variety of natural compounds have been shown to modulate T cell receptor (TCR) activation, including natural sesquiterpene lactones (SLs). In the present studies, we evaluated the biological activity of 11 novel semi-synthetic SLs to determine their ability to modulate TCR activation. Of these compounds, α-epoxyarglabin, cytisinyl epoxyarglabin, 1β,10α-epoxyargolide, and chloroacetate grosheimin inhibited anti-CD3-induced Ca2+ mobilization and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in Jurkat T cells. We also found that the active SLs depleted intracellular glutathione (GSH) in Jurkat T cells, supporting their reactivity towards thiol groups. Because the zeta-chain associated tyrosine kinase 70 kDa (ZAP-70) is essential for TCR signaling and contains a tandem SH2 region that is highly enriched with multiple cysteines, we performed molecular docking of natural SLs and their semi-synthetic derivatives into the ZAP-70 binding site. The docking showed that the distance between the carbon atom of the exocyclic methylene group and the sulfur atom in Cys39 of the ZAP-70 tandem SH2 module was 3.04–5.3 Å for active compounds. Furthermore, the natural SLs and their derivatives could be differentiated by their ability to react with the Cys39 SH-group. We suggest that natural and/or semi-synthetic SLs with an α-methylene-γ-lactone moiety can specifically target GSH and the kinase site of ZAP-70 and inhibit the initial phases of TCR activation.

ZAP-70 Expression in Non Tumoral B Cells: Role in B Tolerance Breakdown?

Abnormal expression of the tyrosine kinase ZAP-70 by tumoral B cells in chronic lymphocytic leukemia (CLL) is associated with bad prognosis, related to B cell receptor (BCR) hypersignalling, clonal expansion and autoimmune cytopenia (AIC) occurrence, these latter being mostly induced by polyclonal IgG from the residual non tumoral B cells. We previously shown that ZAP-70 is expressed by these non tumoral B cells in CLL, positively associated with its expression in CLL B cells and with AIC occurrence (Ghergus et al. Poster ASH 2017). Here, we show for the first time a potential role of ZAP-70 expression in tolerance breakdown in CLL and in an original knock in mouse model overexpressing ZAP-70 conditionally in B cells.First, to assess a potential molecular link between ZAP-70+ CLL and non tumoral B cells, an analysis of their BCR repertoire has performed on FACS-sorting CD19+CD5-IgM-IgD- (non tumoral) and CD19+CD5+IgM-IgD- (tumoral) single B cells from blood samples of CLL patients with AIC. ZAP-70 positivity was screened by RT-PCR, and variable regions of heavy (IGVH) and light (IGVK/VL) immunoglobulin genes amplified by RT-PCR on ZAP-70+ cells. To date, analysis of 24 BCR sequences from 7 patients showed that non tumoral ZAP-70+ B cells were polyclonal, without stereotypy, using different V(D)J and CDR3 in comparison with those of the corresponding CLL B cells. IGVH of non tumoral ZAP-70+ B were mostly mutated, of replacement type, suggesting antigenic contact, contrary to CLL B cells.To determine potential autoreactivity of the non-tumoral ZAP-70+ B cells, IGVH and corresponding IGVK/VL were amplified for production of recombinant antibodies (rAb). To date, among 17 rAB from 7 different patients, 2/13 (15.4%) have an antinuclear autoreactivity on HEp-2 cells and 4/17 (23.5%) were polyreactive on ELISA (DNA, lipopolysaccharide, insulin), compared respectively to 6% and 4,3% of control B cells (Wardemann et al., Science 2003). Production of 7 additional rAb and tests for anti-erythrocytes and anti-platelets reactivity are in process.To study functional consequences of early ZAP-70 expression in B cells in vivo, we generated a knock in Zap-70+/Mb1-Cre+mouse model (KI ZAP), to induce conditional expression of ZAP-70 in the B cell compartment from the proB stage, with KI Zap-70+/Mb1-Cre- mice as controls (CTRL). The ZAP-70 mRNAs levels in B cells from KI ZAP mice were on average 20 times higher than that in CTRL B cells.Up to 20 months-old, KI ZAP mice did not develop signs of lymphoproliferation. KI ZAP mice had hypo-IgG since 16 weeks-old (p<0.001) together with hypo-IgM from 14 months-old (p<0.01). Immunophenotyping revealed a reduction in mature naive, mature switched as well as in germinal center B cells (p<0.001, p=0.002 and p<0.01 respectively) and a trend for plasma cells (p=0.07). Microarrays showed enrichment in circulating IgG and IgM autoantibodies against various antigens in KI ZAP mice. These mice had reduced apoptosis rates of proB (p<0.01), preB (p=0.02), and immatures B cells (p=0.03), together with enrichment in marginal zone (p=0.01), trend for transitional T2/T3, and reduction in B1a cells (p<0.01).After immunization by ovalbumin + Freund's adjuvant, a reduced production of specific IgG and IgM was observed (p=0.01 and p=0.03 respectively) with a trend in decreased number of antibody-secreting cells (p=0.07). KI ZAP B cells shown increased spontaneous activation and proliferation levels holding after BCR stimulation (p<0.01), as well as an increased intracellular calcic flow (p<0.001). Preliminary data suggested a reduced SYK phosphorylation after BCR stimulation in KI ZAP B cells.Our findings highlight for the first time that non tumoral B cells ZAP-70+ are distinct from CLL cells at cellular level, but probably enriched in autoreactive cells. Moreover, we shown that early ZAP-70 expression in normal B cells in vivois associated with autoimmune characteristics, together with partial block in B cells peripheral maturation, and a conversely early increased activation and proliferation status. ZAP-70 could interfere early with SYK leading to an altered BCR signaling responsible for defect in normal B maturation promoting emergence of autoreactive B cells. Mechanistic role of ZAP-70 in BCR signaling has to be further analyzed but our data open new opportunities involving ZAP-70 in the understanding of B cell development and physiopathology of tolerance breakdown. DisclosuresNo relevant conflicts of interest to declare.

GRK2 transactivation of CXCR4 is required for TCR-mediated TCR-CXCR4 complex formation

Abstract T cell cytokine production was recently shown to depend not only on the T cell antigen receptor (TCR), but also on the chemokine receptor CXCR4. Our lab recently published that stimulation of the TCR results in the TCR associating with and transactivating CXCR4, which, in turn, activates a PREX-1/Rac-1 pathway that stabilizes cytokine mRNA. This pathway therefore normally significantly increases IL-2, IL-4, and IL-10 production by activated T cells. Here, we further characterize the molecular mechanisms responsible for the first step of this pathway: TCR-mediated TCR-CXCR4 complex formation. First, we used FRET and proximity ligation assay (PLA) to define the required structural motifs of CXCR4, showing that this pathway requires the CXCR4 cytoplasmic tail domain and phosphorylation of CXCR4-S339. Second, we showed that TCR-induced TCR-CXCR4 complex formation requires TCR-induced activity of Src family and ZAP-70 tyrosine kinases, as well as one or more Ser/Thre kinases. Third, we found that the G protein-coupled receptor kinase-2 (GRK2) is required both for TCR-induced phosphorylation of CXCR4-S339 and for the formation of TCR-CXCR4 complexes. Finally, we found that GRK2 is required for robust IL-2, IL-4, and IL-10 cytokine secretion by normal human T cells. Together these results identify a novel role for TCR-induced GRK-2 in inducing the TCR-CXCR4 complex formation that subsequently signals to increase TCR-mediated cytokine production. Targeting GRK2 or other mechanisms required for TCR-induced TCR-CXCR4 formation may therefore be therapeutically useful for limiting production of T cell cytokines in humans, for example, during graft versus host disease or immunotherapy.

Fine-tuning of substrate preferences of the Src-family kinase Lck revealed through a high-throughput specificity screen.

The specificity of tyrosine kinases is attributed predominantly to localization effects dictated by non-catalytic domains. We developed a method to profile the specificities of tyrosine kinases by combining bacterial surface-display of peptide libraries with next-generation sequencing. Using this, we showed that the tyrosine kinase ZAP-70, which is critical for T cell signaling, discriminates substrates through an electrostatic selection mechanism encoded within its catalytic domain (Shah et al., 2016). Here, we expand this high-throughput platform to analyze the intrinsic specificity of any tyrosine kinase domain against thousands of peptides derived from human tyrosine phosphorylation sites. Using this approach, we find a difference in the electrostatic recognition of substrates between the closely related Src-family kinases Lck and c-Src. This divergence likely reflects the specialization of Lck to act in concert with ZAP-70 in T cell signaling. These results point to the importance of direct recognition at the kinase active site in fine-tuning specificity.

ZAP-70 in Signaling, Biology, and Disease.

T cells possess an array of functional capabilities important for host defense against pathogens and tumors. T cell effector functions require the T cell antigen receptor (TCR). The TCR has no intrinsic enzymatic activity, and thus signal transduction from the receptor relies on additional signaling molecules. One such molecule is the cytoplasmic tyrosine kinase ZAP-70, which associates with the TCR complex and is required for initiating the canonical biochemical signal pathways downstream of the TCR. In this article, we describe recent structure-based insights into the regulation and substrate specificity of ZAP-70, and then we review novel methods for determining the role of ZAP-70 catalytic activity-dependent and -independent signals in developing and mature T cells. Lastly, we discuss the disease states in mouse models and humans, which range from immunodeficiency to autoimmunity, that are caused by mutations in ZAP-70.

Prognosis biomarkers evaluation in chronic lymphocytic leukemia

Objective/BackgroundFrom clinical and biological points of view, chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by a progressive accumulation of lymphocytes in the peripheral blood, bone marrow, and lymphoid organs. New prognostic markers in CLL may be useful to clinicians for predicting outcome and in clinical decision-making. The aim of this study was to evaluate the potential prognostic value of the apoptotic/survival-controlling proteins and protein tyrosine kinase ZAP-70 gene expression in CLL patients and control individuals, correlating such findings with patients’ clinical data. MethodsFifty-three patients diagnosed with CLL attending the hematology service of a clinical hospital, and 24 healthy individuals with no history of leukemia (Control group) were enrolled in this study. Analyses of apoptotic/survival-controlling proteins were performed by western blot and ZAP-70 gene expression was evaluated by real-time polymerase chain reaction. ResultsSignificant differences were observed for the p-p38, Mcl-1 long, and Mcl-1 short proteins when patients were compared with CLL and controls. A positive correlation between the results for Mcl-1 short and Mcl-1 long and lymphocyte count was observed, corroborating the hypothesis of an imbalance between proteins of cell survival pathways/apoptosis in CLL. ConclusionZAP-70 gene expression was not detected as a discriminant biomarker in these CLL patients. An imbalance between apoptosis-related proteins was observed in the present study, corroborating the hypothesis of increased survival of lymphocytes in CLL patients.

Phosphorylation of a Tyrosine Residue on Zap70 by Lck and Its Subsequent Binding via an SH2 Domain May Be a Key Gatekeeper of T Cell Receptor Signaling In Vivo.

The initiation of signaling in T lymphocytes in response to the binding of the T cell receptor (TCR) to cognate ligands is a key step in the emergence of adaptive immune responses. Conventional models posit that TCR signaling is initiated by the phosphorylation of receptor-associated immune receptor activation motifs (ITAMs). The cytoplasmic tyrosine kinase Zap70 binds to phosphorylated ITAMs, is subsequently activated, and then propagates downstream signaling. While evidence for such models is provided by experiments with cell lines, in vivo, Zap70 is bound to phosphorylated ITAMs in resting T cells. However, Zap70 is activated only upon TCR binding to cognate ligand. We report the results of computational studies of a new model for the initiation of TCR signaling that incorporates these in vivo observations. Importantly, the new model is shown to allow better and faster TCR discrimination between self-ligands and foreign ligands. The new model is consistent with many past experimental observations, and experiments that could further test the model are proposed.