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.

TREM-2 promotes Th1 responses by interacting with the CD3ζ-ZAP70 complex following Mycobacterium tuberculosis infection.

Triggering receptor expressed on myeloid cells 2 (TREM-2) is a modulator of pattern recognition receptors on innate immune cells that regulates the inflammatory response. However, the role of TREM-2 in in vivo models of infection and inflammation remains controversial. Here, we demonstrated that TREM-2 expression on CD4+ T cells was induced by Mycobacterium tuberculosis infection in both humans and mice and positively associated with T cell activation and an effector memory phenotype. Activation of TREM-2 in CD4+ T cells was dependent on interaction with the putative TREM-2 ligand expressed on DCs. Unlike the observation in myeloid cells that TREM-2 signals through DAP12, in CD4+ T cells, TREM-2 interacted with the CD3ζ-ZAP70 complex as well as with the IFN-γ receptor, leading to STAT1/-4 activation and T-bet transcription. In addition, an infection model using reconstituted Rag2-/- mice (with TREM-2-KO vs. WT cells or TREM-2+ vs. TREM-2-CD4+ T cells) or CD4+ T cell-specific TREM-2 conditional KO mice demonstrated that TREM-2 promoted a Th1-mediated host defense against M. tuberculosis infection. Taken together, these findings reveal a critical role of TREM-2 in evoking proinflammatory Th1 responses that may provide potential therapeutic targets for infectious and inflammatory diseases.

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.

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.

Adapting T Cell Receptor Ligand Discrimination Capability via LAT.

Self- and non-self ligand discrimination is a core principle underlying T cell-mediated immunity. Mature αß T cells can respond to a foreign peptide ligand presented by major histocompatibility complex molecules (pMHCs) on antigen presenting cells, on a background of continuously sensed self-pMHCs. How αß T cells can properly balance high sensitivity and high specificity to foreign pMHCs, while surrounded by a sea of self-peptide ligands is not well understood. Such discrimination cannot be explained solely by the affinity parameters of T cell antigen receptor (TCR) and pMHC interaction. In this review, we will discuss how T cell ligand discrimination may be molecularly defined by events downstream of the TCR-pMHC interaction. We will discuss new evidence in support of the kinetic proofreading model of TCR ligand discrimination, and in particular how the kinetics of specific phosphorylation sites within the adaptor protein linker for activation of T cells (LAT) determine the outcome of TCR signaling. In addition, we will discuss emerging data regarding how some kinases, including ZAP-70 and LCK, may possess scaffolding functions to more efficiently direct their kinase activities.

Cbl-b deficiency prevents functional but not phenotypic T cell anergy.

T cell anergy is an important peripheral tolerance mechanism. We studied how T cell anergy is established using an anergy model in which the Zap70 hypermorphic mutant W131A is coexpressed with the OTII TCR transgene (W131AOTII). Anergy was established in the periphery, not in the thymus. Contrary to enriched tolerance gene signatures and impaired TCR signaling in mature peripheral CD4 T cells, CD4SP thymocytes exhibited normal TCR signaling in W131AOTII mice. Importantly, the maintenance of T cell anergy in W131AOTII mice required antigen presentation via MHC-II. We investigated the functional importance of the inhibitory receptor PD-1 and the E3 ubiquitin ligases Cbl-b and Grail in this model. Deletion of each did not affect expression of phenotypic markers of anergic T cells or T reg numbers. However, deletion of Cbl-b, but not Grail or PD-1, in W131AOTII mice restored T cell responsiveness and signaling. Thus, Cbl-b plays an essential role in the establishment and/or maintenance of unresponsiveness in T cell anergy.

Demonstrating the effect of SHP2 inhibitor on cervical squamous cell carcinoma from the perspective of ZAP70.

SHP2, encoded by the PTPN11 gene, plays an important role in regulating immune cell functions in tumor microenvironment. Several SHP2 inhibitors have entered the clinical trial stage, but cervical squamous cell carcinoma (CSCC) has not been included in the indications. In Tlymphocytes, SHP2 can promote the dephosphorylation of ZAP70 kinase in the T cell receptor signaling complex after recruitment to the PD-1 cytoplasmic tail. In this study, we used bioinformatics tools to confirm that ZAP70 has a widespread impact on the immunity of CSCC, which is closely related to the survival of CSCC. The effect of ZAP70 on the survival of cervical cancer may not depend on the structure or amplification, but on the enhancement of function. And we identified ZAP70 and PTPN11 protein-protein interactions. SHP2 inhibitor is worth to be studied in the treatment of CSCC.

A disease-associated mutation that weakens ZAP70 autoinhibition enhances responses to weak and self-ligands.

The cytoplasmic kinase ZAP70 is critical for T cell antigen receptor (TCR) signaling. The R360P mutation in ZAP70 is responsible for an early-onset familial autoimmune syndrome. The structural location and biochemical signaling effects of the R360P mutation are consistent with weakening of the autoinhibitory conformation of ZAP70. Mice with a ZAP70 R360P mutation and polyclonal TCR repertoires exhibited relatively normal T cell development but showed evidence of increased signaling. In addition, the R360P mutation resulted in enhanced follicular helper T cell expansion after LCMV infection. To eliminate the possibility of a TCR repertoire shift, the OTI transgenic TCR was introduced into R360P mice, which resulted in enhanced T cell responses to weaker stimuli, including weak agonists and a self-peptide. These observations suggest that disruption of ZAP70 autoinhibition by the R360P mutation enables increased mature T cell sensitivity to self-antigens that would normally be ignored by wild-type T cells, a mechanism that may contribute to the break of tolerance in human patients with R360P mutation.

Signaling from membrane semaphorin 4D in T lymphocytes.

Semaphorin 4D (Sema4D) is widely represented in the immune system in both membrane and soluble form, and controls immune processes through the specific receptors - these are generally accepted views. Here, an alternative way of Sema4D-dependent immunoregulation is presented, suggesting its functioning as a receptor. We have shown that activation of membrane Sema4D induces phosphorylation of Lck/ZAP-70 in intact T lymphocytes and enhances it in stimulated T cells. Since Sema4D is constitutively presented on the membrane of T lymphocytes, and classical Sema4D receptors are highly expressed by antigen-presenting cells, the membrane Sema4D can serve as an obligate costimulatory molecule in T lymphocyte priming or T-dependent B cell activation.

Measuring the T-cell down-regulation of TCR-zeta, ZAP-70 and CD28 in arthritis patients: An old tool for new biomarkers.

Low T-cell receptor (TCR)/CD28 signaling lymphocytes are expanded in arthritis. We asked whether the down-expression of TCR-related molecules correlates with specific arthritis characteristics and if it has clinical implications. TCR-ZETA, ZAP-70 and CD28 expression was measured by flow cytometry in synovial fluid (SF) and peripheral blood (PB)-derived T cells. In PB, ZETA-downregulation in CD4+ CD28+ and consequent CD4+ CD28lowZETAlow cell expansion correlate with CRP elevation, leukocyte recruitment into SF and, primarily, disease activity (DAS). In some patients, ZETA-downregulation extends to CD8+ CD28null and/or CD8+ CD28+ cells, and this correlates with enhanced leukocyte recruitment, multiple joint involvement, and disability index (HAQ). ZETA-downregulation in CD4+ CD28+ may also lead to CD4+ CD28+ ZETAnull cell expansion, which strongly correlates with HAQ. In SF, ZETA-downregulation in CD8+ CD28null and consequent CD8+ CD28nullZETAlow/null cell expansion correlate with CRP elevation and neutrophilic influx into SF, whereas ZAP-downregulation in CD8+ CD28+ and consequent CD8+ CD28lowZAPlow cell expansion strongly correlate with HAQ and DAS. ZETA-downregulation is preponderant in SF of seronegative arthritides, with seronegative rheumatoid arthritis showing significant down-regulation in CD8+ CD28null, and non-rheumatoid arthritides showing significant down-regulation in CD4+ CD28+ . Altogether, we identified new molecular and cellular biomarkers of arthritis-related T-cell inflammation, useful for assessing arthritis activity, predicting polyarticular progression and functional impairment, characterizing seronegative arthritides, and possibly tailoring immunotherapies.

Ablation of core fucosylation attenuates the signal transduction via T cell receptor to suppress the T cell development.

Precise glycosylation plays a crucial and distinctive role in thymic T cell development. The core fucosylation is dramatically up-regulated at the transition from CD4-CD8- (DN) to CD4+CD8+ (DP) in the thymic development. Ablation of core fucosylation in T cells did reduce the size of the thymus due to a significant loss of CD4+ SP, CD8+ SP and DP thymocytes in core fucosyltransferase (Fut8) knockout (Fut8-/-) mice. T cell receptors (TCRs) are heavily core fucosylated glycoproteins. Loss of core fucosylation of TCR contributed to the reduced phosphorylation of ZAP70 (pZAP70) in Fut8-/- DP cells was observed. Compare to the Fut8+/+OT-II DP thymocytes, pZAP70 was significantly reduced in Fut8-/- OT-II DP thymocytes with OVA323-339 stimulation. Also, the pZAP70 of Fut8+/+OT-I DP thymocytes with OVA257-264 stimulation was remarkably attenuated by treatment of the fucosidase. Upon anti-CD3/CD28 Abs stimulation, the increased apoptosis was found in Fut8-/- thymocytes compared with Fut8+/+ thymocytes. Moreover, the TCRhiCD69hi (post-positive selection thymocytes) was markedly depleted in the Fut8-/- thymus without any stimulation. The expression of CD5 was significantly down-regulated on the DP cells in the Fut8-/- thymus. Our results therefore demonstrate that ablation of core fucosylation results in the abnormal T cell development due to the attenuated signaling via TCR.

Combined analysis of ZAP-70 and CD38 expression in sudanese patients with B-cell chronic lymphocytic leukemia.

OBJECTIVE: To investigate the ZAP-70 and CD38 expressions and their combined expressions in Sudanese B-CLL patients and their relationships with clinical and hematological characteristics as well as the disease staging at presentation. RESULTS: In the present cross-sectional descriptive study, analysis of ZAP-70 expression showed that 36/110 (32.7%) patients positively expressed ZAP-70 and insignificant higher presentation in intermediate and at advanced stages as well as no correlation was seen with hematological parameters and clinical features compared with negatively ZAP-70, on the other hand, 41/110 (37.3%) were CD38+ and no significant correlation was shown with the stage at presentation, clinical characteristics (except Splenomegaly, P = 0.02) and hematological parameters. However, in combined expressions of both ZAP-70 and CD38 together, 20/110 (18.2%) were concordantly ZAP-70+/CD38+, 53/110 (48.2%) concordantly ZAP-70-/CD38- and 37/110 (33.6%) either ZAP-70+ or CD38+, and these three groups showed insignificant correlation with clinical (except Splenomegaly, P = 0.03) and hematological parameters, and the stage at presentation. Our data showed the combined analysis of these two markers, lead to classify our patients into three subgroups (either concordant positive, negative or discordant expressions) with statistically insignificant correlation with clinical presentation (except Splenomegaly), hematological parameters and stage at presentation of B-CLL patients.

Altered recruitment of Lyn, Syk and ZAP-70 into lipid rafts of activated B cells in Systemic Lupus Erythematosus.

There is evidence that B cells from patients with Systemic Lupus Erythematosus (SLE) could be hyperactivated due to changes in their lipid rafts (LR) composition, leading to altered BCR-dependent signals. This study aimed to characterize possible alterations in the recruitment of protein tyrosine kinases (PTK) into B cells LR from SLE patients. Fifteen patients with SLE and ten healthy controls were included. Circulating B cells were isolated by negative selection and stimulated with goat Fab´2 anti-human IgM/IgG. LR were isolated with a non-ionic detergent and ultracentrifuged on 5-45% discontinuous sucrose gradients. Proteins from each fraction were analyzed by Western Blot. Total levels of Lyn, Syk, and ZAP-70 in resting B cells were similar in SLE patients and healthy controls. Upon BCR activation, Lyn, Syk and ZAP-70 recruitment into LR increased significantly in B cells of healthy controls and patients with inactive SLE. In contrast, in active SLE patients there was a great heterogeneity in the recruitment of signaling molecules and the recruitment of ZAP-70 was mainly observed in patients with decreased Syk recruitment into LR of activated B cells. The reduction in Flotilin-1 and Lyn recruitment in SLE patients seem to be associated with disease activity. These findings suggest that in SLE patients the PTK recruitment into B cell LR is dysregulated and that B cells are under constant activation through BCR signaling. The decrease of Lyn and Syk, the expression of ZAP-70 by B cells and the increase in Calcium fluxes in response to BCR stimulation in active SLE patients, further support that B cells from SLE patients are under constant activation through BCR signaling, as has been proposed.

TCR microclusters form spatially segregated domains and sequentially assemble in calcium-dependent kinetic steps.

Engagement of the T cell receptor (TCR) by stimulatory ligand results in the rapid formation of microclusters at sites of T cell activation. Whereas microclusters have been studied extensively using confocal microscopy, the spatial and kinetic relationships of their signaling components have not been well characterized due to limits in image resolution and acquisition speed. Here we show, using TIRF-SIM to examine the organization of microclusters at sub-diffraction resolution, the presence of two spatially distinct domains composed of ZAP70-bound TCR and LAT-associated signaling complex. Kinetic analysis of microcluster assembly reveal surprising delays between the stepwise recruitment of ZAP70 and signaling proteins to the TCR, as well as distinct patterns in their disassociation. These delays are regulated by intracellular calcium flux downstream of T cell activation. Our results reveal novel insights into the spatial and kinetic regulation of TCR microcluster formation and T cell activation.

TLR2 Expression on Leukemic B Cells from Patients with Chronic Lymphocytic Leukemia.

Antigenic stimulation is considered as a possible trigger of neoplastic transformation in chronic lymphocytic leukemia (CLL). B-cell receptor plays a key role in the interactions between the microenvironment and leukemic cells; however, an important role has also been attributed to Toll-like receptors (TLRs). It is believed that disorders of TLR expression may play a part in the pathogenesis of CLL. In this study, we investigated the potential role of TLR2 in CLL by analyzing its expression on leukemic B cells in correlation with clinical and laboratory parameters characterizing disease activity and patients' immune status. We assessed the frequencies of TLR2+/CD19+ cells by the flow cytometry method in peripheral blood of 119 patients with CLL. The percentage of TLR2+/CD19+ cells was significantly lower in patients with CLL as compared to the healthy volunteers. There was also a lower percentage of TLR2+/CD19+ cells in CLL patients with poor prognostic factors, such as ZAP70 and/or CD38 expression, 17p and/or 11q deletion. On the other hand, among patients with del(13q14) associated with favorable prognosis, the percentage of TLR2+/CD19+ cells was higher than among those with del(11q22) and/or del(17p13) as well as in the control group. We found an association between low percentage of CD19+/CD5+/TLR2+ cells and shorter time to treatment. We also demonstrated the relationship between low percentage of CD19+/CD5+ TLR2-positive and overall survival (OS) of CLL patients. CLL patients with a proportion of 1.6% TLR2-positive B CD5+ cells (according to the receiver operating characteristic curve analysis) or more had a longer time to treatment and longer OS than the group with a lower percentage of TLR2 positive cells. To sum up, the results of the study suggest that low TLR2 expression is associated with poor prognosis in CLL patients. The monitoring of CD19+/CD5+/TLR2+ cells number may provide useful information on disease activity. Level of TLR2 expression on leukemic B cells may be an important factor of immunological dysfunction for patients with CLL. Our study suggests that TLR2 could becomes potential biological markers for the clinical outcome in patients with CLL.

NF-κB inducing kinase (NIK) is an essential post-transcriptional regulator of T-cell activation affecting F-actin dynamics and TCR signaling.

NF-κB inducing kinase (NIK) is the key protein of the non-canonical NF-κB pathway and is important for the development of lymph nodes and other secondary immune organs. We elucidated the specific role of NIK in T cells using T-cell specific NIK-deficient (NIKΔT) mice. Despite showing normal development of lymphoid organs, NIKΔT mice were resistant to induction of CNS autoimmunity. T cells from NIKΔT mice were deficient in late priming, failed to up-regulate T-bet and to transmigrate into the CNS. Proteomic analysis of activated NIK-/- T cells showed de-regulated expression of proteins involved in the formation of the immunological synapse: in particular, proteins involved in cytoskeleton dynamics. In line with this we found that NIK-deficient T cells were hampered in phosphorylation of Zap70, LAT, AKT, ERK1/2 and PLCγ upon TCR engagement. Hence, our data disclose a hitherto unknown function of NIK in T-cell priming and differentiation.

Regulatory mechanisms in T cell receptor signalling.

The remarkable T cell receptor (TCR) performs essential functions in the initiation of intracellular signals required for T cell development, repertoire selection and effector responses to foreign antigens. How TCR signals elicit such diverse cellular responses and outcomes remains a major question for investigation. Recent years have witnessed important advances in our understanding of the regulatory processes that control and modulate the TCR signalling response. Here, we review newly identified mechanisms for the regulation of TCR signalling and then discuss how the TCR signalling response is regulated to control two critical cellular processes - namely, positive selection and T cell homeostasis.

Plasma membrane LAT activation precedes vesicular recruitment defining two phases of early T-cell activation.

The relative importance of plasma membrane-localized LAT versus vesicular LAT for microcluster formation and T-cell receptor (TCR) activation is unclear. Here, we show the sequence of events in LAT microcluster formation and vesicle delivery, using lattice light sheet microscopy to image a T cell from the earliest point of activation. A kinetic lag occurs between LAT microcluster formation and vesicular pool recruitment to the synapse. Correlative 3D light and electron microscopy show an absence of vesicles at microclusters at early times, but an abundance of vesicles as activation proceeds. Using TIRF-SIM to look at the activated T-cell surface with high resolution, we capture directed vesicle movement between microclusters on microtubules. We propose a model in which cell surface LAT is recruited rapidly and phosphorylated at sites of T-cell activation, while the vesicular pool is subsequently recruited and dynamically interacts with microclusters.

Correction of T cell deficiency in ZAP-70 knock-out mice by simple intraperitoneal adoptive transfer of thymocytes.

The tyrosine kinase zeta chain-associated protein of 70 kDa (ZAP-70) plays a key role in T cell development and signalling. In the absence of ZAP-70, T cell development is arrested in the CD4+ CD8+ double-positive stage, thus ZAP-70 homozygous knockout (ZAP-70-/- ) mice have no mature T cells in their peripheral lymphoid organs and blood, causing severe immunodeficiency. We investigated the early kinetics and long-term effects of wild-type thymocyte transfer on T cell repopulation in ZAP-70-/- mice. We used a single intraperitoneal (i.p.) injection to deliver donor thymocytes to the recipients. Here, we show that after i.p. injection donor thymocytes leave the peritoneum through milky spots in the omentum and home to the thymus, where donor-originated CD4- CD8- double-negative thymocytes most probably restore T cell development and the disrupted thymic architecture. Subsequently, newly developed, donor-originated, single-positive αß T cells appear in peripheral lymphoid organs, where they form organized T cell zones. The established chimerism was found to be stable, as donor-originated cells were present in transferred ZAP-70-/- mice as late as 8 months after i.p. injection. We demonstrate that a simple i.p. injection of ZAP-70+/+ thymocytes is a feasible method for the long-term reconstitution of T cell development in ZAP-70-deficient mice.

CCR7 Is Recruited to the Immunological Synapse, Acts as Co-stimulatory Molecule and Drives LFA-1 Clustering for Efficient T Cell Adhesion Through ZAP70.

The chemokine receptor CCR7 guides T cells and dendritic cells to and within lymph nodes to launch the onset of adaptive immunity. Here, we demonstrate that CCR7 in addition acts as a potent co-stimulatory molecule in T cell activation. We found that antigen recognition and engagement of the TCR results in CCR7 accumulation at the immunological synapse where CCR7 and the TCR co-localize within sub-synaptic vesicles. We demonstrate that CCR7 triggering alone is sufficient to recruit and activate ZAP70, a critical kinase for T cell activation, through Src kinase, whereas TCR CCR7 co-stimulation results in increased and prolonged ZAP70 kinase activity. Finally, we show that ZAP70, acting as adapter molecule, is critical for CCR7-mediated inside-out signaling to integrins, thereby modulating LFA-1 valency regulation to promote cell adhesion, a key step in immunological synapse formation and efficient T cell activation.