Role In TCR Signaling Research Articles

Abstract 6805: CD81 and CD82 in tumor infiltrating lymphocytes within the NSCLC tumor microenvironment play a crucial role in T cell activation and cytokine production

Abstract Background: The role of tetraspanins, particularly CD81 and CD82, in T cell activation, shedding light on their contribution to anti-tumor effects in the tumor microenvironment. The findings provide insights for potential applications of these tetraspanins in immunotherapy. Method: The study employed 10x Genomics for single-cell RNA sequencing and processed the data using Seurat. It focused on the tumor microenvironment, categorizing cell types, especially TIL subsets. T cells were sorted based on CD81 and CD82 expression and activated, and their activation markers and cytokine production were analyzed. Bulk RNA sequencing was performed on both sorted T cells and tumor samples, and the TCR repertoire was examined. Cytotoxicity of CD81 and CD82 transduced cells was measured using the xCELLigence RTCA HT system. Result: Tetraspanins have been linked to T cell co-stimulation and activation, with CD81 and CD82 playing a significant role in TCR signaling and activation. Single-cell and bulk RNA sequencing data from lung cancer patients reveal high expression of CD81 and CD82 in the tumor microenvironment, especially in T cells, correlating with T cell activation and cytokine production. In vitro experiments confirm the activation and effector cytokines, especially IFN-γ, TNF-α, and IL-2, producing potential of T cells expressing CD81 and CD82, suggesting their importance in anti-cancer immunity. Overexpression of CD81 and CD82 enhances T cell cytotoxicity against cancer cells, further supporting their role in T cell function. Despite some limitations, these findings shed light on the significant impact of tetraspanins in modulating immune responses, particularly in the context of cancer immunotherapy. Conclusion: We report that CD81 and CD82 serve as specific markers for activated T cells within the TME. CD81 and CD82 are correlated with the activation and regulation of T cells, and they may also mediate the regulation of relevant cytokines. Overall, our findings provide better insights into the molecular mechanisms regulating TILs present in the TME of non-small cell carcinoma and propose CD81 and CD82 as associated markers. These discoveries could serve as a foundation for the development of new immunotherapies targeting the TME. Citation Format: Kwangmin Na, Seul Lee, Dong Kwon Kim, Young Seob Kim, Joon Yeon Hwang, Seong-san Kang, Sujeong Baek, Chai Young Lee, Seung Min Yang, Yu Jin Han, Mi hyun Kim, Heekyung Han, Youngtaek Kim, Jae Hwan Kim, Seunghyun Jeon, Youngseon Byeon, Jii Bum Lee, Sun Min Lim, Min Hee Hong, Kyoung-Ho Pyo, Byoung Chul Cho. CD81 and CD82 in tumor infiltrating lymphocytes within the NSCLC tumor microenvironment play a crucial role in T cell activation and cytokine production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6805.

The Alternative Splicing of Lck in Treg Induction in Immune Thrombocytopenia

Background： Immune intolerance, causing the production of autoantibodies, is thought to be one of the main pathogenesis of immune thrombocytopenia (ITP). As regulatory T cells (Tregs) are one of the most important cells to maintain immune tolerance and the phenotype, function, and importance of Tregs have been well defined over the past decade, extensive efforts have been devoted to unveiling the dysregulation of Tregs in ITP. It has been reported that the proportion of Tregs in peripheral blood of ITP patients is reduced and the immunosuppressive function of Tregs is impaired, but the mechanism remains unclear. As peripheral Treg production can be induced from CD4+ naive T cells (NTC) with TCR-stimulated activation and TGF-β treatment, how peripheral Treg induction is impaired in patients with ITP remains to be further investigated. Method： We isolated and performed high-throughput sequencing of the CD4+ NTC from the peripheral whole blood of ITP patients and healthy controls. After analyzing the differential expression and splicing pattern, we screened several genes of interest. In addition, a minigene plasmid of the target gene was constructed and RNA-chromatin immunoprecipitation (RNA-ChIP) was performed to explore the upstream splicing factor. PCR，Real-time quantitative PCR and Western Blot were used to validate the expression level and splicing pattern of various genes. Results： Our results revealed the proportion of Tregs induced from CD4+ NTC of ITP patients was decreased in vitro, suggesting the abnormal Treg induction in ITP patients. The differentially expressed genes of the CD4+ NTC between ITP patients and healthy controls were significantly enriched in the alternative splicing pathway based on high-throughput RNA-seq data. Further exploration of the differentially spliced genes revealed significant differences in the exon 8 skipping in the LCK gene between healthy controls and ITP patients, as LCK plays a crucial role in TCR signaling and TCR signaling is essential for Treg induction. Overexpressing the exon 8 spliced LCK, other than overexpressing the exon 8 included LCK in T cells limited the TCR activation. Furthermore, we found splicing factor X could recognize the exon 8 of LCK and promote the inclusion of exon 8 of LCK. Splicing factor X was lowly expressed in the CD4+ NTC of ITP patients. Accordingly, we proposed the hypothesis that the lower expressed splicing factor X in the CD4+ NTC of ITP patients may limit peripheral Treg induction by regulating LCK alternative splicing in ITP. By using RNA-CHIP, we found splicing factor X specifically bound to the exon 8 of LCK. Moreover, the administration of antisense oligos, which could mask the binding site, suppressed the recognition of the exon 8 of LCK and enhanced the exon skipping events of LCK transcripts. As expected, ASO transfection inhibited the Treg induction in CD4+ NTCs from healthy controls, which may mimic the Treg induction in ITP patients. Conclusion： Here we found splicing factor X was downregulated in the CD4+ NTC of ITP patients, which resulted in the exon 8 skipping in the LCK gene and a down-regulated TCR signaling, thereby limiting the induction of peripheral Tregs. This study will explore the pathogenesis of ITP from a novel perspective, and provide new ideas and targets for the clinical treatment of ITP.

Mutation of the Polyproline Sequence in CD3ε Evidences TCR Signaling Requirements for Differentiation and Function of Pro-Inflammatory Tγδ17 Cells.

Tγδ17 cells have emerged as a key population in the development of inflammatory and autoimmune conditions such as psoriasis. Thus, the therapeutic intervention of Tγδ17 cells can exert protective effects in this type of pathologies. Tγδ cells commit to IL-17 production during thymus development, and upon immune challenge, additional extrathymic signals induce the differentiation of uncommitted Tγδ cells into Tγδ17 effector cells. Despite the interest in Tγδ17 cells during the past 20 years, the role of TCR signaling in the generation and function of Tγδ17 cells has not been completely elucidated. While some studies point to the notion that Tγδ17 differentiation requires weak or no TCR signaling, other works suggest that Tγδ17 require the participation of specific kinases and adaptor molecules downstream of the TCR. Here we have examined the differentiation and pathogenic function of Tγδ17 cells in “knockin” mice bearing conservative mutations in the CD3ε polyproline rich sequence (KI-PRS) with attenuated TCR signaling due to lack of binding of the essential adaptor Nck. KI-PRS mice presented decreased frequency and numbers of Tγδ17 cells in adult thymus and lymph nodes. In the Imiquimod model of skin inflammation, KI-PRS presented attenuated skin inflammation parameters compared to wild-type littermates. Moreover, the generation, expansion and effector function Tγδ17 cells were impaired in KI-PRS mice upon Imiquimod challenge. Thus, we conclude that an intact CD3ε-PRS sequence is required for optimal differentiation and pathogenic function of Tγδ17 cells. These data open new opportunities for therapeutic targeting of specific TCR downstream effectors for treatment of Tγδ17-mediated diseases.

ZAP70, too little, too much can lead to autoimmunity.

Establishing both central and peripheral tolerance requires the appropriate TCR signaling strength to discriminate self‐ from agonist‐peptide bound to self MHC molecules. ZAP70, a cytoplasmic tyrosine kinase, directly interacts with the TCR complex and plays a central and requisite role in TCR signaling in both thymocytes and peripheral T cells. By studying ZAP70 hypomorphic mutations in mice and humans with a spectrum of hypoactive or hyperactive activities, we have gained insights into mechanisms of central and peripheral tolerance. Interestingly, both hypoactive and hyperactive ZAP70 can lead to the development of autoimmune diseases, albeit through distinct mechanisms. Immature thymocytes and mature T cells rely on normal ZAP70 function to complete their development in the thymus and to modulate T cell responses in the periphery. Hypoactive ZAP70 function compromises key developmental checkpoints required to establish central tolerance, allowing thymocytes with potentially self‐reactive TCRs a greater chance to escape negative selection. Such ‘forbidden clones’ may escape into the periphery and may pose a greater risk for autoimmune disease development since they may not engage negative regulatory mechanisms as effectively. Hyperactive ZAP70 enhances thymic negative selection but some thymocytes will, nonetheless, escape negative selection and have greater sensitivity to weak and self‐ligands. Such cells must be controlled by mechanisms involved in anergy, expansion of Tregs, and upregulation of inhibitory receptors or signaling molecules. However, such potentially autoreactive cells may still be able to escape control by peripheral negative regulatory constraints. Consistent with findings in Zap70 mutants, the signaling defects in at least one ZAP70 substrate, LAT, can also lead to autoimmune disease. By dissecting the similarities and differences among mouse models of patient disease or mutations in ZAP70 that affect TCR signaling strength, we have gained insights into how perturbed ZAP70 function can lead to autoimmunity. Because of our work and that of others on ZAP70, it is likely that perturbations in other molecules affecting TCR signaling strength will be identified that also overcome tolerance mechanisms and cause autoimmunity. Delineating these molecular pathways could lead to the development of much needed new therapeutic targets in these complex diseases.

Critical Role for TCR Signal Strength and MHC Specificity in ThPOK-Induced CD4 Helper Lineage Choice.

Sustained TCR signaling is critical for ThPOK induction in MHC class II (MHCII)-signaled thymocytes leading to the CD4 helper lineage commitment. ThPOK suppresses the cytotoxic program in the signaled thymocytes and is shown to be necessary and sufficient for the CD4 helper lineage choice. Accordingly, loss and gain of ThPOK function redirects MHCII- and MHC class I (MHCI)-signaled thymocytes into the CD8 cytotoxic and CD4 helper lineage, respectively. However, the impact of a defined ThPOK level on the CD4 helper lineage choice of MHCII- and MHCI-specific thymocytes and the role of TCR signaling in this process is not evaluated. Equally, it is not clear if suppression of the cytotoxic program by ThPOK is sufficient in redirecting MHCI-restricted thymocytes into the CD4 helper lineage. In this study, we have investigated CD8 to CD4 helper lineage redirection in three independent ThPOK overexpressing transgenic mouse lines. Our analysis shows that one of the transgenic lines, despite overexpressing ThPOK compared with wild-type CD4 mature T cells and compromising cytotoxic program, failed to redirect all MHCI-signaled thymocytes into the CD4 helper lineage, resulting in the continued presence of CD8+ mature T cells and the generation of a large number of double negative mature T cells. Critically, the same ThPOK transgene completely restored the CD4 helper lineage commitment of MHCII-specific Thpok -/- thymocytes. Importantly, augmenting TCR signaling significantly enhanced the ThPOK-mediated CD4 helper lineage choice of MHCI-specific thymocytes but was still substantially less efficient than that of MHCII-specific thymocytes expressing the same amount of ThPOK. Together, these data suggest that the ThPOK-induced CD4 helper lineage commitment is strongly influenced by TCR signal strength and MHC specificity of developing thymocytes.

Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells

The protein tyrosine kinase LCK plays a key role in TCR signaling, and its activity is dynamically controlled by the tyrosine kinase C-terminal Src kinase (CSK) and the tyrosine phosphatase CD45. CSK is brought in contiguity to LCK via binding to a transmembrane adaptor known as phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG). The lack of a blatant phenotype in PAG-deficient mice has impeded our understanding of the mechanisms through which PAG exerts its negative-regulatory role in TCR signaling. We used quantitative mass spectrometry and both thymocytes and CD4(+) T cells from mice in which a tag for affinity purification was knocked in the gene coding for PAG to determine the composition and dynamics of the multiprotein complexes that are found around PAG over 5 min of activation. Most of the high-confidence interactions that we observed were previously unknown. Using phosphoproteomic analysis, PAG showed low levels of tyrosine phosphorylation in resting primary mouse CD4(+) T cells; the levels of tyrosine phosphorylation increased and reached a maximum 2 min after stimulation. Analysis of the dynamics of association of the protein tyrosine phosphatase PTPN22 and lipid phosphatase SHIP-1 with PAG following T cell activation suggests that both cooperate with CSK to terminate T cell activation. Our findings provide a model of the role for PAG in mouse primary CD4(+) T cells that is consistent with recent phosphoproteomic studies of the Jurkat T cell line but difficult to reconcile with former biochemical studies indicating that PAG is constitutively phosphorylated in resting T cells and rapidly dephosphorylated once the TCR is engaged.

A Transmembrane Domain GGxxG Motif in CD4 Contributes to Its Lck-Independent Function but Does Not Mediate CD4 Dimerization.

CD4 interactions with class II major histocompatibility complex (MHC) molecules are essential for CD4+ T cell development, activation, and effector functions. While its association with p56lck (Lck), a Src kinase, is important for these functions CD4 also has an Lck-independent role in TCR signaling that is incompletely understood. Here, we identify a conserved GGxxG motif in the CD4 transmembrane domain that is related to the previously described GxxxG motifs of other proteins and predicted to form a flat glycine patch in a transmembrane helix. In other proteins, these patches have been reported to mediate dimerization of transmembrane domains. Here we show that introducing bulky side-chains into this patch (GGxxG to GVxxL) impairs the Lck-independent role of CD4 in T cell activation upon TCR engagement of agonist and weak agonist stimulation. However, using Forster’s Resonance Energy Transfer (FRET), we saw no evidence that these mutations decreased CD4 dimerization either in the unliganded state or upon engagement of pMHC concomitantly with the TCR. This suggests that the CD4 transmembrane domain is either mediating interactions with an unidentified partner, or mediating some other function such as membrane domain localization that is important for its role in T cell activation.

TRAF3 regulates CSK and PTPN22 to enhance T cell receptor signaling (IRM7P.705)

Abstract TCR signaling is critical for T cell activation, so further elucidation of this process is necessary to understanding T cell biology, and to manipulate T cell activation in clinical settings. Extensive research on TCR signaling has revealed many details, but additional information on the molecular mechanisms continues to be revealed. Our group previously produced a T cell-conditional (CD4-Cre) TRAF3-/- mouse, which demonstrated that TRAF3 plays an important positive role in TCR signaling and T cell functions. After TCR engagement, TRAF3 associates with the TCR complex, and TRAF3-/- cells display a marked reduction in the early phosphorylation of Lck and CD3-ζ activation sites. This impact of TRAF3 on proximal TCR signaling events led us to hypothesize that TRAF3 regulates CSK, a T cell membrane-localized protein that binds to and inhibits Src family kinases, such as Lck. Through immunoprecipitation, TRAF3 was shown to interact with CSK upon TCR stimulation. The interaction between TRAF3 and CSK occurs in both the membrane and cytoplasmic fractions. In the absence of TRAF3, elevated amounts of CSK remain in the plasma membrane of stimulated T cells. Another TCR inhibitor, the protein tyrosine phosphatase PTPN22, also interacts with the TRAF3:CSK complex. Similar to its impact on CSK, loss of TRAF3 results in elevated levels of PTPN22 in the plasma membrane. These data identify a new role for TRAF3 in regulating the localization of the TCR signaling inhibitors CSK and PTPN22.

AB0028 Effect of genetic polymorphism of PTPN22 (protein tyrosine phosphatase non-receptor type 22) on progress and course of disease in familial mediterrenean fever

BackgroundPTPN22 gene plays role in TCR signaling. PTPN22 gene polymorphism has been recently found to be related to several autoimmune diseases.ObjectivesThe aim of this study is to investigate the effect...

Critical Roles of the WASP N-Terminal Domain and Btk in LPS-Induced Inflammatory Response in Macrophages

While Wiskott-Aldrich syndrome protein (WASP) plays critical roles in TCR signaling as an adaptor molecule, how it transduces innate immune signals remains to be elucidated. To investigate the roles of WASP in innate immune cells, we established bone marrow-derived macrophage (BMDM) cell lines from WASP15 transgenic (Tg) mice overexpressing the WASP N-terminal region (exons 1–5). Upon LPS stimulation, WASP15 Tg BMDM cell lines produce lower levels of inflammatory cytokines, such as TNF-α, IL-6, and IL-12p40 than the wild-type BMDM cell line. In addition, the production of nitric oxide by WASP15 Tg BMDM cells in response to LPS and IFN-γ was significantly impaired. Furthermore, we uncovered that the WASP N-terminal domain associates with the Src homology (SH) 3 domain of Bruton's tyrosine kinase (Btk). Overexpression of the WASP N-terminal domain diminishes the extent of tyrosine phosphorylation of endogenous WASP in WASP15 Tg BMDM cells, possibly by interfering with the specific binding between endogenous WASP and Btk during LPS signaling. These observations strongly suggest that the interaction between WASP N-terminal domain and Btk plays important roles in the LPS signaling cascade in innate immunity.

Mechanism and function of Vav1 localization in TCR signaling

SummaryThe antigen-specific binding of T cells to antigen presenting cells results in recruitment of signalling proteins to microclusters at the cell-cell interface known as the immunological synapse (IS). The Vav1 guanine nucleotide exchange factor plays a critical role in T cell antigen receptor (TCR) signalling, leading to the activation of multiple pathways. We now show that it is recruited to microclusters and to the IS in primary CD4+ and CD8+ T cells. Furthermore, we show that this recruitment depends on the SH2 and C-terminal SH3 (SH3B) domains of Vav1, and on phosphotyrosines 112 and 128 of the SLP76 adaptor protein. Biophysical measurements show that Vav1 binds directly to these residues on SLP76 and that efficient binding depends on the SH2 and SH3B domains of Vav1. Finally, we show that the same two domains are critical for the phosphorylation of Vav1 and its signalling function in TCR-induced calcium flux. We propose that Vav1 is recruited to the IS by binding to SLP76 and that this interaction is critical for the transduction of signals leading to calcium flux.

Identification of Fyn as the binding partner for the WASP N-terminal domain in T cells

Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling. In transgenic (Tg) mice, over-expression of the WASP N-terminal region (exons 1-5) including the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain and anti-WASP-EVH1 single-chain variable fragment (scFv) intracellular expressed antibodies (intrabodies) impairs IL-2 production in activated T cells. However, it largely remains unknown that how this domain transduces TCR signaling. Here, we demonstrate for the first time that the WASP N-terminal domain specifically associates with the Fyn SH3 domain; the interaction was uncovered by screening a λgt11 cDNA expression library obtained from the mouse T-cell line KKF. The interaction between Fyn and WASP was inhibited by over-expression of the WASP N-terminal domain and anti-WASP-EVH1 scFv intrabodies in gene-transfected NIH3T3 cells and T cells derived from these Tg mice. WASP-interacting protein binding to the EVH1 domain of WASP was also inhibited in these Tg mice T cells. Furthermore, tyrosine phosphorylation of WASP and nuclear translocation of nuclear factor of activated T cells following TCR stimulation was severely inhibited by over-expression of the WASP N-terminal domain. These observations strongly suggest that the WASP N-terminal domain plays a pivotal role in the TCR signaling cascade by binding to Fyn.

TCR-Dependent Translational Control of GATA-3 Enhances Th2 Differentiation

The differentiation of CD4(+) T cells into the Th2 subset is controlled by the transcription factor GATA-3. GATA-3 is both necessary and sufficient for Th2 differentiation and works through the induction of chromatin remodeling at the Th2 effector cytokine loci. We show in this study that IL-4 stimulation induces GATA-3 mRNA upregulation, but the level of GATA-3 protein induced is insufficient for Th2 differentiation. The levels of GATA-3 protein and Th2 differentiation are enhanced by concomitant TCR signaling through the PI3K/mammalian target of rapamycin pathway. The PI3K-mediated increase in GATA-3 protein occurs without increasing the GATA-3 mRNA level. Rather, TCR signaling through PI3K specifically enhances the translation rate of GATA-3 without affecting the protein stability. Importantly, this role of TCR signaling is independent of the effects of TCR signaling in T cell survival and expansion. Thus, TCR signaling through PI3K may play a critical role in Th2 differentiation by the specific enhancement of GATA-3 translation.

Antigen-specific clonal expansion and cytolytic effector function of CD8+ T lymphocytes depend on the transcription factor Bcl11b

CD8+ T lymphocytes mediate the immune response to viruses, intracellular bacteria, protozoan parasites, and tumors. We provide evidence that the transcription factor Bcl11b/Ctip2 controls hallmark features of CD8+ T cell immunity, specifically antigen (Ag)-dependent clonal expansion and cytolytic activity. The reduced clonal expansion in the absence of Bcl11b was caused by altered proliferation during the expansion phase, with survival remaining unaffected. Two genes with critical roles in TCR signaling were deregulated in Bcl11b-deficient CD8+ T cells, CD8 coreceptor and Plcγ1, both of which may contribute to the impaired responsiveness. Bcl11b was found to bind the E8I, E8IV, and E8V, but not E8II or E8III, enhancers. Thus, Bcl11b is one of the transcription factors implicated in the maintenance of optimal CD8 coreceptor expression in peripheral CD8+ T cells through association with specific enhancers. Short-lived Klrg1hiCD127lo effector CD8+ T cells were formed during the course of infection in the absence of Bcl11b, albeit in smaller numbers, and their Ag-specific cytolytic activity on a per-cell basis was altered, which was associated with reduced granzyme B and perforin.

Consequences of Increased CD45RA and RC Isoforms for TCR Signaling and Peripheral T Cell Deficiency Resulting from Heterogeneous Nuclear Ribonucleoprotein L-Like Mutation

CD45 is the most abundant protein tyrosine phosphatase in the plasma membrane of T cells and serves a critical role in TCR signaling. Different CD45 isoforms are made by alternative mRNA splicing depending on the stage of T cell development and activation, yet their role remains unclear. Expression of CD45RA and RC isoforms is increased 20- to 200-fold on T cells from thunder mice with a loss-of-function mutation in the RNA-binding protein, heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL), although total CD45 expression is unaltered. In this study, we test the hypothesis that this shift in CD45 isoform expression alters TCR signaling, thymic selection, and accumulation of peripheral T cells. There was no discernable effect of the change in CD45 isoform expression upon Lck phosphorylation or T cell positive and negative selection, whereas these indices were strongly affected by a decrease in the overall amount of CD45 in Ptprc mutant animals. The one exception to this conclusion was in thymocytes from Ptprc(loc/loc) animals with 4% of normal CD45 protein levels, where Lck505 phosphorylation was increased 25% in Hnrpll mutant cells, suggesting that high m.w. CD45 isoforms had lower Lck505 phosphatase activity in this context. In T cells with no CD45 protein, hnRNPLL mutation still diminished peripheral T cell accumulation, demonstrating that hnRNPLL regulates T cell longevity independently from its effects on CD45 splicing.

The Role of ThPOK in Control of CD4/CD8 Lineage Commitment

During alphabeta T cell development, cells diverge into alternate CD4 helper and CD8(+) cytotoxic T cell lineages. The precise correlation between a T cell's CD8 and CD4 choice and its TCR specificity to class I or class II MHC was noted more than 20 years ago, and establishing the underlying mechanism has remained a focus of intense study since then. This review deals with three formerly discrete topics that are gradually becoming interconnected: the role of TCR signaling in lineage commitment, the regulation of expression of the CD4 and CD8 genes, and transcriptional regulation of lineage commitment. It is widely accepted that TCR signaling exerts a decisive influence on lineage choice, although the underlying mechanism remains intensely debated. Current evidence suggests that both duration and intensity of TCR signaling may control lineage choice, as proposed by the kinetic signaling and quantitative instructive models, respectively. Alternate expression of the CD4 and CD8 genes is the most visible manifestation of lineage choice, and much progress has been made in defining the responsible cis elements and transcription factors. Finally, important clues to the molecular basis of lineage commitment have been provided by the recent identification of the transcription factor ThPOK as a key regulator of lineage choice. ThPOK is selectively expressed in class II-restricted cells at the CD4(+)8(lo) stage and is necessary and sufficient for development to the CD4 lineage. Given the central role of ThPOK in lineage commitment, understanding its upstream regulation and downstream gene targets is expected to reveal further important aspects of the molecular machinery underlying lineage commitment.

CD45 Down-Regulates Lck-Mediated CD44 Signaling and Modulates Actin Rearrangement in T Cells

The tyrosine phosphatase CD45 dephosphorylates the negative regulatory tyrosine of the Src family kinase Lck and plays a positive role in TCR signaling. In this study we demonstrate a negative regulatory role for CD45 in CD44 signaling leading to actin rearrangement and cell spreading in activated thymocytes and T cells. In BW5147 T cells, CD44 ligation led to CD44 and Lck clustering, which generated a reduced tyrosine phosphorylation signal in CD45(+) T cells and a more sustained, robust tyrosine phosphorylation signal in CD45(-) T cells. This signal resulted in F-actin ring formation and round spreading in the CD45(+) cells and polarized, elongated cell spreading in CD45(-) cells. The enhanced signal in the CD45(-) cells was consistent with enhanced Lck Y394 phosphorylation compared with the CD45(+) cells where CD45 was recruited to the CD44 clusters. This enhanced Src family kinase-dependent activity in the CD45(-) cells led to PI3K and phospholipase C activation, both of which were required for elongated cell spreading. We conclude that CD45 induces the dephosphorylation of Lck at Y394, thereby preventing sustained Lck activation and propose that the amplitude of the Src family kinase-dependent signal regulates the outcome of CD44-mediated signaling to the actin cytoskeleton and T cell spreading.

Loss of Protein Kinase Cθ, Bcl10, or Malt1 Selectively Impairs Proliferation and NF-κB Activation in the CD4+ T Cell Subset

The cytosolic proteins protein kinase Ctheta (PKCtheta), Bcl10, and Malt1 play critical roles in TCR signaling to the transcription factor NF-kappaB. Our data confirm that CD4(+) T cells from PKCtheta, Bcl10, and Malt1 knockout mice show severe impairment of proliferation in response to TCR stimulation. Unexpectedly, we find that knockout CD8(+) T cells proliferate to a similar extent as wild-type cells in response to strong TCR signals, although a survival defect prevents their accumulation. Both CD4(+) and CD8(+) knockout T cells express activation markers, including CD25, following TCR stimulation. Addition of exogenous IL-2 rescues survival of knockout CD4(+) and CD8(+) T cells, but fails to overcome the proliferation defect of CD4(+) T cells. CD4(+) T cells from knockout mice are extremely deficient in TCR-induced NF-kappaB activation, whereas NF-kappaB activation is only partially impaired in CD8(+) T cells. Overall, our results suggest that defects in TCR signaling through PKCtheta, Bcl10, and Malt1 predominantly impair NF-kappaB activation and downstream functional responses of CD4(+) T cells. In contrast, CD8(+) T cells maintain substantial NF-kappaB signaling, implying the existence of a significant TCR-regulated NF-kappaB activation pathway in CD8(+) T cells that is independent of PKCtheta, Bcl10, and Malt1.

Structure, organization and expression of common carp ( Cyprinus carpio L.) SLP-76 gene

SLP-76 is an important member of the SLP-76 family of adapters, and it plays a key role in TCR signaling and T cell function. Partial cDNA sequence of SLP-76 of common carp ( Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp SLP-76 was obtained by means of 3′ RACE and 5′ RACE, respectively. The full length cDNA of carp SLP-76 was 2007 bp, consisting of a 5′-terminal untranslated region (UTR) of 285 bp, a 3′-terminal UTR of 240 bp, and an open reading frame of 1482 bp. Sequence comparison showed that the deduced amino acid sequence of carp SLP-76 had an overall similarity of 34–73% to that of other species homologues, and it was composed of an NH2-terminal domain, a central proline-rich domain, and a C-terminal SH2 domain. Amino acid sequence analysis indicated the existence of a Gads binding site R-X-X-K, a 10-aa-long sequence which binds to the SH3 domain of LCK in vitro, and three conserved tyrosine-containing sequence in the NH2-terminal domain. Then we used PCR to obtain a genomic DNA which covers the entire coding region of carp SLP-76. In the 9.2 k-long genomic sequence, twenty one exons and twenty introns were identified. RT-PCR results showed that carp SLP-76 was expressed predominantly in hematopoietic tissues, and was upregulated in thymus tissue of four-month carp compared to one-year old carp. RT-PCR and virtual northern hybridization results showed that carp SLP-76 was also upregulated in thymus tissue of GH transgenic carp at the age of four-months. These results suggest that the expression level of SLP-76 gene may be related to thymocyte development in teleosts.

Impaired LPS-induced signaling in microglia overexpressing the Wiskott Aldrich syndrome protein N-terminal domain

Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling, but its roles in signal transduction in innate immune cells have not been well characterized. As microglia are the primary immune effector cells in the brain, WASP may possibly have important roles in microglial activation, such as production of inflammatory and anti-inflammatory cytokines and neurotoxic factors. Here, we established a microglial cell line from WASP dominant-negative transgenic (Tg) mice overexpressing the N-terminal enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain. WASP Tg microglia were impaired in production of inflammatory cytokines such as tumor necrosis factor-alpha, IL-6 and IL-1beta upon LPS stimulation, whereas anti-inflammatory IL-10 production was significantly enhanced. Also, LPS-induced phosphorylation of nuclear factor kappaB was reduced in WASP Tg microglia. Furthermore, WASP Tg microglia exhibited less cytotoxicity against co-cultured neurons after stimulation by LPS and IFN-gamma, with a concordant decrease in nitric oxide production. These results strongly suggest that WASP may have pivotal roles through the EVH1 domain in the LPS signaling cascade, either directly or indirectly, and modulates inflammatory immune responses in microglia.