TCR-mediated Signaling Research Articles

DIAPH1-Deficiency is Associated with Major T, NK and ILC Defects in Humans

Loss of function mutations in Diaphanous related formin 1 (DIAPH1) are associated with seizures, cortical blindness, and microcephaly syndrome (SCBMS) and are recently linked to combined immunodeficiency. However, the extent of defects in T and innate lymphoid cells (ILCs) remain unexplored. Herein, we characterized the primary T, natural killer (NK) and helper ILCs of six patients carrying two novel loss of function mutation in DIAPH1 and Jurkat cells after DIAPH1 knockdown. Mutations were identified by whole exome sequencing. T-cell immunophenotyping, proliferation, migration, cytokine signaling, survival, and NK cell cytotoxicity were studied via flow cytometry-based assays, confocal microscopy, and real-time qPCR. CD4+ T cell proteome was analyzed by mass spectrometry. p.R351* and p.R322*variants led to a significant reduction in the DIAPH1 mRNA and protein levels. DIAPH1-deficient T cells showed proliferation, activation, as well as TCR-mediated signaling defects. DIAPH1-deficient PBMCs also displayed impaired transwell migration, defective STAT5 phosphorylation in response to IL-2, IL-7 and IL-15. In vitro generation/expansion of Treg cells from naïve T cells was significantly reduced. shRNA-mediated silencing of DIAPH1 in Jurkat cells reduced DIAPH1 protein level and inhibited T cell proliferation and IL-2/STAT5 axis. Additionally, NK cells from patients had diminished cytotoxic activity, function and IL-2/STAT5 axis. Lastly, DIAPH1-deficient patients’ peripheral blood contained dramatically reduced numbers of all helper ILC subsets. DIAPH1 deficiency results in major functional defects in T, NK cells and helper ILCs underlining the critical role of formin DIAPH1 in the biology of those cell subsets.Graphical The summary of findings are presented as a graphical abstract. DIAPH1 deficiency results in multiple defects in CD4+ T, Treg, NK cells and ILCs.

Potential therapeutic applications of targeting signal-transducing adaptor protein-2 in autoimmune diseases

Adaptor proteins are involved in various immune responses via the modulation of many signaling pathways. Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains typical domains such as the pleckstrin homology (PH) domain, Src homology domain, and a proline-rich region from the N-terminal region. In T cells, STAP-2 positively regulates T cell receptor (TCR)-mediated signaling by associating with CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) and lymphocyte-specific protein tyrosine kinase (LCK). Therefore, a peptide that inhibits the interaction between STAP-2 and CD3ζ ITAMs is likely to suppress TCR-mediated T cell activation, as well as T cell-mediated diseases. As expected, the peptide successfully inhibited the STAP-2/CD3ζ ITAM interaction and suppressed TCR-mediated signaling, cell proliferation, and interleukin (IL)-2 production in human/murine T cells. Furthermore, this inhibitor suppressed the pathogenesis of experimental autoimmune encephalomyelitis (EAE), which is widely recognized as a mouse model of multiple sclerosis, via the downregulation of T cell activation and infiltration of T helper (Th) 1/Th17 cells. These results suggest a new strategy for the treatment of multiple sclerosis and other immune diseases.

STAP-2-Derived Peptide Suppresses TCR-Mediated Signals to Initiate Immune Responses.

Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains pleckstrin and Src homology 2-like domains, as well as a proline-rich region in its C-terminal region. Our previous study demonstrated that STAP-2 positively regulates TCR signaling by associating with TCR-proximal CD3ζ ITAMs and the lymphocyte-specific protein tyrosine kinase. In this study, we identify the STAP-2 interacting regions of CD3ζ ITAMs and show that the STAP-2-derived synthetic peptide (iSP2) directly interacts with the ITAM sequence and blocks the interactions between STAP-2 and CD3ζ ITAMs. Cell-penetrating iSP2 was delivered into human and murine T cells. iSP2 suppressed cell proliferation and TCR-induced IL-2 production. Importantly, iSP2 treatment suppressed TCR-mediated activation of naive CD4+ T cells and decreased immune responses in CD4+ T cell-mediated experimental autoimmune encephalomyelitis. It is likely that iSP2 is a novel immunomodulatory tool that modulates STAP-2-mediated activation of TCR signaling and represses the progression of autoimmune diseases.

Antioxidation protein Nrf2 modulates glutamine metabolism to regulate activation-driven expansion of CD4 +T-cells

Abstract Nutrient metabolism governs the effector functions like activation, expansion, and subset differentiation of CD4 +T-cells, the orchestrators of adaptive immunity. The levels of reactive oxygen species (ROS), the metabolic byproducts of cellular metabolism are regulated by the antioxidation pathway, which is composed of Keap1 and Nrf2 proteins. We observed that, the expression of Nrf2 is temporally regulated post-activation in CD4 +T-cells (high on day 1 and low on days 2 and 3) indicating its role in CD4 +T-cell effector functions. It is yet unknown if and how the antioxidation pathway crosstalks with cellular metabolism to shape CD4 +T-cell effector responses. Here, using mice with T-cell specific deletion of Keap1 (Keap1-KO) or Nrf2 (Nrf2-KO) we deciphered the effects of high and low levels of Nrf2 (antioxidation) levels, respectively in CD4 +T-cells. Upon activation in vitro, Keap1-KO CD4 +T-cells with constitutively high levels of Nrf2 showed higher proliferation, increased expression of early activation markers and elevated TCR-mediated signaling compared to wildtype (WT) cells. In contrast, decreased proliferation and lower TCR-mediated signaling was noted in activated Nrf2-KO CD4 +T-cells. To dissect the underlying mechanisms, we performed metabolite analyses and activation in nutrient-limiting conditions. Interestingly, activated Keap1-KO CD4 +T-cells show upregulated glutamine metabolism as measured by metabolic intermediates and rely on glutamine for optimum activation and increased expansion. Together, our findings identify a mechanism by which antioxidation protein Nrf2 metabolically reprograms CD4 +T-cells and modulates their metabolic dependencies to support their activation and expansion.

CBL Syndrome: At the Intersection of Inborn Error of Immunity and Cancer Predisposition

CBL syndrome is a form of RASopathies due to heterozygous germline mutations of the Casitas B-lineage lymphoma (CBL) gene. CBL encodes the cbl family of proteins which function as E3 ubiquitin ligases that negatively regulate intracellular signaling by various receptor tyrosine kinases including RAS-MAPK pathway. Individuals with CBL syndrome have variable clinical presentation but phenotypically resembles Noonan syndrome with predisposition to JMML. Patients affected by RASopathies are at risk to develop autoimmune disorders given the role of Ras-MAPK signaling pathway in peripheral tolerance. Prior mice studies suggested the role of cbl protein as negative regulator of TCR-mediated signaling. We describe a case of CBL syndrome presenting with multiple autoimmune conditions and reviewed reported cases in literature.A 14-year old male presented with pancytopenia, autoimmune thyroiditis and organomegaly. His past medical history was remarkable for ADHD, speech delay, short stature, idiopathic hepatosplenomegaly and history of acute disseminated encephalomyelitis at age 5. His bone marrow evaluation was normal. A somatic NGS panel identified VUS in CBL (c.1193A>G, p.H398R) with VAF of 53%. Autoimmune work up showed elevated ANA, DNA antibody, RNP antibody titer and mildly positive anti-C3 antibody. Immunophenotyping revealed normal Ig profile, NK cytopenia, T cell lymphopenia, decreased naïve T cells and elevated CD8+ effector memory T cell activation. ALPS genetic panel revealed paternally inherited VUS in CTLA4 which was later determined to be benign. Since his cytopenias did not respond to IVIG, he was treated with corticosteroid with improvement in counts although he remained thrombocytopenic. A skin biopsy was performed and sequencing of patient’s fibroblast DNA demonstrated a germline origin of CBL variant. Parental DNA sequencing confirmed a de novo event. Literature review revealed 28 germline CBL mutations (Figure 1), most of which were missense mutations (78%). At least nine of these mutations (32%) have been associated with immune dysregulation including autoimmune cytopenias, aHUS, RA, autoimmune thyroiditis, vasculitis and HLH. JMML has been reported in more than half of these germline mutations. [Display omitted] In conclusion, immune dysregulation is a common feature of CBL syndrome in addition to JMML. CBL syndrome should be considered in the differential diagnosis of individuals with ALPS-like phenotypes.

Termination of TCR-mediated activation signals is regulated by CrkII-dependent Cbl-mediated ubiquitination and degradation of C3G

Crk adaptor proteins are key players in signal transduction from multiple cell surface receptors, including the T cell antigen receptor (TCR). The involvement of CrkII in the early stages of T cell activation is well documented, but little is known about its role during the termination of the activation response. We substantiated findings showing that CrkII utilizes its SH3N and SH2 domains to constitutively associate with C3G and transiently with Cbl in resting and TCR/CD3-stimulated T cells, respectively. Association of CrkII with Cbl peaks within 1 min post-TCR/CD3 stimulation, and involves the formation of multiple CrkII-containing complexes of different molecular mass. Ubiquitination of C3G commences at ∼5 min post TCR/CD3 stimulation concomitantly with its degradation. This entire process conversely correlates with the levels of expression of CrkII and is dependent on the presence of the CrkII-bound Cbl protein. The data suggest that CrkII functions as a scaffold that brings Cbl into close proximity with C3G in TCR/CD3-stimulated T cells and that tyrosine phosphorylation and activation of Cbl promotes C3G ubiquitination and degradation. We suggest that this mechanism contributes to the termination of the TCR/CD3-induced activation signal and helps tune the length and intensity of T cell-mediated immune responses.

Expression of membrane Hsp90 is a molecular signature of T cell activation

Heat shock protein 90 (Hsp90) maintains cellular proteostasis during stress and has been under investigation as a therapeutic target in cancer for over two decades. We and others have identified a membrane expressed form of Hsp90 (mHsp90) that previously appeared to be restricted to rapidly proliferating cells exhibiting a metastatic phenotype. Here, we used HS-131, a fluor-tethered mHsp90 inhibitor, to quantify the effect of T cell activation on the expression of mHsp90 in human and mouse T cells. In cell-based assays, stimulation of human T cells induced a 20-fold increase in mHsp90 expression at the plasma membrane, suggesting trafficking of mHsp90 is regulated by TCR and inflammatory mediated signaling. Following injection of HS-131 in mouse models of human rheumatoid arthritis and inflammatory bowel disease, we detected localization of the probe at sites of active disease, consistent with immune cell invasion. Moreover, despite rapid hepatobiliary clearance, HS-131 demonstrated efficacy in reducing the mean clinical score in the CIA arthritis model. Our results suggest mHsp90 expression on T cells is a molecular marker of T cell activation and potentially a therapeutic target for chronic diseases such as rheumatoid arthritis.

072 The anti-inflammatory cytokine IL-37 inhibits CD4+ T cell activation through the receptor IL-1R8 and supports regulatory T cells

IL-1 family member IL-37 inhibits both innate and adaptive immune responses. Previously, we have shown that intracellular IL-37 expression in monocytes and dendritic cells reduces inflammation and induces immune tolerance. However, the role of extracellular IL-37 on T cell activation remains unknown. Extracellularly, IL-37 has been found to form a tripartite ligand-receptor complex containing IL-1R8 (SIGIRR/TIR8) and IL-18Ra in monocytes. We used human CD4 T cells from healthy donors, transgenic mice expressing human IL-37, and the mice deficient in IL-1R8 to study the role of extracellular IL-37 on T cell activation. Treatment of human CD4+ T cells with recombinant human IL-37 reduced TCR-mediated signaling activation, T-cell proliferation, and pro-inflammatory cytokine production. Because IL-37 was highly expressed in human regulatory T (Treg) cells, we compared the effects of recombinant IL-37 in conventional T (Tconv) cells and Treg cells using human CD4+ T cells, wild-type (WT) mice, IL-37 transgenic mice, and IL-1R8-deficient mice. Recombinant IL-37 directly inhibited the canonical T-cell signaling pathways through IL-1R8 in Tconv cells, indicating a direct role of anti-inflammatory cytokine IL-37 in suppressing CD4+ Tconv cell-mediated immunity. On the other hand, recombinant IL-37 reduced Treg cell activation without affecting Treg-specific gene expression, suggesting that endogenous IL-37 expression helps Treg cells retain high levels of Treg cell gene signatures even in the presence of extracellular IL-37. IL-37’s inhibitory definition now includes a direct role in CD4+ T cell-mediated adaptive immunity.

STAP-2 Is a Novel Positive Regulator of TCR-Proximal Signals.

TCR ligation with an Ag presented on MHC molecules promotes T cell activation, leading to the selection, differentiation, and proliferation of T cells and cytokine production. These immunological events are optimally arranged to provide appropriate responses against a variety of pathogens. We here propose signal-transducing adaptor protein-2 (STAP-2) as a new positive regulator of TCR signaling. STAP-2-deficient T cells showed reduced, whereas STAP-2-overexpressing T cells showed enhanced, TCR-mediated signaling and downstream IL-2 production. For the mechanisms, STAP-2 associated with TCR-proximal CD3ζ immunoreceptor tyrosine activation motifs and phosphorylated LCK, resulting in enhancement of their binding after TCR stimulation. In parallel, STAP-2 expression is required for full activation of downstream TCR signaling. Importantly, STAP-2-deficient mice exhibited slight phenotypes of CD4+ T-cell-mediated inflammatory diseases, such as experimental autoimmune encephalomyelitis, whereas STAP-2-overexpressing transgenic mice showed severe phenotypes of these diseases. Together, STAP-2 is an adaptor protein to enhance TCR signaling; therefore, manipulating STAP-2 will have an ability to improve the treatment of patients with autoimmune diseases as well as the chimeric Ag receptor T cell therapy.

Mechanism of Tim-3 regulation of CD8+ T cell function

Abstract Tim-3 or transmembrane immunoglobulin and mucin domain-3 is a type I membrane protein expressed by various immune cell types, and has been shown to have a co-stimulatory role in T cells through the PI3K pathway. Tim-3 comprises extracellular, transmembrane and intracellular domains, the latter of which contains five tyrosine residues. We and others have shown that these tyrosines participate in TCR-mediated signaling pathways. Using LCMV infection, we also found that Tim-3 expression influences the formation of short-lived effector and memory precursor CD8+ T cells. We hypothesize that Tim-3 signaling through the tyrosine residues in its cytoplasmic domain modulates CD8+ T cell activation and differentiation. To test this hypothesis, we have used LCMV-specific TCR transgenic (P14) mice crossed to truncated version of Tim-3 and CD8+ conditional deletion of Tim-3. CD8+ T cell activation and memory were analyzed by flow cytometry after infection with LCMV Armstrong to induce an acute infection. The effects of Tim-3 on CD8+ T cells were found to be most prominent at the effector stage of infection and RNA sequencing has shown that loss of Tim-3 signaling promotes acquisition of a memory-like phenotype. Transcription factors T-bet and Blimp-1 were found to be lower in Tim-3 KO CD8+ T cells, while Tcf-1 maintained at higher levels in the absence of Tim-3 signaling. Cytokine production by effector CD8+ T cells was also found to be lower when Tim-3 was absent. Thus, we conclude that Tim-3 signaling via its cytoplasmic tyrosine residues plays a role in CD8+ T cells in an acute infection. These results may lead to a better understanding of CD8+ T cell activation and memory development in different settings. Supported by R01 AI138504-01A1

Enhanced cytotoxic CD8+ T cell (CTL) antigen recognition by LCK activation

Abstract The anti-tumor response of cytotoxic T cells requires detection of tumor-derived antigenic peptide-MHC complex (pMHC) by the T cell antigen receptor (TCR). Strategies that boost TCR signaling caused by weak pMHC could augment the recognition of poorly immunogenic tumors by T cells. Because LCK kinase phosphorylates the TCR complex and the recruited ZAP-70 kinase, increased LCK activity has been shown to increase TCR signaling in naïve T cells. Our previous study demonstrated that a small molecule inhibitor of CSK kinase, a negative regulator of LCK, increased LCK kinase activity and sensitized naïve T cells such that they became activated by weak antigens ex vivo. Our goal was to determine whether CD8+ cytotoxic T cells (CTL) could also be sensitized to detect weak antigens, and evaluate whether their capacity to recognize and kill cancer cells could be augmented. Because the Nur77-GFP reporter system responds to TCR-mediated signaling, we used it to determine that LCK activation sensitized OT-1 TCR transgenic CTLs to detect weak (low affinity) antigens displayed by splenocytes. We also observed increased phosphorylation of ZAP-70 and other signaling mediators upon TCR stimulation. We will next evaluate whether CTL-mediated effector functions can be enhanced by monitoring killing of MC38 cancer cells that display a weak antigen. Our findings will inform whether LCK kinase can be targeted to improve the recognition of cancer by T cells.

IL-2 Signaling Couples the MAPK and mTORC1 Axes to Promote T Cell Proliferation and Differentiation in Teleosts.

IL-2 is a pleiotropic cytokine that is critical for T cell immunity. Although the IL-2-mediated regulation of T cell immunity in mammals is relatively well understood, it remains largely unknown whether and how IL-2 regulates T cell immunity in lower vertebrates. To address this knowledge gap, we investigated the role played by IL-2 in the regulation of T cell response, as well as the associated underlying mechanisms in a teleost fish, large yellow croaker (Larimichthys crocea). We found that large yellow croaker (L. crocea) IL-2 (LcIL-2) significantly promoted T cell proliferation both in vivo and in vitro; significantly induced the differentiation of Th1, Th2, regulatory T, and cytotoxic T cells while inhibiting Th17 differentiation; and participated in the elimination of invading pathogenic bacteria. Mechanistically, the binding of LcIL-2 to its heterotrimer receptor complex (LcIL-15Rα/LcIL-2Rβ/Lcγc) triggered the conserved JAK-STAT5 pathway, which in turn regulated the expression of genes involved in T cell expansion, differentiation, and biological function. The MAPK and mammalian target of rapamycin complex 1 (mTORC1) axes, which are involved in TCR-mediated signaling, were also required for LcIL-2-mediated T cell response. Collectively, our results demonstrated that fish IL-2 plays a comprehensive regulatory role in T cell response and highlighted the complex and delicate network regulating T cell-driven immune response. We propose that T cell immunity is regulated by the interplay between TCR signaling and cytokine signaling, and that this basic strategy evolved before the emergence of the tetrapod lineage. Our findings provide valuable insights into the regulatory mechanisms underlying T cell response in teleosts.

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

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

Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

Immune checkpoint blockade (ICB) therapies such as PD-1 antibodies have produced significant clinical responses in treating a variety of human malignancies, yet only a subset of cancer patients benefit from such therapy. To improve the ICB efficacy, combinations with additional therapeutics were under intensive investigation. Recently, special dietary compositions that can lower the cancer risk or inhibit cancer progression have drawn significant attention, although few were reported to show synergistic effects with ICB therapies. Interestingly, Fucoidan is naturally derived from edible brown algae and exhibits antitumor and immunomodulatory activities. Here we discover that fucoidan-supplemented diet significantly improves the antitumor activities of PD-1 antibodies in vivo. Specifically, fucoidan as a dietary ingredient strongly inhibits tumor growth when co-administrated with PD-1 antibodies, which effects can be further strengthened when fucoidan is applied before PD-1 treatments. Immune analysis revealed that fucoidan consistently promotes the activation of tumor-infiltrating CD8+ T cells, which support the evident synergies with ICB therapies. RNAseq analysis suggested that the JAK-STAT pathway is critical for fucoidan to enhance the effector function of CD8+ T cells, which could be otherwise attenuated by disruption of the T-cell receptor (TCR)/CD3 complex on the cell surface. Mechanistically, fucoidan interacts with this complex and augments TCR-mediated signaling that cooperate with the JAK-STAT pathway to stimulate T cell activation. Taken together, we demonstrated that fucoidan is a promising dietary supplement combined with ICB therapies to treat malignancies, and dissected an underappreciated mechanism for fucoidan-elicited immunomodulatory effects in cancer.

Inflammatory signals are sufficient to elicit TOX expression in mouse and human CD8+ T cells.

T cell receptor (TCR) stimulation leads to the expression of the transcription factor thymocyte selection–associated high-mobility group box (TOX). Prolonged TCR signaling, such as encountered during chronic infections or in tumors, leads to sustained TOX expression, which is required for the induction of a state of exhaustion or dysfunction. Although CD8+ memory T (Tmem) cells in mice typically do not express TOX at steady state, some human Tmem cells express TOX but appear fully functional. This seeming discrepancy between mouse and human T cells has led to the speculation that TOX is differentially regulated between these species, which could complicate the interpretation of preclinical mouse model studies. We report here that, similar to TCR-mediated signals, inflammatory cytokines are also sufficient to increase TOX expression in human and mouse Tmem cells. Thus, TOX expression is controlled by the environment, which provides an explanation for the different TOX expression patterns encountered in T cells isolated from specific pathogen–free laboratory mice versus humans. Finally, we report that TOX is not necessary for cytokine-driven expression of programmed cell death 1. Overall, our data highlight that the mechanisms regulating TOX expression are conserved across species and indicate that TOX expression reflects a T cell’s activation state and does not necessarily correlate with T cell dysfunction.

Wiskott-Aldrich Syndrome Protein: Roles in Signal Transduction in T Cells.

Signal transduction regulates the proper function of T cells in an immune response. Upon binding to its specific ligand associated with major histocompatibility complex (MHC) molecules on an antigen presenting cell, the T cell receptor (TCR) initiates intracellular signaling that leads to extensive actin polymerization. Wiskott-Aldrich syndrome protein (WASp) is one of the actin nucleation factors that is recruited to TCR microclusters, where it is activated and regulates actin network formation. Here we highlight the research that has focused on WASp-deficient T cells from both human and mice in TCR-mediated signal transduction. We discuss the role of WASp in proximal TCR signaling as well as in the Ras/Rac-MAPK (mitogen-activated protein kinase), PKC (protein kinase C) and Ca2+-mediated signaling pathways.

The transcription factor T cell factor 1 (TCF1) expression is elevated in vaccine-elicited CD8+ T cells and negatively regulated by T-bet

Abstract T cells that express high levels of TCF1 display stem cell-like properties and give rise to effector-like cells that protect during chronic infections and cancer. Due to their increased responsiveness to checkpoint blockade, TCF1hi T cells are now recognized as promising targets for immunotherapies. We found that adjuvanted subunit vaccine-elicited T cells (Tvac) express significantly higher levels of TCF1 than T cells responding to infections (Tinf). In exploring the underlying causes for such high expression of TCF1 in Tvac, we examined the role of sustained TCR and inflammatory mediators in Tvac TCF1 regulation. Using Nr4a3-Tocky reporter mice and pMHCI antibody blockade, we found that TCR mediated signals regulate Tvac TCF1 to a limited degree. In line with previous reports, TCF1 expression in Tvac inversely correlated with adjuvant-elicited pro-inflammatory signals, increasing concomitantly with the rapid decline of cytokines after adjuvant administration. Further, the addition of TLR9 ligand CpG increased inflammatory cytokine production as it reduced TCF1 expression. However, in contrast to previous work, anti-IL-12 or anti-IFNγ treatment failed to reverse the lower TCF1 expression induced by CpG. More surprisingly, we found that Tvac TCF1 is regulated independently of Foxo1 even while Foxo1 plays its canonical role in facilitating TCF1 expression under inflammatory conditions. Inflammation does however negatively regulate TCF1 in a T-bet dependent manner, such that T-bet KO Tvac maintained high TCF1 expression even when inflammation was increased. Our data highlight differences in the transcriptional regulation of vaccine- and infection-elicited T cells and provide insights into the mechanism of TCF1 regulation.

Engineering T cells with improved TCR-CD3 interaction for optimal tumor killing

Abstract T cell activation requires extracellular stimulatory signals mainly mediated by T cell receptor complex (TCR/CD3). Obtaining detailed mechanistic knowledge of the TCR-mediated signaling pathway holds significant importance in understanding immune diseases, formulating new immunotherapies, and overcoming immunosuppression in current therapies. Here, we used a novel strategy to overcome ineffective anti-tumor responses by modifying the TCR interaction with CD3 subunits without affecting the binding affinity and/or specificity of the TCR-antigen interaction. Based on NMR and mutational studies, we introduced specific alanine mutations in the Cβ helix 3 and Cβ helix 4 – F strand of the TCR extracellular domain. We demonstrated varied functionality in terms of IL-2 production as well as altered bond lifetime measured by biomembrane force probe assay (BFP). Selected mutations retained or displayed enhanced CD3 binding ability in a CD3 tetramer assay. Based on these results we generated retroviral TCR libraries by random mutagenesis at specific extracellular TCR-CD3 interaction sites, expressed them in a T cell hybridoma system and selected TCRs with novel binding specificity using soluble CD3γɛ- and CD3δɛ-tetramers. We are currently determining the correlation between CD3 tetramer binding, bond lifetime, and functionality (kinase activity, IL-2 production, and tumor rejection) of selected TCR mutants. By integrating binding data, T cell signaling data, and functional outcomes, we expect to be able to manipulate the human immune system via the extracellular TCR-CD3 interaction site to be utilized in novel human T cell therapies.

The membrane proximal proline-rich region and correct order of C-terminal tyrosines on the adaptor protein LAT are required for TCR-mediated signaling and downstream functions

The primary activating receptor for T cells is the T cell receptor (TCR), which is stimulated upon binding to an antigen/MHC complex. TCR activation results in the induction of regulated signaling pathways vital for T cell differentiation, cellular adhesion and cytokine release. A critical TCR-induced signaling protein is the adaptor protein LAT. Upon TCR stimulation, LAT is phosphorylated on conserved tyrosines, which facilitates the formation of multiprotein complexes needed for propagation of signaling pathways. Although the role of the conserved tyrosines in LAT-mediated signaling has been investigated, few studies have examined the role of larger regions of LAT in TCR-induced pathways. In this study, a sequence alignment of 97 mammalian LAT proteins was used to identify several “functional” domains on LAT. Using LAT mutants expressed in Jurkat E6.1 cells, we observed that the membrane proximal, proline-rich region of LAT and the correct order of domains containing conserved tyrosines are necessary for optimal TCR-mediated early signaling, cytokine production, and cellular adhesion. Together, these data show that LAT contains distinct regions whose presence and correct order are required for the propagation of TCR-mediated signaling pathways.

Tonic TCR Signaling Inversely Regulates the Basal Metabolism of CD4+ T Cells.

The contribution of self-peptide-MHC signaling in CD4+ T cells to metabolic programming has not been definitively established. In this study, we employed LLO118 and LLO56, two TCRtg CD4+ T cells that recognize the same Listeria epitope. We previously have shown that LLO56 T cells are highly self-reactive and respond poorly in a primary infection, whereas LLO118 cells, which are less self-reactive, respond well during primary infection. We performed metabolic profiling and found that naive LLO118 had a dramatically higher basal respiration rate, a higher maximal respiration rate, and a higher glycolytic rate relative to LLO56. The LLO118 cells also exhibited a greater uptake of 2-NBD-glucose, in vitro and in vivo. We extended the correlation of low self-reactivity (CD5lo) with high basal metabolism using two other CD4+ TCRtg cells with known differences in self-reactivity, AND and Marilyn. We hypothesized that the decreased metabolism resulting from a strong interaction with self was mediated through TCR signaling. We then used an inducible knock-in mouse expressing the Scn5a voltage-gated sodium channel. This channel, when expressed in peripheral T cells, enhanced basal TCR-mediated signaling, resulting in decreased respiration and glycolysis, supporting our hypothesis. Genes and metabolites analysis of LLO118 and LLO56 T cells revealed significant differences in their metabolic pathways, including the glycerol phosphate shuttle. Inhibition of this pathway reverts the metabolic state of the LLO118 cells to be more LLO56 like. Overall, these studies highlight the critical relationship between peripheral TCR-self-pMHC interaction, metabolism, and the immune response to infection.