Abstract Upon T cell receptor (TCR) engagement, phosphorylation of the LAT adapter protein enables binding of the enzyme PLC-γ1 to a Gads/SLP-76 dimer, forming a tetrameric structure. The interaction between SLP-76 and PLC-γ1 is weak within this heterotetramer, and the relevant binding sites of SLP-76 and PLC-γ1 are highly conserved in vertebrates. We generated a mouse with a T cell-specific mutation in the SLP-76 that enhanced its affinity for PLC-γ1, thereby increasing PLC-γ1 activity and TCR signal strength. This mutation not only altered the development of αβTCR thymocytes, invariant NKT cells, and intraepithelial lymphocyte precursors but also impaired the generation of central memory CD8 + T cells upon acute viral infection and the humoral immune response mediated by germinal center T follicular helper T cells. These findings suggest that the conserved weak SLP-76/PLC-γ1 interaction is important for the controlled activation of PLC-γ1, thus fine-tuning TCR signal strength to optimize T cell-mediated immunity.

An early step in the activation of T cells via the T cell antigen receptor is the phosphorylation and activation of phospholipase C-γ1 (PLC-γ1) by the TEC family tyrosine kinase, interleukin-2 (IL-2) inducible T cell kinase (ITK). PLC-γ1 activation occurs within a multi-protein complex comprised of the enzymes ITK, PLC-γ1, and VAV, and the adapter molecules, LAT, Gads, SLP-76, and NCK. Studies of ITK activation and the role of this heptameric complex in regulating ITK activation and function have not been possible due to the lack of success in the expression and purification of full-length, monomeric ITK protein. In this study, we have produced soluble full-length wild-type ITK protein by co-expressing an N-terminal solubility-tagged ITK construct with a kinase-specific co-chaperone CDC37 in an insect cell line. Although the majority of the purified ITK protein is oligomerized, there is a 13-fold increase in the yield of monomeric protein production compared to the last reported purification. Previous studies suggest that the ITK oligomerization is mediated by intermolecular interactions. We created several mutants to disrupt these self-associations. Expression of one of these, the C96E/T110I mutant, produced 20 times more monomer than the wild-type construct. The in vitro characterization of these protein constructs showed that the purified protein is stable and functional. This successful purification and in vitro characterization of full-length monomeric ITK protein will aid in understanding the mechanism by which ITK is recruited into the heptameric complex and is enabled to phosphorylate and activate PLC-γ1.

Upon TCR engagement several protein tyrosine kinases are recruited and activated, and adapter proteins and enzymes are phosphorylated on tyrosine residues, leading to further events characterizing activated T cells. Phosphorylation of the LAT adapter protein enables binding of the enzyme PLC-γ1 and of a dimer of two additional adapter proteins Gads and SLP-76, forming a tetrameric structure. Within this heterotetramer there is a weak interaction between SLP-76 and PLC-γ1, and the relevant binding sites of SLP-76 and PLC-γ1 are highly conserved in vertebrates. To address the biological relevance of this weak interaction, we introduced a mutation in the SLP-76 that enhanced its affinity for PLC-γ1 and found that this mutation increased PLC-γ1 activity and altered thymocyte development and peripheral T cell responses due to enhanced TCR signal strength. The conserved weak SLP-76-PLC-γ1 interaction is critical for the controlled activation of PLC-γ1, thus fine-tuning TCR signal strength to optimize T cell-mediated immunity.

IntroductionT cell activation requires T cell receptor (TCR) engagement by its specific ligand. This interaction initiates a series of proximal events including tyrosine phosphorylation of the CD3 and TCRζ chains, recruitment, and activation of the protein tyrosine kinases Lck and ZAP70, followed by recruitment of adapter and signaling proteins. CD28 co-stimulation is also required to generate a functional immune response. Currently we lack a full understanding of the molecular mechanism of CD28 activation.MethodsWe employed TIRF microscopy to establish detailed spatial and kinetic relationships among these molecules in live Jurkat and murine primary T cells. We used anti-TCR (CD3) antibodies to trigger formation of TCR microclusters (MC), which are submicron-sized basic signaling units formed during T cell activation. Using this model, we aimed to delineate how the CD28 co-stimulatory signal alters the kinetics and molecular stoichiometry of TCR proximal signaling events, and how these effects could affect the immune response.ResultsOur results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in MCs and increased ZAP70 activation. CD28-mediated acceleration of ZAP70 recruitment was driven by enhanced Lck recruitment to the MCs. A greater spatial separation between active and inactive species of Lck was also observed in the MCs as a consequence of CD28 co-stimulation.ConclusionThese results suggest that CD28 co- stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs.

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.

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

TCR signal strength regulates T cell fate at various stages of thymocyte development and the differentiation of peripheral T cells into distinct T cell subsets. We previously showed that TCR engagement forms a heterotetrametric complex composed of LAT, Gads, SLP-76, PLC-γ1 and that this complex is essential to activate PLC-γ1 to further transduce the TCR signal. The interaction of the SLP-76 proline-rich region (PRR) with the PLC-γ1 SH3 domain is relatively weak, and these two domains are highly conserved amongst many mammalian species. Therefore, we hypothesized that the SLP-76 PRR sequence with low affinity for PLC-γ1 might be evolutionarily favored to prevent excess TCR signals. To test this hypothesis, we developed mice carrying a conditional T cell-specific knock-in mutation in the SLP-76 PRR with a 2.5-fold increased affinity for PLC-γ1 by CRISPR/Cas9-mediated gene editing and Cre/LoxP system (cKI). Notably, cKI increased the expression of active caspase-3 in signaled SP4 and SP8 thymocytes and decreased the numbers of mature medullary thymocytes, suggestive of enhanced negative selection. Under steady-state conditions, cKI enhanced the generation of CD8 +central memory T cells, implying an increased sensitivity to self-Ag/MHC I in the periphery. Moreover, cKI rendered CD8 +T cells more sensitive to OVA altered peptide ligands with a weaker affinity for OT-I TCR. Finally, cKI enhanced priming of OT-I CD8 +T cells that had not undergone massive cell divisions for IFNγ-producing effectors in vivo, implying an increased TCR signal strength in weakly activated CD8 +T cells by cognate Ag. Our data indicate that the SLP-76 PRR/PLC-γ1 SH3 interaction plays a critical role in fine-tuning TCR signal strength to avoid excess signals via TCR. NCI/NIH Intramural Support

Chimeric antigen receptors (CAR) are molecules with an antibody-derived extracellular domain combined intracellularly with T cell receptor (TCR) signaling proteins. Although CAR-T cells have been a breakthrough clinically, several challenges persist: adverse effects from cytokine secretion, CAR-T cell exhaustion, and limited sensitivity in tumor microenvironments with low density of target antigen (i.e. tumor relapse and solid tumors). While many efforts have focused on identifying new cell surface targets, we have modified CAR intracellular domains. This idea arose from our super-resolution microscopy study that revealed that adapter molecules form clusters distinct from the TCR complex and that full activation necessitates passing a signaling threshold. From these findings, novel Chimeric Adapter Proteins (CAPs) were designed to trigger signaling downstream of the TCRζ chain. CAPs replace the TCRζ with ZAP70 intracellularly and are fused to an extracellular targeting domain. Thus, the potency problem associated with CARs would be ameliorated as CAPs would bypass kinetic proofreading steps defining the signaling threshold and the inhibitory regulation of upstream molecules. Indeed, CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo persistence of tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19–28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.

Activation of the T cell antigen receptor (TCR) is a key step in initiating the adaptive immune response. Single-molecule localization techniques have been used to investigate the arrangement of proteins within the signaling complexes formed around activated TCRs, but a clear picture of nanoscale organization in stimulated T cells has not emerged. Here, we have improved the examination of T cell nanostructure by visualizing individual molecules of six different proteins in a single sample of activated Jurkat T cells using the multiplexed antibody-size limited direct stochastic optical reconstruction microscopy (madSTORM) technique. We formally define irregularly shaped regions of interest, compare areas where signaling complexes are concentrated with other areas, and improve the statistical analyses of the locations of molecules. We show that nanoscale organization of proteins is mainly confined to the areas with dense concentrations of TCR-based signaling complexes. However, randomly distributed molecules are also found in some areas containing concentrated signaling complexes. These results are consistent with the view that the proteins within signaling complexes are connected by numerous weak interactions, leading to flexible, dynamic, and mutable structures which produce large variations in the nanostructure found in activated T cells.

Activation of T cells upon engagement of the T cell antigen receptor rapidly leads to a number of phosphorylation and plasma membrane recruitment events. For example, trans location of phospholipase-C gamma 1 (PLC-gamma 1) to the plasma membrane and its association with the transmembrane adapter protein LAT and two other adapter proteins, Gads and SLP-76, are critical events in the early T cell activation process. We have previously characterized the formation of a tetrameric LAT-Gads-SLP-76-PLC-gamma 1 complex by reconstitution in vitro and have also characterized the thermodynamics of tetramer formation. In the current study, we define how PLC-gamma 1 recruitment to liposomes, which serve as a plasma membrane surrogate, and PLC-gamma 1 activation are regulated both independently and additively by recruitment of PLC-gamma 1 to phosphorylated LAT, by formation of the LATGads-SLP-76-PLC-gamma 1 tetramer, and by tyrosine phosphorylation of PLC-gamma 1. The recently solved structure of PLC-gamma 1 indicates that, in the resting state, several PLC-gamma 1 domains inhibit its enzymatic activity and contact with the plasma membrane. We propose the multiple cooperative steps that we observed likely lead to conformational alterations in the regulatory domains of PLC-gamma 1, enabling contact with its membrane substrate, disinhibition of PLC-gamma 1 enzymatic activity, and production of the phosphoinositide cleavage products necessary for T cell activation.

T cell receptor (TCR) engagement initiates a chain of proximal TCR signaling events including phosphorylation of TCRζ, recruitment of upstream kinases, followed by recruitment of signaling and adapter proteins. These events occur within seconds of TCR engagement and are important determinants of the T cell response. Yet, we lack a precise understanding of the spatiotemporal relationships between these molecular events due to the limitations of conventional light microscopy. We used stimulatory antibodies and ligands against the TCR to trigger formation of TCR microclusters (MC), which are submicron-sized basic signaling units formed during T cell activation. Formation of these signaling subunits was then studied using TIRF and TIRF-SIM to delineate how the CD28 co-stimulatory signal alters the kinetics and molecular stoichiometry of TCR proximal signaling events and subsequent effects on the immunoresponse. Our results show that CD28 co-stimulation specifically accelerated recruitment of ZAP70 to the TCRζ chain in the MCs, as revealed by a decreased kinetic lag between detection of TCRζ and the recruitment of ZAP70. We also observed that CD28 co-stimulation markedly changed Lck dynamics in T cells, resulting in Lck enrichment within MCs and a concomitant increase in co-localization of Lck with ZAP70 and TCRζ. CD28 co-stimulation led to spatial segregation between activated and inhibited species of Lck, raising the possibility of formation of localized Lck activation sites within MCs. These results suggest that CD28 co-stimulation may lower the TCR activation threshold by enhancing the activated form of Lck in the TCR MCs, thereby decreasing the energy barrier for a rate-limiting kinetic step in TCR proximal signaling.

We describe the first cases of germline biallelic null mutations in ARPC5, part of the Arp2/3 actin nucleator complex, in two unrelated patients presenting with recurrent and severe infections, early-onset autoimmunity, inflammation, and dysmorphisms. This defect compromises multiple cell lineages and functions, and when protein expression is reestablished in-vitro, the Arp2/3 complex conformation and functions are rescued. As part of the pathophysiological evaluation, we also show that interleukin (IL)−6 signaling is distinctively impacted in this syndrome. Disruption of IL-6 classical but not trans-signaling highlights their differential roles in the disease and offers perspectives for therapeutic molecular targets.

Chimeric antigen receptors (CAR) T cells have been successfully used to treat lymphoma, leukemia, and multiple myeloma, but adverse effects due to cytokine secretion, CAR-T cell exhaustion, and loss of target antigen have limited their potential. Furthermore, while CARs have been designed to harness T Cell Receptor (TCR) signaling, they are significantly less sensitive than TCRs, resulting in suboptimal signaling. We have developed novel Chimeric Adapter Proteins (CAPs) that are designed to trigger signaling downstream of the TCRζ chain. CAPs are chimeric molecules that contain adapter domains in tandem with the kinase domain of ZAP70, fused to an extracellular targeting domain. We hypothesized that CAPs would be more potent than CARs because kinetic proofreading steps that define the signaling threshold and the inhibitory regulation of upstream molecules are bypassed. Indeed, second generation CAPs exhibited high anti-tumor efficacy, and significantly enhanced long-term in vivo tumor clearance in leukemia-bearing NSG mice as compared with conventional CD19-28ζ CAR-T. Mechanistically, CAPs were activated in an Lck-independent manner and displayed slower phosphorylation kinetics and a longer duration of signaling compared with 28ζ-CAR. The unique signaling properties of CAPs may therefore be harnessed to improve the in vivo efficacy of T cells engineered to express an anti-tumor chimeric receptor.

The TMC genes encode a set of homologous transmembrane proteins whose functions are not well understood. Biallelic mutations in either TMC6 or TMC8 are detected in more than half of cases of the pre-malignant skin disease epidermodysplasia verruciformis (EV). It is controversial whether EV induced by mutations in TMC6 or TMC8 originates from keratinocyte or lymphocyte defects. Quantification of TMC6 and TMC8 RNA levels in various organs revealed that lymphoid tissues have the highest levels of expression of both genes, and custom antibodies confirmed protein expression in mouse lymphocytes. To study the function of these proteins we generated mice with targeted deletion mutant alleles of Tmc6 or Tmc8. Either TMC6 or TMC8 deficiency induced a reduction in apparent molecular weight and/or amount of the other TMC molecule. Co-immunoprecipitation experiments indicated that TMC6 and TMC8 formed a protein complex in mouse and human T cells. MS and biochemical analysis demonstrated that TMC6 and TMC8 additionally interacted with the CIB1 protein to form TMC6-TMC8-CIB1 trimers. We demonstrated that TMC6 and TMC8 regulated CIB1 levels by protecting CIB1 from ubiquitination and proteasomal degradation. Reciprocally, CIB1 was needed for stabilizing TMC6 and TMC8 levels. These results suggest why inactivating mutations in any of the three human genes leads to similar clinical presentations. We also demonstrated that TMC6 and TMC8 levels are drastically lower and the proteins are less active in regulating CIB1 in keratinocytes than in T cells. Our study suggests that defects in lymphocytes may contribute to the etiology and pathogenesis of EV.

LAT molecules defective in ubiquitination have an increased half-life and induce enhanced signaling when expressed in T cells. In this study, we have examined the role of ubiquitination in regulating LAT endocytosis, recycling, and degradation in resting and stimulated T cells. By tracking and comparing plasma membrane-labeled wild type and ubiquitination-resistant 2KR LAT, we find that ubiquitination promotes the degradation of surface LAT in T cells. Activation of T cells increases LAT ubiquitination and promotes trafficking of internalized LAT to lysosomes for degradation. Ubiquitination of LAT does not change internalization rates from the cell surface, but prevents efficient recycling of LAT to the surface of T cells. Our study demonstrates that surface LAT levels are tightly controlled by ubiquitination. LAT in unstimulated cells lacks ubiquitin allowing for increased LAT stability and efficient T cell activation upon TCR triggering; ubiquitination leads to efficient removal of LAT after activation.

Normal function of the adaptive immune system requires trafficking of T cells between the blood and lymphoid organs. Lymphocyte homing to lymph nodes requires that they cross endothelial barriers present in blood vessels and lymphatics. This multi-step process requires a remodeling of the lymphocyte plasma membrane, which is mediated by the dynamic re-arrangement of the actin cytoskeleton. Pak1 plays a central role in cell morphology, adhesion and migration in various cell types. Here we demonstrate that Pak1 is required for activated CD4+ T cell trafficking to lymph nodes. Pak1 deficiency in T cells causes a defect in the transcription of CCR7 and L-selectin, thereby altering lymphocyte trafficking. Additionally, we report an increase in L-selectin shedding in Pak1-deficient T cells, which correlates with a decrease in the recruitment of calmodulin to the cytoplasmic tail of L-selectin during T cell activation. Overall, our findings demonstrate that by regulating the expression of two major lymph node homing molecules, L-selectin and CCR7, Pak1 mediates activated CD4+ T cell trafficking.

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.

Cish, participates within a multi-molecular E3 ubiquitin ligase complex, which ubiquitinates target proteins. It has an inhibitory effect on T cell activation mediated by PLC-γ1 regulation, and it functions as a potent checkpoint in CD8+ T cell tumor immunotherapy. To study the structural and functional relationships between Cish and PLC-γ1 during CD8+ T cell activation, we tested mutants of the Cish-SH2 (R107K) and D/BC (L222Q, C226Q) domains. We confirmed that Cish-SH2-specific binding was essential for PLC-γ1 ubiquitination and degradation. This domain was essential for the Cish-mediated inhibition of Ca2+ release upon TCR stimulation. No effect on inhibition of cytokine release was observed with SH2 or D/BC mutants, although the absence of Cish led to an increased release of IFN-γ and TNF-α. Using imaging we showed that Cish was expressed mostly in the cytoplasm and we did not see any Cish clustering at the plasma membrane upon stimulation. We conclude that the Cish-SH2 domain is essential for PLC-γ1 regulation in TCR-stimulated CD8+ T cells.

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.