T Cell Receptor Signal Strength Research Articles

TH2 heterogeneity: Does function follow form?

T(H)2 immune responses are required for the 2 fundamental pathological processes characteristic of allergic disease: IgE-mediated hypersensitivity and eosinophilic inflammation. The 3 established T(H)2 cytokines, IL-4, IL-5, and IL-13, each play a nonredundant role in allergic disease pathology. The recent explosion of T(H) subpopulations combined with the wide availability of polychromatic cytokine staining has facilitated the discovery of T(H)2 lineage heterogeneity. In this article we review T(H)2 heterogeneity and ask the following question: At what point do these subpopulations graduate from in vitro curiosities to immunologically robust therapeutic targets? We propose criteria to establish a T-cell subset as a biologically relevant entity and address the evidence to support these T(H)2 subpopulations having a unique function or specific contribution to allergic pathology or host defense.

Coordination of IL-7 receptor and T-cell receptor signaling by cell-division cycle 42 in T-cell homeostasis

T-cell homeostasis is essential for normal functioning of the immune system. IL-7 receptor (IL-7R) and T-cell receptor (TCR) signaling are pivotal for T-cell homeostatic regulation. The detailed mechanisms regulating T-cell homeostasis and how IL-7R and TCR signaling are coordinated are largely unknown. Here we demonstrate that T cell-specific deletion of cell-division cycle 42 (Cdc42) GTPase causes a profound loss of mature T cells. Deletion of Cdc42 leads to a markedly increased expression of growth factor independence-1 (Gfi-1) and represses expression of IL-7Rα. In the absence of Cdc42, aberrant ERK1/2 MAP kinase activity results in enhanced, TCR-mediated T-cell proliferation. In vivo reconstitution of effector-binding-defective Cdc42 mutants and the effector p21 protein-activated kinase 1 (PAK1) into Cdc42-deficient T cells showed that PAK1 is both necessary and sufficient for Cdc42-regulated T-cell homeostasis. Thus, T-cell homeostasis is maintained through a concerted regulation of Gfi-1-IL-7R-controlled cytokine responsiveness and ERK-mediated TCR signaling strength by the Cdc42-PAK1 signaling axis.

Differential expression of IL-17A and IL-17F is coupled to TCR signaling via Itk-mediated regulation of NFATc1 (139.4)

Abstract Th17 CD4+ effector T cells play important roles in autoimmunity and responses to bacterial infections. The roles of cytokines in regulating the transcription factors STAT3 and RORγT in Th17 differentiation have been extensively studied. However, the role of T cell receptor (TCR) signaling in Th17 differentiation is less appreciated. To examine this issue, we evaluated IL17 production from T cells lacking Itk, a tyrosine kinase required for full TCR-induced activation of PLC-γ and downstream pathways. We find that Itk-/- CD4+T cells exhibit specific defects in the expression of IL-17A despite relatively normal expression of RORγT, RORα and the other Th17 cytokines, including the closely related IL-17F. Defects in IL-17A expression and differential regulation of IL-17A and IL-17F were also observed in vivo in Itk-/- mice challenged with an allergic asthma model. Although Itk-deficient cells have slightly depressed phosphorylation of STAT-3 in response to IL-6, expression of constitutively activated STAT-3 failed to rescue their IL-17A production. In contrast, expression of IL-17A could be rescued by pharmacologically-induced Ca2+ influx or by a constitutively active NFATc1. The effects of Itk on transcriptional complexes and chromatin at the IL-17 locus are under further investigation. Our results suggest that Itk specifically couples TCR signaling strength to IL-17A expression through NFATc1 and that TCR signaling pathways differentially influence Th17 cytokine production.

C-Cbl Promotes T Cell Receptor-induced Thymocyte Apoptosis by Activating the Phosphatidylinositol 3-Kinase/Akt Pathway

The ability of thymocytes to assess T cell receptor (TCR) signaling strength and initiate the appropriate downstream response is crucial for determining their fate. We have previously shown that a c-Cbl RING finger mutant knock-in mouse, in which the E3 ubiquitin ligase activity of c-Cbl is inactivated, is highly sensitive to TCR-induced death signals that cause thymic deletion. This high intensity signal involves the enhanced tyrosine phosphorylation of the mutant c-Cbl protein promoting a marked increase in the activation of Akt. Here we show that this high intensity signal in c-Cbl RING finger mutant thymocytes also promotes the enhanced induction of two mediators of TCR-directed thymocyte apoptosis, Nur77 and the pro-apoptotic Bcl-2 family member, Bim. In contrast, a knock-in mouse harboring a mutation at Tyr-737, the site in c-Cbl that activates phosphatidylinositol 3-kinase, shows reduced TCR-mediated responses including suppression of Akt activation, a reduced induction of Nur77 and Bim, and greater resistance to thymocyte death. These findings identify tyrosine-phosphorylated c-Cbl as a critical sensor of TCR signal strength that regulates the engagement of death-promoting signals.

What determines Th2 differentiation, in vitro and in vivo?

We have known since 1991 how to induce naive CD4 T cells to differentiate in vitro into Th2 cells and, over the ensuing years, a comprehensive picture of the molecules involved in this important process has emerged. GATA3 and STAT5 are both essential for in vitro differentiation, stimulating naive cells through a process involving induction, which is T-cell receptor (TCR) dependent but interleukin (IL)-4 independent, and commitment, which is IL-4 dependent. Th2 differentiation in vivo appears more complex. GATA3 and probably STAT5 are required in vivo but, at least for certain helminth infections, the IL-4/IL-4Ra/STAT6 pathway is dispensable. The role of thymic stromal lymphopoietin and of low TCR signal strength and the participation of basophils in establishing a Th2-baising in vivo environment have achieved considerable attention. Here I discuss the major players in Th2 differentiation particularly as they may exert their effects in vivo.

The Tec kinases Itk and Rlk regulate conventional versus innate T‐cell development

Tec family kinases are important components of antigen receptor signaling pathways in B cells, T cells, and mast cells. In T cells, three members of this family, inducible T-cell kinase (Itk), resting lymphocyte kinase (Rlk), and Tec, are expressed. In the absence of Itk and Rlk, T-cell receptor signaling is impaired, with defects in mitogen-activated protein kinase activation, Ca(2+) mobilization, and actin polymerization. During T-cell development in the thymus, no role has been found for these kinases in the CD4(+) versus CD8(+) T-cell lineage decision; however, several studies indicate that Itk and Rlk contribute to the signaling leading to positive and negative selection. In addition, we and others have recently described an important role for Itk and Rlk in the development of conventional as opposed to innate CD4(+) and CD8(+) T cells. Natural killer T and gammadelta T-cell populations are also altered in Itk- and Rlk/Itk-deficient mice. These findings strongly suggest that the strength of T-cell receptor signaling during development determines whether T cells mature into conventional versus innate lymphocyte lineages. This lineage decision is also influenced by signaling via signaling lymphocytic activation molecule (SLAM) family receptors. Here we discuss these two signaling pathways that each contribute to conventional versus innate T-cell lineage commitment.

Differential impact of the CD45 juxtamembrane wedge on central and peripheral T cell receptor responses

The cooperative activity of protein tyrosine kinases and phosphatases plays a central role in regulation of T cell receptor (TCR) signal strength. Perturbing this balance, and thus the threshold for TCR signals, has profound impacts on T cell development and function. We previously generated mice containing a point mutation in the juxtamembrane wedge of the receptor-like protein tyrosine phosphatase CD45. Demonstrating the critical negative regulatory function of the wedge, the CD45 E613R (WEDGE) mutation led to a lymphoproliferative disorder (LPD) and a lupus-like autoimmune syndrome. Using genetic, cellular, and biochemical approaches, we now demonstrate that the CD45 wedge influences T cell development and function. Consistent with increased TCR signal strength, WEDGE mice have augmented positive selection and enhanced sensitivity to the CD4-mediated disease experimental autoimmune encephalitis (EAE). These correspond with hyperresponsive calcium and pERK responses to TCR stimulation in thymocytes, but surprisingly, not in peripheral T cells, where these responses are actually depressed. Together, the data support a role for the CD45 wedge in regulation of T cell responses in vivo and suggest that its effects depend on cellular context.

CD28 enhances in vivo clonal expansion by CD4+ T cells without increasing sensitivity to antigen

Costimulatory signals are required for optimal CD4+ T cell immunity. In vitro studies and experiments with monoclonal T cells have shown that ligation of CD28 by B7 enhances CD4+ T cell proliferation, effector function, and survival upon priming. One model posits that CD28 acts by amplifying signals transduced through the T cell receptor (TCR). A prediction of this model is that increasing TCR signal strength would compensate for the defective T cell proliferation observed in the absence of CD28. We tested this prediction by using peptide:major histocompatibility complex II tetramers and magnetic bead enrichment to measure the clonal expansion of polyclonal normal or CD28‐deficient T cells in response to subcutaneous immunization with a wide range of antigen doses emulsified in Freund's Adjuvant. CD28‐deficient and normal T cells accumulated to a peak level at a similar antigen dose. However, the number of CD28‐deficient T cells present at the peak dose was 5‐fold lower than the number of normal T cells. Furthermore, intravenous or subcutaneous immunization with a high dose of soluble antigen plus lipopolysaccharide did not increase the numbers of antigen‐specific CD28‐deficient T cells above that of naïve mice. These data suggest that CD28 is essential for T cells to respond at all to soluble antigen and enhances T cell responsiveness to emulsified antigen by a mechanism that does not involve TCR signal amplification.

Toxins that affect the cardiovascular system

<h3>Summary</h3> How T cell receptor (TCR) signal strength modulates T cell function and to what extent this is modified by immune checkpoint blockade (ICB) are key questions in immunology. Using Nr4a3-Tocky mice as a digital read-out of NFAT pathway activity, we identify the rapid quantitative and qualitative changes that occur in CD4⁺ T cells in response to a range of TCR signalling strengths. We demonstrate that the time and dose dependent programming of distinct co-inhibitory receptors rapidly re-calibrates T cell activation thresholds. By developing a new <i>in vivo</i> model, we analyse the immediate effects of ICB on T cell re-activation. Our findings reveal that anti-PD1 but not anti-Lag3 immunotherapy leads to an increased TCR signal strength. We define a strong TCR signal metric of five genes specifically upregulated by anti-PD1 in T cells (TCR.strong), which can stratify clinical outcomes during anti-PD1 monotherapy in melanoma patients. Our study therefore reveals how analysis of TCR signal strength – and its manipulation – can provide powerful metrics for monitoring outcomes to immunotherapy. <h3>Key Points</h3> TCR signal strength-dependent programming of CD4⁺ T cells revealed over time in vivo Inhibitory receptor expression is dynamic, TCR signal strength dependent, and rapidly re-calibrates T cell activation thresholds PD1 but not Lag3 blockade leads to a unique and increased TCR signal strength signature (coined TCR.strong) TCR.strong metric stratifies melanoma patient survival in response to Nivolumab (anti-PD1) therapy

Loss of c-Cbl RING finger function results in high-intensity TCR signaling and thymic deletion

Signaling from the T-cell receptor (TCR) in thymocytes is negatively regulated by the RING finger-type ubiquitin ligase c-Cbl. To further investigate this regulation, we generated mice with a loss-of-function mutation in the c-Cbl RING finger domain. These mice exhibit complete thymic deletion by young adulthood, which is not caused by a developmental block, lack of progenitors or peripheral T-cell activation. Rather, this phenotype correlates with greatly increased expression of the CD5 and CD69 activation markers and increased sensitivity to anti-CD3-induced cell death. Thymic loss contrasts the normal fate of the c-Cbl-/- thymus, even though thymocytes from both mutant mice show equivalent enhancement in proximal TCR signaling, Erk activation and calcium mobilization. Remarkably, only the RING finger mutant thymocytes show prominent TCR-directed activation of Akt. We show that the mutant c-Cbl protein itself is essential for activating this pathway by recruiting the p85 regulatory subunit of PI 3-kinase. This study provides a unique model for analyzing high-intensity TCR signals that cause thymocyte deletion and highlights multiple roles of c-Cbl in regulating this process.

Celebrating T Cell Diversity

The duration of signals emanating from the T cell receptor (TCR) is finely regulated through several processes that control protein half-life, posttranslational modification of proteins, and the recruitment of inhibitory proteins. Egen and Allison have found that the inhibitory protein cytotoxic T lymphocyte antigen-4 (CTLA-4) is recruited to the immunological synapse, in direct proportion to the strength of the TCR signal, where it acts to extinguish TCR-dependent signals. CTLA-4 in T cells that were cultured with antigen-presenting cells (APCs) bearing agonistic or weakly agonistic peptides translocated to microtubule-organizing centers (MTOCs) close to the site of T cell-APC apposition. However, only T cells stimulated with the stronger agonist peptides led to the movement of CTLA-4 to the immune synapse at the plasma membrane. These data suggest that the location of CTLA-4 is dependent on the strength of the signal sent by the TCR. More important, the data suggest a reason why T cells would need a mechanism to inhibit TCR-dependent signaling after a strong signal is elicited. It is in an organism's best interest to have a widely varied immune response to a pathogen. Strong signals by a few T cells (that have high affinity for an antigen) could quickly lead to the proliferation of a very small subset of T cells at the expense of a diverse group of proliferating T cells with varying degrees of affinity for the antigen. Thus, a large number of T cells would recognize a very narrow repertoire of antigen--clearly a situation to the pathogen's advantage. However, by recruiting CTLA-4 to the immune synapse, the prolonged signaling that would be mediated by strongly agonistic antigens is attenuated, such that weakly stimulated T cells have an opportunity to proliferate and increase the diversity of T cell-mediated responses. J. G. Egen, J. P. Allison, Cytotoxic T lymphocyte antigen-4 accumulation in the immunological synapse is regulated by TCR signal strength. Immunity 16 , 23-35 (2002). [Online Journal]

Role of the T Cell Receptor Ligand Affinity in T Cell Activation by Bacterial Superantigens

Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3 (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency of the SEC3 variants correlated with enhanced binding without any optimum in the binding range covered by native TCR ligands. Comparable studies using anti-TCR antibodies of known affinity confirmed these observations. By comparing the biological potency of the two sets of ligands, we found a significant correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength.