T-cell Receptor Restimulation Research Articles

Dynamic chromatin accessibility licenses STAT5- and STAT6-dependent innate-like function of Th9 cells to promote allergic inflammation

Abstract Allergic diseases are a major global health issue, causing significant morbidity and mortality. Interleukin-9 (IL-9) producing T helper 9 (Th9) cells promote allergic inflammation, yet Th9 effector functions are incompletely understood because the heterogeneity and instability of Th9 cells makes them challenging to study. Although the significance and mechanisms of Th9 instability are unknown, T cell receptor (TCR) activation is hypothesized to play a key role. Conversely to this paradigm, we found that resting Th9 cells did not require TCR restimulation for IL-9 production, which was induced by STAT5- and STAT6-dependent paracrine cytokines. This mechanism was seen in circulating T cells from allergic patients and was restricted to recently activated cells. Analysis of IL-9 +T cells from allergic subjects revealed a transcriptional program that was enriched for activation pathways and unstable over time, with IL9 amongst the least stable genes. Prolonged resting of cells decreased the accessibility of STAT5-dependent IL9 enhancers, inactivating the locus. In vivo, Th9 cells induced airway inflammation via TCR-independent, STAT-dependent mechanisms. In allergic patients, Th9 expansion was associated with STAT activation and responsiveness to JAK inhibitors. These findings suggest that Th9 instability is a negative checkpoint on TCR-independent inflammation that breaks down in allergic disease, and that JAK inhibitors should be considered for allergic patients with a Th9 signature. Supported by the NIAID intramural research program (2018-2022), University of Pittsburgh start-up funds (2022-current), and the National Psoriasis Foundation (Robertson Fellowship, 2018-2021; Translational Grant, 2022-current)

FOXP3 protects conventional human T cells from premature restimulation-induced cell death.

The adaptive immune response relies on specific apoptotic programs to maintain homeostasis. Conventional effector T cell (Tcon) expansion is constrained by both forkhead box P3 (FOXP3)+-regulatory T cells (Tregs) and restimulation-induced cell death (RICD), a propriocidal apoptosis pathway triggered by repeated stimulation through the T-cell receptor (TCR). Constitutive FOXP3 expression protects Tregs from RICD by suppressing SLAM-associated protein (SAP), a key adaptor protein that amplifies TCR signaling strength. The role of transient FOXP3 induction in activated human CD4 and CD8 Tcons remains unresolved, but its expression is inversely correlated with acquired RICD sensitivity. Here, we describe a novel role for FOXP3 in protecting human Tcons from premature RICD during expansion. Unlike FOXP3-mediated protection from RICD in Tregs, FOXP3 protects Tcons through a distinct mechanism requiring de novo transcription that does not require SAP suppression. Transcriptome profiling and functional analyses of expanding Tcons revealed that FOXP3 enhances expression of the SLAM family receptor CD48, which in turn sustains basal autophagy and suppresses pro-apoptotic p53 signaling. Both CD48 and FOXP3 expression reduced p53 accumulation upon TCR restimulation. Furthermore, silencing FOXP3 expression or blocking CD48 decreased the mitochondrial membrane potential in expanding Tcons with a concomitant reduction in basal autophagy. Our findings suggest that FOXP3 governs a distinct transcriptional program in early-stage effector Tcons that maintains RICD resistance via CD48-dependent protective autophagy and p53 suppression.

CD4+ T cells from patients with primary sclerosing cholangitis exhibit reduced apoptosis and down-regulation of proapoptotic Bim in peripheral blood.

The pathogenesis of the progressive liver disease, primary sclerosing cholangitis (PSC), remains largely elusive. The strong genetic association with HLA loci suggests that T cell-dependent, adaptive immune reactions could contribute to disease pathogenesis. Recent studies have indicated that PSC is also associated with polymorphisms in the locus encoding for proapoptotic Bim (BCL2L11). Bim is crucial for the maintenance of immunologic tolerance through induction of apoptosis in activated T cells. Of interest with regard to PSC is the finding that BCL2L11-deficient mice develop periductular infiltrates. We, therefore, investigated, whether defective apoptosis of T cells might contribute to the phenotype of PSC. Thus, we induced apoptosis of T cells from patients with PSC and controls by repeated T cell receptor (TCR) stimulation or cytokine withdrawal. We found that CD4+ T cells, but not CD8+ T cells, from patients with PSC exhibited significantly reduced apoptosis in response to both, TCR restimulation or cytokine withdrawal. This increased apoptosis resistance was associated with significantly reduced up-regulation of proapoptotic Bim in T cells from patients with PSC. However, T cell apoptosis did not seem to be influenced by the previously described BCL2L11 polymorphisms. Reduced CD4+ T cell apoptosis in patients with PSC was not due to reduced cell activation, as indicated by a similar surface expression of the activation markers CD69, CD25, and CD28 in T cells from patients and controls. Thus, decreased apoptosis of activated CD4+ T cells may be part of the immune dysregulation observed in patients with PSC.

Sensitivity to restimulation-induced cell death is linked to glycolytic metabolism in human T cells

Abstract Restimulation-induced cell death (RICD) helps to regulate immune responses by setting an upper threshold for effector T cell expansion and limiting nonspecific damage to the host. RICD is triggered by re-engagement of the T cell receptor (TCR) on a cycling, activated T cell, resulting in apoptosis. However, it remains unclear how RICD sensitivity is calibrated in T cells derived from different individuals and subsets. As effector T cells expand and become responsive to RICD, they have also switched from a quiescent metabolic state into active aerobic glycolysis (Warburg effect). We hypothesized that in addition to facilitating proliferation and effector function, metabolic programming directly influences RICD sensitivity. We found that secretion of L-lactate, the end product of glycolysis, positively correlated with greater RICD sensitivity in CD8+ effector T cells from different human donors. Strikingly, replacing glucose in culture media for galactose, which results in almost exclusive use of oxidative phosphorylation (OXPHOS), rendered T cells significantly less sensitive to RICD. Consistent with this, blocking glycolysis with 2-deoxy-glucose (2-DG) significantly reduced RICD sensitivity, while inhibiting OXPHOS increased RICD sensitivity. Glycolysis promoted RICD sensitivity in part through mTOR signaling. Moreover, we found that active glycolysis facilitated induction of pro-apoptotic effector molecules like Fas ligand upon TCR restimulation, which was blocked with 2-DG treatment. Collectively, these data suggest that RICD susceptibility is dependent on glycolytic metabolism in effector T cells, and that switching back to OXPHOS may help shield cells transitioning into the memory pool from RICD.

Induction of cell death following restimulation of the T cell receptor results in activation of the DNA damage response pathway in a JNK and ROS-dependent manner (TUM7P.1026)

Abstract The p53 protein is activated by various forms of cellular stress and its function is modulated though a multiplicity of posttranslational modifications to dictate signaling outcomes. A recent study demonstrated that inhibition of p53 by pifithrin-m effectively blocked cell death caused by restimulation of the T cell receptor (TCR). Using human T cells, we investigated the phosphorylation status of p53 following TCR restimulation and detected a time-dependent increase in phosphorylation on Ser15, which correlated with onset of cell death. Phospho-p53 (Ser15) was limited to the nucleus and coincided with phosphorylation of ATM. Blocking ATM function through KU55933 or caffeine prevented cell death. Phosphorylation of ATM suggested activation of the DNA damage response pathway and we confirmed coordinate increases in both yH2AX and SMC-1. Activation of the ATM-p53-dependent DNA damage response pathway following restimulation of the TCR was reduced by inhibition of JNK by SP600125 as well as the anti-oxidant N-acetyl cysteine (NAC). Activation of the DNA damage pathway occurred in an antigen-specific manner and was also observed in T cells derived from melanoma patients undergoing adoptive T cell therapy. In vitro treatment with NAC improved survival of T cells while increasing melanoma cell killing. Thus our data highlight a role for activation of the DNA damage response pathway following TCR restimulation and its potential translational significance in adoptive T cell therapy.

Critical role for BIM in T cell receptor restimulation-induced death

BackgroundUpon repeated or chronic antigen stimulation, activated T cells undergo a T cell receptor (TCR)-triggered propriocidal cell death important for governing the intensity of immune responses. This is thought to be chiefly mediated by an extrinsic signal through the Fas-FasL pathway. However, we observed that TCR restimulation still potently induced apoptosis when this interaction was blocked, or genetically impaired in T cells derived from autoimmune lymphoproliferative syndrome (ALPS) patients, prompting us to examine Fas-independent, intrinsic signals.ResultsUpon TCR restimulation, we specifically noted a marked increase in the expression of BIM, a pro-apoptotic Bcl-2 family protein known to mediate lymphocyte apoptosis induced by cytokine withdrawal. In fact, T cells from an ALPS type IV patient in which BIM expression is suppressed were more resistant to restimulation-induced death. Strikingly, knockdown of BIM expression rescued normal T cells from TCR-induced death to as great an extent as Fas disruption.ConclusionOur data implicates BIM as a critical mediator of apoptosis induced by restimulation as well as growth cytokine withdrawal. These findings suggest an important role for BIM in eliminating activated T cells even when IL-2 is abundant, working in conjunction with Fas to eliminate chronically stimulated T cells and maintain immune homeostasis.ReviewersThis article was reviewed by Dr. Wendy Davidson (nominated by Dr. David Scott), Dr. Mark Williams (nominated by Dr. Neil Greenspan), and Dr. Laurence C. Eisenlohr.

CD4+ CD5+ regulatory T cells render naive CD4+ CD25– T cells anergic and suppressive

SummaryCD4+ CD25+ Foxp3+ naturally occurring regulatory T cells (nTreg) are potent inhibitors of almost all immune responses. However, it is unclear how this minor population of cells is capable of exerting its powerful suppressor effects. To determine whether nTreg mediate part of their suppressor function by rendering naive T cells anergic or by converting them to the suppressor phenotype, we cocultured mouse nTreg with naive CD4+ CD25– T cells from T‐cell receptor (TCR) transgenic mice on a RAG deficient (RAG–/–) background in the presence of anti‐CD3 and interleukin‐4 (IL‐4) to promote cell viability. Two distinct responder cell populations could be recovered from the cocultures. One population remained undivided in the coculture and was non‐responsive to restimulation with anti‐CD3 or exogenous IL‐2, and could not up‐regulate IL‐2 mRNA or CD25 expression upon TCR restimulation. Those responder cells that had divided in the coculture were anergic to restimulation with anti‐CD3 but responded to restimulation with IL‐2. The undivided population was capable of suppressing the response of fresh CD4+ CD25– T cells and CD8+ T cells, while the divided population was only marginally suppressive. Although cell contact between the induced regulatory T cell (iTreg) and the responders was required for suppression to be observed, anti‐transforming growth factor‐β partially abrogated their suppressive function. The iTreg did not express Foxp3. Therefore nTreg are not only able to suppress immune responses by inhibiting cytokine production by CD4+ CD25– responder cells, but also appear to modulate the responder cells to render them both anergic and suppressive.

Activation or suppression of NFκB by HPK1 determines sensitivity to activation-induced cell death

Restimulation of the T-cell receptor (TCR) in activated T cells induces CD95 (Fas/Apo-1)-mediated activation-induced cell death (AICD). The TCR-proximal mechanisms leading to AICD are elusive. Here we characterize hematopoietic progenitor kinase 1 (HPK1) as a differentially regulated TCR-proximal signaling protein involved in AICD of primary T cells. We show that HPK1 is a functional component of the endogenous IkappaB kinase (IKK) complex and is crucial for TCR-mediated NFkappaB activation. While full-length HPK1 enhances IKKbeta phosphorylation, siRNA-mediated knockdown of HPK1 blunts TCR-mediated NFkappaB activation and increases cell death. We also demonstrate proteolytic processing of HPK1 into HPK1-C, specifically in AICD-sensitive primary T cells. The cleavage product HPK1-C sequesters the inactive IKK complex and suppresses NFkappaB upon TCR restimulation by binding to IKKalpha and IKKbeta. T cells of HPK1-C transgenic mice are sensitized towards TCR-mediated AICD. Consequently, preventing HPK1-C generation in primary T cells by siRNA-mediated knockdown results in decreased AICD. Thus, these results show a novel mechanism of sensitization of T lymphocytes towards AICD by suppression of NFkappaB, and propose that HPK1 is a life/death switch in T lymphocytes.

Ligand-independent redistribution of Fas (CD95) into lipid rafts mediates clonotypic T cell death

Clonotypic elimination of activated T cells through Fas-Fas ligand (CD95-CD95L) interactions is one mechanism of peripheral self-tolerance. T cell receptor (TCR) stimuli trigger FasL synthesis but also sensitize activated T cells to Fas-mediated apoptosis through an unknown mechanism. Here we show that TCR restimulation of activated human CD4(+) T cells resulted in Fas translocation into lipid raft microdomains before binding FasL, rendering these cells sensitive to apoptosis after stimulation with bivalent antibody or FasL. Disruption of lipid rafts reduced sensitivity to Fas-mediated apoptosis after TCR restimulation. Thus, the redistribution of Fas and other tumor necrosis factor family receptors into and out of lipid rafts may dynamically regulate the efficiency and outcomes of signaling by these receptors.

Enhancement of calcium signaling and proliferation responses in activated human T lymphocytes Inhibitory effects of K + channel block by charybdotoxin depend on the T cell activation state

T cell receptor (TCR) stimulation, leading to T cell activation and ultimately to cell proliferation and differentiation, evokes elevations of [Ca 2+] i with a high variability between individual T lymphocytes. We have used ca 2+-imaging of Fura-2 loaded cells to study the origin of the variation in Ca 2+ signals and its consequences for the final cellular response. We found that, compared to resting cells, the percentage of responding cells and the average amplitude of the Ca 2+ signal upon TCR re-stimulation by PHA increases in the first 5 days of T cell activation and declines thereafter, with more pronounced [Ca 2+] i oscillations in later stages. In parallel, an enhancement of T cell proliferation is observed. Stronger stimulation of the TCR/CD3 complex by co-crosslinking CD3 with CD4/CD8 molecules evokes oscillating Ca 2+ responses irrespective of the activation state, indicating that the basic capacity for Ca 2+ signaling is essentially the same in resting and activated cells. Nevertheless, also the amplitude of the CD3+CD4/8 response shows a transient additional increase during the first days of T cell activation. Experiments with the K + channel blocker charybdotoxin (CTX) indicate that [Ca 2+] i oscillations depend critically on K + channel functioning, but suppression of these oscillations by CTX does not significantly affect the average amplitude of the Ca 2+ signal nor PHA-induced proliferation. However, when applied during the first 4–5 days of activation, CTX reduces in addition the average level of the TCR evoked Ca 2+ response and inhibits subsequent proliferation.