Restimulation-induced Cell Death Research Articles

A20 intrinsically influences human effector T-cell survival and function by regulating both NF-κB and JNK signaling.

A20 is a dual-function ubiquitin-editing enzyme that maintains immune homeostasis by restraining inflammation. Although A20 serves a similar negative feedback function for T-cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T-cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-κB and c-Jun/AP-1. Utilizing WT and A20 knockout Jurkat T cells, we found that A20 is required to negatively regulate NF-κB and JNK. Utilizing a novel set of A20 mutants in NF-κB and AP-1-driven reporter systems, we discovered the ZnF7 domain is crucial for negative regulatory capacity, while deubiquitinase activity is dispensable. Successful inactivation of A20 in human primary effector T cells congruently conferred sustained NF-κB and JNK signaling, including enhanced upregulation of activation markers, and increased secretion of several cytokines including IL-9. Finally, loss of A20 in primary human T cells resulted in decreased sensitivity to restimulation-induced cell death and increased sensitivity to cytokine withdrawal-induced death. These findings demonstrate the importance of A20 in maintaining T-cell homeostasis via negative regulation of both NF-κB and JNK signaling.

Lentiviral vectors for precise expression to treat X-linked lymphoproliferative disease

X-linked lymphoproliferative disease (XLP1) results from SH2D1A gene mutations affecting the SLAM-associated protein (SAP). A regulated lentiviral vector (LV), XLP-SMART LV, designed to express SAP at therapeutic levels in T, NK, and NKT cells, is crucial for effective gene therapy. We experimentally identified 34 genomic regulatory elements of the SH2D1A gene and designed XLP-SMART LVs to emulate the lineage and stage-specific control of SAP. We screened them for their on-target enhancer activity in T, NK, and NKT cells and their off-target enhancer activity in B cell and myeloid populations. In combination, three enhancer elements increased SAP promoter expression up to 4-fold in on-target populations invitro. NSG-Tg(Hu-IL15) xenograft studies with XLP-SMART LVs demonstrated up to 7-fold greater expression in on-target cells over a control EFS-LV, with no off-target expression. The XLP-SMART LVs exhibited stage-specific T and NK cell expression in peripheral blood, bone marrow, spleen, and thymic tissues (mimicking expression patterns of SAP). Transduction of XLP1 patient CD8+ Tcells or BM CD34+ cells with XLP-SMART LVs restored restimulation-induced cell death and NK cytotoxicity to wild-type levels, respectively. These data demonstrate that it is feasible to create a lineage and stage-specific LV to restore the XLP1 phenotype by gene therapy.

IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells.

IL-2Rα knock out (KO) mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine, IL-2 and IL-2Rα KO mice revealed that this cytokine-receptor system controls immune responses through restimulation-induced cell death and by promoting the survival of T regulatory cells. Although described mostly in the context of lymphocytes, recent studies by our laboratory showed that IL-2R is expressed in smooth muscle cells. Given this finding, we sought to use IL-2Rα KO to determine the function of this receptor in vascular smooth muscle cells. Surprisingly, we found that IL-2Rα KO vascular smooth muscle cells had detectable IL-2Rα. We used multiple gene and protein-based methods to determine why IL-2Rα KO vascular smooth muscle cells exhibited IL-2Rα protein. These methods included: genomic sequencing, assessing cells and tissues for evidence of maternal microchimerism, and determining the half-life of IL-2Rα protein. Our studies demonstrated the following: (1) in addition to the cell surface, IL-2Rα is localized to the nucleus; (2) the genetic deletion of IL-2Rα is intact in IL-2Rα KO mice; (3) both IL-2Rα KO and WT tissues show evidence of maternal microchimerism, the likely source of IL-2Rα (4) IL-2Rα is transmitted between cells; (5) IL-2Rα has a long half-life; and (6) nuclear IL-2Rα contributes to the regulation of cell proliferation and size. Our findings suggest that the phenotype of complete IL-2Rα loss is more severe than demonstrated by IL-2Rα KO mice, and that IL-2Rα plays a here-to-fore unrecognized role in regulating cell proliferation in non-lymphoid cells.

A20 is a negative regulator of both NF-κB and JNK signaling following T cell receptor stimulation in humans

Abstract A20 is a ubiquitously expressed, inducible ubiquitin-editing enzyme that restrains NF-kB-dependent inflammatory signaling throughout the body. In T cell receptor (TCR) signaling, the molecular mechanisms utilized and their ultimate impact on human T cell function remain unclear. TCR engagement triggers the assembly of the CARD11-BCL10-MALT1 (CBM) protein complex, a signaling platform that governs the activation of downstream transcription factors including NF-kB and c-Jun partner AP-1. Utilizing WT and A20KO Jurkat T cells, we demonstrate A20 is required to negatively regulate both NF-kB and JNK/AP-1 signaling. By employing a novel set of A20 mutant constructs with NF-kB and AP-1 driven reporter systems in Jurkat transfectants, we discovered the ZnF7 domain is essential for CBM localization and signal modulation. We confirmed these findings in CRISPR-mediated A20 knockout human primary effector T cells, demonstrating sustained TCR-induced NF-kB and JNK signaling, enhanced upregulation of activation markers, and increased cytokine secretion associated with a skewed Th9/Th17 phenotype. Moreover, loss of A20 altered apoptosis sensitivity in primary human T cells, resulting in decreased restimulation induced cell death (RICD) and increased cytokine withdrawal induced death (CWID). These findings further elucidate the mechanism of A20-dependent regulation of both NF-kB and c-Jun in T cells, and highlight the functional importance of A20 in maintaining adaptive immune homeostasis.

Molecular and temporal control of restimulation-induced cell death (RICD) in T lymphocytes

For effective adaptive immunity, T lymphocytes must rapidly expand and contract in an antigen-specific manner to effectively control invading pathogens and preserve immunological memory, without sustaining excessive collateral damage to host tissues. Starting from initial antigen encounter, carefully calibrated programmed cell death pathways are critical for maintaining homeostasis over distinct phases of the T cell response. Restimulation-induced cell death (RICD), a self-regulatory apoptosis pathway triggered by re-engagement of the T cell receptor (TCR), is particularly important for constraining effector T cell expansion to preclude overt immunopathology; indeed, genetic disorders affecting key molecules involved in RICD execution can manifest in excessive lymphoproliferation, malignancy, and autoimmunity. Herein we review our current knowledge of how RICD sensitivity is ultimately regulated over the course of an immune response, including recent revelations on molecules that tune RICD by enforcing resistance or promoting susceptibility in expanding versus mature effector T cells, respectively. Detailed dissection of the molecular and temporal control of RICD also illuminates novel therapeutic strategies for correcting abnormal T cell responses noted in various immune disorders by ultimately tuning RICD sensitivity.

Complementary HLH susceptibility factors converge on CD8 T-cell hyperactivation

AbstractHemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening hyperinflammatory syndromes. Familial HLH is caused by genetic impairment of granule-mediated cytotoxicity (eg, perforin deficiency). MAS is linked to excess activity of the inflammasome-activated cytokine interleukin-18 (IL-18). Though individually tolerated, mice with dual susceptibility (Prf1⁻/⁻Il18tg; DS) succumb to spontaneous, lethal hyperinflammation. We hypothesized that understanding how these susceptibility factors synergize would uncover key pathomechanisms in the activation, function, and persistence of hyperactivated CD8 T cells. In IL-18 transgenic (Il18tg) mice, IL-18 effects on CD8 T cells drove MAS after a viral (lymphocytic choriomeningitis virus), but not innate (toll like receptor 9), trigger. In vitro, CD8 T cells also required T-cell receptor (TCR) stimulation to fully respond to IL-18. IL-18 induced but perforin deficiency impaired immunoregulatory restimulation-induced cell death (RICD). Paralleling hyperinflammation, DS mice displayed massive postthymic oligoclonal CD8 T-cell hyperactivation in their spleens, livers, and bone marrow as early as 3 weeks. These cells increased proliferation and interferon gamma production, which contrasted with increased expression of receptors and transcription factors associated with exhaustion. Broad-spectrum antibiotics and antiretrovirals failed to ameliorate the disease. Attempting to genetically “fix” TCR antigen-specificity instead demonstrated the persistence of spontaneous HLH and hyperactivation, chiefly on T cells that had evaded TCR fixation. Thus, drivers of HLH may preferentially act on CD8 T cells: IL-18 amplifies activation and demand for RICD, whereas perforin supplies critical immunoregulation. Together, these factors promote a terminal CD8 T-cell activation state, combining features of exhaustion and effector function. Therefore, susceptibility to hyperinflammation may converge on a unique, unrelenting, and antigen-dependent state of CD8 T-cell hyperactivation.

Engineering donor lymphocytes with Fas ligand protein effectively prevents acute graft-versus-host disease.

Alloreactive T-effector cells (Teffs) are the major culprit of acute graft-versus-host disease (aGVHD) associated with hematopoietic stem cell transplantation. Exvivo nonspecific depletion of T cells from the donor graft impedes stem cell engraftment and posttransplant immune reconstitution. Teffs upregulate Fas after activation and undergo Fas ligand (FasL)-mediated restimulation-induced cell death (RICD), an important mechanism of immune homeostasis. We targeted RICD as a means to eliminate host-reactive Teffs invivo for the prevention of aGVHD. A novel form of FasL protein chimeric with streptavidin (SA-FasL) was transiently displayed on the surface of biotinylated lymphocytes, takingadvantage of the high-affinity interaction between biotin and streptavidin. SA-FasL-engineered mouse and human T cells underwent apoptosis after activation in response to alloantigens invitro and invivo. SA-FasL on splenocytes was effective in preventing aGVHD in >70% of lethally irradiated haploidentical mouse recipients after cotransplantation with bone marrow cells, whereas all controls that underwent transplantation with nonengineered splenocytes developed aGVHD. Prevention of aGVHD was associated with an increased ratio of CD4+CD25+FoxP3+ T regulatory (Tregs) to Teffs and significantly reduced transcripts for proinflammatory cytokines in the lymphoid organs and target tissues. Depletion of Tregs from the donor graft abrogated the protection conferred by SA-FasL. This approach was also effective in a xenogeneic aGVHD setting where SA-FasL-engineered human PBMCs were transplanted into NSG mice. Direct display of SA-FasL protein on donor cells as an effective means of eliminating alloreactive Teffs in the host represents a practical approach with significant translation potential for the prevention of aGVHD.

PD-1 protects expanding human T cells from premature restimulation-induced cell death.

Abstract Programmed cell death-1 (PD-1) is a co-inhibitory protein that dampens TCR/CD28 signaling in part by recruiting the phosphatase SHP2 to the immunological synapse. Chronic antigenic restimulation of T cells can induce apoptosis or elicit PD-1-mediated exhaustion, rendering them hyporesponsive and ineffectual. While the checkpoint function of PD-1 has profound implications for cancer immunotherapy, the role of PD-1 in conventional effector T cell responses remains unclear. Restimulation-induced cell death (RICD) is a critical propriocidal apoptosis program triggered in activated T cells upon robust TCR re-engagement, constraining effector T cell expansion and longevity to prevent collateral tissue damage. We hypothesized that PD-1 attenuates RICD sensitivity in human effector T cells by modulating TCR signal strength. Here we show that transient upregulation of PD-1 helps to protect newly activated human CD4+ and CD8+ T cells from premature RICD during clonal expansion, with lesser effects noted in terminally differentiated T cells. Both antibody-mediated blockade of PD-1-PD-L1 and SHP2 inhibition increased RICD sensitivity to a similar extent. Conversely, concomitant addition of recombinant PD-L1 with TCR restimulation significantly reduced RICD in a dose-dependent manner, with more pronounced protection noted uopn CD28 coligation. Our ongoing work aims to define the role of recently discovered downstream PD-1 signaling nodes (e.g. PAG, VRK2) and specific pro/anti-apoptotic proteins in this process. These findings present significant implications for understanding how PD-1 blockade may influence apoptosis sensitivity in both normal and exhausted T cells to alter adaptive immune responses. Supported by grants from NIH (R35GM139619)

Hemophagocytic Lymphohistiocytosis Susceptibility (HLH) Factors IL-18 and Cytotoxic Impairment Converge on Pathologic CD8 T-cell Hyperactivation

Excess IL-18 and graded defects in cytotoxicity are fundamental susceptibility factors for HLH and MAS. In mice, neither excess IL-18 (18tg mice) nor perforin-deficiency individually cause immunopathology without inflammatory challenge. However, 18tg mice lacking perforin (18tg; Prf1-/-) develop spontaneous lethal hyperinflammation, while subclinical MAS was found in 18tg;Prf1+/- mice. HLH and MAS patients demonstrate CD8 T-cell activation in tissue & peripheral blood. We sought to understand how IL-18 and cytotoxic impairment may interact in driving spontaneous hyperinflammation.We examined mice bearing single- and dual-susceptibility factors for “clinical” features of hyperinflammation, cytokine levels, and flow cytometric, transcriptional, functional, and TCR-seq phenotypes.Flow cytometric analysis of dual susceptibility mice showed spontaneous expansion of CD8 T-cells (and not CD4 T- or NK-cells), which expressed the IL-18 receptor and multiple inhibitory receptors (PD-1, Lag-3, CD39), yet overproduced IFNg. Such “hyperinflammatory CD8 T-cells” were present in reticuloendothelial organs (spleen, bone marrow, and liver) but not lymph nodes, peripheral blood, or thymus. Transcriptional analysis demonstrated hyperactivated CD8 T cells showed features of both terminal effector function and exhaustion when compared to well-described datasets. This analysis also revealed a strong signature for recent antigen activation. BulkCD8 TCR sequencing showed dramatic oligoclonal hyper-expansion of TCR sequences from dual susceptibility mice with no significant clonal overlap between biologic replicate samples. In vitro, TCR stimulation was necessary for IL-18 to promote IFNg production and restimulation-induced cell death (RICD), whereas perforin-deficiency prevented RICD. Broad-spectrum antibiotics did not rescue spontaneous hyperinflammation in dual-susceptibility mice, suggesting T-cell responses were not directed at commensal bacteria. Attempts to circumvent CD8 T-cell activation, using inducible CD8 T-cell-specific deletion of Il18r1 or by promoting allelic exclusion by fixing the T-cell receptor, showed breakthrough expansion of hyperactivated CD8 T-cells.Thus, IL-18 and cytotoxicity drive progressive, post-thymic, oligoclonal CD8 T-cell expansion that may underly life-threatening immunopathology in HLH and MAS. Expansion of hyperactivated CD8T cells in reticuloendothelial organs aligns with sites of hemophagocytosis. These data together implicate HLH/MAS susceptibility factors interact with TCR stimulation, whether by infectious triggers or as-yet undetermined self antigens, to drive potentially-targetable mechanisms of T-cell activation. [Display omitted] Supporting figures.

Wiskott-Aldrich syndrome protein interacts and inhibits diacylglycerol kinase alpha promoting IL-2 induction.

Phosphorylation of diacylglycerol by diacylglycerol-kinases represents a major inhibitory event constraining T cell activation upon antigen engagement. Efficient TCR signalling requires the inhibition of the alpha isoform of diacylglycerol kinase, DGKα, by an unidentified signalling pathway triggered by the protein adaptor SAP. We previously demonstrated that, in SAP absence, excessive DGKα activity makes the T cells resistant to restimulation-induced cell death (RICD), an apoptotic program counteracting excessive T cell clonal expansion. Herein, we report that the Wiskott-Aldrich syndrome protein (WASp) inhibits DGKα through a specific interaction of the DGKα recoverin homology domain with the WH1 domain of WASp. Indeed, WASp is necessary and sufficient for DGKα inhibition, and this WASp function is independent of ARP2/3 activity. The adaptor protein NCK-1 and the small G protein CDC42 connect WASp-mediated DGKα inhibition to SAP and the TCR signalosome. In primary human T cells, this new signalling pathway is necessary for a full response in terms of IL-2 production, while minimally affecting TCR signalling and restimulation-induced cell death. Conversely, in T cells made resistant to RICD by SAP silencing, the enhanced DAG signalling due to DGKα inhibition is sufficient to restore apoptosis sensitivity. We discover a novel signalling pathway where, upon strong TCR activation, the complex between WASp and DGKα blocks DGKα activity, allowing a full cytokine response.

Allosteric inhibition of SHP2 rescues functional T-cell abnormalities in SAP deficiency

X-linked lymphoproliferative disease (XLP) is a primary immunodeficiency arising from SH2D1A mutations leading to loss of SLAM-associated protein (SAP). SAP is an intracellular adaptor protein that binds to SLAM family receptors and is expressed in specific lymphoid lineages. In T cells, SAP relays activatory signals from the T-cell receptor but in its absence SH2 containing protein tyrosine phosphase-1 (SHP1), SH2 containing protein tyrosine phosphase-2 (SHP2), and SH2 containing inositol 5'-phosphatase proteins (SHIP) induce T-cell inhibitory signals leading to abnormal T-cell responses. This results in severe clinical manifestations including immune dysregulation, dysgammaglobulinemia, lymphoma, and hemophagocytic lymphohistiocytosis. Current treatment relies on supportive therapies including immunoglobulin replacement and symptom-directed therapy, with hematopoietic stem cell transplant offering the only curative option. As most XLP symptoms are due to defective T-cell function, this study investigated whether inhibition of SHP2 can restore cellular function in the absence of SAP. Healthy donor and XLP patient T cells were activated with anti-CD3/CD28 in T-cell media supplemented with a SHP2 inhibitor (RMC-4550 invitro for 24 hours) and functional assays were performed to assess follicular TH (TFH) cell function, CD8 cytotoxicity, and sensitivity to restimulation-induced cell death. Additionally, SAP-deficient (SAPy/-) mice were treated with RMC-4550 before T-cell mediated challenge with 4-hydroxy-3-nitrophenylacetly conjugated chicken gammaglobulin and subsequent assessment of humoral immunity analyzing TFH cell population, germinal center formation, and antigen-dependent immunoglobulin secretion. This study shows that the use of RMC-4550 restores T-cell function in XLP patient cells and a SAPy/- model, demonstrating restoration of TFH cell function through immunoglobulin and cytokine secretion analysis alongside rescue of cytotoxicity and restimulation-induced cell death. These data suggest that SHP2 inhibitors could offer a novel and effective targeted treatment approach for patients with XLP.

Restimulation-Induced Cell Death (RICD): Methods for Modeling, Investigating, and Quantifying RICD Sensitivity in Primary Human T Cells via Flow Cytometric Analysis.

When the body mounts an immune response against a foreign pathogen, the adaptive arm of the immune system relies upon clonal expansion of antigen-specific T cell populations to exercise acquired effector and cytotoxic functions to clear it. However, T cell expansion must be modulated to effectively combat the perceived threat without inducing excessive collateral damage to host tissues. Restimulation-induced cell death (RICD) is an apoptotic program triggered in activated T cells when an abundance of antigen and IL-2 are present, imposing a negative feedback mechanism that constrains the growing T cell population. This autoregulatory process can be detected via increases in caspase activation, Annexin V binding, and loss of mitochondrial membrane potential. However, simple changes in T cell viability through flow cytometric analysis can reliably measure RICD sensitivity in response to T-cell receptor (TCR) restimulation. This protocol describes the in vitro polyclonal activation, expansion, and restimulation of human primary T cells isolated from donor peripheral blood mononuclear cells (PBMC). This simple procedure allows for accurate quantification of RICD via flow cytometry. We also describe strategies for interrogating the role of specific proteins and pathways that may alter RICD sensitivity. This straightforward protocol provides a quick and dependable tool to track RICD sensitivity in culture over time while probing critical factors that control the magnitude and longevity of an adaptive immune response. Graphic abstract: In-vitro simulation of restimulation-induced cell death in activated human T cells.

TIM-3 drives temporal differences in restimulation-induced cell death sensitivity in effector CD8+ T cells in conjunction with CEACAM1

Abstract Immune homeostasis depends upon effective clearance of pathogens while simultaneously preventing immunopathology in the host. Restimulation-induced cell death (RICD) is one such mechanism where by activated T cells receive subsequent antigenic stimulation, reach a critical signal threshold through the T cell receptor (TCR), and commit to apoptosis. Many details of this process remain unclear, including the role of proteins that influence the TCR signaling cascade. Here we characterize the role of T cell immunoglobulin and mucin domain containing 3 (TIM-3) in RICD regulation. TIM-3 protected newly activated CD8+ effector T cells from premature RICD during clonal expansion. Surprisingly, however, we found that TIM-3 potentiated RICD in late-stage effector T cells. The presence of TIM-3 increased proximal TCR signaling and pro-apoptotic protein expression in late-stage effector T cells, with no consistent signaling effects in newly activated cells. We found that TIM-3 was expressed on the surface of newly activated effector T cells, but remained largely intracellular in late-stage effector cells. Consistent with this, TIM-3 required a ligand to prevent early RICD only immediately after activation. We found that CEACAM1, a known ligand of TIM-3, protected newly activated cells from premature RICD, with no measurable effects in late-stage effectors. Indeed, CEACAM1 enabled TIM-3 surface expression on T cells, implying a co-dependency for these proteins in protecting expanding T cells from premature RICD. Our findings suggest that co-signaling proteins like TIM-3 and CEACAM1 can alter RICD sensitivity at different stages of the effector T cell response, with important implications for checkpoint blockade therapy.

TIM-3 drives temporal differences in restimulation-induced cell death sensitivity in effector CD8+ T cells in conjunction with CEACAM1

Immune homeostasis depends upon effective clearance of pathogens while simultaneously preventing autoimmunity and immunopathology in the host. Restimulation-induced cell death (RICD) is one such mechanism where by activated T cells receive subsequent antigenic stimulation, reach a critical signal threshold through the T cell receptor (TCR), and commit to apoptosis. Many details of this process remain unclear, including the role of co-stimulatory and co-inhibitory proteins that influence the TCR signaling cascade. Here we characterize the role of T cell immunoglobulin and mucin domain containing 3 (TIM-3) in RICD regulation. TIM-3 protected newly activated CD8+ effector T cells from premature RICD during clonal expansion. Surprisingly, however, we found that TIM-3 potentiated RICD in late-stage effector T cells. The presence of TIM-3 increased proximal TCR signaling and proapoptotic protein expression in late-stage effector T cells, with no consistent signaling effects noted in newly activated cells with or without TIM-3. To better explain these differences in TIM-3 function as T cells aged, we characterized the temporal pattern of TIM-3 expression in effector T cells. We found that TIM-3 was expressed on the surface of newly activated effector T cells, but remained largely intracellular in late-stage effector cells. Consistent with this, TIM-3 required a ligand to prevent early RICD, whereas ligand manipulation had no effects at later stages. Of the known TIM-3 ligands, carcinoembryonic antigen‐related cell adhesion molecule (CEACAM1) showed the greatest difference in surface expression over time and also protected newly activated cells from premature RICD, with no measurable effects in late-stage effectors. Indeed, CEACAM1 enabled TIM-3 surface expression on T cells, implying a co-dependency for these proteins in protecting expanding T cells from premature RICD. Our findings suggest that co-signaling proteins like TIM-3 and CEACAM1 can alter RICD sensitivity at different stages of the effector T cell response, with important implications for checkpoint blockade therapy.

Haploinsufficiency of A20 Due to Novel Mutations in TNFAIP3.

Haploinsufficiency of A20 (HA20) is a newly described immune dysregulation disease due to the loss-of-function mutation in TNFAIP3. In the present study, we report six patients from four unrelated Chinese families with distinct pathogenic mutations in TNFAIP3, including three novel variants. All of the patients presented with early-onset autoimmune/auto-inflammatory diseases, including Crohn's disease, Behcet's disease, systemic lupus erythematosus, and unclassified auto-inflammatory syndrome. Immunological phenotype tests showed elevated levels of serum pro-inflammatory cytokines, reduced naïve B cells and TFH cells, an inverted CD4:CD8 ratio, and increased susceptibility to restimulation-induced cell death (RICD) and FASL-induced apoptosis in derived T cells. Insufficient expression of A20 was found in these patients. A20 truncated protein was detected in mutant-transfected 293T cells. Upon TNF-α stimulation, the NF-κB pathway was over-activated in both derived T cells of these patients and mutant-transfected Hela cells. In conclusion, clinical manifestations are diverse in patients with HA20, even in those with the same TNFAIP3 mutation. A20 inhibits the NF-κB pathway and plays a crucial role in the regulation of cell death. Haploinsufficiency of A20 leads to defects in both innate and adaptive immunity.

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.

Fatty Acid Synthase Contributes to Restimulation-Induced Cell Death of Human CD4 T Cells.

Restimulation-induced cell death (RICD) is an apoptotic pathway triggered in activated effector T cells after T cell receptor (TCR) re-engagement. RICD operates at the peak of the immune response to ensure T cell expansion remains in check to maintain immune homeostasis. Understanding the biochemical regulation of RICD sensitivity may provide strategies for tuning the magnitude of an effector T cell response. Metabolic reprogramming in activated T cells is not only critical for T cell differentiation and effector functions, but also influences apoptosis sensitivity. We previously demonstrated that aerobic glycolysis correlates with optimum RICD sensitivity in human effector CD8 T cells. However, metabolic programming in CD4 T cells has not been investigated in this context. We employed a pharmacological approach to explore the effects of fatty acid and glycolytic metabolism on RICD sensitivity in primary human CD4 T cells. Blockade of fatty acid synthase (FASN) with the compound C75 significantly protected CD4 effector T cells from RICD, suggesting that fatty acid biosynthesis contributes to RICD sensitivity. Interestingly, sphingolipid synthesis and fatty acid oxidation (FAO) were dispensable for RICD. Disruption of glycolysis did not protect CD4 T cells from RICD unless glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymatic activity was targeted specifically, highlighting important differences in the metabolic control of RICD in effector CD4 vs. CD8 T cell populations. Moreover, C75 treatment protected effector CD4 T cells derived from naïve, effector memory, and central memory T cell subsets. Decreased RICD in C75-treated CD4 T cells correlated with markedly reduced FAS ligand (FASL) induction and a Th2-skewed phenotype, consistent with RICD-resistant CD4 T cells. These findings highlight FASN as a critical metabolic potentiator of RICD in human effector CD4 T cells.

FOXP3, CD48 and autophagy confer the protection of CD4 and CD8 human T cells from T cell receptor restimulation-induced cell death

Abstract The proliferation and contraction of activated effector T cells must be carefully coordinated to maintain immune homeostasis. This is achieved in part through restimulation-induced cell death (RICD), a pre-programmed apoptosis program triggered by antigen restimulation through the T cell receptor (TCR). Forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) also constrain conventional T cell (Tcon) responses, but can resist RICD themselves despite frequent TCR stimulation. We previously showed that FOXP3 protects Tregs from RICD by suppressing SLAM-associated protein (SAP), a key adaptor protein that amplifies TCR signal strength. Mysteriously, FOXP3 expression is also transiently induced in human Tcons after activation, with a kinetic expression profile that correlates inversely with acquired RICD sensitivity. Hence we asked whether FOXP3 protects expanding human effector T cells from premature RICD by modulating SAP expression. Our results show that although siRNA-mediated FOXP3 knockdown sensitizes early effector CD4 and CD8 T cells to RICD, SAP expression remains unaffected. Unlike late stage effector T cells, a low level of RICD in expanding Tcons was entirely dependent on de novo transcription, and knockdown of SAP failed to reduce death. Subsequent RNA-Seq analyses revealed that CD48, a SLAM family receptor, was markedly reduced upon FOXP3 knockdown. Blockade or knockdown of CD48 also increased RICD in CD4 and CD8 T cells. We now show that CD48 protects early effector T cells both by promoting autophagy and upregulation of pro-survival genes such as BATF. These findings implicate FOXP3 as the central governor of a distinct transcriptional program that promotes RICD resistance early in the effector T cell response.

TIM-3-dependent regulation of restimulation-induced cell death sensitivity flips changes in early versus late-stage human effector T cells

Abstract In healthy individuals, the balance between immune-mediated pathogen clearance and tissue damage is tightly regulated through multiple processes. One such mechanism is restimulation-induced cell death (RICD), a propriocidal apoptotic mechanism that constrains T cell responses. Activated T cells must surpass a high threshold of TCR signal strength to induce pro-apoptotic genes responsible for executing RICD. Because co-receptors such as TIM-3 are thought to modulate TCR signal strength, we hypothesized that TIM-3 can shape the T cell response by tuning RICD sensitivity. We utilized siRNA knockdowns and transfection of wild-type and mutant TIM-3 expression constructs to assay for changes in RICD sensitivity in both immortalized and primary human T cells in vitro. Strikingly, we observed that the effect of altering TIM-3 expression varied temporally during the effector T cell response. Whereas TIM-3 promoted RICD resistance in newly activated T cells, TIM-3 enhanced RICD sensitivity in late-stage effector T cells by boosting TCR-induced FASL and BIM expression in a ligand-independent fashion. Preliminary evidence suggests that this dichotomy may relate to substantial temporal changes in the expression, cellular localization, and glycosylation of TIM-3 in early versus late-stage effector T cells. Overall, these results indicate that TIM-3 influences apoptosis sensitivity differently at distinct phases of the human T cell response, with important implications for the use of checkpoint blockade immunotherapies targeting TIM-3. Our findings may also help to clarify discrepancies in the literature regarding TIM-3 signaling and function in T cells.

FOXP3 protects human T cells from restimulation-induced cell death via CD48 upregulation and autophagy

Abstract The adaptive immune response relies on specific apoptosis programs to regulate effector T cell expansion and maintain homeostasis. Restimulation-induced cell death (RICD) is an apoptotic pathway triggered by repeated stimulation through the T cell receptor (TCR), which ensures that effector T cell proliferation remains in check. Constitutive fork head box P3 (FOXP3) expression renders regulatory T cells (Tregs) resistant to RICD. Although FOXP3 is also expressed transiently in human conventional T cells (Tcons) during initial rounds of activation-induced proliferation, its function in this context remains unclear. Here we describe a novel role for FOXP3 in protecting both CD4+ and CD8+ human Tcons from premature RICD during the expansion phase. SiRNA-mediated silencing of FOXP3 sensitized early effector Tcons to RICD, and was dependent on de novo transcription. RNA-Seq analysis revealed FOXP3-dependent effects on genes involved in metabolic pathways, autophagy, and cell surface receptors. FOXP3 silencing increased glycolysis and reduced autophagy in Tcons, which has been linked to changes in apoptosis sensitivity. Indeed, blocking autophagy increased RICD in control Tcons without augmenting death of FOXP3-silenced cells, suggesting FOXP3 reduces RICD sensitivity through an autophagy-dependent mechanism. Additionally, FOXP3 knockdown markedly reduced expression of CD48, a SLAM-family receptor. We now show that CD48 protects expanding Tcons from RICD. Surprisingly, CD48 also promoted protective autophagy, illuminating a novel connection between SLAM receptor signaling, autophagy regulation, and RICD sensitivity that is governed by FOXP3 expression in early Tcons.