Antigen Receptor Stimulation Research Articles

Autoregulated splicing of TRA2β programs T cell fate in response to antigen-receptor stimulation.

T cell receptor (TCR) sensitivity to peptide-major histocompatibility complex (MHC) dictates T cell fate. Canonical models of TCR sensitivity cannot be fully explained by transcriptional regulation. In this work, we identify a posttranscriptional regulatory mechanism of TCR sensitivity that guides alternative splicing of TCR signaling transcripts through an evolutionarily ultraconserved poison exon (PE) in the RNA-binding protein (RBP) TRA2β in mouse and human. TRA2β-PE splicing, seen during cancer and infection, was required for TCR-induced effector T cell expansion and function. Tra2β-PE skipping enhanced T cell response to antigen by increasing TCR sensitivity. As antigen levels decreased, Tra2β-PE reinclusion allowed T cell survival. Finally, we found that TRA2β-PE was first included in the genome of jawed vertebrates that were capable of TCR gene rearrangements. We propose that TRA2β-PE splicing acts as a gatekeeper of TCR sensitivity to shape T cell fate.

Metabolic regulation of forkhead box P3 alternative splicing isoforms and their impact on health and disease.

Forkhead Box P3 (FOXP3) is crucial for the development and suppressive function of human regulatory T cells (Tregs). There are two predominant FOXP3 splicing isoforms in healthy humans, the full-length isoform and the isoform lacking exon 2, with different functions and regulation mechanisms. FOXP3 splicing isoforms show distinct abilities in the cofactor interaction and the nuclear translocation, resulting in different effects on the differentiation, cytokine secretion, suppressive function, linage stability, and environmental adaptation of Tregs. The balance of FOXP3 splicing isoforms is related to autoimmune diseases, inflammatory diseases, and cancers. In response to environmental challenges, FOXP3 transcription and splicing can be finely regulated by T cell antigen receptor stimulation, glycolysis, fatty acid oxidation, and reactive oxygen species, with various signaling pathways involved. Strategies targeting energy metabolism and FOXP3 splicing isoforms in Tregs may provide potential new approaches for the treatment of autoimmune diseases, inflammatory diseases, and cancers. In this review, we summarize recent discoveries about the FOXP3 splicing isoforms and address the metabolic regulation and specific functions of FOXP3 splicing isoforms in Tregs.

Hallmarks of CD8+ T cell dysfunction are established within hours of tumor antigen encounter before cell division.

Tumor-specific CD8+ T cells (TST) in patients with cancer are dysfunctional and unable to halt cancer progression. TST dysfunction, also known as exhaustion, is thought to be driven by chronic T cell antigen receptor (TCR) stimulation over days to weeks. However, we know little about the interplay between CD8+ T cell function, cell division and epigenetic remodeling within hours of activation. Here, we assessed early CD8+ T cell differentiation, cell division, chromatin accessibility and transcription in tumor-bearing mice and acutely infected mice. Surprisingly, despite robust activation and proliferation, TST had near complete effector function impairment even before undergoing cell division and had acquired hallmark chromatin accessibility features previously associated with later dysfunction/exhaustion. Moreover, continued tumor/antigen exposure drove progressive epigenetic remodeling, 'imprinting' the dysfunctional state. Our study reveals the rapid divergence of T cell fate choice before cell division in the context of tumors versus infection.

Stimulation of ectopically expressed muscarinic receptors induces IFN-γ but suppresses IL-2 production by inhibiting activation of pAKT pathways in primary T cells

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCβ1 is coexpressed. Resting peripheral hM3Dq+PLCβ1 (hM3Dq/β1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCβ1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/β1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/β1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/β1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

CARD11 gain-of-function mutation drives cell-autonomous accumulation of PD-1+ ICOShigh activated T cells, T-follicular, T-regulatory and T-follicular regulatory cells.

Germline CARD11 gain-of-function (GOF) mutations cause B cell Expansion with NF-κB and T cell Anergy (BENTA) disease, whilst somatic GOF CARD11 mutations recur in diffuse large B cell lymphoma (DLBCL) and in up to 30% of the peripheral T cell lymphomas (PTCL) adult T cell leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL) and Sezary Syndrome. Despite their frequent acquisition by PTCL, the T cell-intrinsic effects of CARD11 GOF mutations are poorly understood. Here, we studied B and T lymphocytes in mice with a germline Nethyl-N-nitrosourea (ENU)-induced Card11M365K mutation identical to a mutation identified in DLBCL and modifying a conserved region of the CARD11 coiled-coil domain recurrently mutated in DLBCL and PTCL. Our results demonstrate that CARD11.M365K is a GOF protein that increases B and T lymphocyte activation and proliferation following antigen receptor stimulation. Germline Card11M365K mutation was insufficient alone to cause B or T-lymphoma, but increased accumulation of germinal center (GC) B cells in unimmunized and immunized mice. Card11M365K mutation caused cell-intrinsic over-accumulation of activated T cells, T regulatory (TREG), T follicular (TFH) and T follicular regulatory (TFR) cells expressing increased levels of ICOS, CTLA-4 and PD-1 checkpoint molecules. Our results reveal CARD11 as an important, cell-autonomous positive regulator of TFH, TREG and TFR cells. They highlight T cell-intrinsic effects of a GOF mutation in the CARD11 gene, which is recurrently mutated in T cell malignancies that are often aggressive and associated with variable clinical outcomes.

A selective LIS1 requirement for mitotic spindle assembly discriminates distinct T-cell division mechanisms within the T-cell lineage.

The ability to proliferate is a common feature of most T-cell populations. However, proliferation follows different cell-cycle dynamics and is coupled to different functional outcomes according to T-cell subsets. Whether the mitotic machineries supporting these qualitatively distinct proliferative responses are identical remains unknown. Here, we show that disruption of the microtubule-associated protein LIS1 in mouse models leads to proliferative defects associated with a blockade of T-cell development after b-selection and of peripheral CD4+ T cell expansion after antigen priming. In contrast, cell divisions in CD8+ T cells occurred independently of LIS1 following T-cell antigen receptor stimulation, although LIS1 was required for proliferation elicited by pharmacological activation. In thymocytes and CD4+ T cells, LIS1-deficiency did not affect signaling events leading to activation but led to an interruption of proliferation after the initial round of division and to p53-induced cell death. Proliferative defects resulted from a mitotic failure, characterized by the presence of extra-centrosomes and the formation of multipolar spindles, causing abnormal chromosomes congression during metaphase and separation during telophase. LIS1 was required to stabilize dynein/dynactin complexes, which promote chromosome attachment to mitotic spindles and ensure centrosome integrity. Together, these results suggest that proliferative responses are supported by distinct mitotic machineries across T-cell subsets.

The mitochondrial sodium/calcium exchanger NCLX (Slc8b1) in B lymphocytes

Antigen receptor stimulation triggers cytosolic Ca2+ signals, which activate transcriptional and metabolic programs critical for immune function. B-cell receptor (BCR) engagement causes rapid cytosolic Ca2+ rise through the ubiquitous store-operated calcium entry (SOCE) pathway. Slc8b1, which encodes the mitochondrial Na+/Ca2+ exchanger (NCLX), extrudes Ca2+ out of the mitochondria and maintains optimal SOCE activity. Inhibition of NCLX in DT40 and A20 B lymphocyte lines was recently shown to impair cytosolic Ca2+ transients in response to antigen-receptor stimulation, however the downstream functional consequences of this impairment remain unclear. Here, we generated Slc8b1 knockout A20 B-cell lines using CRISPR/Cas9 technology and B-cell specific Slc8b1 knockout mice. Surprisingly, while loss of Slc8b1 in B lymphocytes led to reduction in SOCE, it had a marginal effect on mitochondrial Ca2+ extrusion, suggesting that NCLX is not the major mitochondrial Ca2+ extrusion mechanism in B cells. Furthermore, endoplasmic reticulum (ER) Ca2+ content and rates of ER depletion and refilling remained unaltered in Slc8b1 knockout B cells. Slc8b1 deficiency increased mitochondrial production of oxidants, reduced mitochondrial bioenergetics and altered mitochondrial ultrastructure. B-cell specific Slc8b1 knockout mice showed reduced germinal center B cell responses following foreign antigen and pathogen driven immune responses. Our studies provide novel insights into the function of Slc8b1 in germinal center B cells and its contribution to B-cell signaling and effector function.

Spatial and Functional Crosstalk between the Mitochondrial Na+-Ca2+ Exchanger NCLX and the Sarcoplasmic Reticulum Ca2+ Pump SERCA in Cardiomyocytes.

The mitochondrial Na+-Ca2+ exchanger, NCLX, was reported to supply Ca2+ to sarcoplasmic reticulum (SR)/endoplasmic reticulum, thereby modulating various cellular functions such as the rhythmicity of cardiomyocytes, and cellular Ca2+ signaling upon antigen receptor stimulation and chemotaxis in B lymphocytes; however, there is little information on the spatial relationships of NCLX with SR Ca2+ handling proteins, and their physiological impact. Here we examined the issue, focusing on the interaction of NCLX with an SR Ca2+ pump SERCA in cardiomyocytes. A bimolecular fluorescence complementation assay using HEK293 cells revealed that the exogenously expressed NCLX was localized in close proximity to four exogenously expressed SERCA isoforms. Immunofluorescence analyses of isolated ventricular myocytes showed that the NCLX was localized to the edges of the mitochondria, forming a striped pattern. The co-localization coefficients in the super-resolution images were higher for NCLX–SERCA2, than for NCLX–ryanodine receptor and NCLX–Na+/K+ ATPase α-1 subunit, confirming the close localization of endogenous NCLX and SERCA2 in cardiomyocytes. The mathematical model implemented with the spatial and functional coupling of NCLX and SERCA well reproduced the NCLX inhibition-mediated modulations of SR Ca2+ reuptake in HL-1 cardiomyocytes. Taken together, these results indicated that NCLX and SERCA are spatially and functionally coupled in cardiomyocytes.

Schnurri 3 promotes Th2 cytokine production during the late phase of T-cell antigen stimulation.

Th1 and Th2 polarization is determined by the coordination of numerous factors including the affinity and strength of the antigen‐receptor interaction, predominant cytokine environment, and costimulatory molecules present. Here, we show that Schnurri (SHN) proteins have distinct roles in Th1 and Th2 polarization. SHN2 was previously found to block the induction of GATA3 and Th2 differentiation. We found that, in contrast to SHN2, SHN3 is critical for IL‐4 production and Th2 polarization. Strength of stimulation controls SHN2 and SHN3 expression patterns, where higher doses of antigen receptor stimulation promoted SHN3 expression and IL‐4 production, along with repression of SHN2 expression. SHN3‐deficient T cells showed a substantial defect in IL‐4 production and expression of AP‐1 components, particularly c‐Jun and Jun B. This loss of early IL‐4 production led to reduced GATA3 expression and impaired Th2 differentiation. Together, these findings uncover SHN3 as a novel, critical regulator of Th2 development.

Siglec-5 is an inhibitory immune checkpoint molecule for human T cells.

Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunoglobulin-type lectins that mediate protein-carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33-related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec-5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec-5suppresses T cell activation. In support of these findings, we found that Siglec-5 overexpression abrogates antigen receptor induced activation of NFAT and AP-1. Furthermore, we show that GBS β-protein, a known bacterial ligand of Siglec-5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec-5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec-5ligand(s), and that the presence of soluble Siglec-5 enhances tumor-cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec-5. Together, our data demonstrate that Siglec-5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggest that blockade of Siglec-5 could serve as a new strategy to enhance anti-tumor T cell functions.

Potential role of inducible GPR3 expression under stimulated T cell conditions

G protein-coupled receptor 3 (GPR3) constitutively activates Gαs proteins without any ligands and is predominantly expressed in neurons. Since the expression and physiological role of GPR3 in immune cells is still unknown, we examined the possible role of GPR3 in T lymphocytes. The expression of GPR3 was upregulated 2 h after phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation and was sustained in Jurkat cells, a human T lymphocyte cell line. In addition, the expression of nuclear receptor 4 group A member 2 (NR4A2) was highly modulated by GPR3 expression. Additionally, GPR3 expression was linked with the transcriptional promoter activity of NR4A in Jurkat cells. In mouse CD4+ T cells, transient GPR3 expression was induced immediately after the antigen receptor stimulation. However, the expression of NR4A2 was not modulated in CD4+ T cells from GPR3-knockout mice after stimulation, and the population of Treg cells in thymocytes and splenocytes was not affected by GPR3 knockout. By contrast, spontaneous effector activation in both CD4+ T cells and CD8+ T cells was observed in GPR3-knockout mice. In summary, GPR3 is immediately induced by T cell stimulation and play an important role in the suppression of effector T cell activation.

Genetic Ablation of the Mitochondrial Calcium Uniporter (MCU) Does not Impair T Cell-Mediated Immunity In Vivo.

T cell activation and differentiation is associated with metabolic reprogramming to cope with the increased bioenergetic demand and to provide metabolic intermediates for the biosynthesis of building blocks. Antigen receptor stimulation not only promotes the metabolic switch of lymphocytes but also triggers the uptake of calcium (Ca2+) from the cytosol into the mitochondrial matrix. Whether mitochondrial Ca2+ influx through the mitochondrial Ca2+ uniporter (MCU) controls T cell metabolism and effector function remained, however, enigmatic. Using mice with T cell-specific deletion of MCU, we here show that genetic inactivation of mitochondrial Ca2+ uptake increased cytosolic Ca2+ levels following antigen receptor stimulation and store-operated Ca2+ entry (SOCE). However, ablation of MCU and the elevation of cytosolic Ca2+ did not affect mitochondrial respiration, differentiation and effector function of inflammatory and regulatory T cell subsets in vitro and in animal models of T cell-mediated autoimmunity and viral infection. These data suggest that MCU-mediated mitochondrial Ca2+ uptake is largely dispensable for murine T cell function. Our study has also important technical implications. Previous studies relied mostly on pharmacological inhibition or transient knockdown of mitochondrial Ca2+ uptake, but our results using mice with genetic deletion of MCU did not recapitulate these findings. The discrepancy of our study to previous reports hint at compensatory mechanisms in MCU-deficient mice and/or off-target effects of current MCU inhibitors.

ER stress and its PERK branch enhance TCR-induced activation in regulatory T cells

Although accumulating evidence indicates participation of endoplasmic reticulum (ER) stress pathway or the unfolded protein response (UPR) in immunity, there still exists little information linking ER stress to regulatory T cells (Tregs). To evaluate the potential contribution of the UPR, we tested the effects of thapsigargin (TG), an ER stress inducer, on the function of Tregs. Here we reported that TG stimulation induced the up-regulation of the endoplasmic reticulum (ER)-stress chaperon Glucose-Regulated Protein 78 (GRP78), which is a master regulator of the UPR, the phosphorylation of eukaryotic initiation factor 2 alpha (elF2α) and the induction of activating transcription factor 4 (ATF4), which are both protein kinase R (PKR)-like ER kinase (PERK) downstream targets in Tregs. Simultaneously, we demonstrated that, under ER stress conditions, Tregs presented enhanced functional activity upon TCR stimulation, as illustrated with forkhead box transcription factor (Foxp3) expression, interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) production and suppressive functional analysis. Notably, pretreatment with GSK2656157, a potent and selective PERK inhibitor, markedly diminished the TG-induced hyperresponsiveness of Tregs upon T cell antigen receptor (TCR) stimulation. Therefore, our findings illustrated the inter-connection and coordination of the evolutionarily conserved ER stress response and TCR signaling in Tregs and uncover a critical new role of the PERK branch of UPR in the regulation of Tregs function.

Calcium flux control by Pacs1-Wdr37 promotes lymphocyte quiescence and lymphoproliferative diseases.

Endoplasmic reticulum (ER) calcium (Ca2+ ) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca2+ handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca2+ release from the ER after antigen receptor stimulation. Pacs1-deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1-/- B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1-Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1-Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca2+ handling and presents a new target for lymphoproliferative disease therapy.

T-bet is a critical determinant in the instability of the IL-17-secreting T-helper phenotype

IL-23, an IL-12-related cytokine, induces an IL-17-secreting T-helper phenotype that is involved in autoimmune diseases and host defense against certain pathogens. Although the transcription factors required for development of IL-23-stimulated cells are unknown, we show that T-bet is a critical negative regulator of the IL-23-primed T-cell phenotype, which we term Th1beta. Th1 or Th1beta Tbx21-/- cultures secrete higher than WT levels of IL-17 in response to T-cell receptor (TCR) or IL-23 + IL-18 stimulation. Ectopic T-bet expression in Th1beta cells promotes IFN-gamma secretion but decreases IL-17 production. Although antigen-receptor stimulation of Th1beta cells stimulates IL-17 production, it also induces the IFN-gamma-independent expression of T-bet and progression to a Th1 cytokine secretion pattern. T-bet is required for the progression to the Th1 phenotype, because Tbx21-/- Th1beta cultures maintain the IL-17-secreting phenotype after 2 weeks of culture. Addition of IFN-gamma to Tbx21-/- Th1beta cultures cannot recover the progression to the Th1 phenotype, suggesting T-bet, rather than IFN-gamma, mediates Th1beta to Th1 progression. The transient nature of the Th1beta phenotype suggests that these cells are a component of type I immunity and that T-bet expression is a critical determinant of Th1 versus Th1beta cell fate.

NR4A nuclear receptors restrain B cell responses to antigen when second signals are absent or limiting.

Antigen stimulation (signal 1) triggers B cell proliferation, and primes B cells to recruit, engage, and respond to T cell help (signal 2). Failure to receive signal 2 within a defined time window results in B cell apoptosis, yet the mechanisms that enforce dependence upon co-stimulation are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell antigen receptor (BCR) stimulation. Here we showed that Nr4a1 and Nr4a3 play partially redundant roles to restrain B cell responses to antigen in the absence of co-stimulation, and do so in part by repressing expression of BATF and consequently MYC. The NR4A family also restrains B cell access to T cell help by repressing expression of the T cell chemokines CCL3 and CCL4, as well as CD86 and ICAM1. Such NR4A-mediated regulation plays a role specifically under conditions of competition for limiting T cell help.

Gain-of-function mutations in CARD11 promote enhanced aggregation and idiosyncratic signalosome assembly

BENTA (B cell Expansion with NF-κB and T cell Anergy) is a novel lymphoproliferative disorder caused by germline, gain-of-function (GOF) mutations in the lymphocyte-restricted scaffolding protein CARD11. Similar somatic CARD11 mutations are found in lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL). Normally, antigen receptor (AgR) engagement converts CARD11 into an active conformation that nucleates a signalosome required for IκB kinase (IKK) activation and NF-κB nuclear translocation. However, GOF CARD11 mutants drive constitutive NF-κB activity without AgR stimulation. Here we show that unlike wild-type CARD11, GOF CARD11 mutants can form large, peculiar cytosolic protein aggregates we term mCADS (mutant CARD11 dependent shells). MALT1 and phospho-IKK are reliably colocalized with mCADS, indicative of active signaling. Moreover, endogenous mCADS are detectable in ABC-DLBCL lines harboring similar GOF CARD11 mutations. The unique aggregation potential of GOF CARD11 mutants may represent a novel therapeutic target for treating BENTA or DLBCL.

Downstream effectors of BCR signaling control transcription of autoimmunity-associated transcription factor Ets1

Abstract The transcription factor (TF) Ets1 is a critical regulator of B cell quiescence. Its expression must be maintained at a high level in resting B cells to prevent inappropriate differentiation and autoimmune responses, and its downregulation in response to antigen receptor stimulation is vital for proper B cell responses. How Ets1 expression is controlled remains unstudied. Quantification of newly-transcribed Ets1 pre-mRNA shows that Ets1 gene transcription is reduced upon BCR stimulation. Inhibition of new protein synthesis did not prevent repression of Ets1 transcription in response to stimulation, suggesting that the phenomenon depends on modulation of existing TF binding to Ets1 regulatory elements (REs) rather than de novo synthesis of a repressor protein. Epigenetic analyses identified numerous putative REs in the mouse Ets1 locus. A 217 kb BAC transgenic construct containing potential RE sequences drives eGFP reporter expression in a manner that recapitulates that of endogenous Ets1. To identify REs that may be important for regulating Ets1 expression and stimulation-induced repression, we performed ATAC-seq in untreated and BCR-stimulated B cells, and observed potentially meaningful changes to chromatin accessibility. Motifs for highly relevant TFs are enriched in accessible regions in the Ets1 locus, including members of the Smad, ETS, AP-1, and NFκB families. Following up on NFκB as a potential regulator of Ets1 expression, downregulation of Ets1 transcription was found to be dependent upon IKK2 activity but did not require RelA. These analyses suggest a potentially complex regulatory network behind the expression of Ets1 as it safeguards the resting B cell state. Funded by NIH R01 AI122720 and Lupus Research Alliance.

Targeting metabolic dysregulation of T cells in kidney cancer.

722 Background: Cancer cells can inhibit effector T cells through both immunomodulatory receptors and alteration of the tumor microenvironment. Rather than efficient use of aerobic glycolysis by activated effector T cells, Renal Cell Carcinoma (RCC) infiltrating T cells (TIL) fail to increase glucose metabolism, and instead display increased reactive oxygen species (ROS) and mitochondrial dysfunction. CD8 RCC TIL also have notable differences in mitochondrial morphology compared to healthy control CD8 T cells and were punctate and dispersed rather than fused in networks. Here we test if RCC TIL can be re-activated and identify metabolic requirements for inflammatory TIL function. Methods: De-identified samples from tumor or adjacent normal tissue donations from patients with RCC were collected under an approved IRB protocol and processed into single cell suspensions of tumor and associated cells by mechanical dissociation to test TIL activation and metabolic requirements upon in vitro re-stimulation. Results: RNA-seq data suggested that CD8 from TIL rely on distinct metabolic pathways compared to control. While control T cells increased effector cytokine production and glycolysis with antigen receptor alone and further augmented this pathway with co-stimulation, single cell RNAseq showed that CD8 RCC TIL required co-stimulation for this transition. Co-stimulation can promote T cell glycolysis and we found antigen receptor stimulation with CD28-mediated co-stimulation increased function of CD8 RCC TIL as indicated by increased surface markers of activation and IFNγproduction. This was accompanied by rescue of metabolic markers, including increased mitochondrial mass and markers of electron transport. Improved functional capacity was dependent upon glycolysis because inhibition with 2-deoxyglucose limited CD8 RCC TIL activation following CD28 co-stimulation. Conclusions: Bypassing metabolic defects restore markers of TIL activation and effector function.These datademonstrate that CD8 RCC TIL can be functionally restored but that this requires the ability to increase glucose metabolism. These findings may allow for combined therapies to improve response rates of checkpoint inhibition in this disease.

Interleukin-23 engineering improves CAR T cell function in solid tumors.

Cytokines that stimulate T cell proliferation, such as interleukin (IL)-15, have been explored as a means of boosting the antitumor activity of chimeric antigen receptor (CAR) T cells. However, constitutive cytokine signaling in T cells and activation of bystander cells may cause toxicity. IL-23 is a two-subunit cytokine known to promote proliferation of memory T cells and T helper type 17 cells. We found that, upon T cell antigen receptor (TCR) stimulation, T cells upregulated the IL-23 receptor and the IL-23α p19 subunit, but not the p40 subunit. We engineered expression of the p40 subunit in T cells (p40-Td cells) and obtained selective proliferative activity in activated T cells via autocrine IL-23 signaling. In comparison to CAR T cells, p40-Td CAR T cells showed improved antitumor capacity in vitro, with increased granzyme B and decreased PD-1 expression. In two xenograft and two syngeneic solid tumor mouse models, p40-Td CAR T cells showed superior efficacy in comparison to CAR T cells and attenuated side effects in comparison to CAR T cells expressing IL-18 or IL-15.