Wild-type CD4 Research Articles

Flotillin-2 dampens T cell antigen-sensitivity and functionality.

T cell receptor (TCR) engagement triggers T cell responses, yet how TCR-mediated activation is regulated at the plasma membrane remains unclear. Here, we report that deleting the membrane scaffolding protein Flotillin-2 (Flot2) increases T cell antigen-sensitivity, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice exhibited reduced tumor growth and enhanced immunity to infection. Flot2-null CD4+ T cells exhibited increased T helper 1 polarization, proliferation, Nur77 induction, and phosphorylation of ZAP70 and ERK1/2 upon weak TCR stimulation, indicating a sensitized TCR-triggering threshold. Single cell-RNA sequencing suggested that Flot2-null CD4+ T cells follow a similar route of activation as wild-type CD4+ T cells but exhibit higher occupancy of a discrete activation state under weak TCR stimulation. Given prior reports that TCR clustering influences sensitivity of T cells to stimuli, we evaluated TCR distribution with super-resolution microscopy. Flot2 ablation increased the number of surface TCR nanoclusters on naïve CD4+ T cells. Collectively, we posit that Flot2 modulates T cell functionality to weak TCR stimulation, at least in part, by regulating surface TCR clustering. Our findings have implications for improving T cell reactivity in diseases with poor antigenicity, such as cancer and chronic infections.

Reduced function of the adaptor SH2B3 promotes T1D via altered gc cytokine-regulated, T cell intrinsic immune tolerance.

The rs3184504 polymorphism, encoding a hypomorphic variant of the negative regulator SH2B3, strongly associates with T1D.SH2B3 deficiency results in hypersensitivity to cytokines, including IL-2, in murine CD4+ and CD8+ T cells.SH2B3 deficient CD8+ T cells exhibit a comparable transcriptome to wild-type CD8+ T cells at baseline, but upon antigen stimulation SH2B3 deficient cells upregulate genes characteristic of enhanced JAK/STAT signaling and effector functions.We found a T-cell intrinsic role of SH2B3 leading to severe islet destruction in an adoptive transfer murine T1D model, while global SH2B3 deficiency accelerated spontaneous NOD diabetes across sexes.

Chronic TNF in the aging microenvironment exacerbates Tet2 loss-of-function myeloid expansion

AbstractSomatic mutations in the TET2 gene occur more frequently with age, imparting an intrinsic hematopoietic stem cells (HSCs) advantage and contributing to a phenomenon termed clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutant CHIP have a higher risk of developing myeloid neoplasms and other aging-related conditions. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing tumor necrosis factor (TNF) favors TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSCs with TET2 mutations in vivo. To test this hypothesis, we generated mixed bone marrow chimeric mice of old wild-type (WT) and TNF–/– genotypes reconstituted with WT CD45.1+ and Tet2–/– CD45.2+ HSCs. We show that age-associated increases in TNF dramatically increased the expansion of Tet2–/– cells in old WT recipient mice, with strong skewing toward the myeloid lineage. This aberrant myelomonocytic advantage was mitigated in old TNF–/– recipient mice, suggesting that TNF signaling is essential for the expansion Tet2-mutant myeloid clones. Examination of human patients with rheumatoid arthritis with clonal hematopoiesis revealed that hematopoietic cells carrying certain mutations, including in TET2, may be sensitive to reduced TNF bioactivity following blockade with adalimumab. This suggests that targeting TNF may reduce the burden of some forms of CHIP. To our knowledge, this is the first evidence to demonstrate that TNF has a causal role in driving TET2-mutant CHIP in vivo. These findings highlight TNF as a candidate therapeutic target to control TET2-mutant CHIP.

VISTA Deficiency Exacerbates the Development of Pulmonary Fibrosis by Promoting Th17 Differentiation.

Interstitial lung disease (ILD), characterized by pulmonary fibrosis (PF), represents the end-stage of various ILDs. The immune system plays an important role in the pathogenesis of PF. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is an immune checkpoint with immune suppressive functions. However, its specific role in the development of PF and the underlying mechanisms remain to be elucidated. We assessed the expression of VISTA in CD4 T cells from patients with connective tissue disease-related interstitial lung disease (CTD-ILD). Spleen cells from wild-type (WT) or Vsir -/- mice were isolated and induced for cell differentiation in vitro. Additionally, primary lung fibroblasts were isolated and treated with interleukin-17A (IL-17A). Mice were challenged with bleomycin (BLM) following VISTA blockade or Vsir knockout. Moreover, WT or Vsir -/- CD4 T cells were transferred into Rag1 -/- mice, which were then challenged with BLM. VISTA expression was decreased in CD4 T cells from patients with CTD-ILD. Vsir deficiency augmented T-helper 17 (Th17) cell differentiation in vitro. Furthermore, IL-17A enhanced the production of inflammatory cytokines, as well as the differentiation and migration of lung fibroblasts. Both VISTA blockade and knockout of Vsir increased the percentage of IL-17A-producing Th17 cells and promoted BLM-induced PF. In addition, mice receiving Vsir -/- CD4 T cells exhibited a higher percentage of Th17 cells and more severe PF compared to those receiving WT CD4 T cells. These findings demonstrate the significant role of VISTA in modulating the development of PF by controlling Th17 cell differentiation. These insights suggest that targeting VISTA could be a promising therapeutic strategy for PF.

Abstract 2654: HPK1 inhibits CD8+ T cell effector gene expression following T cell activation

Abstract Hematopoietic progenitor kinase 1 (HPK1) has been shown to act as a negative regulator of T cell receptor signaling and of subsequent effector T cell function. Inhibiting HPK1 to enhance T cell activity has emerged as a promising strategy for cancer immunotherapy. Upon T cell receptor engagement, the kinase domain of HPK1 is activated and targets components of the T cell receptor (TCR) signaling pathway for degradation, including SLP76. However, the molecular events connecting HPK1 kinase activity to observed T cell functions downstream of proximal TCR signaling are not well understood. Through transcriptional profiling of HPK1-kinase-dead (HPK1-KD) versus wild-type CD8+ T cells following an acute, attenuated Listeria monocytogenes infection, we observed increased expression of many genes associated with T cell activation and effector function, including changes in expression of several key transcription factors and their targets. Upon activation, HPK1-KD T cells, as well as T cells treated with our small-molecule HPK1 inhibitor, produce higher levels of a wide range of effector cytokines in vitro and in vivo. Treatment of mice with the HPK1 inhibitor also inhibited tumor growth in several syngeneic tumor models. These data expand our understanding of the role of HPK1 in inhibiting CD8+ T cell effector programs, and, importantly, the impact of HPK1 inhibitors on T cell function. Citation Format: Rachel Y. Ames, Rongqi Zhao, Parker Mace, Adam Grant, Guorui Xie, Heather Milestone, Molly Grandcolas, Eva Fang, Nicolae Kiosea, Stephen Wong, Martin Brovarney, Scott Jacobson, Danilo Nebalasca, Jorge Arguello, Blanca Gomez, Hiranmayee Kandala, Michelle Y. Ko, Lan Nguyen, Omar Robles, Grant Shibuya, Anton A. Shakhmin, Parcharee Tivitmahaisoon, Vi-Anh Vu, Ashkaan Younai, Mikhail Zibinsky, Babu Subramanyam, Daniel Poon, Mohsen Sabouri Ghomi, David J. Wustrow, Paul D. Kassner, George E. Katibah, Dirk G. Brockstedt. HPK1 inhibits CD8+ T cell effector gene expression following T cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2654.

In Vivo Stimulation of Therapeutic Antigen-Specific T Cells in an Artificial Lymph Node Matrix.

T cells are critical mediators of antigen-specific immune responses and are common targets for immunotherapy. Biomaterial scaffolds have previously been used to stimulate antigen-presenting cells to elicit antigen-specific immune responses; however, structural and molecular features that directly stimulate and expand naïve, endogenous, tumor-specific T cells in vivo have not been defined. Here, an artificial lymph node (aLN) matrix is created, which consists of an extracellular matrix hydrogel conjugated with peptide-loaded-MHC complex (Signal 1), the co-stimulatory signal anti-CD28 (Signal 2), and a tethered IL-2 (Signal 3), that can bypass challenges faced by other approaches to activate T cells in situ such as vaccines. This dynamic immune-stimulating platform enables direct, in vivo antigen-specific CD8+ T cell stimulation, as well as recruitment and coordination of host immune cells, providing an immuno-stimulatory microenvironment for antigen-specific T cell activation and expansion. Co-injecting the aLN with naïve, wild-type CD8+ T cells results in robust activation and expansion of tumor-targeted T cells that kill target cells and slow tumor growth in several distal tumor models. The aLN platform induces potent in vivo antigen-specific CD8+ T cell stimulation without the need for ex vivo priming or expansion and enables in situ manipulation of antigen-specific responses for immunotherapies.

CRL4b Inhibition Ameliorates Experimental Autoimmune Encephalomyelitis Progression.

Multiple sclerosis, and its murine model experimental autoimmune encephalomyelitis (EAE), is a neurodegenerative autoimmune disease of the CNS characterized by T cell influx and demyelination. Similar to other autoimmune diseases, therapies can alleviate symptoms but often come with side effects, necessitating the exploration of new treatments. We recently demonstrated that the Cullin-RING E3 ubiquitin ligase 4b (CRL4b) aided in maintaining genome stability in proliferating T cells. In this study, we examined whether CRL4b was required for T cells to expand and drive EAE. Mice lacking Cul4b (Cullin 4b) in T cells had reduced EAE symptoms and decreased inflammation during the peak of the disease. Significantly fewer CD4+ and CD8+ T cells were found in the CNS, particularly among the CD4+ T cell population producing IL-17A, IFN-γ, GM-CSF, and TNF-α. Additionally, Cul4b-deficient CD4+ T cells cultured invitro with their wild-type counterparts were less likely to expand and differentiate into IL-17A- or IFN-γ-producing effector cells. When wild-type CD4+ T cells were activated invitro in the presence of the recently developed CRL4 inhibitor KH-4-43, they exhibited increased apoptosis and DNA damage. Treatment of mice with KH-4-43 following EAE induction resulted in stabilized clinical scores and significantly reduced numbers of T cells and innate immune cells in the CNS compared with control mice. Furthermore, KH-4-43 treatment resulted in elevated expression of p21 and cyclin E2 in T cells. These studies support that therapeutic inhibition of CRL4 and/or CRL4-related pathways could be used to treat autoimmune disease.

The chemokine receptor CXCR3 promotes CD8+ T cell-dependent lung pathology during influenza pathogenesis.

The dual role of CD8+ T cells in influenza control and lung pathology is increasingly appreciated. To explore whether protective and pathological functions can be linked to specific subsets, we dissected CD8+ T responses in influenza-infected murine lungs. Our single-cell RNA-sequencing (scRNA-seq) analysis revealed notable diversity in CD8+ T subpopulations during peak viral load and infection-resolved state. While enrichment of a Cxcr3hi CD8+ T effector subset was associated with a more robust cytotoxic response, both CD8+ T effector and central memory exhibited equally potent effector potential. The scRNA-seq analysis identified unique regulons regulating the cytotoxic response in CD8+ T cells. The late-stage CD8+ T blockade in influenza-cleared lungs or continuous CXCR3 blockade mitigated lung injury without affecting viral clearance. Furthermore, adoptive transfer of wild-type CD8+ T cells exacerbated influenza lung pathology in Cxcr3-/- mice. Collectively, our data imply that CXCR3 interception could have a therapeutic effect in preventing influenza-linked lung injury.

TCF-1is Required By CD8 + T Cells for the Maintenance of Alloimmune Responses in Graft-Vs-Host Disease

Graft-vs-host disease (GVHD) is a common complication of allogeneic stem cell transplant (alloSCT) wherein donor T cells target alloantigens on recipient tissues. It is unclear how alloimmune responses are maintained in GVHD despite abundant antigen, which causes T cell anergy, deletion and exhaustion. Previously, we identified alloreactive TCF-1 high T cells arising post-transplant that resemble exhausted progenitors (T EXP) capable of propagating immune responses in other chronic antigen models. Here, we sought to further characterize these cells in the B6→129 MHC-matched GVHD mouse model, in which 129 recipients express the immunodominant H-2K b-restricted minor histocompatibility antigen (miHA) H60. At day +7 post-transplant, alloreactive CD8 + cells specific to H60 (as determined by MHC-I-tetramer staining; Tet H60+) were nearly uniformly PD-1 hiTox hi whereas Tet H60- cells displayed a bimodal distribution into discrete PD-1 hiTox hi and PD-1 loTox lo populations, indicative of more diverse antigen experiences. Among these both Tet H60+ and Tet H60- cells were TCF-1 hi cells. TCF-1 hi Tet H60+ cells were uniformly CD39 loTox hiPD-1 hi, which is a canonical T EXP phenotype. In contrast, among activated Tet H60- cells there were TCF-1 hi cells that were CD39 loTox hiPD-1 hi and Tox loPD-1 lo. At later times in spleen and lymph node, and in GVHD target tissues, these populations of TCF-1 + Tet H60+ and Tet H60- were found. To test if these CD39 loTCF-1 hi T EXP had proliferative advantages in GVHD, we sorted congenic TCF-1 hiCD39 lo and TCF-1 loCD39 hi CD8 + cells from recipient spleens 14-days post-transplant and adoptively transferred them in competition in a 1:1 ratio (of Tet H60+ cells) into newly transplanted recipients. Among Tet H60+ cells in all tissues at day 14 post-transfer, TCF-1 hiCD39 lo-sorted progeny greatly outperformed TCF-1 loCD39 hi-sorted progeny. In line with their role as a source of GVHD effectors, progeny of TCF-1 hiCD39 lo cells were mostly TCF-1 loCD39 hi; however, a fraction remained TCF-1 hi consistent with their being able to undergo self-renewal. Conversely, we observed few if any TCF-1 + progeny of CD39 hi cells. We next tested whether TCF-1 was an important mediator of T cell fitness or whether it was only a marker for functionality. To do so we competed congenic wild-type (WT) and Tcf7 p45-/- (p45 -/-) donor CD8 cells, which lack the N-terminal β-catenin binding domain of TCF-1, in allogeneic (129) and syngeneic (B6) recipients. Strikingly, p45 -/- CD8 cells were greatly outcompeted by WT CD8 cells in 129 recipients in all tissues and at all times post-transplant, among both Tet H60+ and Tet H60- cells. In contrast, in B6 recipients, WT and p45 -/- cells remained evenly matched, suggesting that full-length TCF-1 isoforms are dispensable for lymphopenia-induced T cell expansion. Further, p45 -/- cells were also not disadvantaged when adoptively transferred into B6 mice and acutely challenged with H60 antigen by vaccination. Together these data suggest a model wherein TCF-1 hi progenitor like T cells are seeded in GVHD target organs where they may serve as a key local source for GVHD effectors, and moreover, full-length TCF-1 is itself critical for alloreactive T cell fitness in GVH responses.

Piezo1 facilitates optimal T cell activation during tumor challenge

ABSTRACT Functional effector T cells in the tumor microenvironment (TME) are critical for successful anti-tumor responses. T cell anti-tumor function is dependent on their ability to differentiate from a naïve state, infiltrate into the tumor site, and exert cytotoxic functions. The factors dictating whether a particular T cell can successfully undergo these processes during tumor challenge are not yet completely understood. Piezo1 is a mechanosensitive cation channel with high expression on both CD4+ and CD8+ T cells. Previous studies have demonstrated that Piezo1 optimizes T cell activation and restrains the CD4+ regulatory T cell (Treg) pool in vitro and under inflammatory conditions in vivo. However, little is known about the role Piezo1 plays on CD4+ and CD8+ T cells in cancer. We hypothesized that disruption of Piezo1 on T cells impairs anti-tumor immunity in vivo by hindering inflammatory T cell responses. We challenged mice with T cell Piezo1 deletion (P1KO) with tumor models dependent on T cells for immune rejection. P1KO mice had the more aggressive tumors, higher tumor growth rates and were unresponsive to immune-mediated therapeutic interventions. We observed a decreased CD4:CD8 ratio in both the secondary lymphoid organs and TME of P1KO mice that correlated inversely with tumor size. Poor CD4+ helper T cell responses underpinned the immunodeficient phenotype of P1KO mice. Wild type CD8+ T cells are sub-optimally activated in vivo with P1KO CD4+ T cells, taking on a CD25loPD-1hi phenotype. Together, our results suggest that Piezo1 optimizes T cell activation in the context of a tumor response.

Chronic TNF in the Aging Microenvironment Exacerbates TET2-loss-of-Function Myeloid Expansion

Introduction: Age-associated TET2 somatic mutations impart an intrinsic hematopoietic stem cell (HSC) advantage and contribute to the phenomenon of clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutantCHIP have a higher risk of developing myeloid neoplasms and other age-related conditions, including heart, lung, liver, kidney and infectious disease, and have increased risk of all-cause mortality. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing TNF favours TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSC and progeny with TET2-mutations in vivo. Methods: All human and mouse studies met ethics approval. C57Bl/6 background wildtype (WT), TNF-α knockout ( TNF-/-), Tet2 hematopoietic knockout (Vav1-iCre-mediated; Tet2-/-) and floxed control mice ( Tet2f/f) mice were obtained from the Jackson Laboratory. Young (6-mo [n=9]) and old (18-22 mo [n=9]) WT mice, and old TNF-/- mice (n=7) were subjected to nonirradiative myeloablation with busulfan, and retro-orbital injections of 8x10 6 cells/ml T-cell-depleted bone marrow (BM), equally harvested from 4-mo-old WT CD45.1 (n=5) and Tet2-/- CD45.2 donor mice (n=5). Flow cytometry was used to confirm and monitor engraftment. Mice were harvested 8 weeks post-transplant for analysis of HSC, progenitor, monocyte and neutrophil populations, and cytokine analyses. Consenting human research participants diagnosed with rheumatoid arthritis (RA) were recruited from the Greater Hamilton Area (Ontario, Canada) from 2016-2018. Blood draws occurred prior to any immunomodulatory treatment (baseline), and at 3- and 6-months following treatment with Adalimumab (Humira®), an anti-TNF agent. CHIP status was determined with our successful 48-gene, targeted, Ion-Torrent based sequencing approach to isolated genomic DNA from PBMC. Confirmation of calls and increased sensitivity to detect clones with VAF < 0.02 employed our established single molecule molecular inversion probes (smMIP)-targeted genomic capture technique and high-depth (47,500X avg. coverage) paired-end sequencing, employing high-stringency filters for error-suppression. Statistical analyses were performed in GraphPad Prism V9.2 or R 4.1.2. Results: 1. Mixed BM chimeric mice of WT and TNF -/- genotypes reconstituted with WT CD45.1 + and Tet2 -/- CD45.2 + HSC showed that age-associated increases in TNF significantly increased by 2- to 2.5-fold the proportions of HSC in old recipient mice, with myeloid lineage skewing (2- to 2.5-fold more granulocyte-monocyte, monocyte-dendritic and common monocyte progenitor cells in BM, and dramatic expansion of Tet2-/- monocytes and neutrophils in blood). This aberrant myelomonocytic advantage was mitigated in old TNF -/- recipient mice, suggesting that TNF signalling in the BM is essential for Tet2-mutant myeloid expansion. 2. Age-associated TNF predisposed Tet2-/- HSC to the development of Ly6C high inflammatory monocytes, including increased intracellular and serum TNF levels, further exacerbating an inflammatory environment in favor of Tet2-mutantexpansion. 3. Examination of human RA patients (n=7; avg. age 57y) with serial PBMC sampling revealed one patient with CHIP driven by CBL and PPM1D variants, with a 32% reduction in clone size between 3 and 6 months of anti-TNF therapy (adalimumab). Two additional variants were detected at baseline in this RA patient, TP53 R196* (VAF = 1.34%) and ASXL1 P689L (VAF = 3.13%), but these were not detected at 3- and 6-months post-treatment even with high-depth, error suppression sequencing. Two additional patients had detectible low-level CH clones prior to anti-TNF treatment - one with a STAG2 R1033Q variant at baseline (VAF = 1.65%) and the other with a TET2 baseline E798K variant (VAF = 1.68%) - but not detected during anti-TNF treatment. Conclusions: We present novel findings that TNF in the aging BM environment has a causal role in driving TET2-mutant CHIP in vivo, and the first examination of the impact of TNF blockade on CH dynamics in humans, suggesting promise for reducing clonal burden. Larger, prospective studies are required to confirm these findings and to determine if inflammatory cytokine blockade may improve CHIP-comorbid conditions and myeloid cancer risk.

Gab3 Plays an Essential Role in CD8 + T-Cell Expansion through Regulating IL-2-Mediated Activation of PI3K/AKT/mTOR and Erk/FoxO Pathways

Grb2-associated binding protein 3 (Gab3) is a member of the Gab family of scaffolding proteins. Its role in T cells is largely unknown. In the current study, we investigated its in vivo function in CD8 + T cells using the lymphocytic choriomeningitis virus (LCMV) Armstrong infection model. We created BM chimera mice with a specific deficiency of Gab3 in CD8 + T cells. This was achieved by transplanting a mixture of BM cells obtained from CD8-deficient mice and Gab3-deficient (Gab3 -/-) mice (1) into lethally irradiated CD45.1 mice. The control chimera mice were lethally irradiated CD45.1 mice transplanted with a mixture of BM cells from CD8-deficient mice and WT mice. Twelve weeks after the BM transplantation, the Gab3 -/- and control BM chimera mice were infected with Armstrong. On day eight following infection, Gab3 -/- CD8 + T cells exhibited reduced expansion compared to WT CD8 + T cells. Importantly, Gab3 -/- LCMV antigen specific CD8 + T cells, GP33 + and NP396 + CD8 + T cells, were significantly reduced compared to their WT counterparts. Furthermore, CD8 + T cells in Gab3 -/- BM chimera mice showed significant increase of apoptosis compared to that in WT BM chimera mice following LCMV infection. To explore the mechanism underlying the observed phenotype, we used a primary CD8 + T-cell culture system, in which we stimulated CD8 + T cells with anti-CD3 and anti-CD28 followed by expanding the cells in IL-2. We observed that Gab3 deficiency severely impaired IL-2-induced CD8 + T cell expansion. Gab3 -/- CD8 + T cells in this culture system displayed increased apoptosis and reduced proliferation compared to wild-type (WT) controls, which may account for the reduced CD8 + T cell expansion. We then performed Seahorse analysis of the cultured CD8 + T cells after IL-2 stimulation. We found that IL-2-mediated extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were largely impaired in Gab3 -/- CD8 + T cells compared to WT CD8 + T cells. This data indicates a crucial role of Gab3 in both glycolysis and mitochondrial respiration. We further investigated how Gab3 regulates the IL-2 signaling pathway by conducting transcriptomic and phosphoproteomic analysis. RNA-seq analysis revealed that genes associated with mTOR signaling, glucose metabolism, cell cycle, and Erk/MAPK targets were highly enriched in WT CD8 + T cells, whereas genes related to apoptosis, autophagy, and FoxO3 targets were highly enriched in Gab3 -/- CD8 + T cells. In contrast, the gene set enrichment analysis (GSEA) showed that IL-2-induced Stat5 target gene expression was comparable between WT and Gab3 -/- CD8 + T cells, indicating an important role of Gab3 specifically in the activation of the mTOR and Erk/MAPK pathways downstream of IL-2 signaling. Consistent with that observed in the transcriptomic analysis, phosphoproteomics analysis revealed a significant reduction in IL-2-induced phosphorylation of key components in ERK/FOXO signaling pathway, including RafA, MAPK1, FoxO3, AMPK, and mTOR downstream molecule Eif4B in Gab3 -/- CD8 + T cells compared to WT CD8 + cells. Furthermore, phosphorylation of Stat5 at different sites was comparable between Gab3 -/- and WT CD8 + T cells, further supporting an important role of Gab3 specifically in IL-2 mediated mTOR and Erk/MAPK activation. Finally, we validated the results obtained from the transcriptomic and phosphoproteomic analyses using western blot analysis. Our findings revealed that in Gab3 -/- CD8 + T cells, the IL-2-induced phosphorylation of signaling molecules in the mTOR pathway (S6K, S6, 4EBP1, AKT) and the Erk pathway (Erk) was impaired. Notably, the phosphorylation of Stat5 remained intact despite the absence of Gab3. In summary, our studies have demonstrated that Gab3 plays an essential role in CD8 + T-cell expansion by promoting cell proliferation and prohibiting cell apoptosis through regulating IL-2-mediated activation of PI3K/AKT/mTOR and Erk-FoxO3 pathways. (1) We gratefully acknowledge that Gab3-deficient mice were originally generated by Dr. Larry Rohrschneider at Fred Hutchinson Cancer Research Center ( Mol Cell Biol 2003 Apr;23(7):2415-24. doi: 10.1128/MCB.23.7.2415-2424.2003.). The mice were subsequently backcrossed to C57BL/6.

PD-1 Impairs CD8+ T Cell Granzyme B Production in Aged Mice during Acute Viral Respiratory Infection.

CD8+ T cell dysfunction contributes to severe respiratory viral infection outcomes in older adults. CD8+ T cells are the primary cell type responsible for viral clearance. With increasing age, CD8+ T cell function declines in conjunction with an accumulation of cytotoxic tissue-resident memory (TRM) CD8+ T cells. We sought to elucidate the role of PD-1 signaling on aged CD8+ T cell function and accumulation of CD8+ TRM cells during acute viral respiratory tract infection, given the importance of PD-1 regulating CD8+ T cells during acute and chronic infections. PD-1 blockade or genetic ablation in aged mice yielded improved CD8+ T cell granzyme B production comparable to that in young mice during human metapneumovirus and influenza viral infections. Syngeneic transplant and adoptive transfer strategies revealed that improved granzyme B production in aged Pdcd1-/- CD8+ T cells was primarily cell intrinsic because aged wild-type CD8+ T cells did not have increased granzyme B production when transplanted into a young host. PD-1 signaling promoted accumulation of cytotoxic CD8+ TRM cells in aged mice. PD-1 blockade of aged mice during rechallenge infection resulted in improved clinical outcomes that paralleled reduced accumulation of CD8+ TRM cells. These findings suggest that PD-1 signaling impaired CD8+ T cell granzyme B production and contributed to CD8+ TRM cell accumulation in the aged lung. These findings have implications for future research investigating PD-1 checkpoint inhibitors as a potential therapeutic option for elderly patients with severe respiratory viral infections.

Tespa1 deficiency reduces the antitumour immune response by decreasing CD8+T cell activity in a mouse Lewis lung cancer model

Thymocyte-expressed, positive selection-associated 1 (Tespa1) is a key molecule in T-cell development and has been linked to immune diseases. However, its role in antitumour CD8+T cell immunity remains unclear. Here, we demonstrated that Tespa1 plays an important role in antitumour CD8+T cell immunity. First, compared with wild-type (WT) mice, Lewis lung cancer cells grew faster in Tespa1 knockout (Tespa1−/−) mice, with reduced apoptosis, and decreased CD8+T cells in peripheral blood and tumor tissues. Second, the proportion of CD8+T and Th1 cells in the splenocytes of Tespa1−/− mice was lower than that in WT mice. Third, Tespa1−/− CD8+ tumor-infiltrating lymphocytes (TILs) showed weakened proliferation, invasion, cytotoxicity, and protein expression of IL-2 signalling pathway components compared to WT CD8+TILs. Furthermore, PD-1 expression in CD8+TILs was higher in Tespa1−/− than in WT mice. Lastly, CD8+TILs in WT mice improved the antitumour ability of Tespa1−/− mice. In conclusion, these findings suggest that Tespa1 plays a critical role in the tumor immune system by regulating CD8+T cells.

High-affinity CD8 variants enhance the sensitivity of pMHCI antigen recognition via low-affinity TCRs

CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of high-affinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs.

Cutting Edge: Optimal Formation of Hepatic Tissue-Resident Memory CD4 T Cells Requires T-bet Regulation of CD18.

CD4 tissue-resident memory T cells (TRMs) allow robust protection of barrier surfaces against pathogens. We investigated the role of T-bet in the formation of liver CD4 TRMs using mouse models. T-bet-deficient CD4 T cells did not efficiently form liver TRMs when compared with wild-type (WT). In addition, ectopic expression of T-bet enhanced the formation of liver CD4 TRMs, but only when in competition with WT CD4 T cells. Liver TRMs also expressed higher levels of CD18, which was T-bet dependent. The WT competitive advantage was blocked by Ab neutralization of CD18. Taken together, our data show that activated CD4 T cells compete for entry to liver niches via T-bet-induced expression of CD18, allowing TRM precursors to access subsequent hepatic maturation signals. These findings uncover an essential role for T-bet in liver TRM CD4 formation and suggest targeted enhancement of this pathway could increase the efficacy of vaccines that require hepatic TRMs.

STAT1 expression controls CD4 T cell Th1 and Th17 effector functionality during influenza virus infection

Abstract Most CD4 T cells primed by influenza A virus (IAV) express the transcription factor T-bet, the Th1 ‘master regulator’, and are marked by phenotypic and functional Th1 hallmarks. The extent to which STAT1 and STAT4 activation, both needed for maximal T-bet induction in vitro, are required to direct IAV-primed CD4 T cell responses is not clear. Here, we transferred STAT-deficient (STAT −/−) or wildtype CD4 T cells recognizing IAV to congenic wildtype mice, challenged them with IAV, and analyzed the donor cells in the infected lungs 7 days later. STAT1 −/−cells were barely detected in host mice but were rescued when NK cells were depleted in host mice prior to infection. Functional analysis revealed compromised Th1 identity in STAT1 −/−effectors mirroring that of T-bet −/−cells. However, while T-bet −/−cells develop Th17 functions, STAT1 −/−cells do not. We show that this is due to alternative receipt of type I IFN signals by the STAT1 −/−cells, as blocking type I IFN receptor in IAV-primed mice promotes robust Th17 identity in STAT1 −/−but not in WT cells. In contrast, STAT4 −/−donor cells closely resemble WT cells. To ask if IAV-primed CD4 T cells can respond to STAT4-activating signals we treated mice receiving STAT4 −/−donor cells with IL-12 during IAV infection. IL-12 dramatically increased T-bet and Th1 cytokine production of wildtype, but not of STAT4 −/−or T-bet −/−cells. Our findings thus indicate that STAT1 activation is sufficient to direct prototypical antiviral effector identity, but that this response is not fully Th1-polarized without therapeutic STAT4 activation. This work impacts vaccine strategies that aim to promote protective T cell immunity against IAV by shaping the priming environment with STAT-activating cytokine signals.

CD8 +T cells require CCR5 expression to mediate immunopathology in cutaneous leishmaniasis

Abstract Cytolytic CD8 +T cells mediate immunopathology in cutaneous leishmaniasis by a mechanism dependent on degranulation and lysis of Leishmania-infected cells, culminating in NLRP3 activation and IL-1b release. Here, we sought to identify chemokine receptors involved in CD8 +T cell migration to the lesion that could be employed as a treatment target to ameliorate disease severity. A transcriptional study identified the profile of chemokine receptors that predicts treatment failure in Leishmania braziliensispatients and identifies possible targets that may be involved in CD8 +T cell migration. Using murine models of cutaneous leishmaniasis, we found that around 20% of CD8 +T-cells express CCR5 in lesions at the peak of the disease. In previous studies, we found that CD8 +T cells transferred to Rag1 −/−mice mediated increased NLRP3 and IL-1b dependent disease. Therefore, to test if CCR5 expression on CD8 +T cells was involved in their migration to lesions, we transferred wild-type or CCR5 −/−CD8 +T cells to L. braziliensisinfected Rag1 −/−mice. While infected Rag1 −/−mice reconstituted with wild-type CD8 +T cells developed severe pathology, Rag1 −/−mice that received CCR5 −/−CD8 +T cells developed smaller lesions accompanied by a significant reduction in the number of CD8 +T cells in the lesions. To test whether CCR5 blockade would control disease severity, we used maraviroc (MVC), a selective inhibitor of CCR5 approved by the FDA. MVC treatment significantly reduced lesion development without affecting parasite number in our murine models. Collectively, these results demonstrate that cytolytic CD8 +T cells migrate to leishmania lesions in a CCR5-dependent manner, and MVC treatment efficiently prevents CD8 +T-cell mediated pathology. R01 AI106842 and U01 AI08865006

Chemokine CCL19 promotes type 2 helper T cell differentiation and allergic airway inflammation

Abstract Allergic asthma is largely driven by the actions of allergen-specific CD4 +type 2 T helper (Th2) cells. Following allergen inhalation, these cells are induced by the interaction of naïve CD4 +T cells with antigen-bearing dendritic cells (DCs) in lung-draining lymph nodes (LNs). The molecular mechanisms underlying the induction of T helper cells have been well studied and include the migration of allergen-bearing DCs from the lung to regional LNs in a manner dependent on the chemokine receptor, CCR7. Previous studies have demonstrated that CCL21, a CCR7 ligand, is required for the accumulation of naïve T cells and activated DCs in the LNs, but the function of another ligand, CCL19, has been unknown. In the present study, we investigated the role of CCL19 in allergic airway inflammation using CCL19-deficient mice. In a mouse model of asthma, eosinophils and lymphocytes were significantly reduced in airways of CCL19-deficient mice compared with those of wildtype (WT) animals, and production of IL-4 and IL-13 in the lung was significantly diminished, suggesting that CCL19 is required for type 2 immune responses. Co-cultures of naïve WT CD4 +T cells with CCL19-deficient DCs produced less type 2 cytokines than did T cells co-cultured with WT DCs. Further, fibroblastic reticular cells (FRCs) from CCL19-deficient mouse LNs produced less Th2 cytokine production compared with WT FRCs. Addition of recombinant CCL19 to the culture of naïve CD4 +T cells increased phosphorylation of STAT5 and gene expression of Il2, Il2ra, Il2rb, Il2rg, Sta5a, Stat5b and Stat6. Together, our findings suggest that CCL19 produced by DCs and FRCs promotes Th2 differentiation through the activation of STAT5-signaling, and that this promotes allergic airway inflammation. This work was supported by Intramural Research Program of NIEHS, NIH (ZIA ES102025-09).

ILC3s restrict intestinal inflammation via IL-10 production.

Abstract In this study, we investigated the role of IL-10 production by group 3 innate lymphoid cells (ILC3s) in regulating intestinal immunity. Prior work has established the importance of IL-10 as well as ILC3s in promoting immune homeostasis in the gut. However, the requirement of IL-10 in ILC3s for homeostasis remains unknown. We examined an ILC3 focused cluster in a publicly available single-cell RNA sequencing dataset from patients with ileal Crohn’s disease and detected ILC3s expressing IL10. We then developed Rag1−/−; Il10GFP; Rosa26lsltdTomato; RorcCremice and injected them with PBS or anti-CD40 to induce intestinal inflammation and determine if putative ILC3s expressed IL-10. IL-10 was significantly upregulated following treatment with anti-CD40 7 days following disease initiation. To assess whether ILC3 production of IL-10 played a functional role, we targeted IL-10 specifically in ILC3s by generating Rag1−/−; Il10flox/flox; Rosa26lsl−tdTomato; RorcCre(Il10ΔILC3) mice. Il10ΔILC3mice displayed significantly worse colitis induced by anti-CD40 that was attributed to an increase in Ly6C hiMHCII hiintestinal macrophages (Mϕ) at baseline. Given the alterations in the phenotype of intestinal Mϕ, we examined whether unfractionated wild-type CD4 +T cells would be sufficient to cause colitis when transferred to Il10ΔILC3mice despite the presence of regulatory T cells. Mice were monitored weekly for weight loss and colons harvested for histology at 8 weeks. While IL-10-sufficient littermate controls did not develop inflammation as expected, Il10ΔILC3mice developed severe colitis. Collectively, our findings identify an essential role for IL-10 production by ILC3s to maintain intestinal immune homeostasis. Vanderbilt Digestive Disease Research Center Grant (P30DK058404) The National Institute of Diabetes and Digestive and Kidney Diseases R03DK123489