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BACKGROUND: Acute lung allograft dysfunction (ALAD) is an imprecise syndrome denoting concern for the onset of chronic lung allograft dysfunction (CLAD). Mechanistic biomarkers are needed that stratify risk of ALAD progression to CLAD. We hypothesized that single cell investigation of bronchoalveolar lavage (BAL) cells at the time of ALAD would identify immune cells linked to progressive graft dysfunction. METHODS: We prospectively collected BAL from consenting lung transplant recipients for single cell RNA sequencing. ALAD was defined by a ≥10% decrease in FEV1 not caused by infection or acute rejection and samples were matched to BAL from recipients with stable lung function. We examined cell compositional and transcriptional differences across control, ALAD with decline, and ALAD with recovery groups. We also assessed cell-cell communication. RESULTS: BAL was assessed for 17 ALAD cases with subsequent decline (ALAD declined), 13 ALAD cases that resolved (ALAD recovered), and 15 cases with stable lung function. We observed broad differences in frequencies of the 26 unique cell populations across groups (p = 0.02). A CD8 T cell (p = 0.04) and a macrophage cluster (p = 0.01) best identified ALAD declined from the ALAD recovered and stable groups. This macrophage cluster was distinguished by an anti-inflammatory signature and the CD8 T cell cluster resembled a Tissue Resident Memory subset. Anti-inflammatory macrophages signaled to activated CD8 T cells via class I HLA, fibronectin, and galectin pathways (p

Signals emanating from the T-cell receptor (TCR), co-stimulatory receptors, and cytokine receptors each influence CD8 T-cell fate. Understanding how these signals respond to homeostatic and microenvironmental cues can reveal new ways to therapeutically direct T-cell function. Through forward genetic screening in mice, we discover that loss-of-function mutations in LDL receptor-related protein 10 (Lrp10) cause naive and central memory CD8 T cells to accumulate in peripheral lymphoid organs. Lrp10 encodes a conserved cell surface protein of unknown immunological function. T-cell activation induces Lrp10 expression, which post-translationally suppresses IL7 receptor (IL7R) levels. Accordingly, Lrp10 deletion enhances T-cell homeostatic expansion through IL7R signaling. Lrp10 -deficient mice are also intrinsically resistant to syngeneic tumors. This phenotype depends on dense tumor infiltration of CD8 T cells, which display increased memory cell characteristics, reduced terminal exhaustion, and augmented responses to immune checkpoint inhibition. Here, we present Lrp10 as a new negative regulator of CD8 T-cell homeostasis and a host factor that controls tumor resistance with implications for immunotherapy. Synopsis: Lrp10 is a putative cell surface receptor with unknown functions in immunity. This study shows that Lrp10 prevents accumulation of peripheral CD8 T cells, suppresses IL7R expression to limit homeostatic expansion, and impairs anti-tumor immune responses. Genetic deletion of Lrp10 causes an increased number of peripheral CD8 T cells in mice. Lrp10 is induced upon T cell activation and post-translationally suppresses IL7R expression. Lrp10 reduces CD8 central memory differentiation, T cell homeostatic expansion, and IL7 responsiveness. Lrp10 deletion promotes CD8 T cell infiltration, resulting in enhanced anti-tumor immunity and susceptibility to immune checkpoint inhibition. Lrp10 is a putative cell surface receptor with unknown functions in immunity. This study shows that Lrp10 prevents accumulation of peripheral CD8 T cells, suppresses IL7R expression to limit homeostatic expansion, and impairs anti-tumor immune responses. [ABSTRACT FROM AUTHOR]

Introduction: Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Methods: We developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). Results: The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Discussion: ARMATA does not only allow same day identification of antiviral Tcell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum stress occurs due to large amounts of misfolded proteins, hypoxia, nutrient deprivation, and more. The unfolded protein is a complex intracellular signaling network designed to operate under this stress. Composed of three individual arms, inositol-requiring enzyme 1, protein kinase RNA-like ER kinase, and activating transcription factor-6, the unfolded protein response looks to resolve stress and return to proteostasis. The CD8+ T cell is a critical cell type for the adaptive immune system. The unfolded protein response has been shown to have a wide-ranging spectrum of effects on CD8+ T cells. CD8+ T cells undergo cellular stress during activation and due to environmental insults. However, the magnitude of the effects this response has on CD8+ T cells is still understudied. Thus, studying these pathways is important to unraveling the inner machinations of these powerful cells. In this review, we will highlight the recent literature in this field, summarize the three pathways of the unfolded protein response, and discuss their roles in CD8+ T cell biology and functionality. [ABSTRACT FROM AUTHOR]

Microglia, the resident macrophages of the central nervous system, exhibit altered gene expression in response to various neurological conditions. This study investigates the relationship between West Nile Virus infection and microglial senescence, focusing on the role of LGALS3BP, a protein implicated in both antiviral responses and aging. Using spatial transcriptomics, RNA sequencing and flow cytometry, we characterized changes in microglial gene signatures in adult and aged mice following recovery from WNV encephalitis. Additionally, we analyzed Lgals3bp expression and generated Lgals3bp-deficient mice to assess the impact on neuroinflammation and microglial phenotypes. Our results show that WNV-activated microglia share transcriptional signatures with aged microglia, including upregulation of genes involved in interferon response and inflammation. Lgals3bp was broadly expressed in the CNS and robustly upregulated during WNV infection and aging. Lgals3bp-deficient mice exhibited reduced neuroinflammation, increased homeostatic microglial numbers, and altered T cell populations without differences in virologic control or survival. These data indicate that LGALS3BP has a role in regulating neuroinflammation and microglial activation and suggest that targeting LGALS3BP might provide a potential route for mitigating neuroinflammation-related cognitive decline in aging and post-viral infections. [ABSTRACT FROM AUTHOR]

Acute lung allograft dysfunction (ALAD) is an imprecise syndrome denoting concern for the onset of chronic lung allograft dysfunction (CLAD). Mechanistic biomarkers are needed that stratify risk of ALAD progression to CLAD. We hypothesized that single cell investigation of bronchoalveolar lavage (BAL) cells at the time of ALAD would identify immune cells linked to progressive graft dysfunction. We prospectively collected BAL from consenting lung transplant recipients for single cell RNA sequencing. ALAD was defined by a ≥10% decrease in FEV 1 not caused by infection or acute rejection and samples were matched to BAL from recipients with stable lung function. We examined cell compositional and transcriptional differences across control, ALAD with decline, and ALAD with recovery groups. We also assessed cell-cell communication. BAL was assessed for 17 ALAD cases with subsequent decline (ALAD declined), 13 ALAD cases that resolved (ALAD recovered), and 15 cases with stable lung function. We observed broad differences in frequencies of the 26 unique cell populations across groups (p = 0.02). A CD8 T cell (p = 0.04) and a macrophage cluster (p = 0.01) best identified ALAD declined from the ALAD recovered and stable groups. This macrophage cluster was distinguished by an anti-inflammatory signature and the CD8 T cell cluster resembled a Tissue Resident Memory subset. Anti-inflammatory macrophages signaled to activated CD8 T cells via class I HLA, fibronectin, and galectin pathways (p < 0.05 for each). Recipients with discordance between these cells had a nearly 5-fold increased risk of severe graft dysfunction or death (HR 4.6, 95% CI 1.1–19.2, adjusted p = 0.03). We validated these key findings in 2 public lung transplant genomic datasets. BAL anti-inflammatory macrophages may protect against CLAD by suppressing CD8 T cells. These populations merit functional and longitudinal assessment in additional cohorts. [ABSTRACT FROM AUTHOR]

Infection with Mycobacterium tuberculosis (Mtb) in people with HIV (PWH) is associated with depletion of Mtb-specific CD4 T cell responses, increased risk of progression to active tuberculosis (TB) disease, and increased immune activation. Although higher HIV viral loads have been reported in Mtb/HIV co-infection, the extent to which Mtb infection and TB disease impact the frequency and phenotype of HIV-specific T cell responses has not been well described. We enrolled a cohort of PWH in Kenya across a spectrum of Mtb infection states, including those with no evidence of Mtb infection, latent Mtb infection (LTBI), and active pulmonary TB disease, and evaluated the frequency, immune activation, and cytotoxicity phenotype of HIV-specific CD4 and CD8 T cell responses in peripheral blood by flow cytometry. We found evidence of depletion of HIV-specific CD4 and CD8 T cells in people with TB, but not with LTBI. Expression of the immune activation markers human leukocyte antigen-DR isotype (HLA-DR) and Ki67 and of the cytotoxic molecules granzyme B and perforin were increased in total CD4 and CD8 T cell populations in individuals with TB, although expression of these markers by HIV-specific CD4 and CD8 T cells did not differ by Mtb infection status. These data suggest that TB is associated with overall increased T cell activation and cytotoxicity and with depletion of HIV-specific CD4 and CD8 T cells, which may contribute to further impairment of T cell–mediated immune control of HIV replication in the setting of TB. [ABSTRACT FROM AUTHOR]

Objectives: Gout is characterized by hyperuricemia and recurrent inflammatory episodes caused by intra-articular crystal deposition of monosodium urate (MSU). There is a clear relationship between gout and metabolic syndrome. Recent evidence indicates that perforin plays a role in regulating glucose homeostasis and provides protection in diet-induced non-alcoholic steatohepatitis models. However, the impact of perforin on immune inflammation in gout remains unclear. Methods: We induced acute gout models in both wild-type (WT) mice and Prf1null mice by administering intra-articular injections of MSU crystals. We compared the ankle joint swelling and the histological score between the two groups. Furthermore, we investigated underlying mechanisms through in vitro co-culture experiments involving CD8 T cells and macrophages. Results: In this study, Prf1null mice showed significantly more pronounced ankle swelling with increased inflammatory cell infiltrations compared with WT mice 24 h after local MSU injection. Moreover, MSU-induced Prf1null mice exhibited increased accumulation of CD8 T cells but not NK cells. Perforin-deficient CD8 T cells displayed reduced cytotoxicity towards bone marrow–derived M0 and M1 macrophages and promoted TNF-α secretion from macrophage. Conclusions: Perforin from CD8 T cells limits joint inflammation in mice with acute gout by downregulating macrophage-mediated inflammation. Key Points • Perforin deficiency increased swelling in the ankle joints of mice upon MSU injection. • Perforin deficiency is associated with increased immune cell recruitment and severe joint damage in gout. • Perforin regulated CD8 T cell accumulation in gout and promoted CD8 T cell cytotoxicity towards M0 and M1 macrophages. • CD8 T cell-derived perforin regulated pro-inflammatory cytokine secretion of macrophage. [ABSTRACT FROM AUTHOR]

Pigs are the most important source of meat and valuable biomedical models. However, the porcine immune system, especially the heterogeneity of CD8 T cell subtypes, has not been fully characterized. Here, using single-cell RNA sequencing, we identified 14 major cell types from peripheral blood circulating cells of pigs and observed remarkable heterogeneity among CD8 T cell types. Upon re-clustering of CD8+ T cells, we defined four CD8 T cell subtypes and revealed their potential differentiation trajectories and transcriptomic differences among them. Additionally, we identified transcription factors with potential regulatory roles in maintaining CD8 T cell differentiation. The cell-cell communication analysis inferred an extensive interaction between CD8 T cells and other immune cells. Finally, cross-species analysis further identified species-specific and conserved cell types across different species. Overall, our study provides the first insight into the extensive functional heterogeneity and state transitions among porcine CD8 T cell subtypes in pig peripheral blood, complements the knowledge of porcine immunity, and enhances its potential as a biomedical model. [ABSTRACT FROM AUTHOR]

Suboptimal CD4+ T cell activation leads to a hyporesponsive state in mice characterized by incomplete differentiation, limited cytokine production, and changes in T helper (Th) subsets. Together, these factors impair CD4+ T cell trafficking to the tumor and enable the immune evasion of tumor cells. Sustained inflammation, antigen stimulation, and suppressive factors induce CD4+ T cell dysfunctions during tumor progression in murine models. Despite sharing some phenotypic similarities with CD8+ T cells in mouse and human, CD4+ T cell dysfunctions are distinct and should be viewed through the lens of Th subset differentiation. Since CD4+ T cells can help multiple parameters of the innate and adaptive immune response, reinvigorating CD4+ T cells holds great potential in enhancing various immunotherapies, including immune checkpoint blockade, cancer vaccines, and the use of CAR T cells. CD4+ T cells are crucial for enhancing the functions of antigen-presenting cells, increasing CD8+ T cell effector differentiation, driving B cell activation and antibody affinity maturation, and temporally sustaining immune efficacy during cancer progression. As a result, CD4+ T cells serve as a central nexus that directs the initiation and coordination of an immune response. Thus, a deeper understanding of the nature, impact, and restorability of CD4+ T cell dysfunction holds vast potential in guiding therapeutic strategies to effectively harness the potential of these cells in cancer treatments. CD4+ T cells are crucial in generating and sustaining immune responses. They orchestrate and fine-tune mammalian innate and adaptive immunity through cell-based interactions and the release of cytokines. The role of these cells in contributing to the efficacy of antitumor immunity and immunotherapy has just started to be uncovered. Yet, many aspects of the CD4+ T cell response are still unclear, including the differentiation pathways controlling such cells during cancer progression, the external signals that program them, and how the combination of these factors direct ensuing immune responses or immune-restorative therapies. In this review, we focus on recent advances in understanding CD4+ T cell regulation during cancer progression and the importance of CD4+ T cells in immunotherapies. [ABSTRACT FROM AUTHOR]

IntroductionHuman Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods.MethodsWe developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi).ResultsThe timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes.DiscussionARMATA does not only allow same day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

Endoplasmic reticulum stress occurs due to large amounts of misfolded proteins, hypoxia, nutrient deprivation, and more. The unfolded protein is a complex intracellular signaling network designed to operate under this stress. Composed of three individual arms, inositol-requiring enzyme 1, protein kinase RNA-like ER kinase, and activating transcription factor-6, the unfolded protein response looks to resolve stress and return to proteostasis. The CD8+ T cell is a critical cell type for the adaptive immune system. The unfolded protein response has been shown to have a wide-ranging spectrum of effects on CD8+ T cells. CD8+ T cells undergo cellular stress during activation and due to environmental insults. However, the magnitude of the effects this response has on CD8+ T cells is still understudied. Thus, studying these pathways is important to unraveling the inner machinations of these powerful cells. In this review, we will highlight the recent literature in this field, summarize the three pathways of the unfolded protein response, and discuss their roles in CD8+ T cell biology and functionality.

BackgroundThere is considerable evidence that epigenetic mechanisms and DNA methylation are critical drivers of immune cell lineage differentiation and activation. However, there has been limited coordinated investigation of common epigenetic pathways among cell lineages. Further, it remains unclear if long-lived memory cell subtypes differentiate distinctly by cell lineages.ResultsWe used the Illumina EPIC array to investigate the consistency of DNA methylation in B cell, CD4 T, and CD8 T naïve and memory cells states. In the process of naïve to memory activation across the three lineages, we identify considerable shared epigenetic regulation at the DNA level for immune memory generation. Further, in central to effector memory differentiation, our analyses revealed specific CpG dinucleotides and genes in CD4 T and CD8 T cells with DNA methylation changes. Finally, we identified unique DNA methylation patterns in terminally differentiated effector memory (TEMRA) CD8 T cells compared to other CD8 T memory cell subtypes.ConclusionsOur data suggest that epigenetic alterations are widespread and essential in generating human lymphocyte memory. Unique profiles are involved in methylation changes that accompany memory genesis in the three subtypes of lymphocytes.

Primary biliary cholangitis (PBC) is a chronic autoimmune disease characterized by immune-mediated destruction of intrahepatic small bile ducts. CD8 T cells play a critical role in biliary destruction. However, regulatory T cells (Tregs) have also been identified in the portal tracts of PBC patients. This study tested the hypothesis that hepatic Tregs in PBC were dysfunctional in suppressing immune responses in disease by using our human PBC-like autoimmune cholangitis (AIC) mouse model induced by 2-octynoic acid-conjugated ovalbumin (2-OA-OVA). Our results showed that female and male mice immunized with 2-OA-OVA developed AIC; however, female AIC mice had more severe liver inflammation and fibrosis than male AIC mice. Levels of functional effector CD8 T cells and their chemoattractants, CXCL9 and CXCL10, in the liver were markedly elevated in female AIC mice than in male AIC mice. These results reinforce that CD8 T cells are the primary effector cells in PBC. The number of hepatic Tregs in AIC mice was also higher than in saline-treated mice, but there was no difference between male and female AIC mice. The suppressive function of AIC Tregs was evident despite a discrepancy in the changes in their co-inhibitory receptors and inhibitory cytokines. However, the expansion of hepatic Tregs by low-dose IL-2 treatment did not reduce immune responses to AIC, which may be due to the dysfunction of Tregs in inhibiting T cells. In conclusion, the function of Tregs in the inflamed liver of PBC was insufficient, and low-dose IL-2 treatment could not restore their function to suppress pathological immune responses. Transferring normal Tregs or directly targeting effector CD8 T cells may be beneficial for treating PBC. In 2-OA-OVA immunized autoimmune cholangitis (AIC) mice, a model resembling primary biliary cholangitis, elevated levels of functional CD8 T cells and related chemoattractants in the liver were observed, potentially contributing to biliary epithelial cell damage. Additionally, regulatory T cells (Tregs) in the AIC liver demonstrated inadequate suppressive function, even when expanded through low-dose IL-2 treatment. Graphical Abstract [ABSTRACT FROM AUTHOR]

An aberrant MHC-I antigen presentation pathway has been closely linked to low tumor immunogenicity and immunotherapy resistance. Genetic alteration or reduced expression of MHC-I positive regulators has been observed in certain cancers, but is considered a challenge for the development of targeted therapies. MHC-I modulation by cancer-associated inhibitory mechanisms is becoming an emerging field of study, calling for a search for specific modulators and their underlying biology and function in MHC-I modulation and tumor immunity. A membrane-associated MHC-I inhibitory axis was recently documented, mediating MHC-I ubiquitination and subsequent lysosomal degradation in leukemia and solid cancers. We posit that the future exploration of MHC-I inhibitory pathways and their potential application represents an exciting direction for cancer immunotherapy. Cancer cells efficiently hijack the MHC-I antigen presentation (AP) pathway to escape from immunosurveillance and cause resistance to immunotherapy. Exploring specific mechanisms that actively inhibit MHC-I expression and/or AP in tumors can improve our understanding of immune regulation in cancers and provide potential targets for next-generation cancer immunotherapy. The MHC-I antigen presentation (AP) pathway is key to shaping mammalian CD8+ T cell immunity, with its aberrant expression closely linked to low tumor immunogenicity and immunotherapy resistance. While significant attention has been given to genetic mutations and downregulation of positive regulators that are essential for MHC-I AP, there is a growing interest in understanding how tumors actively evade MHC-I expression and/or AP through the induction of MHC-I inhibitory pathways. This emerging field of study may offer more viable therapeutic targets for future cancer immunotherapy. Here, we explore potential mechanisms by which cancer cells evade MHC-I AP and function and propose therapeutic strategies that might target these MHC-I inhibitors to restore impaired T cell immunity within the tumor microenvironment (TME). [ABSTRACT FROM AUTHOR]

Diabetic foot ulcers (DFUs) are one of the most common and challenging complications of diabetes, yet our understanding of their pathogenesis remains limited. We collected gene expression data of DFU patients from public databases. Bioinformatics tools were applied for systematic analysis, including the identification of differentially expressed genes (DEGs), weighted gene co‐expression network analysis (WGCNA) and enrichment analysis. We further used single‐cell RNA sequencing to identify the distribution of different cell populations in DFU. Finally, key results were validated using reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) and flow cytometry. We identified 217 DEGs between ulcerated and healthy skin, and 37 DEGs between healing ulcers and ulcers. WGCNA revealed that the cyan module had the highest positive correlation with healthy skin and negative correlation with ulcers. The black module had the highest negative correlation with healthy skin and positive correlation with ulcers. Enrichment analysis showed that the genes in the cyan module were mainly associated with complement and coagulation cascades, while the genes in the black module were mainly associated with the IL‐17 signalling pathway. In addition, CD8 T cells were significantly lower in ulcers than in healthy and healing ulcers. By comparing marker genes of CD8 T cells, we identified key genes in the cyan and black modules and validated their expression using RT‐qPCR. The proportion of CD8 T cells was increased in healing ulcers. Flow cytometry detected increased levels of CD8 T, B and natural killer cells in healing ulcers. CD8 T cells and related key genes play an important role in the healing process of DFU. The results of this study provide a new perspective for understanding the pathogenesis and treatment of DFU. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease virus (FMDV) remains a challenge for clovenhooved animals. The currently licensed FMDV vaccines induce neutralizing antibody (NAb)-mediated protection but show defects in the early protection. Dendritic cell (DC) vaccines have shown great potency in inducing rapid T-cell immunity in humans and mice. Whether DC vaccination could enhance early protection against FMDV has not been elaborately explored in domestic pigs. In this study, we employed DC vaccination as an experimental approach to study the roles of cellular immunity in the early protection against FMDV in pigs. Autologous DCs were differentiated from the periphery blood mononuclear cells of each pig, pulsed with inactivated FMDV (iFMDV-DC) and treated with LPS, and then injected into the original pigs. The cellular immune responses and protective efficacy elicited by the iFMDV-DC were examined by multicolor flow cytometry and tested by FMDV challenge. The results showed that autologous iFMDV-DC immunization induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells (CTLs), high NAb titers, compared to the inactivated FMDV vaccine, and accelerated the development of memory CD4 and CD8 T cells, which was concomitantly associated with early protection against FMDV virulent strain in pigs. Such early protection was associated with the rapid proliferation of secondary T-cell response after challenge and significantly contributed by secondary CD8 effector memory T cells. These results demonstrated that rapid induction of cellular immunity through DC immunization is important for improving early protection against FMDV. Enhancing cytotoxic CD8+ T cells may facilitate the development of more effective FMDV vaccines. IMPORTANCE Although the currently licensed FMDV vaccines provide NAb-mediated protection, they have defects in early immune protection, especially in pigs. In this study, we demonstrated that autologous swine DC immunization augmented the cellular immune response and induced an early protective response against FMDV in pigs. This approach induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells, high NAb titers, and rapid development of memory CD4 and CD8 T cells. Importantly, the early protection conferred by this DC immunization is more associated with secondary CD8+ T response rather than NAbs. Our findings highlighted the importance of enhancing cytotoxic CD8+ T cells in early protection to FMDV in addition to Th1 response and identifying a strategy or adjuvant comparable to the DC vaccine might be a future direction for improving the current FMDV vaccines. [ABSTRACT FROM AUTHOR]

Expression levels of the chemokine receptor CX3CR1 serve as high‐resolution marker delineating functionally distinct antigen‐experienced T‐cell states. The factors that influence CX3CR1 expression in T cells are, however, incompletely understood. Here, we show that in vitro priming of naïve CD8+ T cells failed to robustly induce CX3CR1, which highlights the shortcomings of in vitro priming settings in recapitulating in vivo T‐cell differentiation. Nevertheless, in vivo generated memory CD8+ T cells maintained CX3CR1 expression during culture. This allowed us to investigate whether T‐cell receptor ligation, cell death, and CX3CL1 binding influence CX3CR1 expression. T‐cell receptor stimulation led to downregulation of CX3CR1. Without stimulation, CX3CR1+ CD8+ T cells had a selective survival disadvantage, which was enhanced by factors released from necrotic but not apoptotic cells. Exposure to CX3CL1 did not rescue their survival and resulted in a dose‐dependent loss of CX3CR1 surface expression. At physiological concentrations of CX3CL1, CX3CR1 surface expression was only minimally reduced, which did not hamper the interpretability of T‐cell differentiation states delineated by CX3CR1. Our data further support the broad utility of CX3CR1 surface levels as T‐cell differentiation marker and identify factors that influence CX3CR1 expression and the maintenance of CX3CR1 expressing CD8+ T cells. [ABSTRACT FROM AUTHOR]

Abstract Rare cases of paraneoplastic obesity in children suggest sporadic obesity might also arise from an adaptive immune cell-mediated mechanism. Since the hypothalamus is a central regulator of feeding behavior and energy expenditure, we quantified lymphocytic inflammation in this region in a cohort of obese and non-obese human post-mortem brains. We report that CD8-positive cytotoxic T-cells are increased in hypothalamic median eminence/arcuate nucleus (ME/Arc) and bed nucleus of the stria terminalis in 40% of obese compared to non-obese patients, but not in other hypothalamic nuclei or brain regions. CD8 T-cells were most abundant in individuals with concurrent obesity and diabetes. Markers of cytotoxic T-cell induced damage, activated caspase 3 and poly-ADP ribose, were also elevated in the ME/Arc of obese patients. To provoke CD8 cytotoxic T-cell infiltrates in ventromedial region of hypothalamus in mice we performed stereotactic injections of an adeno-associated virus expressing immunogenic green fluorescent protein or saline. AAV but not saline injections triggered hypothalamic CD8 T-cell infiltrates associated with a rapid weight gain in mice recapitulating the findings in human obesity. This is the first description of the neuropathology of human obesity and when combined with its reconstitution in a mouse model suggests adaptive immunity may drive as much as 40% of the human condition.

Transforming growth factor β (TGF-β) directly acts on naive, effector, and memory T cells to control cell fate decisions, which was shown using genetic abrogation of TGF-β signaling. TGF-β availability is altered by infections and cancer; however, the dose-dependent effects of TGF-β on memory CD8 T cell (Tmem) reactivation are still poorly defined. We examined how activation and TGF-β signals interact to shape the functional outcome of Tmem reactivation. We found that TGF-β could suppress cytotoxicity in a manner that was inversely proportional to the strength of the activating TCR or proinflammatory signals. In contrast, even high doses of TGF-β had a comparatively modest effect on IFN-γ expression in the context of weak and strong reactivation signals. Since CD8 Tmem may not always receive TGF-β signals concurrently with reactivation, we also explored whether the temporal order of reactivation versus TGF-β signals is of importance. We found that exposure to TGF-β before or after an activation event were both sufficient to reduce cytotoxic effector function. Concurrent ATAC-seq and RNA-seq analysis revealed that TGF-β altered ~10% of the regulatory elements induced by reactivation and also elicited transcriptional changes indicative of broadly modulated functional properties. We confirmed some changes on the protein level and found that TGF-β-induced expression of CCR8 was inversely proportional to the strength of the reactivating TCR signal. Together, our data suggest that TGF-β is not simply suppressing CD8 Tmem but modifies functional and chemotactic properties in context of their reactivation signals and in a dose-dependent manner. [ABSTRACT FROM AUTHOR]

Rare cases of paraneoplastic obesity in children suggest sporadic obesity might also arise from an adaptive immune cell-mediated mechanism. Since the hypothalamus is a central regulator of feeding behavior and energy expenditure, we quantified lymphocytic inflammation in this region in a cohort of obese and non-obese human post-mortem brains. We report that CD8-positive cytotoxic T-cells are increased in hypothalamic median eminence/arcuate nucleus (ME/Arc) and bed nucleus of the stria terminalis in 40% of obese compared to non-obese patients, but not in other hypothalamic nuclei or brain regions. CD8 T-cells were most abundant in individuals with concurrent obesity and diabetes. Markers of cytotoxic T-cell induced damage, activated caspase 3 and poly-ADP ribose, were also elevated in the ME/Arc of obese patients. To provoke CD8 cytotoxic T-cell infiltrates in ventromedial region of hypothalamus in mice we performed stereotactic injections of an adeno-associated virus expressing immunogenic green fluorescent protein or saline. AAV but not saline injections triggered hypothalamic CD8 T-cell infiltrates associated with a rapid weight gain in mice recapitulating the findings in human obesity. This is the first description of the neuropathology of human obesity and when combined with its reconstitution in a mouse model suggests adaptive immunity may drive as much as 40% of the human condition. [ABSTRACT FROM AUTHOR]

The poor prognosis of immunotherapy in patients with colorectal cancer (CRC) necessitates a comprehensive understanding of the immunosuppressive mechanisms within tumor microenvironment (TME). Undoubtedly, the anti‐tumor immune cells play an indispensable role in immune tolerance. Therefore, it is imperative to investigate novel immune‐related factors that have the capacity to enhance anti‐tumor immunity. Here, we employed bioinformatic analysis using R and Cytoscape to identify the hub gene chemokine (C‐X‐C motif) ligand 8 (CXCL8), which is overexpressed in CRC, in the malignant progression of CRC. However, its specific role of CXCL8 in CRC immunity remains to be elucidated. For this purpose, we evaluated how tumor‐derived CXCL8 promotes M2 macrophage infiltration by in vivo and in vitro, which can be triggered by IL‐1β within TME. Mechanistically, CXCL8‐induced polarization of M2 macrophages depends on the activation of the STAT3 signaling. Finally, immunohistochemistry and multiplexed immunohistochemistry analysis identified that CXCL8 not only enhances PD‐L1+ M2 macrophage infiltration but also attenuates the recruitment of PD‐1+CD8+ T cells in murine CRC models. Together, these findings emphasize the critical role for CXCL8 in promoting M2 macrophage polarization and inhibiting CD8+ T cell infiltration, thereby links CXCL8 to the emergency of immunosuppressive microenvironment facilitating tumor evasion. Overall, these findings may provide novel strategy for CRC immunotherapy. [ABSTRACT FROM AUTHOR]

Abstract Background Many studies have utilized computational methods, including cell composition deconvolution (CCD), to correlate immune cell polarizations with the survival of cancer patients, including those with hepatocellular carcinoma (HCC). However, currently available cell deconvolution estimated (CDE) tools do not cover the wide range of immune cell changes that are known to influence tumor progression. Results A new CCD tool, HCCImm, was designed to estimate the abundance of tumor cells and 16 immune cell types in the bulk gene expression profiles of HCC samples. HCCImm was validated using real datasets derived from human peripheral blood mononuclear cells (PBMCs) and HCC tissue samples, demonstrating that HCCImm outperforms other CCD tools. We used HCCImm to analyze the bulk RNA‐seq datasets of The Cancer Genome Atlas (TCGA)‐liver hepatocellular carcinoma (LIHC) samples. We found that the proportions of memory CD8+ T cells and Tregs were negatively associated with patient overall survival (OS). Furthermore, the proportion of naïve CD8+ T cells was positively associated with patient OS. In addition, the TCGA‐LIHC samples with a high tumor mutational burden had a significantly high abundance of nonmacrophage leukocytes. Conclusions HCCImm was equipped with a new set of reference gene expression profiles that allowed for a more robust analysis of HCC patient expression data. The source code is provided at https://github.com/holiday01/HCCImm.

Pigs are the most important source of meat and valuable biomedical models. However, the porcine immune system, especially the heterogeneity of CD8 T cell subtypes, has not been fully characterized. Here, using single-cell RNA sequencing, we identified 14 major cell types from peripheral blood circulating cells of pigs and observed remarkable heterogeneity among CD8 T cell types. Upon re-clustering of CD8+ T cells, we defined four CD8 T cell subtypes and revealed their potential differentiation trajectories and transcriptomic differences among them. Additionally, we identified transcription factors with potential regulatory roles in maintaining CD8 T cell differentiation. The cell-cell communication analysis inferred an extensive interaction between CD8 T cells and other immune cells. Finally, cross-species analysis further identified species-specific and conserved cell types across different species. Overall, our study provides the first insight into the extensive functional heterogeneity and state transitions among porcine CD8 T cell subtypes in pig peripheral blood, complements the knowledge of porcine immunity, and enhances its potential as a biomedical model.

IntroductionMucosal immunization eliciting local T-cell memory has been suggested for improved protection against respiratory infections caused by viral variants evading pre-existing antibodies. However, it remains unclear whether T-cell targeted vaccines suffice for prevention of viral transmission and to which extent local immunity is important in this context.MethodsTo study the impact of T-cell vaccination on the course of viral respiratory infection and in particular the capacity to inhibit viral transmission, we used a mouse model involving natural murine parainfluenza infection with a luciferase encoding virus and an adenovirus based nucleoprotein targeting vaccine.Results and discussionPrior intranasal immunization inducing strong mucosal CD8+ T cell immunity provided an almost immediate shut-down of the incipient infection and completely inhibited contact based viral spreading. If this first line of defense did not operate, as in parentally immunized mice, recirculating T cells participated in accelerated viral control that reduced the intensity of inter-individual transmission. These observations underscore the importance of pursuing the development of mucosal T-cell inducing vaccines for optimal protection of the individual and inhibition of inter-individual transmission (herd immunity), while at the same time explain why induction of a strong systemic T-cell response may still impact viral transmission.

ABSTRACTGroup 2 innate lymphoid cells (ILC2s) are essential for orchestrating type 2 immune responses during allergic airway inflammation and infection. ILC2s have been reported to play a regulatory role in tumors; however, this conclusion is controversial. In this study, we showed that IL-33-activated ILC2s could boost CD8+ T-cell function through direct antigen cross-presentation. After activation by IL-33, ILC2s showed an enhanced potential to process antigens and prime CD8+ T cell activation. Activated ILC2s could phagocytose exogenous antigens in vivo and in vitro, promoting antigen-specific CD8+ T cell function to enhance antitumor immune responses. Administration of OVA-loaded ILC2s induces robust antitumor effects on the OVA-expressing tumor model. These findings suggested that the administration of tumor antigen-loaded ILC2s might serve as a potential strategy for cancer treatment.

During recent years, evidence has emerged that immune privileged sites such as the CNS and the retina may be more integrated in the systemic response to infection than was previously believed. In line with this, it was recently shown that a systemic acute virus infection leads to infiltration of CD8 T cells in the brains of immunocompetent mice. In this study, we extend these findings to the neurological tissue of the eye, namely the retina. We show that an acute systemic virus infection in mice leads to a transient CD8 T cell infiltration in the retina that is not directed by virus infection inside the retina. CD8 T cells were found throughout the retinal tissue, and had a high expression of CXCR6 and CXCR3, as also reported for tissue residing CD8 T cells in the lung and liver. We also show that the pigment epithelium lining the retina expresses CXCL16 (the ligand for CXCR6) similar to epithelial cells of the lung. Thus, our results suggest that the retina undergoes immune surveillance during a systemic infection, and that this surveillance appears to be directed by mechanisms similar to those described for non-privileged tissues. [ABSTRACT FROM AUTHOR]

Background: Many studies have utilized computational methods, including cell composition deconvolution (CCD), to correlate immune cell polarizations with the survival of cancer patients, including those with hepatocellular carcinoma (HCC). However, currently available cell deconvolution estimated (CDE) tools do not cover the wide range of immune cell changes that are known to influence tumor progression. Results: A new CCD tool, HCCImm, was designed to estimate the abundance of tumor cells and 16 immune cell types in the bulk gene expression profiles of HCC samples. HCCImm was validated using real datasets derived from human peripheral blood mononuclear cells (PBMCs) and HCC tissue samples, demonstrating that HCCImm outperforms other CCD tools. We used HCCImm to analyze the bulk RNA‐seq datasets of The Cancer Genome Atlas (TCGA)‐liver hepatocellular carcinoma (LIHC) samples. We found that the proportions of memory CD8+ T cells and Tregs were negatively associated with patient overall survival (OS). Furthermore, the proportion of naïve CD8+ T cells was positively associated with patient OS. In addition, the TCGA‐LIHC samples with a high tumor mutational burden had a significantly high abundance of nonmacrophage leukocytes. Conclusions: HCCImm was equipped with a new set of reference gene expression profiles that allowed for a more robust analysis of HCC patient expression data. The source code is provided at https://github.com/holiday01/HCCImm. [ABSTRACT FROM AUTHOR]

Summary: Cytotoxic CD8+ T cells recognize and eliminate infected or cancerous cells. A subset of CD8+ memory T cells called tissue‐resident memory T cells (TRM) resides in peripheral tissues, monitors the periphery for pathogen invasion, and offers a rapid and potent first line of defense at potential sites of re‐infection. TRM cells are found in almost all tissues and are transcriptionally and epigenetically distinct from circulating memory populations, which shows their ability to acclimate to the tissue environment to allow for long‐term survival. Recent work and the broader availability of single‐cell profiling have highlighted TRM heterogeneity among different tissues, as well as identified specialized subsets within individual tissues, that are time and infection dependent. TRM cell phenotypic and transcriptional heterogeneity has implications for understanding TRM function and longevity. This review aims to summarize and discuss the latest findings on CD8+ TRM heterogeneity using single‐cell molecular profiling and explore the potential implications for immune protection and the design of immune therapies. [ABSTRACT FROM AUTHOR]

Memory T cells are thought to rely on oxidative phosphorylation and short-lived effector T cells on glycolysis. Here, we investigated how T cells arrive at these states during an immune response. To understand the metabolic state of rare, early-activated T cells, we adapted mass cytometry to quantify metabolic regulators at single-cell resolution in parallel with cell signaling, proliferation, and effector function. We interrogated CD8+ T cell activation in vitro and in response to Listeria monocytogenes infection in vivo. This approach revealed a distinct metabolic state in early-activated T cells characterized by maximal expression of glycolytic and oxidative metabolic proteins. Cells in this transient state were most abundant 5 days post-infection before rapidly decreasing metabolic protein expression. Analogous findings were observed in chimeric antigen receptor (CAR) T cells interrogated longitudinally in advanced lymphoma patients. Our study demonstrates the utility of single-cell metabolic analysis by mass cytometry to identify metabolic adaptations of immune cell populations in vivo and provides a resource for investigations of metabolic regulation of immune responses across a variety of applications.

Infections elicit immune adaptations to enable pathogen resistance and/or tolerance and are associated with compositional shifts of the intestinal microbiome. However, a comprehensive understanding of how infections with pathogens that exhibit distinct capability to spread and/or persist differentially change the microbiome, the underlying mechanisms, and the relative contribution of individual commensal species to immune cell adaptations is still lacking. Here, we discovered that mouse infection with a fast-spreading and persistent (but not a slow-spreading acute) isolate of lymphocytic choriomeningitis virus induced large-scale microbiome shifts characterized by increased Verrucomicrobia and reduced Firmicute/Bacteroidetes ratio. Remarkably, the most profound microbiome changes occurred transiently after infection with the fast-spreading persistent isolate, were uncoupled from sustained viral loads, and were instead largely caused by CD8 T cell responses and/or CD8 T cell-induced anorexia. Among the taxa enriched by infection with the fast-spreading virus, Akkermansia muciniphila, broadly regarded as a beneficial commensal, bloomed upon starvation and in a CD8 T cell-dependent manner. Strikingly, oral administration of A. muciniphila suppressed selected effector features of CD8 T cells in the context of both infections. Our findings define unique microbiome differences after chronic versus acute viral infections and identify CD8 T cell responses and downstream anorexia as driver mechanisms of microbial dysbiosis after infection with a fast-spreading virus. Our data also highlight potential context-dependent effects of probiotics and suggest a model in which changes in host behavior and downstream microbiome dysbiosis may constitute a previously unrecognized negative feedback loop that contributes to CD8 T cell adaptations after infections with fast-spreading and/or persistent pathogens.

During recent years, evidence has emerged that immune privileged sites such as the CNS and the retina may be more integrated in the systemic response to infection than was previously believed. In line with this, it was recently shown that a systemic acute virus infection leads to infiltration of CD8 T cells in the brains of immunocompetent mice. In this study, we extend these findings to the neurological tissue of the eye, namely the retina. We show that an acute systemic virus infection in mice leads to a transient CD8 T cell infiltration in the retina that is not directed by virus infection inside the retina. CD8 T cells were found throughout the retinal tissue, and had a high expression of CXCR6 and CXCR3, as also reported for tissue residing CD8 T cells in the lung and liver. We also show that the pigment epithelium lining the retina expresses CXCL16 (the ligand for CXCR6) similar to epithelial cells of the lung. Thus, our results suggest that the retina undergoes immune surveillance during a systemic infection, and that this surveillance appears to be directed by mechanisms similar to those described for non-privileged tissues.

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing b-cells is thought to be mediated primarily by antigenspecific CD8+ T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)- based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRa chain gene (TRAC) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8+ T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHCdextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-g production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201+ human b-cell line by HLA-A*02:01 restricted CD8+ T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8+ T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigenspecific regulatory T cells. [ABSTRACT FROM AUTHOR]

Introduction: Toxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic “Regulator Of CD8 T cell Response” (ROCTR). Methods: Therefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages. Results: Genetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii. Discussion: Collectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii. [ABSTRACT FROM AUTHOR]

Muscle-directed gene therapy with adeno-associated viral (AAV) vectors is undergoing clinical development for treating neuromuscular disorders and for systemic delivery of therapeutic proteins. Although these approaches show considerable therapeutic benefits, they are also prone to induce potent immune responses against vector or transgene products owing to the immunogenic nature of the intramuscular delivery route, or the high doses required for systemic delivery to muscle. Major immunological concerns include antibody formation against viral capsid, complement activation, and cytotoxic T cell responses against capsid or transgene products. They can negate therapy and even lead to life-threatening immunotoxicities. Herein we review clinical observations and provide an outlook for how the field addresses these problems through a combination of vector engineering and immune modulation. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) possess mutations that confer resistance to neutralizing antibodies within the Spike protein and are associated with breakthrough infection and reinfection. By contrast, less is known about the escape from CD8+ T cell-mediated immunity by VOC. Here, we demonstrated that all SARS-CoV-2 VOCs possess the ability to suppress major histocompatibility complex class I (MHC-I) expression. We identified several viral genes that contribute to the suppression of MHC I expression. Notably, MHC-I upregulation was strongly inhibited after SARS-CoV-2 but not influenza virus infection in vivo. While earlier VOCs possess similar capacity as the ancestral strain to suppress MHC-I, the Omicron subvariants exhibited a greater ability to suppress surface MHC-I expression. We identified a common mutation in the E protein of Omicron that further suppressed MHC-I expression. Collectively, our data suggest that in addition to escaping from neutralizing antibodies, the success of Omicron subvariants to cause breakthrough infection and reinfection may in part be due to its optimized evasion from T cell recognition. [ABSTRACT FROM AUTHOR]

Formation of long-lasting memory lymphocytes is one of the foundational characteristics of adaptive immunity and the basis of many vaccination strategies. Following the rapid expansion and contraction of effector CD8 T cells, the surviving antigen (Ag)-specific cells give rise to the memory CD8 T cells that persist for a long time and are phenotypically and functionally distinct from their naïve counterparts. Significant heterogeneity exists within the memory CD8 T cell pool, as different subsets display distinct tissue localization preferences, cytotoxic ability, and proliferative capacity, but all memory CD8 T cells are equipped to mount an enhanced immune response upon Ag re-encounter. Memory CD8 T cells demonstrate numerical stability under homeostatic conditions, but sepsis causes a significant decline in the number of memory CD8 T cells and diminishes their Ag-dependent and -independent functions. Sepsis also rewires the transcriptional profile of memory CD8 T cells, which profoundly impacts memory CD8 T cell differentiation and, ultimately, the protective capacity of memory CD8 T cells upon subsequent stimulation. This review delves into different aspects of memory CD8 T cell subsets as well as the immediate and long-term impact of sepsis on memory CD8 T cell biology. [ABSTRACT FROM AUTHOR]

Group 2 innate lymphoid cells (ILC2s) are essential for orchestrating type 2 immune responses during allergic airway inflammation and infection. ILC2s have been reported to play a regulatory role in tumors; however, this conclusion is controversial. In this study, we showed that IL-33-activated ILC2s could boost CD8+ T-cell function through direct antigen cross-presentation. After activation by IL-33, ILC2s showed an enhanced potential to process antigens and prime CD8+ T cell activation. Activated ILC2s could phagocytose exogenous antigens in vivo and in vitro, promoting antigen-specific CD8+ T cell function to enhance antitumor immune responses. Administration of OVA-loaded ILC2s induces robust antitumor effects on the OVA-expressing tumor model. These findings suggested that the administration of tumor antigen-loaded ILC2s might serve as a potential strategy for cancer treatment. [ABSTRACT FROM AUTHOR]

The autoimmune pathogenesis of type 1 diabetes (T1D) involves cellular infiltration from innate and adaptive immune subsets into the islets of Langerhans within the pancreas; however, the direct cytotoxic killing of insulin-producing β-cells is thought to be mediated primarily by antigen-specific CD8+ T cells. Despite this direct pathogenic role, key aspects of their receptor specificity and function remain uncharacterized, in part, due to their low precursor frequency in peripheral blood. The concept of engineering human T cell specificity, using T cell receptor (TCR) and chimeric antigen receptor (CAR)-based approaches, has been demonstrated to improve adoptive cell therapies for cancer, but has yet to be extensively employed for modeling and treating autoimmunity. To address this limitation, we sought to combine targeted genome editing of the endogenous TCRα chain gene (TRAC) via CRISPR/Cas9 in combination with lentiviral vector (LV)-mediated TCR gene transfer into primary human CD8+ T cells. We observed that knockout (KO) of endogenous TRAC enhanced de novo TCR pairing, which permitted increased peptide:MHC-dextramer staining. Moreover, TRAC KO and TCR gene transfer increased markers of activation and effector function following activation, including granzyme B and interferon-γ production. Importantly, we observed increased cytotoxicity toward an HLA-A*0201+ human β-cell line by HLA-A*02:01 restricted CD8+ T cells engineered to recognize islet-specific glucose-6-phosphatase catalytic subunit (IGRP). These data support the notion of altering the specificity of primary human T cells for mechanistic analyses of autoreactive antigen-specific CD8+ T cells and are expected to facilitate downstream cellular therapeutics to achieve tolerance induction through the generation of antigen-specific regulatory T cells.

IntroductionToxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic “Regulator Of CD8 T cell Response” (ROCTR).MethodsTherefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages.ResultsGenetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii.DiscussionCollectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii.

Control of CNS pathogens by CD8 T cells is key to avoid fatal neuroinflammation. Yet, the modalities of MHC I presentation in the brain are poorly understood. Here, we analyze the antigen presentation mechanisms underlying CD8 T cell-mediated control of the Toxoplasma gondii parasite in the CNS. We show that MHC I presentation of an efficiently processed model antigen (GRA6-OVA), even when not expressed in the bradyzoite stage, reduces cyst burden and dampens encephalitis in C57BL/6 mice. Antigen presentation assays with infected primary neurons reveal a correlation between lower MHC I presentation of tachyzoite antigens by neurons and poor parasite control in vivo. Using conditional MHC I-deficient mice, we find that neuronal MHC I presentation is required for robust restriction of T. gondii in the CNS during chronic phase, showing the importance of MHC I presentation by CNS neurons in the control of a prevalent brain pathogen.

Formation of long-lasting memory lymphocytes is one of the foundational characteristics of adaptive immunity and the basis of many vaccination strategies. Following the rapid expansion and contraction of effector CD8 T cells, the surviving antigen (Ag)-specific cells give rise to the memory CD8 T cells that persist for a long time and are phenotypically and functionally distinct from their naïve counterparts. Significant heterogeneity exists within the memory CD8 T cell pool, as different subsets display distinct tissue localization preferences, cytotoxic ability, and proliferative capacity, but all memory CD8 T cells are equipped to mount an enhanced immune response upon Ag re-encounter. Memory CD8 T cells demonstrate numerical stability under homeostatic conditions, but sepsis causes a significant decline in the number of memory CD8 T cells and diminishes their Ag-dependent and -independent functions. Sepsis also rewires the transcriptional profile of memory CD8 T cells, which profoundly impacts memory CD8 T cell differentiation and, ultimately, the protective capacity of memory CD8 T cells upon subsequent stimulation. This review delves into different aspects of memory CD8 T cell subsets as well as the immediate and long-term impact of sepsis on memory CD8 T cell biology.

Despite the therapeutic success of immune checkpoint blockade (ICB) therapy against multiple tumors, many patients still do not benefit from ICB. In particular, high-grade brain tumors, such as glioblastoma multiforme (GBM), have a very low response rate to ICB, resulting in several failed clinical trials. This low response rate might be caused by a lack of understanding of the unique characteristics of brain immunity. To overcome this knowledge gap, macroscopic studies of brain immunity are needed. We use single cell RNA sequencing to analyze the immune landscape of the tumor microenvironment (TME) under anti-PD-1 antibody treatment in a murine GBM model. We observe that CD8 T cells show a mixed phenotype overall that includes reinvigoration and re-exhaustion states. Furthermore, we find that CCL5 induced by anti-PD- 1 treatment might be related to an increase in the number of anti-inflammatory macrophages in the TME. Therefore, we hypothesize that CCL5-mediated recruitment of anti-inflammatory macrophages may be associated with re-exhaustion of CD8 T cells in the TME. We compare our observations in the murine GBM models with publicly available data from human patients with recurrent GBM. Our study provides critical information for the development of novel immunotherapies to overcome the limitations of anti-PD-1 therapy. [ABSTRACT FROM AUTHOR]

Background: Lymphocytic thyroiditis (LT) is frequently seen in the tumor microenvironment (TME) of papillary thyroid carcinomas (PTCs). However, the characteristic of these tumor-infiltrating lymphocytes (TILs) is not well understood. Objective: We aim to define the TME of PTC cases by characterizing the TILs. Design: This is a cross-sectional observational study. Patients: We enrolled 29 PTC (23 having concurrent LT), 14 LT, and 13 hyperplastic nodules with LT (HN) patients from January 2016 to December 2020. Measurements: Immunohistochemical (IHC) expression of CD8, FoxP3, PD-1, and PD-L1 was studied in PTC with LT and compared with HN. PD-1 and PD-L1 expression was correlated at the mRNA level by quantitative real-time PCR. Immunophenotyping of TILs was done in FNAC samples of PTC and LT by flow cytometry. Results: IHC revealed the presence of CD8+ cytotoxic T lymphocytes (CTLs) and FoxP3+ T regulatory cells (Tregs) in 83% and 52% of PTC with LT cases, respectively. Flow cytometric analysis of the PTC samples revealed a significant abundance of CTL compared with Treg and a higher CTL with lower Treg counts compared with LT. On IHC, PD-1 positivity was noted in 56.5% of PTC with LT cases, while intermediate PD-L1 positivity was found in 70% of the cases. There was a significant upregulation of PD-1 mRNA in PTC with LT. A significant correlation was noted with PD-L1 expression with lymph node metastasis and presence of Treg cells. Conclusions: Increased expression of PD-1 and PD-L1 in the TME of PTC may provide a potential molecular mechanism for tumor survival despite the predominance of CTLs, possibly through their inactivation or exhaustion. [ABSTRACT FROM AUTHOR]

Introduction: The frequencies and functions of T stem cell memory (TSCM) subsets vary in autoimmune diseases. We evaluated the frequencies of CD4+ and CD8+ TSCM subsets as well as their PD‐1 expression levels in patients with T1D. Methods: Blood samples were collected from new case (NC) (n = 15), and long‐term (LT) (n = 15) groups and healthy controls (n = 15). Five subsets of T cells including TCM(CD4+/CD8+ CCR7+ CD45RO+ CD95+), TCMhi (CD4+/CD8+ CCR7+ CD45ROhi CD95+), TEM(CD4+/CD8+ CCR7− CD45RO+ CD95+), TSCM(CD4+/CD8+ CCR7+ CD45RO− CD95+), and T naive (CD4+/CD8+ CCR7+ CD45RO− CD95−) were detected by flow‐cytometry. Results: The frequency of CD4+ TSCM was higher in NC patients than LT patients and controls (p <.0001 and p =.0086, respectively). A higher percentage of the CD8+ T naive cells was shown in NC patients as compared with LT and healthy individuals (p =.0003 and p =.0002, respectively). An increased level of PD‐1 expression was observed on the CD4+TCM and TCMhi cells in LT patients as compared with healthy controls (p =.0037 and p =.0145, respectively). Also, the higher PD‐1 expression was observed on the CD8+ TCM and TCMhi in NC and LT patients as compared with controls (p =.0068 and p <.0001; p =.0012 and p =.0012, respectively). Conclusion: Considering TSCMs' capacities to generate all memory and effector T cells, our results may suggest a potential association between the increased frequencies of TSCMs and T1D progression. [ABSTRACT FROM AUTHOR]

• ESM1-SCD1 axis in OC. • Lactate drives ESM1-SCD1 axis to inhibit the antitumor CD8 + T cell response by activating Wnt/β-catenin pathway in ovarian cancer cells and to induce Cisplatin resistance. • Summary of our study has indicated that the induction of SCD1 by lactic acid-induced ESM1 can impede the CD8 + T cell response against tumors and promote resistance to cisplatin by activating the Wnt/β-catenin pathway in ovarian cancer. Ovarian cancer (OC) is a gynecological malignancy that results in a global threat to women's lives. Lactic acid, a key metabolite produced from the glycolytic metabolism of glucose molecules, is correlated with tumor immune infiltration and platinum resistance. In our previous study, we found that endothelial cell-specific molecule 1 (ESM1) plays a key role in OC progression. This study revealed that lactate could upregulate ESM1, which enhances SCD1 to attenuate the antitumor CD8+ T-cell response. ESM1 and SCD1 expression levels were significantly greater in OC patients with high lactic acid levels than in those with low lactic acid levels. Further mechanistic studies suggested that the Wnt/β-catenin pathway was inactivated after ESM1 knockdown and rescued by SCD1 overexpression. IC50 analysis indicated that the ESM1–SCD1 axis induces the resistance of OC cells to platinum agents, including cisplatin, carboplatin, and oxaliplatin, by upregulating P-gp. In conclusion, our study indicated that the induction of SCD1 by lactic acid-induced ESM1 can impede the CD8+ T-cell response against tumors and promote resistance to cisplatin by activating the Wnt/β–catenin pathway in ovarian cancer. Consequently, targeting ESM1 may have considerable therapeutic potential for modulating the tumor immune microenvironment and enhancing drug sensitivity in OC patients. [ABSTRACT FROM AUTHOR]

Human CD8 tumor-infiltrating lymphocytes (TILs) with impaired effector functions and PD-1 expression are categorized as exhausted. However, the exhaustion-like features reported in TILs might stem from their activation rather than the consequence of T cell exhaustion itself. Using CRISPR-Cas9 and lentiviral overexpression in CD8 T cells from non-cancerous donors, we show that the T cell receptor (TCR)-induced transcription factor interferon regulatory factor 4 (IRF4) promotes cell proliferation and PD-1 expression and hampers effector functions and expression of nuclear factor κB (NF-κB)-regulated genes. While CD8 TILs with impaired interferon γ (IFNγ) production exhibit activation markers IRF4 and CD137 and exhaustion markers thymocyte selection associated high mobility group box (TOX) and PD-1, activated T cells in patients with COVID-19 do not demonstrate elevated levels of TOX and PD-1. These results confirm that IRF4+ TILs are exhausted rather than solely activated. Our study indicates, however, that PD-1 expression, low IFNγ production, and active cycling in TILs are all influenced by IRF4 upregulation after T cell activation. [Display omitted] • IRF4 impairs cytokine production in human CD8 T cells • IRF4 promotes PD-1 expression and T cell proliferation • IRF4 regulates cell cycle genes and impairs NF-κB-regulated gene expression • Exhaustion features in TILs are correlated to their activation and IRF4 expression Hirsch et al. subjected primary human CD8 T cells to IRF4 overexpression or knockout and assessed T cell functions and the transcriptome. The TCR-induced transcription factor IRF4 promotes proliferation and PD-1 expression but hampers effector functions and expression of NF-κB-regulated genes. They also compared CD8 tumor-infiltrating lymphocytes to activated T cells in patients with COVID-19. [ABSTRACT FROM AUTHOR]

Tumor-specific CD8+ T cells are frequently dysfunctional and unable to halt tumor growth. We investigated whether tumor-specific CD4+ T cells can be enlisted to overcome CD8+ T cell dysfunction within tumors. We find that the spatial positioning and interactions of CD8+ and CD4+ T cells, but not their numbers, dictate anti-tumor responses in the context of adoptive T cell therapy as well as immune checkpoint blockade (ICB): CD4+ T cells must engage with CD8+ T cells on the same dendritic cell during the effector phase, forming a three-cell-type cluster (triad) to license CD8+ T cell cytotoxicity and cancer cell elimination. When intratumoral triad formation is disrupted, tumors progress despite equal numbers of tumor-specific CD8+ and CD4+ T cells. In patients with pleural mesothelioma treated with ICB, triads are associated with clinical responses. Thus, CD4+ T cells and triads are required for CD8+ T cell cytotoxicity during the effector phase and tumor elimination. [Display omitted] • Intratumoral immune triads are required for cancer elimination • CD4+ T cells within triads reprogram CD8+ T cells and license them to kill • CD4+ T cells must engage with CD8+ T cells on same antigen-presenting cell • Triads are associated with clinical responses to immunotherapy in humans Tumor-specific CD8+ T cells are dysfunctional within tumors. Espinosa-Carrasco et al. show that CD4+ T cells must engage with CD8+ T cells on the same antigen-presenting cell (APC) during the effector phase, forming a three-cell-cluster (triad) to license CD8+ T cell cytotoxicity and CD8+ T cell-mediated cancer cell elimination. [ABSTRACT FROM AUTHOR]