Exploitation of CD3ζ to enhance TCR expression levels and antigen-specific T cell function.

The expression levels of TCRs on the surface of human T cells define the avidity of TCR-HLA/peptide interactions. In this study, we have explored which components of the TCR-CD3 complex are involved in determining the surface expression levels of TCRs in primary human T cells. The results show that there is a surplus of endogenous TCR α/ß chains that can be mobilised by providing T cells with additional CD3γ,δ,ε,ζ chains, which leads to a 5-fold increase in TCR α/ß surface expression. The analysis of individual CD3 chains revealed that provision of additional ζ chain alone was sufficient to achieve a 3-fold increase in endogenous TCR expression. Similarly, CD3ζ also limits the expression levels of exogenous TCRs transduced into primary human T cells. Interestingly, transduction with TCR plus CD3ζ not only increased surface expression of the introduced TCR, but it also reduced mispairing with endogenous TCR chains, resulting in improved antigen-specific function. TCR reconstitution experiments in HEK293T cells that do not express endogenous TCR or CD3 showed that TCRα/ß and all four CD3 chains were required for optimal surface expression, while in the absence of CD3ζ the TCR expression was reduced by 50%. Together, the data show that CD3ζ is a key regulator of TCR expression levels in human T cells, and that gene transfer of exogenous TCR plus CD3ζ improved TCR surface expression, reduced TCR mispairing and increased antigen-specific function.

Characterization of atypical T cells generated during ex vivo expansion process for T cell-based adoptive immunotherapy.

Engineered T cell-based adoptive immunotherapies met promising success for the treatment of hematological malignancies. Nevertheless, major hurdles remain to be overcome regarding the management of relapses and the translation to solid tumor settings. Properties of T cell-based final product should be appropriately controlled to fine-tune the analysis of clinical trial results, to draw relevant conclusions, and finally to improve the efficacy of these immunotherapies. For this purpose, we addressed the existence of atypical T cell subsets and deciphered their phenotypic and functional features in an HPV16-E7 specific and MHC II-restricted transgenic-TCR-engineered T cell setting. To note, atypical T cell subsets include mismatched MHC/co-receptor CD8 or CD4 and miscommitted CD8+ or CD4+ T cells. We generated both mismatched and appropriately matched MHC II-restricted transgenic TCR on CD8 and CD4-expressing T cells, respectively. We established that CD4+ cultured T cells exhibited miscommitted phenotypic cytotoxic pattern and that both interleukin (IL)-2 or IL-7/IL-15 supplementation allowed for the development of this cytotoxic phenotype. Both CD4+ and CD8+ T cell subsets, transduced with HPV16-E7 specific transgenic TCR, demonstrated cytotoxic features after exposure to HPV-16 E7-derived antigen. Ultimately, the presence of such atypical T cells, either mismatched MHC II-restricted TCR/CD8+ T cells or cytotoxic CD4+ T cells, is likely to influence the fate of patient-infused T cell product and would need further investigation.

Maladaptive T-Cell Metabolic Fitness in Autoimmune Diseases.

Immune surveillance and adaptive immune responses, involving continuously circulating and tissue-resident T-lymphocytes, provide host defense against infectious agents and possible malignant transformation while avoiding autoimmune tissue damage. Activation, migration, and deployment of T-cells to affected tissue sites are crucial for mounting an adaptive immune response. An effective adaptive immune defense depends on the ability of T-cells to dynamically reprogram their metabolic requirements in response to environmental cues. Inability of the T-cells to adapt to specific metabolic demands may skew cells to become either hyporesponsive (creating immunocompromised conditions) or hyperactive (causing autoimmune tissue destruction). Here, we review maladaptive T-cell metabolic fitness that can cause autoimmune diseases and discuss how T-cell metabolic programs can potentially be modulated to achieve therapeutic benefits.

A novel homozygous mutation in RASGRP1 that predisposes to immune dysregulation and immunodeficiency associated with uncontrolled Epstein-Barr virus-induced B cell proliferation.

BACKGROUND: RASGRP1-deficiency results in an immune dysregulation and immunodeficiency that manifest as autoimmunity, lymphoproliferation, lymphopenia, defective T cell function, and increased incidence of Epstein-Bar Virus infections and lymphomas. OBJECTIVE: To investigate the mechanism of autoimmune hemolytic anemia and infections in a male patient of consanguineous parents from Lebanon. METHODS: Genetic diagnosis was obtained using next generation and Sanger sequencing. Protein expression and phosphorylation were determined by immunoblotting. T and B cell development and function were studied by flow cytometry. Cytokine and immunoglobulin secretions were quantified by enzyme-linked immunosorbent assay. RESULTS: The patient suffered from severe lymphopenia especially affecting the T cell compartment. Genetic analysis revealed a homozygous insertion of adenine at position 1396_1397 in RASGRP1 that abolished protein expression and downstream Ras signaling. T cells from the patient showed severe activation defects resulting in uncontrolled Epstein-Bar Virus-induced B cell proliferation. B cells from the patient were normal. CONCLUSION: This report expands the spectrum of mutations in patients with RasGRP1 deficiency, and provides evidence for the important role RasGRP1 plays in the ability of T cells to control Epstein-Bar Virus-induced B cell proliferation. CLINICAL IMPLICATIONS: Following diagnosis, the patient will be maintained on oral valganciclovir and monitored regularly for Epstein-Bar Virus infections to avoid the development of Epstein-Bar Virus- induced B cell lymphoma. He is also candidate for hematopoietic stem cell transplantation.

The two enantiomers of 2-hydroxyglutarate differentially regulate cytotoxic T cell function.

2-Hydroxyglutarate (2HG) is a byproduct of the tricarboxylic acid (TCA) cycle and is readily detected in the tissues of healthy individuals. 2HG is found in two enantiomeric forms: S-2HG and R-2HG. Here, we investigate the differential roles of these two enantiomers in cluster of differentiation (CD)8+ T cell biology, where we find they have highly divergent effects on proliferation, differentiation, and T cell function. We show here an analysis of structural determinants that likely underlie these differential effects on specific α-ketoglutarate (αKG)-dependent enzymes. Treatment of CD8+ T cells with exogenous S-2HG, but not R-2HG, increased CD8+ T cell fitness in vivo and enhanced anti-tumor activity. These data show that S-2HG and R-2HG should be considered as two distinct and important actors in the regulation of T cell function.

Modulation of T cell function and survival by the tumor microenvironment.

Cancer immunotherapy is shifting paradigms in cancer care. T cells are an indispensable component of an effective antitumor immunity and durable clinical responses. However, the complexity of the tumor microenvironment (TME), which consists of a wide range of cells that exert positive and negative effects on T cell function and survival, makes achieving robust and durable T cell responses difficult. Additionally, tumor biology, structural and architectural features, intratumoral nutrients and soluble factors, and metabolism impact the quality of the T cell response. We discuss the factors and interactions that modulate T cell function and survive in the TME that affect the overall quality of the antitumor immune response.

Modifications outside CDR1, 2 and 3 of the TCR variable ß domain increase TCR expression and antigen-specific function.

T cell receptor (TCR) gene modified T cells are a promising form of adoptive cellular therapy against human malignancies and viral infections. Since the first human clinical trial was carried out in 2006, several strategies have been developed to improve the efficacy and safety of TCR engineered T cells by enhancing the surface expression of the introduced therapeutic TCRs whilst reducing the mis-pairing with endogenous TCR chains. In this study, we explored how modifications of framework residues in the TCR variable domains affect TCR expression and function. We used bioinformatic and protein structural analyses to identify candidate amino acid residues in the framework of the variable ß domain predicted to drive high TCR surface expression. Changes of these residues in poorly expressed TCRs resulted in improved surface expression and boosted target cell specific killing by engineered T cells expressing the modified TCRs. Overall, these results indicate that small changes in the framework of the TCR variable domains can result in improved expression and functionality, while at the same time reducing the risk of toxicity associated with TCR mis-pairing.

New insights into follicular regulatory T cells in the intestinal and tumor microenvironments.

Follicular regulatory T (Tfr) cells are a novel and unique subset of effector regulatory T (Treg) cells that are located in germinal centers (GCs). Tfr cells express transcription profiles that are characteristic of both follicular helper T (Tfh) cells and Treg cells and negatively regulate GC reactions, including Tfh cell activation and cytokine production, class switch recombination and B cell activation. Evidence also shows that Tfr cells have specific characteristics in different local immune microenvironments. This review focuses on the regulation of Tfr cell differentiation and function in unique local immune microenvironments, including the intestine and tumor.

Soluble immune checkpoints as correlates for HIV persistence and T cell function in people with HIV on antiretroviral therapy.

Introduction: In people with HIV (PWH) both off and on antiretroviral therapy (ART), the expression of immune checkpoint (IC) proteins is elevated on the surface of total and HIV-specific T-cells, indicating T-cell exhaustion. Soluble IC proteins and their ligands can also be detected in plasma, but have not been systematically examined in PWH. Since T-cell exhaustion is associated with HIV persistence on ART, we aimed to determine if soluble IC proteins and their ligands also correlated with the size of the HIV reservoir and HIV-specific T-cell function. Methods: We used multiplex bead-based immunoassay to quantify soluble programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), T cell immunoglobulin domain and mucin domain 3 (TIM-3), PD-1 Ligand 1 (PD-L1) and PD-1 Ligand 2 (PD-L2) in plasma from PWH off ART (n=20), on suppressive ART (n=75) and uninfected controls (n=20). We also quantified expression of membrane-bound IC and frequencies of functional T-cells to Gag and Nef peptide stimulation on CD4+ and CD8+ T-cells using flow cytometry. The HIV reservoir was quantified in circulating CD4+ T-cells using qPCR for total and integrated HIV DNA, cell-associated unspliced HIV RNA and 2LTR circles. Results: Soluble (s) PD-L2 level was higher in PWH off and on ART compared to uninfected controls. Higher levels of sPD-L2 correlated with lower levels of HIV total DNA and higher frequencies of gag-specific CD8+ T-cells expressing CD107a, IFNγ or TNFα. In contrast, the concentration of sLAG-3 was similar in uninfected individuals and PWH on ART, but was significantly elevated in PWH off ART. Higher levels of sLAG-3 correlated with higher levels of HIV total and integrated DNA, and lower frequency of gag-specific CD4+ T cells expressing CD107a. Similar to sLAG-3, levels of sPD-1 were elevated in PWH off ART and normalized in PWH on ART. sPD-1 was positively correlated with the frequency of gag-specific CD4+ T cells expressing TNF-a and the expression of membrane-bound PD-1 on total CD8+ T-cells in PWH on ART. Discussion: Plasma soluble IC proteins and their ligands correlate with markers of the HIV reservoir and HIV-specific T-cell function and should be investigated further in in large population-based studies of the HIV reservoir or cure interventions in PWH on ART.

Tumor-Infiltrating T Cells in EBV-Associated Gastric Carcinomas Exhibit High Levels of Multiple Markers of Activation, Effector Gene Expression, and Exhaustion.

Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with 10% of all gastric cancers (GCs) and 1.5% of all human cancers. EBV-associated GCs (EBVaGCs) are pathologically and clinically distinct entities from EBV-negative GCs (EBVnGCs), with EBVaGCs exhibiting differential molecular pathology, treatment response, and patient prognosis. However, the tumor immune landscape of EBVaGC has not been well explored. In this study, a systemic and comprehensive analysis of gene expression and immune landscape features was performed for both EBVaGC and EBVnGC. EBVaGCs exhibited many aspects of a T cell-inflamed phenotype, with greater T and NK cell infiltration, increased expression of immune checkpoint markers (BTLA, CD96, CTLA4, LAG3, PD1, TIGIT, and TIM3), and multiple T cell effector molecules in comparison with EBVnGCs. EBVaGCs also displayed a higher expression of anti-tumor immunity factors (PDL1, CD155, CEACAM1, galectin-9, and IDO1). Six EBV-encoded miRNAs (miR-BARTs 8-3p, 9-5p, 10-3p, 22, 5-5p, and 14-3p) were strongly negatively correlated with the expression of immune checkpoint receptors and multiple markers of anti-tumor immunity. These profound differences in the tumor immune landscape between EBVaGCs and EBVnGCs may help explain some of the observed differences in pathological and clinical outcomes, with an EBV-positive status possibly being a potential biomarker for the application of immunotherapy in GC.

Assessing the Immune Regulatory Role of Metabolites In Vitro.

One method of immune evasion that cancer cells employ is the secretion of immune regulatory metabolites into the tumor microenvironment (TME). These metabolites can promote immunosuppressive cell subsets, while inhibiting key tumor-killing subsets, such as T cells. Thus, the identification of these metabolites may help develop methods for improving cell-based therapy. However, after identifying a potential immune regulatory metabolite, it is crucial to assess the impacts of the metabolite on T cell immunobiology. In this chapter, we describe an in vitro method of testing and analyzing the influence of a specific metabolite on T cell proliferation and function.

Immunophenotype and function of peripheral blood lymphocyte subsets in Chinese rhesus macaques / 中华微生物学和免疫学杂志

Objective:To provide data reference for using Chinese rhesus macaques as research model by studying the immunophenotype and function of peripheral blood lymphocytes in Chinese rhesus macaques.Methods:By optimizing antibody clones and fluorescent colors, the lymphocyte subset assay and T cell function assay panels were determined. Then the panels were used to analyze the proportion of T, B, NK and other cell subsets in peripheral blood mononuclear cells (PBMCs) in 15 healthy Chinese rhesus monkeys, and the ability of T cells to secrete cytokines after non-specific stimulation.Results:Two multi-color flow cytometry analytic panels were established. Panel 1 could simultaneously detect a variety of lymphocyte subsets, including cytotoxic T lymphocytes, follicular helper T cells, regulatory T cells, B cells and NK cells. Panel 2 could detect the functions of multiple T cell subsets and the expression of immune checkpoint moleculars. The mean percentages of T, B, NK, Tfh, Treg, CD16 + NK and CD56 + NK cells in PBMCs of the Chinese rhesus macaques were (75.32±7.73)%, (13.22±7.50)%, (0.88±0.48)%, (0.73±0.27)%, (0.75±0.43)%, (47.87±22.35)% and (10.69±12.41)%. After non-specific stimulation, the proportion of CD4 + T cells secreting IL-2 and TNF-α was higher than that of CD8 + T cells, and the proportion of CD8 + T cells secreting CD107a and IFN-γ was higher than that of CD4 + T cells, while the proportion of CD4 + and CD8 + T cells secreting IL-17A was low. Conclusions:This study established a multi-color flow detection scheme that could simultaneously detect multiple cellular surface molecules and cytokines at the single cell level and could accurately and comprehensively analyze the immune cell subsets, functions and the immune checkpoint molecules in peripheral blood of Chinese rhesus macaques, providing a new experimental method and basic data for the development of vaccines and drugs against infectious diseases.

Nutrition deprivation affects the cytotoxic effect of CD8 T cells in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and the third leading cause of cancer-related death worldwide. Factors including carcinogens, infection of hepatitis viruses, alcohol abuse, and metabolic disorders such as non-alcoholic fatty liver disease mainly contribute to HCC initiation and progression. Immunotherapy is one of the most powerful tools for unresectable HCC treatment in patients. CD8+ T cells are a major immune component in the tumor microenvironment with cytotoxic effects against cancer cells. However, these CD8+ T cells commonly display an exhaustion phenotype with high expression of programmed cell death protein 1, T-cell immunoglobulin and mucin-domain containing-3, and/or lymphocyte-activation gene 3, producing low levels of perforin (PRF1) and granzyme B (GZMB), as well as anti-tumor cytokines, such as interferon gamma and tumor necrosis factor alpha. In the referenced study, the authors also showed that deprivation of glutamine decreased the antitumor function of CD8+ T cells, as well as the production of PRF1 and GZMB. However, the role of each amino acid in T cell function and exhaustion may depend on tumor type and tumor microenvironment, including the source of other nutrients. Overall, amino acids or other nutrient metabolites in the tumor microenvironment play a pivotal role in both tumor growth and immune response.

Survival prediction optimization of acute myeloid leukaemia based on T-cell function-related genes and plasma proteins.

Interactions between acute myeloid leukaemia (AML) cells and immune cells are postulated to corelate with outcomes of AML patients. However, data on T-cell function-related signature are not included in current AML survival prognosis models. We examined data of RNA matrices from 1611 persons with AML extracted from public databases arrayed in a training and three validation cohorts. We developed an eight-gene T-cell function-related signature using the random survival forest variable hunting algorithm. Accuracy of gene identification was tested in a real-world cohort by quantifying cognate plasma protein concentrations. The model had robust prognostic accuracy in the training and validation cohorts with five-year areas under receiver-operator characteristic curve (AUROC) of 0.67-0.76. The signature was divided into high- and low-risk scores using an optimum cut-off value. Five-year survival in the high-risk groups was 6%-23% compared with 42%-58% in the low-risk groups in all the cohorts (all p values <0.001). In multivariable analyses, a high-risk score independently predicted briefer survival with hazard ratios of death in the range 1.28-2.59. Gene set enrichment analyses indicated significant enrichment for genes involved in immune suppression pathways in the high-risk groups. Accuracy of the gene signature was validated in a real-world cohort with 88 pretherapy plasma samples. In scRNA-seq analyses most genes in the signature were transcribed in leukaemia cells. Combining the gene expression signature with the 2017 European LeukemiaNet classification significantly increased survival prediction accuracy with a five-year AUROC of 0.82 compared with 0.76 (p < 0.001). Our T-cell function-related risk score complements current AML prognosis models.

T-cell activation state differentially contributes to neuropsychiatric complications in women with HIV.

Neuropsychiatric complications are common among women with HIV (WWH). The pathophysiological mechanisms underlying these complications are not fully known but likely driven in part by immune modulation. We examined associations between T-cell activation states which are required to mount an effective immune response (activation, co-stimulation/normal function, exhaustion, senescence) and neuropsychiatric complications in WWH. 369 WWH (78% HIV RNA undetectable/<20cp/mL) enrolled in the Women's Interagency HIV Study completed neuropsychological testing and measures of depression (Center for Epidemiological Studies Depression Scale-CES-D), self-reported stress levels (Perceived Stress Scale-10), and post-traumatic stress (PTSD Checklist-Civilian Scale). Multiparametric flow cytometry evaluated T-cell activation state. Partial least squares regressions were used to examine T-cell phenotypes and neuropsychiatric outcome associations after confounder adjustment. In the total sample and among virally suppressed (VS)-WWH, CD4+ T-cell exhaustion was associated with poorer learning and attention/working memory (P's < 0.05). In the total sample, CD4+ T-cell activation was associated with better attention/working memory and CD8+ T-cell co-stimulation and senescence was associated with poorer executive function (P's < 0.05). For mental health outcomes, in the total sample, CD4+ T-cell activation was associated with more perceived stress and CD4+ T-cell exhaustion was associated with less depressive symptoms (P's < 0.05). Among VS-WWH, CD4+ senescence was associated with less perceive stress and CD8+ T-cell co-stimulation and senescence was associated with higher depression (P's < 0.05). Together, results suggest the contribution of peripheral CD4+ and CD8+ T-cell activation status to neuropsychiatric complications in WWH.

Cycling and activated CD8+ T lymphocytes and their association with disease severity in influenza patients.

BACKGROUND: T cell lymphopenia was a significant characteristic of severe influenza infection and it was associated with the functional changes of T cells. It is necessary to clarify the T cells characteristics of kinetic changes and their correlation with disease severity. METHODS: In a cohort of hospitalized influenza patients with varying degrees of severity, we characterized lymphocyte populations using flow cytometry. RESULTS: The numbers of cycling (Ki67+) T cells at the acute phase of severe influenza were higher, especially in the memory (CD45RO+) T cell subsets. T cells from hospitalized influenza patients also had significantly higher levels of the exhausted marker PD-1. Cycling status of T cells was associated with T cell activation during the acute phase of influenza infection. The recruitment of cycling and activated (CD38+HLA-DR+) CD8+ T cells subset is delayed in severe influenza patients. CONCLUSIONS: The increased numbers of cycling memory (Ki67+CD45RO+) T cells subsets and delayed kinetics of activated (CD38+HLA-DR+) CD8+ T cells, could serve as possible biological markers for disease severity.

Immune function in newborns with in-utero exposure to anti-TNFα therapy.

Background and aim: Anti-TNFα is measurable in infants exposed in utero up to 12 months of age. Data about the exposure effect on the infant's adaptive immunity are limited. We aimed to prospectively evaluate the distribution and function of T and B cells, in infants of females with inflammatory bowel disease, in utero exposed to anti-TNFα or azathioprine. Methods: A prospective multi-center study conducted 2014-2017. Anti-TNFα levels were measured in cord blood, and at 3 and 12 months. T-cell repertoire and function were analyzed at 3 and 12 months by flow-cytometry, expression of diverse T cell receptors (TCR) and T-cell receptor excision circles (TREC) quantification assay. Serum immunoglobulins and antibodies for inactivated vaccines were measured at 12 months. Baseline clinical data were retrieved, and 2-monthly telephonic interviews were performed regarding child infections and growth. Results: 24 pregnant females, age 30.6 (IQR 26.5-34.5) years were recruited, 20 with anti-TNFα (infliximab 8, adalimumab 12), and 4 with azathioprine treatment. Cord blood anti-TNFα was higher than maternal blood levels [4.3 (IQR 2.3-9.2) vs. 2.5 (IQR 1.3-9.7) mcg/ml], declining at 3 and 12 months. All infants had normal number of B-cells (n = 17), adequate levels of immunoglobulins (n = 14), and protecting antibody levels to Tetanus, Diphtheria, Hemophilus influenza-B and hepatitis B (n = 17). All had normal CD4+, CD8+ T-cells, and TREC numbers. TCR repertoire was polyclonal in 18/20 and slightly skewed in 2/20 infants. No serious infections requiring hospitalization were recorded. Conclusion: We found that T-cell and B-cell immunity is fully mature and immune function is normal in infants exposed in utero to anti-TNFα, as in those exposed to azathioprine. Untreated controls and large-scale studies are needed to confirm these results.

Signaling networks controlling ID and E protein activity in T cell differentiation and function.

E and inhibitor of DNA binding (ID) proteins are involved in various cellular developmental processes and effector activities in T cells. Recent findings indicate that E and ID proteins are not only responsible for regulating thymic T cell development but also modulate the differentiation, function, and fate of peripheral T cells in multiple immune compartments. Based on the well-established E and ID protein axis (E-ID axis), it has been recognized that ID proteins interfere with the dimerization of E proteins, thus restricting their transcriptional activities. Given this close molecular relationship, the extent of expression or stability of these two protein families can dynamically affect the expression of specific target genes involved in multiple aspects of T cell biology. Therefore, it is essential to understand the endogenous proteins or extrinsic signaling pathways that can influence the dynamics of the E-ID axis in a cell-specific and context-dependent manner. Here, we provide an overview of E and ID proteins and the functional outcomes of the E-ID axis in the activation and function of multiple peripheral T cell subsets, including effector and memory T cell populations. Further, we review the mechanisms by which endogenous proteins and signaling pathways alter the E-ID axis in various T cell subsets influencing T cell function and fate at steady-state and in pathological settings. A comprehensive understanding of the functions of E and ID proteins in T cell biology can be instrumental in T cell-specific targeting of the E-ID axis to develop novel therapeutic modalities in the context of autoimmunity and cancer.

HPV-Positive and -Negative Cervical Cancers Are Immunologically Distinct.

Although infection with human papillomavirus (HPV) is associated with nearly all cervical cancers (CC), a small proportion are HPV-negative. Recently, it has become clear that HPV-negative CC represent a distinct disease phenotype compared to HPV-positive disease and exhibit increased mortality. In addition, variations between different HPV types associated with CC have been linked to altered molecular pathology and prognosis. We compared the immune microenvironments of CC caused by HPV α9 species (HPV16-like), HPV α7 species (HPV18-like) and HPV-negative disease. HPV-negative CC appeared distinct from other subtypes, with greatly reduced levels of lymphocyte infiltration compared to either HPV α9 or α7 CC. Besides reduced levels of markers indicative of B, T, and NK lymphocytes, the expression of T-cell effector molecules, activation/exhaustion markers, and T-cell receptor diversity were also significantly lower in HPV-negative CC. Interestingly, HPV-negative CC expressed much higher levels of potential neoantigens than HPV-positive CC. These results identify profound differences between the immune landscape of HPV-positive and HPV-negative CC as well as modest differences between HPV α9 and α7 CC. These differences may contribute to altered patient outcomes between HPV-negative and HPV-positive CC and potentially between CC associated with different HPV types.

Glutamine deprivation impairs function of infiltrating CD8+ T cells in hepatocellular carcinoma by inducing mitochondrial damage and apoptosis.

BACKGROUND: The functions of infiltrating CD8+ T cells are often impaired due to tumor cells causing nutrient deprivation in the tumor microenvironment. Thus, the mechanisms of CD8+ T cell dysfunction have become a hot research topic, and there is increased interest on how changes in metabolomics correlate with CD8+ T cell dysfunction. AIM: To investigate whether and how glutamine metabolism affects the function of infiltrating CD8+ T cells in hepatocellular carcinoma. METHODS: Immunohistochemical staining and immunofluorescence were performed on surgically resected liver tissues from patients. Differentially expressed genes in infiltrating CD8+ T cells in hepatocellular carcinoma were detected using RNA sequencing. Activated CD8+ T cells were co-cultured with Huh-7 cells for 3 d. The function and mitochondrial status of CD8+ T cells were analyzed by flow cytometry, quantitative real-time polymerase chain reaction, and transmission electron microscopy. Next, CD8+ T cells were treated with the mitochondrial protective and damaging agents. Functional alterations in CD8+ T cells were detected by flow cytometry. Then, complete medium without glutamine was used to culture cells and their functional changes and mitochondrial status were detected. RESULTS: There were a large number of infiltrating PD-1+CD8+ T cells in liver cancer tissues. Next, we co-cultured CD8+ T cells and Huh-7 cells to explore the regulatory effect of hepatoma cells on CD8+ T cells. Flow cytometry results revealed increased PD-1 expression and decreased secretion of perforin (PRF1) and granzyme B (GZMB) by CD8+ T cells in the co-culture group. Meanwhile, JC-1 staining was decreased and the levels of reactive oxygen species and apoptosis were increased in CD8+ T cells of the co-culture group; additionally, the mitochondria of these cells were swollen. When CD8+ T cells were treated with the mitochondrial protective and damaging agents, their function was restored and inhibited, respectively, through the mitochondrial damage and apoptotic pathways. Subsequently, complete medium without glutamine was used to culture cells. As expected, CD8+ T cells showed functional downregulation, mitochondrial damage, and apoptosis. CONCLUSION: Glutamine deprivation impairs the function of infiltrating CD8+ T cells in hepatocellular carcinoma through the mitochondrial damage and apoptotic pathways.