T Cells Research Articles

Single-cell RNA sequencing uncovers heterogenous immune cell responses upon exposure to food additive (E171) titanium dioxide

The prospective use of food additive titanium dioxide (E171 TiO2) in a variety of fields (food, pharmaceutics, and cosmetics) prompts proper cellular cytotoxicity and transcriptomic assessment. Interestingly, smaller-sized E171 TiO2 can translocate in bloodstream and induce a diverse immunological response by activating the immune system, which can be either pro-inflammatory or immune-suppressive. Nevertheless, their cellular or immunologic responses in a heterogeneous population of the immune system following exposure of food additive E171 TiO2 is yet to be elucidated. For this purpose, we have used male Sprague-Dawley rats to deliver E171 TiO2 (5 mg/kg bw per day) via non-invasive intratracheal instillation for 13 weeks. After the 4 weeks recovery period, 3 mL of blood samples from both treated and untreated groups were collected for scRNAseq analysis. Firstly, granulocyte G1 activated innate immune response through the upregulation of genes involved in pro-inflammatory cytokine mediated cytotoxicity. Whereas NK cells resulted in heterogeneity role depending on the subsets where NK1 significantly inhibited cytotoxicity, whereas NK2 and NK3 subsets activated pro-B cell population & inhibited T cell mediated cytotoxicity respectively. While NKT_1 activated innate inflammatory responses which was confirmed by cytotoxic CD8+ T killer cell suppression. Similarly, NKT_2 cells promote inflammatory response by releasing lytic granules and MHC-I complex inhibition to arrest cytotoxic T killer cell responses. Conversely, NKT_3 suppressed inflammatory response by release of anti-inflammatory cytokines suggesting the functional heterogeneity of NKT subset. The formation of MHC-I or MHC-II complexes with T-cell subsets resulted in neither B and T cell dysfunction nor cytotoxic T killer cell inhibition suppressing adaptive immune response. Overall, our research offers an innovative high-dimensional approach to reveal immunological and transcriptomic responses of each cell types at the single cell level in a complex heterogeneous cellular environment by reassuring a precise assessment of immunological response of E171 TiO2.Graphical

Differential Control of T-Cell Subsets by Recombinant Human PLD2 in a Mouse Model of Allergic Asthma

ABSTRACT Background Phospholipase D2 (PLD2) enzymes are expressed on the cytoplasmic membrane of bacteria, fungi, plants, and animals. Recently, extensive research has linked PLD2 to the chronic inflammatory activity of cells. Allergic asthma is a chronic airway inflammation disease. In this context, a recombinant human phospholipase D2 (rhPLD2) was designed and modified from the wild-type PLD2 to study its effects in an ovalbumin (OVA) induced murine model of asthma. Methods Hematoxylin and eosin staining was used for lung histopathology. Cytokine concentrations in bronchoalveolar lavage fluid (BALF) were measured using ELISA kits. The ratio of T-bet and GATA-3 expression level in spleen and lymph nodes following rhPLD2 administration was assessed through RT-PCR. Phenotyping analysis of Treg cells from peripheral blood was performed by flow cytometry. Results It indicated that OVA-induced mice exhibited elevated pulmonary eosinophilia and allergic inflammation in the airways, along with increased expression of IFN-γ, IL-4 in the lung BALF. Administration of rhPLD2 alleviated lung inflammation and significantly reduce the number of eosinophils in peripheral blood and BALF. RhPLD2 also reversed the IFN-γ/IL-4 ratio at the molecular level in BALF and the T-bet/GATA-3 ratio in lymphocytes of the lung, spleen, lymph nodes at the genetic level. Furthermore, FACS analysis demonstrated that rhPLD2 increased the frequency of both IL-10+Treg cells and CD25+ Treg cells. Conclusion From a therapeutic perspective, rhPLD2 alleviates allergic airway inflammation by balancing Th1/Th2 homeostasis and increasing Treg cells. It has been shown to function in immunoregulatory activities in OVA-induced asthma mice.

Microenvironmental Traits of Classical Hodgkin’s Lymphoma in Adolescents and Their Prognostic Impact

Background. Classical Hodgkin’s lymphoma (cHL) in adolescents between 15 and 18 years old shows a higher disease-related mortality, and the overall prognosis is worse than in both children and adults. Objectives. We investigated the immune checkpoint inhibitors (ICPIs) therapeutic targets and specific T-regulatory and cytotoxic T-cell subsets in the subgroup of adolescent cHL patients, and we challenged their prognostic power. Methods. We retrieved formalin-fixed paraffin-embedded (FFPE) tissue of adolescent patients diagnosed with cHL and tested by immunohistochemistry the immune checkpoint molecules CTLA-4, LAG-3, PD-1, and PDL1 as well as the biological markers FOXP3 and CD8. Results. All the cases of our cohort expressed the immune checkpoint molecules CTLA-4, LAG-3, and PD-1 in microenvironment (ME), and the number of PD1+ cells was strongly associated with advanced disease, being higher in stage III/IV, indicating a possible role in the progression of cHL. A higher risk of recurrence and progression occurred in patients with lower amount of CD8+ microenvironmental T-cells at diagnosis (67.14 ± 27.23 vs. 42.86 ± 17.33 p = 0.032 and 65.59 ± 26.68 vs. 37 ± 17.45 p = 0.046, respectively). Conclusions. We showed that microenvironment of cHL in adolescent patients is enriched with potential therapeutic targets of ICPI that may be considered for therapeutic applications. Furthermore, the presence of PD-1 expressing T-cells strongly relates to advanced stage disease and a low density of CD8+ T lymphocytes is associated with recurrence and progression of disease.

Multiomic profiling of chronically activated CD4+ T cells identifies drivers of exhaustion and metabolic reprogramming.

Repeated antigen exposure leads to T-cell exhaustion, a transcriptionally and epigenetically distinct cellular state marked by loss of effector functions (e.g., cytotoxicity, cytokine production/release), up-regulation of inhibitory receptors (e.g., PD-1), and reduced proliferative capacity. Molecular pathways underlying T-cell exhaustion have been defined for CD8+ cytotoxic T cells, but which factors drive exhaustion in CD4+ T cells, that are also required for an effective immune response against a tumor or infection, remains unclear. Here, we utilize quantitative proteomic, phosphoproteomic, and metabolomic analyses to characterize the molecular basis of the dysfunctional cell state induced by chronic stimulation of CD4+ memory T cells. We identified a dynamic response encompassing both known and novel up-regulated cell surface receptors, as well as dozens of unexpected transcriptional regulators. Integrated causal network analysis of our combined data predicts the histone acetyltransferase p300 as a driver of aspects of this phenotype following chronic stimulation, which we confirmed via targeted small molecule inhibition. While our integrative analysis also revealed large-scale metabolic reprogramming, our independent investigation confirmed a global remodeling away from glycolysis to a dysfunctional fatty acid oxidation-based metabolism coincident with oxidative stress. Overall, these data provide both insights into the mechanistic basis of CD4+ T-cell exhaustion and serve as a valuable resource for future interventional studies aimed at modulating T-cell dysfunction.

Using T-Cell Subsets to Better Characterize Immunoresiliency and Immunodeficiency in Patients with Recurrent Infections

Background/Objectives: Common Variable Immunodeficiency Disease (CVID) and other immunodeficiencies can present in subtle and variable ways. Whether or not a genetic lesion can be identified, there are not well understood biomarkers that quantitatively describe how severe a deficiency is. Here we discuss two possible ranking systems, CD4/CD8 T cell ratios and Immune Health Grades, and how such data maybe applicable to some immunodeficiencies. Methods: This is not a systematic review, but we identify papers relating to immunodeficiencies with enough data to comment on the CD4/CD8 and Immune Health Grade. We also summarized relevant data publicly available from USIDNET, a website that compiles data on immunodeficiencies, and provide two new cases that illustrate ways that this information can alter clinical assessment. Results: We review the HIV literature on CD4/CD8 T cell data and how this correlates with both immunologic function and comorbidity better than CD4 count alone. The ratio aslso relates to a new system called Immune Health Grades (IHG) derived from young adult to elderly subjects from many NIH cohorts without HIV. CVID is often thought of as an antibody problem, but in fact most patients also have low CD4/CD8 ratio and other cellular abnormalities. We review IDNET to categorize nine molecular immunodeficiencies including two subcategories of CVID into low, normal, or high ratios. Finally, we present two new cases in the literature of patients with recurrent infection and discuss how viewing the cases through the “lens” of CD4/CD8 ratio and IHG can facilitate clinical decisions. Conclusions: Emerging data suggests at least some immunodeficiencies can be grouped by how abnormal their CD4/CD8 ratio or IHG. This represents a clinically available biomarker that can be tracked to see if the condition is worsening or not.

Infection of nonclassic monocytes by respiratory syncytial virus induces an imbalance in the CD4+ T-cell subset response.

Respiratory syncytial virus (RSV) causes lower respiratory tract infections in infants and young children, leading to a pathogenesis-associated imbalance in CD4+ T-cell subsets and monocyte subsets. To investigate whether RSV affects the imbalance of CD4+ T-cells through monocytes, we examined the effects of the RSV-infected monocyte subset on CD4+ T-cell subsets, namely, Th1, Th2, Th17, and regulatory T (Treg) subsets, on proliferation in vitro and identified key monocyte-derived cytokines. We found that RSV efficiently infects CD16+ monocytes, but not CD16- monocytes, via cocultures of CD4+ T-cells with RSV-infected CD16+ monocytes, inhibits Treg cell proliferation and increases Th2 cell frequency, suggesting that RSV causes an imbalance in the CD4+ T-cell subset by primarily infecting CD16+ monocytes. Our data also revealed that IL-1β and IL-10 are key cytokines responsible for the activities of RSV-infected CD16+ monocytes. In a mouse model, we found that high-efficiency RSV infection of mouse Ly6C- monocytes, corresponding to CD16+ monocytes in humans, and adoptive transfer of Ly6C- monocytes during RSV infection decreased the Treg frequency in the lungs and aggravated pneumonia. Our data indicate that RSV can increase its pathogenesis through infection of nonclassic monocytes, leading to a CD4+ T-cell imbalance.IMPORTANCEThis study identified a pathogenesis pathway related to the RSV-nonclassic monocyte-IL-1β/IL-10-CD4+ T-cell subset balance, which links the heterogeneity of monocytes to RSV pathogenesis and elucidates a new mechanism by which RSV infection disrupts the balance of CD4+ T cells during RSV infection. These new findings provide potential therapeutic targets for RSV infection.

CD8+ T cell exhaustion in the tumor microenvironment of breast cancer

CD8+ T cells are crucial cytotoxic components of the tumor immune system. In chronic inflammation, they become low-responsive, a state known as T cell exhaustion (TEX). The aim of immune checkpoint blockade is to counteract TEX, yet its dynamics in breast cancer remain poorly understood. This review defines CD8+ TEX and outlines its features and underlying mechanisms. It also discusses the primary mechanisms of CD8+ TEX in breast cancer, covering inhibitory receptors, immunosuppressive cells, cytokines, transcriptomic and epigenetic alterations, metabolic reprogramming, and exosome pathways, offering insights into potential immunotherapy strategies for breast cancer.

Novel T-cell subsets as non-invasive biomarkers of vascular damage along the predialysis stages of chronic kidney disease

IntroductionCardiovascular disease is the major cause of premature death in chronic kidney disease (CKD) and vascular damage is often detected belatedly, usually evaluated by expensive and invasive techniques. CKD involves specific risk factors that lead to vascular calcification and atherosclerosis, where inflammation plays a critical role. However, there are few inflammation-related markers to predict vascular damage in CKD. This study aimed to investigate immune populations in pre-dialysis patients to (i) identify subset alterations, (ii) assess longitudinal changes, and (iii) evaluate their applicability as biomarkers of subclinical vascular indices.Methods43 pre-dialysis CKD patients in stages CKD-2 to CKD-5 and 38 controls were recruited at baseline and after 18-month follow-up. Aortic stiffness was determined by carotid-femoral pulse wave velocity (PWV) and abdominal aortic calcification was quantified by the Kauppila index on X-rays. Carotid intima-media thickness, the number of carotid plaques and adventitial neovascularization were evaluated by Superb Microvascular Imaging. Peripheral blood mononuclear cells were isolated and immune cell populations were assessed by flow cytometry: senescent T cells (CD4+CD28null), Tang (CD3+CD31+CD184+) and derived subsets, and monocyte subsets (classical, intermediate and non-classical; and ACE expression).ResultsSenescent T cells were increased in CKD. Despite Tang levels were unchanged compared to controls, this subset exhibited enhanced immunosenescence traits (CD28null and inverted CD4+CD8+ ratio) in CKD. Furthermore, Tang were negatively correlated with CKD progression. Slight alterations within monocyte subsets were observed. These findings were validated at the 18-month follow-up. Tang were correlated with several subclinical indices, and further analyses revealed an independent effect on PWV and their potential value as biomarkers. Intermediate monocytes were positively correlated with PWV.ConclusionPre-dialysis CKD stages are hallmarked by alterations in immune cell populations related to vascular homeostasis, including early T-cell immunosenescence traits and a stage-dependent Tang depletion, which was independently related to vascular stiffness. All these features were replicated upon follow-up, thus providing validation toward our results. Our findings pave the ground for future studies addressing the functional contribution of these cellular mediators at the local level, assessing their potential predictive value in the long-term and implementing preventive strategies in the clinical setting.

Transient Anti-TCRβ mAb Treatment Induces CD4+ T Cell Exhaustion and Prolongs Survival in a Mouse Model of Systemic Lupus Erythematosus.

T cells play a critical role in the pathogenesis of systemic lupus erythematosus (SLE). Chronic T cell receptor (TCR) signalling induces T cell exhaustion, characterised by reduced capacity to induce tissue damage. Here, we investigated the therapeutic potential of the anti-TCRβ (H57-597) monoclonal antibody (mAb) in a mouse model of SLE. Four-month-old MRL/lpr mice exhibiting SLE phenotypes received 5 weekly doses of anti-TCRβ mAb or phosphate-buffered saline (PBS) vehicle control. Subsequently, mouse survival was monitored daily. On day 1 post the final dose of treatment, SLE pathogenesis was determined using histological staining and spot urine test. T and B cell states in the brain, kidney, and secondary lymphoid organs were determined by flow cytometry. Transient treatment of anti-TCRβ mAb significantly prolonged the survival of MRL/lpr mice. Accordingly, MRL/lpr mice in the anti-TCRβ mAb group exhibited decreased proteinuria scores and minimal renal pathological damage compared to the PBS control group. Flow cytometric analysis revealed that anti-TCRβ mAb treatment resulted in a reduction in the frequencies of CD4+ T cells and CD138+B220lo/- plasma cells, plus an increase in Foxp3+ regulatory T cell frequency. Furthermore, CD4+ T cells from anti-TCRβ mAb treated mice exhibited elevated expression levels of PD-1 and TIM-3, with reduced IFN-γ production, indicative of an exhaustion-like phenotype. Therefore, transient administration of anti-TCRβ mAb treatment induces an exhaustion-like phenotype in CD4+ T cells, resulting in prolonged survival of MRL/lpr mice. Inducing autoreactive T-cell exhaustion holds promise as an attractive therapeutic approach for SLE.

A 16-week progressive exercise training intervention in treatment-naïve chronic lymphocytic leukaemia: a randomised-controlled pilot study

BackgroundChronic lymphocytic leukaemia (CLL) typically presents with asymptomatic, early-stage disease that is monitored until disease progression (‘treatment-naïve’ CLL). The objective of this pilot study was to assess the feasibility and preliminary safety of an exercise program in treatment-naïve CLL. We also sought to preliminarily assess the impact of the exercise program on disease activity, as it has been proposed that exercise training may reduce disease outgrowth in treatment-naïve CLL.MethodsA total of 40 treatment-naïve CLL patients were recruited into this randomised-controlled pilot study, and after screening, n = 28 were randomised into a 16-week, home-based, partially supervised, personalised, progressive exercise intervention (n = 14: mean ± SD: age = 62 ± 12 years) or 16 weeks of usual care, control group (n = 14: mean ± SD: age = 61 ± 10 years). The primary outcome measures were safety (number and severity of adverse events) and feasibility (uptake, retention, and adherence to the trial). Disease activity (CD5+/CD19+ CLL cells clonally restricted to kappa or lambda) and other immune cell phenotypes, with a principal focus on T cells, were measured by flow cytometry. Other secondary outcomes included DEXA-derived body composition, cardiorespiratory and functional fitness, resting cardiovascular measures.ResultsTrial uptake was 40%, and the overall retention rate was 86%, with 79% of the exercise group and 93% of the control group completing the trial. Adherence to the exercise intervention was 92 ± 8%. One serious adverse event was reported unrelated to the trial, and one adverse event related to the trial was reported. The exercise intervention elicited a 2% increase in DEXA-derived lean mass in the exercise group compared with a 0.4% decrease in the control group (p = 0.01). No between-group differences were observed over time for whole-body mass, BMI, bone mineral density, body fat, blood pressure resting heart rate, or measures of cardiorespiratory or functional fitness (all p > 0.05). No between-group differences were observed over time for clonal CLL cells and CD4+ or CD8+ T-cell subsets (all p > 0.05).ConclusionThe exercise training program used in this study was feasible in people with treatment-naïve CLL who passed pre-trial screening, and we preliminarily conclude that the exercise training program was safe and also resulted in an increase in lean mass.Clinical trial registrationhttps://doi.org/10.1186/ISRCTN55166064, identifier ISRCTN 55166064.

FC03 T17 polarization of skin-homing (CLA-positive) T cells is maintained by activation-dependent, AP-1-mediated chromatin remodelling

Abstract Background T cells undergo polyclonal expansion in psoriasis and PsA, with persistence of ‘driver clones’ after effective treatment (JCI 127:4031; JI 172:1935). A recent study found roughly equally sized compartments of clonally expanded and polyclonal Th17 and Tc17 cells in psoriasis (Science 371:364). Memory T17 cells undergo monocyte-dependent expansion in CD3/CD28-activated PBMC cultures (PNAS 104:17034; JID 139:1245). Methods We stimulated PBMC from 85 psoriatic and 68 controls for 1 day with anti-CD3/CD28 Dynabeads (24 h), or without beads (0 h), followed on Day 0 or Day 1 by flow sorting (CD3+CD45RO+, CD4/CD8 × CLA+/CLA- × 0/24 h), RNA-seq and ATAC-seq. Results CD3/CD28 activation provoked marked (>100-fold) upregulation of the T17 signature transcripts IL17A, IL17F, IL22, and CCL22, and of the T1 signature transcript IFNG. However, stratified analysis of CLAP vs. CLAN in activated T cells revealed 2.9- to 12.1-fold upregulation of the four T17 signature mRNAs, without a corresponding increase in IFNG. Notably, IL17A and IL17F were overexpressed in activated T cells from psoriatic vs. control donors (each 1.9-fold, P = 4.7E−4). Occupancy analysis of 702 transcription factor (TF) motifs using CENTIPEDE revealed remarkable correlations between activation (CLAN at 24 vs. 0 h) and skin-homing status (CLAP vs. CLAN at 0 h) [Spearman’s rho = 0.94 for CD4 and 0.93 for CD8; rho(expected)∼0.5]. Compared with CLAP/CLAN, correlations with activation were much weaker for CD8/CD4, the other dimension in our 2 × 2 × 2 study design (rho = 0.41 for CLAN and 0.29 for CLAP). AP-1 motifs were significantly enriched among CLAP vs. CLAN differentially occupied motifs (P < 2.2E−16 for CD4 and CD8, Fisher’s exact). Discussion CLAP are effector memory T cells (Tem) arising after injury/infection of skin (or tonsils), whereas CLAN are central memory T cells (Tcm) that may arise in the skin, lung, or gut (JID 130:362; STM 7:269). Our results support findings that human Th17 cells are long-lived Tem (STM 3:104) and that Tem are more responsive than Tcm due to increased TF occupancy driven by AP-1 TFs (Commun Biol 6:363). Our results nominate repetitive activation of memory T cells (whether clonally via TCR activation or polyclonally via cytokines) as a mechanism for increased responsiveness of CLAP/Tem vs. CLAN/Tcm. They also advance a plausible explanation for the presence of polyclonal T cells in psoriasis lesions (‘chromatin memory’ of secondarily activated T cells), suggesting a molecular basis of the Koebner phenomenon (heterologous activation of poised chromatin, Nature 580:475), and provide a rationale for implementing highly effective treatment early in the course of psoriasis (to prevent expansion of memory T cells, clonal or not, with chromatin poised for T17 reactivation).

NOD alleles at Idd1 and Idd2 loci drive exocrine pancreatic inflammation.

Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes and have enabled the identification of several loci associated with diabetes susceptibility, termed insulin-dependent diabetes (Idd). The generation of congenic mice has allowed the characterization of the impact of several loci on disease susceptibility. For instance, NOD.B6-Idd1 and B6.NOD-Idd1 congenic mice were instrumental in demonstrating that susceptibility alleles at the MHC locus (known as Idd1) are necessary but not sufficient for autoimmune diabetes progression. We previously showed that diabetes resistance alleles at the Idd2 locus provide significant protection from autoimmune diabetes onset, second to Idd1. In search of the minimal genetic factors required for T1D onset, we generated B6.Idd1.Idd2 double-congenic mice. Although the combination of Idd1 and Idd2 is not sufficient to induce diabetes onset, we observed immune infiltration in the exocrine pancreas of B6.Idd2 mice, as well as an increase in neutrophils and pancreatic tissue fibrosis. In addition, we observed phenotypic differences in T-cell subsets from B6.Idd1.Idd2 mice relative to single-congenic mice, suggesting epistatic interaction between Idd1 and Idd2 in modulating T-cell function. Altogether, these data show that Idd1 and Idd2 susceptibility alleles are not sufficient for autoimmune diabetes but contribute to inflammation and immune infiltration in the pancreas.

Immunomodulatory effects of inulin-type fructans from Arctium lappa L. by targeting gut microbiota and their metabolites

This study aimed to examine the in vitro digestion properties and immunomodulatory effects of inulin-type fructans (ALP-1) from Arctium lappa L. on immunosuppressive mice and to explore the underlying mechanisms. The simulated gastrointestinal digestion showed that ALP-1 underwent slight degradation during gastric and intestinal fluid digestion, with most of it reaching the gut as long-chain structures. The administration of ALP-1 effectively improved overall health and regulated immune function according to the spleen index, thymus index, splenic T-lymphocyte subsets, and other immune-related cytokines. Besides, 16S rDNA sequencing, ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry imaging technique revealed fructan-induced changes in gut microbiota composition, metabolic processes, and spatial information of key metabolites. These changes likely contributed to the immunomodulatory effects of ALP-1 in immunosuppressive mice. Therefore, ALP-1 shows promise as an immunomodulator for use in functional foods and nutraceuticals.

T-Switch: A specificity-based engineering platform for developing safe and effective Tcell therapeutics.

Many promising targets for adoptive Tcell therapy (ACT) are self-antigens, but self-reactive Tcells are generally eliminated during thymic selection or diverted to regulatory phenotypes. To bypass Tcell tolerance and obtain potent and safe Tcell therapeutics, we developed T-Switch, an invitro Tcell receptor (TCR) engineering platform for the creation, modification, and comprehensive profiling of TCRs that can target self-antigens. T-Switch first expands Tcells that recognize a "foreign" peptide closely related to a self-antigen. The fine specificity of the TCR is then modified by directed evolution of the peptide binding region to switch its specificity to the self-antigen of interest. We applied T-Switch to engineer synthetic TCRs reactive to a tumor-associated self-antigen, validated the safety and efficacy of this approach, and detected no off-target recognition as measured against the human proteome. Thus, T-Switch represents a resource for the creation of collections of highly sensitive synthetic TCRs for Tcell-based immunotherapies.

In human CD4+ T-Cells, omeprazole suppresses proliferation, downregulates V-ATPase, and promotes differentiation toward an autoimmunity-favoring phenotype

BackgroundProton pump inhibitors (PPIs) represent a commonly prescribed class of medications. Triggered by findings indicating a correlation between PPI usage and susceptibility to infectious or autoimmune diseases, we studied the impact of a pharmacological concentration of omeprazole on human CD4+ T-cells. MethodsIn mixed lymphocyte reactions (MLRs), we analyzed the proliferation index and measured the concentration of key cytokines representative of distinct CD4+ T-cell subsets. In CD4+ T-cells isolated from the MLRs, we evaluated proliferation markers and pathways, the expression of signature transcription factors of CD4+ T-cell subsets, vacuolar H+- ATPase (V-ATPase) levels, and the activation status of AMP-activated kinase (AMPK) and mammalian target of rapamycin complex-1 (mTORC1). ResultsOmeprazole reduced proliferation index in MLRs, and in isolated CD4+ T-cells, it downregulated the proliferation marker Ki-67, possibly mediated by the p53- p21 pathway. Analysis of cytokines and signature transcription factors of CD4+ T-cell subsets indicated that omeprazole decreased T helper 1 (Th1) differentiation, had negligible impact on Th2 differentiation, increased Th17 differentiation, and reduced regulatory T-cell (Treg) differentiation. Omeprazole also decreased V-ATPase, a known target of PPIs and a site for AMPK and mTORC1 activation. Consequently, this led to diminished activation of these kinases, potentially elucidating the mechanism by which omeprazole influences CD4+ T-cell differentiation. ConclusionOmeprazole downregulates V-ATPase and inhibits activation of AMPK and mTORC1. As a result, omeprazole suppresses CD4+ T-cell clonal expansion, potentially contributing to the observed association between PPIs and susceptibility to infections. Additionally, it modulates CD4+ T-cell differentiation in a manner that favors autoimmunity.

Identification of the CD8+ T-cell exhaustion signature of hepatocellular carcinoma for the prediction of prognosis and immune microenvironment by integrated analysis of bulk- and single-cell RNA sequencing data.

Hepatocellular carcinoma (HCC) is a prevalent type of cancer with high incidence and mortality rates. It is the third most common cause of cancer-related deaths. CD8+ T cell exhaustion (TEX) is a progressive decline in T cell function due to sustained T cell receptor stimulation from continuous antigen exposure. Studies have shown that CD8+ TEX plays an important role in the anti-tumor immune process and is significantly correlated with patient prognosis. The aim of the research is to establish a reliable CD8+ TEX-based signature using single-cell RNA sequencing (scRNA-seq) and high-throughput RNA sequencing (RNA-seq), providing a new approach to evaluate HCC patient prognosis and immune microenvironment. The RNA-seq data of HCC patients were download from three different databases: The Cancer Genome Atlas (TCGA), the Gene Expression Omnibus (GEO), and the International Cancer Genome Consortium (ICGC). HCC's 10× scRNA data were acquired from GSE149614. Based on single-cell sequencing data, CD8+ TEX-related genes were identified using uniform manifold approximation and projection (UMAP) algorithm, singleR, and marker gene methods. Afterwards, we proceeded to construct CD8+ TEX signature using differential gene analysis, univariate Cox regression analysis, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression analysis. We also validated the CD8+ TEX signature in GEO and ICGC external cohorts and investigated clinical characteristics, chemotherapy sensitivity, mutation landscape, functional analysis, and immune cell infiltration in different risk groups. The CD8+ TEX signature, consisting of 13 genes (HSPD1, UBB, DNAJB4, CALM1, LGALS3, BATF, COMMD3, IL7R, FDPS, DRAP1, RPS27L, PAPOLA, GPR171), was found to have a strong predictive effect on the prognosis of HCC. The Kaplan-Meier (KM) analysis showed that the overall survival (OS) rate of patients in the low-risk group was higher than that of patients in the high-risk group across different datasets and specific populations. The research findings suggested that the risk score was an independent predictor of HCC prognosis. The model based on clinical features and risk score has a strong predictive effect. We observed significant differences among various risk groups in terms of clinical characteristics, functional analysis, mutation landscape, chemotherapy sensitivity, and immune cell infiltration. We constructed a CD8+ TEX signature to predict the survival probability of patients with HCC. We also found that the model could predict the sensitivity of targeted drugs and immune cell infiltration, and the risk score was negatively correlated with CD8+ T cell infiltration. In summary, the CD8+ TEX signature of HCC was constructed for the prediction of prognosis and immune microenvironment by integrated analysis of bulk and scRNA-seq data.

Associations of circulating T-cell subsets with carotid artery stiffness: the Multi-Ethnic Study of Atherosclerosis.

Background: Arterial stiffness measured by total pulse wave velocity (T-PWV) is associated with increased risk of multiple age-related diseases. T-PWV can be described by structural (S-PWV) and load-dependent (LD-PWV) arterial stiffening. T-cells have been implicated in arterial remodeling, arterial stiffness, and hypertension in humans and animals; however, it is unknown whether T-cells are risk factors for T-PWV or its components. Therefore, we evaluated the cross-sectional associations of peripheral T-cell subpopulations with T-PWV, S-PWV, and LD-PWV. Methods: Peripheral blood T-cells were characterized using flow cytometry, and carotid artery stiffness was measured using B-mode ultrasound to calculate T-PWV at the baseline examination in a participant subset of the Multi-Ethnic Study of Atherosclerosis (MESA, n=1,984). A participant-specific exponential model was used to calculate S-PWV and LD-PWV based on elastic modulus and blood pressure gradients. The associations between five primary (p-significance<0.01) and twenty-five exploratory (p-significance<0.05) immune cell subpopulations, per 1-SD increment, and arterial stiffness measures were assessed using adjusted linear regression models. Results: For the primary analysis, higher CD4+CD28-CD57+, but not CD8+CD28-CD57+, T-cells were associated with higher LD-PWV (β=0.04 m/s, p<0.01) after adjusting for co-variates. None of the remaining T-cell subpopulations in the primary analysis were associated with T-PWV or S-PWV. For the exploratory analysis, several memory and differentiated/senescence-associated CD4+ and CD8+ T-cell subpopulations were associated with greater T-PWV, S-PWV, and LD-PWV after adjusting for co-variates. Conclusions: We highlight novel associations in humans between CD4+ and CD8+ memory and differentiated/senescence-associated T-cell subpopulations and measures of arterial stiffness in MESA. These results warrant longitudinal, prospective studies that examine changes in T-cell subpopulations and arterial stiffness in humans.

Cancer Cell-Derived Exosomal miR-500a-3p Modulates Hepatic Stellate Cell Activation and the Immunosuppressive Microenvironment.

Hepatocellular carcinoma (HCC) mainly depends on liver fibrosis/cirrhosis, which is regulated by tumor cells and the tumor microenvironment (TME), and is a crucial factor in tumor progression. This study aimed to identify abnormally expressed miR-500a-3p in the hepatitis-cirrhosis-HCC pathway and explored the roles of miR-500a-3p in HCC progression. A clinical cohort of patients with HCC is studied retrospectively. Subsequently, the role of miR-500a-3p transported by HCC exosomes in hepatic stellate cell (HSC) activation, hepatoma growth and invasion, and immune cell differentiation is determined by in vitro and in vivo experiments. In clinical tissues, miR-500a-3p is significantly enriched in HCC and cirrhosis tissues, and co-expression of the immune marker CD4 or PD-L1 significantly correlates with low survival rates in patients. Extracellular miR-500a-3p is taken up by HSC and PBMC, which promotes the secretion of the cytokines TGF-β1 and IL-10, increases PD-L1 expression in HSC, and stabilizes PD-1 expression in PBMC to affect the TME. Moreover, miR-500a-3p is associated with CD4+ T-cell exhaustion and Treg differentiation and is significantly associated with increased tumorigenicity in in situ mouse HCC models. Mechanistically, HCC-derived exosomal miR-500a-3p directly influences SOCS2 to regulate the JAK3/STAT5A/STAT5B signaling pathway. MiR-500a-3p promotes the growth and migration of HCC through the SOCS2/JAK3/STAT5A/STAT5B axis.

Temporal genomic analysis of homogeneous tumor models reveals key regulators of immune evasion in melanoma

Abstract Low intra-tumor heterogeneity (ITH) correlates with increased patient survival and immunotherapy response. However, even highly homogeneous tumors are variably aggressive, and the immunological factors impacting aggressiveness remain understudied. Here, we analyzed the mechanisms underlying immune escape in murine tumors with low ITH. We used immunophenotyping and single-cell RNA sequencing to compare the temporal growth of in-vivo transplanted, genetically similar rejected vs. non-rejected single-cell clones. Non-rejected clones showed high infiltration of tumor-associated macrophages (TAMs), lower T-cell infiltration, and increased T-cell exhaustion compared to rejected clones. Comparative analysis of rejection-associated gene expression programs, combined with in-vivo CRISPR knockout screens of candidate regulators, identified Mif (macrophage migration inhibitory factor) as a major contributor to immune rejection. Mif knockout resulted in smaller tumors and reduced TAM infiltration. These results were validated in melanoma patient data. Overall, our homogeneous tumor system can uncover factors regulating growth variability and identifies Mif as critical in aggressive melanoma.

Unveiling the hidden link: elevated platelets and T cell subsets in 5% of moderate COVID-19 patients 48 days post-onset.

Platelets are hyperactived during acute COVID-19, promoting clotting and modulating immune-cell responses. Immune thrombocytopenia in adults can manifest as an uncommon complication resulting from various viral infections or as a rare adverse event associated with vaccination. However, their role in convalescent COVID-19 patients remains underexplored. This study examines platelet dynamics early in the pandemic, 48 days post-symptom onset, in unvaccinated patients. This longitudinal study included 298 unvaccinated COVID-19 patients (17 mild, 281 moderate) from multiple centers. Clinical evaluations and peripheral lymphocyte subset analyses via flow cytometry were conducted upon admission and on day 48 post-symptom onset (DPSO 48). At DPSO 48, 5.3% of moderate COVID-19 patients exhibited high platelet counts (>300×109/L), associated with elevated total T-cells (26.4%), CD4 T-cells (24.4%), CD8 T-cells (36.9%), and Tregs (33.9%) compared to patients with normal platelet counts. However, the CD4/CD8 T-cell ratio and T-cell subset frequencies remained unaffected, indicating ongoing T-cell homeostasis restoration. Additionally, a significant positive correlation (r=0.636, p=0.03) was found between platelet counts and B cells in patients with elevated platelet counts. Platelets may play a pivotal role in immune regulation during the recovery phase of COVID-19. Targeting platelets and their secreted mediators could improve immune balance in patients with immune disorders, highlighting a potential therapeutic approach for enhancing recovery in post-COVID-19 patients.