Single cell RNA sequencing analysis of CD8+ T cell subsets following DENV-vaccine administration
ABSTRACT: PBMC-derived CD8 T cell activation and gene expression profiles were assessed 14 days following a single dose of a candidate live-attenuated tetravalent denge virus vaccine (DENVax). Three distinct populations of CD8+ T cells were isolated by flow cytometric sorting: 1) CD3+ CD8+ CD38- HLA-DR- T cells, 2) CD3+ CD8+ CD38+ HLA-DR+ T cells, and 3) CD3+ CD8+ CD71+ HLA-DR+ T cells Overall design: Single cell RNA sequencing analysis of 3 different CD8 T cell populations present in a vaccine recpient 14 days post vaccine administration
Project description:BACKGROUND:HIV controllers (HIC) are rare HIV-1-infected patients who exhibit spontaneous viral control. HIC have high frequency of CD38-/HLA-DR+ HIV-specific CD8+ T cells. Here we examined the role of this subset in HIC status. MATERIALS AND METHODS:We compared CD38-/HLA-DR+ CD8+ T cells with the classical CD38+/HLA-DR+ activated phenotype in terms of 1) their activation status, reflected by CD69, CD25, CD71, CD40 and Ki67 expression, 2) functional parameters: Bcl-2 expression, proliferative capacity, and IFN-? and IL-2 production, and 3) cytotoxic activity. We also investigated how this particular profile is generated. RESULTS:Compared to CD38+/HLA-DR+ cells, CD38-/HLA-DR+ cells exhibited lower expression of several activation markers, better survival capacity (Bcl-2 MFI, 367 [134-462] vs 638 [307-747], P?=?0.001), higher frequency of polyfunctional cells (15% [7%-33%] vs 21% [16%-43%], P?=?0.0003), greater proliferative capacity (0-fold [0-2] vs 3-fold -, P?=?0.007), and higher cytotoxicity in vitro (7% [3%-11%] vs 13% [6%-22%], P?=?0.02). The CD38-/HLA-DR+ profile was preferentially generated in response to low viral antigen concentrations. CONCLUSIONS:These data highlight the role of CD38-/HLA-DR+ HIV-specific CD8+ T cell cytotoxicity in HIC status and provide insights into the mechanism by which they are generated. Induction of this protective CD8+ subset may be important for vaccine strategies.
Project description:Human glioma facilitates an impaired anti-tumor immunity response, including defects in circulation of T lymphocytes. The level of CD8+ T-cell activation acts as an immune regulator associated with disease progression. However, little is known about the characteristics of peripheral and tumor-infiltrating CD8+ T cells in patients with glioma. In this study, we examined the level of CD8+ T-cell activation in a group of 143 patients with glioma and determined that peripheral CD3+ T cells decreased in accordance with disease severity. The patients' peripheral CD8+ T-cell populations were similar to that of healthy donors, and a small amount of CD8+ tumor-infiltrating lymphocytes was identified in glioma tissues. An increase in activated CD8+ T cells, characterized as CD38+HLA-DR+, and their association with disease progression were identified in the patients' peripheral blood and glioma, and shown to display enriched CCR5+ and TNFR2+ expression levels. Ex vivo examination of CD38+HLA-DR+CD8+ T cells indicated that this subset of cells displayed stronger secretion of IFN-? and IL-2 before and after a 6-h stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) relative to healthy CD38+HLA-DR+CD8+ T cells, indicating the functional feasibility of CD38+HLA-DR+CD8+ T cells. Higher CCL5 protein and mRNA levels were identified in glioma tissues, which was consistent with the immunohistochemistry results revealing both CCL5 and CD38+HLA-DR+CD8+ T cell expression. Patients' CCR5+CD38+HLA-DR+CD8+ T cells were further validated and shown to display increases in CD45RA+CCR7- and T-bet+ accompanied by substantial CD107-a, IFN-?, and Granzyme B levels in response to glioma cells.
Project description:An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8+ T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8+ and CD4+ T cells in vitro, with CD4+ T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8+ T cell epitopes, A2/S269-277 and A2/Orf1ab3183-3191 Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/S269 +CD8+ and A2/Orf1ab3183 +CD8+ populations indicated that A2/S269 +CD8+ T cells were detected at comparable frequencies (?1.3 × 10-5) in acute and convalescent HLA-A*02:01+ patients. These frequencies were higher than those found in uninfected HLA-A*02:01+ donors (?2.5 × 10-6), but low when compared to frequencies for influenza-specific (A2/M158) and Epstein-Barr virus (EBV)-specific (A2/BMLF1280) (?1.38 × 10-4) populations. Phenotyping A2/S269 +CD8+ T cells from COVID-19 convalescents ex vivo showed that A2/S269 +CD8+ T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8+ T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/S269 +CD8+ T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8+ T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8+ T cell immunity in COVID-19.
Project description:BACKGROUND: Chronic persistent infections have been associated with T lymphocytes functional impairment. The aim of this study was to compare the activation status, the proliferative potential and the expression of CD28 and CD3? chain on T lymphocytes between chronic chagasic patients and uninfected controls. METHODOLOGY/PRINCIPAL FINDINGS: Forty-two chronic chagasic patients, 28 healthy individuals and 32 non-chagasic cardiomyopathy donors were included. Peripheral blood was marked for CD3, CD4, CD8, HLA-DR, CD28, CD38 and intracellular CD3?. Peripheral blood mononuclear cells were stained with carboxyfluorescein diacetate succinimidylester and incubated with T. cruzi lysate or phytohemagglutinin for five days. Cells from 3 healthy controls were incubated with T. cruzi trypomastigotes separated with transwells; and the expression of CD3? chain and proliferation index was determined. Heart-infiltrating cells from two chronic chagasic patients were tested for the aforementioned cellular markers. Chagasic patients displayed higher frequencies of CD4+/HLA-DR+/CD38+ (8.1% ± 6.1) and CD8+/HLA-DR+/CD38+ (19.8 ± 8.9) T cells in comparison with healthy (1.6 ± 1.0; 10.6 ± 8.0) and non-chagasic cardiomyopathy donors (2.9 ± 2.9; 5.8 ± 6.8). Furthermore, the percentage of CD4+ activated T cells was higher in chagasic patients with cardiac involvement. CD8+ T cells proliferation index in chagasic donors (1.7 ± 0.3) was lower when compared with healthy (2.3 ± 0.3) and non-chagasic cardiomyopathy individuals (3.1 ± 1.1). The frequencies of CD4+/CD28+ and CD8+/CD28+ T cells, as well as the CD3?(bright)/CD3?(dim)% ratios in CD4+ and CD8+ were lower in chagasic patients when compared with both control groups. The CD3?(bright)/CD3?(dim)% ratio and proliferative indexes for CD4+ and CD8+ T lymphocytes decreased gradually in those cells cultivated with parasites and displayed lower values than those incubated with medium alone. Finally, heart-infiltrating T cells from two T. cruzi infected patients also expressed activation markers and down-regulate CD28 and CD3?. CONCLUSIONS: CD8+ T lymphocytes from chagasic donors displayed reduced proliferative capacity, which might be associated with CD3? down-regulation and diminished CD28 expression on CD4 T cells.
Project description:People living with HIV-1 experience an accelerated aging due to the persistent and chronic activation of the immune system. This phenomenon conduces to immune exhaustion and precipitate immunosenescence. In general, frailty is defined as a syndrome of physiological degeneration in the elderly. Circulating naïve and memory T cells were studied by flow cytometry in non-frail and frail HIV-1-infected groups. Thymopoiesis, cell activation, senescence and cell proliferation were analyzed by CD31, HLA-DR/CD38, CD28/CD57 and Ki-67 expression, respectively. Plasma levels of sCD14 and MDA were measured by ELISA. Frail infected individuals showed a reduced number of memory T cells, both CD4+ and CD8+ populations. Activated CD3+CD4+HLA-DR+ T cells were lower in frail individuals, and directly correlated with CD3+CD8+HLA-DR+ and CD8M cells. Senescent CD8+CD28-CD57+ cells were reduced in frail HIV-1 infected individuals and inversely correlated with CD8RTE, CD8N and CD3+CD4+HLA-DR+. Higher plasma levels of sCD14 and MDA were found in HIV-1 infected frail individuals. Our data show association among frailty, markers of immune activation and oxidative stress. Understanding the immune mechanisms underlying frailty status in HIV-1 population is of high relevance not only for the prediction of continuing longevity but also for the identification of potential strategies for the elderly.
Project description:BACKGROUND:Vulvar squamous cell carcinoma (VSCC) has been suggested to consist of three subtypes; HPV-positive, HPV-negative mutated TP53 or HPV-negative TP53 wildtype, with different clinical courses. To analyze the immune infiltrate in these molecular subtypes and its impact on clinical outcome, an in-depth study of the tumor immune microenvironment was performed. METHODS:Sixty-five patients with invasive VSCC matched for age, FIGO stage and treatment modality, were grouped according to the presence of HPV and p53 protein expression status. Archived tissues were analyzed for intraepithelial and stromal expression of CD3, CD8, Foxp3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC (n =?14) and blood samples by flow cytometry. Healthy vulvar samples and blood served as controls. RESULTS:Based on T-cell infiltration patterns about half of the VSCC were classified as inflamed or altered-excluded while one-third was immune-deserted. High intraepithelial helper T cell infiltration was observed in 78% of the HPV-induced VSCC, 60% of the HPVnegVSCC/p53wildtype and 40% of the HPVnegVSCC with abnormal p53 expression. A high intraepithelial infiltration with activated (CD3+PD-1+), specifically helper T cells (CD3+CD8-Foxp3-), was associated with a longer recurrence-free period and overall survival, irrespective of HPV and p53 status. Flow cytometry confirmed the tumor-specific presence of activated (CD4+PD-1++CD161-CD38+HLA-DR+ and CD8+CD103+CD161-NKG2A+/-PD1++CD38++HLA-DR+) effector memory T cells. CONCLUSION:This is the first study demonstrating an association between intraepithelial T cells and clinical outcome in VSCC. Our data suggest that abnormal p53 expressing VSCCs mostly are cold tumors whereas HPV-driven VSCCs are strongly T-cell infiltrated.
Project description:Severe influenza A virus (IAV) infection is associated with immune dysfunction. Here, we show circulating CD8+ T-cell profiles from patients hospitalized with avian H7N9, seasonal IAV, and influenza vaccinees. Patient survival reflects an early, transient prevalence of highly activated CD38+HLA-DR+PD-1+ CD8+ T cells, whereas the prolonged persistence of this set is found in ultimately fatal cases. Single-cell T cell receptor (TCR)-?? analyses of activated CD38+HLA-DR+CD8+ T cells show similar TCR?? diversity but differential clonal expansion kinetics in surviving and fatal H7N9 patients. Delayed clonal expansion associated with an early dichotomy at a transcriptome level (as detected by single-cell RNAseq) is found in CD38+HLA-DR+CD8+ T cells from patients who succumbed to the disease, suggesting a divergent differentiation pathway of CD38+HLA-DR+CD8+ T cells from the outset during fatal disease. Our study proposes that effective expansion of cross-reactive influenza-specific TCR?? clonotypes with appropriate transcriptome signatures is needed for early protection against severe influenza disease.
Project description:Infection with co-pathogens is one of the postulated factors contributing to persistent inflammation and non-AIDS events in virologically-suppressed HIV-infected patients. We aimed to investigate the relationship of human herpesvirus-8 (HHV-8), a vasculotropic virus implicated in the pathogenesis of Kaposi's sarcoma, with inflammation and subclinical atherosclerosis in HIV-infected patients.Prospective study including virologically suppressed HIV-infected patients. Several blood biomarkers (highly-sensitive C-reactive protein [hsCRP], tumour necrosis factor-α, interleukin-6, monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, intercellular cell adhesion molecule-1, malondialdehyde, plasminogen activator inhibitor [PAI-1], D-dimer, sCD14, sCD163, CD4/CD38/HLA-DR, and CD8/CD38/HLA-DR), serological tests for HHV-8 and the majority of herpesviruses, carotid intima-media thickness, and endothelial function through flow-mediated dilatation of the brachial artery were measured.A total of 136 patients were included, 34.6% of them infected with HHV-8. HHV-8-infected patients were more frequently co-infected with herpes simplex virus type 2 (HSV-2) (P<0.001), and less frequently with hepatitis C virus (HCV) (P = 0.045), and tended to be older (P = 0.086). HHV-8-infected patients had higher levels of hsCRP (median [interquartile range], 3.63 [1.32-7.54] vs. 2.08 [0.89-4.11] mg/L, P = 0.009), CD4/CD38/HLA-DR (7.67% [4.10-11.86]% vs. 3.86% [2.51-7.42]%, P = 0.035) and CD8/CD38/HLA-DR (8.02% [4.98-14.09]% vs. 5.02% [3.66-6.96]%, P = 0.018). After adjustment for the traditional cardiovascular risk factors, HCV and HSV-2 infection, the associations remained significant: adjusted difference between HHV-8 positive and negative patients (95% confidence interval) for hsCRP, 74.19% (16.65-160.13)%; for CD4/CD38/HLA-DR, 89.65% (14.34-214.87)%; and for CD8/CD38/HLA-DR, 58.41% (12.30-123.22)%. Flow-mediated dilatation and total carotid intima-media thickness were not different according to HHV-8 serostatus.In virologically suppressed HIV-infected patients, coinfection with HHV-8 is associated with increased inflammation and immune activation. This might contribute to increase the risk of non-AIDS events, including accelerated atherosclerotic disease.
Project description:Biological aging is associated with immune activation (IA) and declining immunity due to systemic inflammation. It is widely accepted that HIV infection causes persistent IA and premature immune senescence despite effective antiretroviral therapy and virologic suppression; however, the effects of combined HIV infection and aging are not well defined. Here, we assessed the relationship between markers of IA and inflammation during biological aging in HIV-infected and -uninfected populations. Antibody response to seasonal influenza vaccination was implemented as a measure of immune competence and relationships between IA, inflammation, and antibody responses were explored using statistical modeling appropriate for integrating high-dimensional data sets. Our results show that markers of IA, such as coexpression of HLA antigen D related (HLA-DR) and CD38 on CD4+ T cells, exhibit strong associations with HIV infection but not with biological age. Certain variables that showed a strong relationship with aging, such as declining naive and CD38+ CD4 and CD8+ T cells, did so regardless of HIV infection. Interestingly, the variable of biological age was not identified in a predictive model as significantly impacting vaccine responses in either group, while distinct IA and inflammatory variables were closely associated with vaccine response in HIV-infected and -uninfected populations. These findings shed light on the most relevant and persistent immune defects during virological suppression with antiretroviral therapy.
Project description:Epidemiological studies suggest that India has the largest number of dengue virus infection cases worldwide. However, there is minimal information about the immunological responses in these patients. CD8 T cells are important in dengue, because they have been implicated in both protection and immunopathology. Here, we provide a detailed analysis of HLA-DR+ CD38+ and HLA-DR- CD38+ effector CD8 T cell subsets in dengue patients from India and Thailand. Both CD8 T cell subsets expanded and expressed markers indicative of antigen-driven proliferation, tissue homing, and cytotoxic effector functions, with the HLA-DR+ CD38+ subset being the most striking in these effector qualities. The breadth of the dengue-specific CD8 T cell response was diverse, with NS3-specific cells being the most dominant. Interestingly, only a small fraction of these activated effector CD8 T cells produced gamma interferon (IFN-?) when stimulated with dengue virus peptide pools. Transcriptomics revealed downregulation of key molecules involved in T cell receptor (TCR) signaling. Consistent with this, the majority of these CD8 T cells remained IFN-? unresponsive even after TCR-dependent polyclonal stimulation (anti-CD3 plus anti-CD28) but produced IFN-? by TCR-independent polyclonal stimulation (phorbol 12-myristate 13-acetate [PMA] plus ionomycin). Thus, the vast majority of these proliferating, highly differentiated effector CD8 T cells probably acquire TCR refractoriness at the time the patient is experiencing febrile illness that leads to IFN-? unresponsiveness. Our studies open novel avenues for understanding the mechanisms that fine-tune the balance between CD8 T cell-mediated protective versus pathological effects in dengue.Dengue is becoming a global public health concern. Although CD8 T cells have been implicated both in protection and in the cytokine-mediated immunopathology of dengue, how the balance is maintained between these opposing functions remains unknown. We comprehensively characterized CD8 T cell subsets in dengue patients from India and Thailand and show that these cells expand massively and express phenotypes indicative of overwhelming antigenic stimulus and tissue homing/cytotoxic-effector functions but that a vast majority of them fail to produce IFN-? in vitro Interestingly, the cells were fully capable of producing the cytokine when stimulated in a T cell receptor (TCR)-independent manner but failed to do so in TCR-dependent stimulation. These results, together with transcriptomics, revealed that the vast majority of these CD8 T cells from dengue patients become cytokine unresponsive due to TCR signaling insufficiencies. These observations open novel avenues for understanding the mechanisms that fine-tune the balance between CD8-mediated protective versus pathological effects.