Timing of CD8+ T cell responses in relation to commencement of capillary leakage in children with dengue.
ABSTRACT: Immune activation is a feature of dengue hemorrhagic fever (DHF) and CD8+ T cell responses in particular have been suggested as having a role in the vasculopathy that characterizes this disease. By phenotyping CD8+ T cells (CD38+/HLA-DR+, CD38+/Ki-67+, or HLA-DR+/Ki-67+) in serial blood samples from children with dengue, we found no evidence of increased CD8+ T cell activation prior to the commencement of resolution of viremia or hemoconcentration. Investigations with MHC class I tetramers to detect NS3(133-142)-specific CD8+ T cells in two independent cohorts of children suggested the commencement of hemoconcentration and thrombocytopenia in DHF patients generally begins before the appearance of measurable frequencies of NS3(133-142)-specific CD8+ T cells. The temporal mismatch between the appearance of measurable surface activated or NS3(133-142)-specific CD8+ T cells suggests that these cells are sequestered at sites of infection, have phenotypes not detected by our approach, or that other mechanisms independent of CD8+ T cells are responsible for early triggering of capillary leakage in children with DHF.
Project description:INTRODUCTION:Although the role of dengue virus (DENV)-specific T cells in the pathogenesis of acute dengue infection is emerging, the functionality of virus-specific T cells associated with milder clinical disease has not been well studied. We sought to investigate how the functionality of DENV-NS3 and DENV-NS5 protein-specific T cells differ in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). METHODS:Using intracellular cytokine assays, we assessed the production of interferon ? (IFN?), tumor necrosis factor-? (TNF-?), macrophage inflammatory protein-1? (MIP-1?), and CD107a expression in adult patients with acute DF (n?=?21) and DHF (n?=?22). RESULTS:Quadruple cytokine-producing, polyfunctional DENV-NS3- and DENV-NS5-specific T cells were more frequent in those with DF when compared to those with DHF. While DENV-NS3- and DENV-NS5-specific T cells in patients with DF expressed IFN??>?TNF-??>?MIP-??>?CD107a, T cells of those with DHF predominantly expressed CD107a?>?MIP-1??>?IFN??>?TNF-?. Overall production of IFN? or TNF-? by DENV-NS3- and DENV-NS5-specific T cells was significantly higher in patients with DF. The majority of NS3-specific T cells in patients with DF (78.6%) and DHF (68.9%) were single-cytokine producers; 76.6% of DENV-NS5-specific T cells in those with DF and 77.1% of those with DHF, produced only a single cytokine. However, no significant association was found with polyfunctional T-cell responses and the degree of viraemia. CONCLUSIONS:Our results suggest that the functional phenotype of DENV-specific T cells are likely to associate with clinical disease severity.
Project description:Serotype-cross-reactive memory T cells responding to secondary dengue virus (DENV) infection are thought to contribute to disease. However, epitope-specific T cell responses have not been thoroughly compared between subjects with primary versus secondary DENV infection. We studied CD8(+) T cells specific for the HLA-A*1101-restricted NS3(133) epitope in a cohort of A11(+) DENV-infected patients throughout acute illness and convalescence. We compared the expansion, serotype-cross-reactivity, and activation of these cells in PBMC from patients experiencing primary or secondary infection and mild or severe disease by flow cytometry. Our results show expansion and activation of DENV-specific CD8(+) T cells during acute infection, which are predominantly serotype-cross-reactive regardless of DENV infection history. These data confirm marked T cell activation and serotype-cross-reactivity during the febrile phase of dengue; however, A11-NS3(133)-specific responses did not correlate with prior antigenic exposure or current disease severity.
Project description:Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3(422-431) were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3(422-431) and NS5(563-571) in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3(422-431). A large number of patients' T cells also responded to the NS2B(97-106) region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.
Project description:Both HIV and TB cause a state of heightened immune activation. Immune activation in HIV is associated with progression to AIDS. Prior studies, focusing on persons with advanced HIV, have shown no decline in markers of cellular activation in response to TB therapy alone.This prospective cohort study, composed of participants within a larger phase 3 open-label randomized controlled clinical trial, measured the impact of TB treatment on immune activation in persons with non-advanced HIV infection (CD4>350 cells/mm3) and pulmonary TB. HIV load, CD4 count, and markers of immune activation (CD38 and HLA-DR on CD4 and CD8 T cells) were measured prior to starting, during, and for 6 months after completion of standard 6 month anti-tuberculosis (TB) therapy in 38 HIV infected Ugandans with smear and culture confirmed pulmonary TB.Expression of CD38, and co-expression of CD38 and HLA-DR, on CD8 cells declined significantly within 3 months of starting standard TB therapy in the absence of anti-retroviral therapy, and remained suppressed for 6 months after completion of therapy. In contrast, HIV load and CD4 count remained unchanged throughout the study period.TB therapy leads to measurable decreases in immune activation in persons with HIV/TB co-infection and CD4 counts>350 cells/mm3.
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.
Project description:What governs the increased apoptosis sensitivity of HIV-specific CD8(+) T cells is poorly understood. Here, we examined the involvement of mitochondria in this apoptosis. Remarkably higher mitochondrial mass (MM) was found in HIV-specific compared with CMV-specific CD8(+) T cells from HIV(+) patients and this could not be attributed to their different differentiation status. MM(High) phenotype characterized those CD8(+) T cells from HIV(+) patients that are sensitive to spontaneous and CD95/Fas-induced apoptosis. CD38 expression did not correlate with high MM, whereas Bcl-2 levels were significantly reduced in both CD38(+) and CD38(-) HIV-specific CD8(+) T cells. Although CD38(+) HIV-specific CD8(+) T cells were more susceptible to apoptosis, CD38 expression does not explain on its own the selective apoptosis sensitivity of HIV-specific CD8(+) T cells, as CD38(-) HIV-specific CD8(+) T cells were more apoptotic than CD38(+) CMV-specific ones. Proapoptotic HIV-specific CD8(+) T cells were CD38(+)Bcl-2(Low)MM(High). Copolarization of mitochondria with CD95/Fas capping, very early in CD95/Fas-induced apoptosis of HIV-specific CD8(+) T cells, suggests that mitochondria act as an amplification step for this apoptosis. Thus, an extensive mitochondrial network contributes to apoptosis sensitivity of CD8(+) T cells and, when this occurs together with reduced levels of Bcl-2 and chronic activation, determines the proapoptotic state of HIV-specific CD8(+) T cells.
Project description:Regulatory CD8(+) T cells are critical for self-tolerance and restricting excessive immune responses. The variety of immune functions they fulfill, the heterogeneity of their phenotype, and the mechanism of action are still poorly understood. Here we describe that regulatory CD8(+) T cells exhibiting immunosuppressive actions in vitro and in vivo are recognized as CD38(high) T cells and present in naive mice. CD38 is a glycosylated membrane protein with ectonucleotidase properties. CD8(+)CD38(high) (CD44(+)CD122(+)CD62L(high)) lymphocytes suppress CD4(+) effector T-cell proliferation in an antigen-non specific manner via IFN-?. While direct cell-to-cell contact is needed for this suppressor activity, it is independent of membrane-bound TGF-? and granzyme B release. IL-15 potentiates the suppressive activity of CD8(+)CD38(high) T cells and controls their survival and expansion. In humans CD8(+)CD38(high) T cells inhibit CD4(+) effector T cell proliferation. In vivo, CD8(+)CD38(high), but not CD8(+)CD38(-) T cells mitigate murine experimental autoimmune encephalomyelitis (EAE) by reducing the clinical score and delaying disease occurrence. EAE suppression is enhanced by pre-treatment of CD8(+)CD38(high) T cells with IL-15. These findings add evidence that the expression of ectoenzyme receptor family members positively correlates with suppressor functions and identifies CD8(+)CD38(high) T cells as potential inhibitors of excessive immune responses.
Project description:Determinants of HIV-infected women's genital tract mucosal immune health are not well understood. Because raltegravir (RAL) achieves relatively higher genital tract concentrations than ritonavir-boosted atazanavir (ATV), we examined whether an RAL-based regimen is associated with improved cervical immune reconstitution and less activation in HIV(+) women compared to an ATV-based regimen. Peripheral blood, cervical brushings, cervical-vaginal lavage (CVL), and cervical biopsies were collected from HIV(+) women on tenofovir disoproxil fumarate and emtricitabine (TDF/FTC) and either RAL (n=14) or ATV (n=19) with CD4(+) T cells>300 cells/mm(3) and HIV RNA<48 copies/ml. HLA-DR(+)CD38(+) T cells were measured in blood and cervical cells using flow cytometry, CD4(+) and CD8(+) T cells were quantified in cervical biopsies by immunofluorescent analysis, and HIV RNA (VL), ATV, and RAL concentrations were measured in CVL. In a linear regression model of log(CVL concentration) versus both log(plasma concentration) and treatment group, the RAL CVL level was 519% (95% CI: 133, 1,525%) higher than for ATV (p<0.001). Genital tract VL was undetectable in 90% of subjects and did not differ by regimen. There were no significant differences between groups in terms of cervical %HLA-DR(+)CD38(+)CD4(+) or CD8(+) T cells, CD4(+) or CD8(+) T cells/mm(2), or CD4:CD8 ratio. After adjusting for treatment time and group, the CVL:plasma drug ratio was not associated with the cervical CD4:CD8 ratio or immune activation (p>0.6). Despite significantly higher genital tract penetration of RAL compared to ATV, there were no significant differences in cervical immune activation or reconstitution between women on these regimens, suggesting both drug regimens achieve adequate genital tract levels to suppress virus replication.
Project description:Dengue infections are increasing at an alarming rate in many tropical and subtropical countries, where epidemics can put health care systems under extreme pressure. The more severe infections lead to dengue hemorrhagic fever (DHF), which can be life threatening. A variety of viral and host factors have been associated with the severity of dengue infections. Because secondary dengue infection is more commonly associated with DHF than primary infections, the acquired immune response to dengue, both B cells and T cells have been implicated. In this study, we set out to study T-cell responses across the entire dengue virus proteome and to see whether these were related to disease severity in a cohort of dengue-infected children from Thailand. Robust responses were observed in most infected individuals against most viral proteins. Responses to NS3 were the most frequent, and there was a very strong association between the magnitude of the response and disease severity. Furthermore, in DHF, cytokine-high CD107a-negative cells predominated.
Project description:Previous studies using mouse liver tumor models have indicated that coexpression of CD38 and CD101 in programmed cell death-1 (PD-1)+CD8+ T cells may reflect fixed dysregulation of CD8+ T cells and thus indicate a poor response to anti-PD-1 immunotherapy. However, whether CD38 and CD101 expression in PD-1+CD8+ T cells can predict the clinical status and efficacy of chemotherapy for various cancer types, including ovarian cancer (OC), remains unclear. In the present study, peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll-Hypaque gradient centrifugation from 96 fresh samples from healthy adult volunteers and patients with epithelial OC, aged 55.21±9.91 years. Additionally, tumor-infiltrating lymphocytes (TILs) were separated using a combination of mechanical, chemical and enzymatic digestion from fresh surgically removed tumor tissues from 15 patients with epithelial OC. The expression of CD38 and CD101 in PD-1+CD8+ T cells or TILs was detected by flow cytometry or immunofluorescence (IF) staining, respectively. The association between the level of CD38/CD101 expression and clinicopathological parameters or postoperative chemotherapy in patients with OC was statistically analyzed. The levels of CD38/CD101-coexpressing PD-1+CD8+ T cells were significantly increased in PBMCs and TILs of patients with OC compared with those of healthy volunteers. The frequency of PD-1+CD38+CD101+CD8+ T cells among the total PD-1+CD8+ T cell subpopulation was negatively associated with clinical stage, lymph node metastasis and postoperative chemotherapy prognosis in patients with OC. Furthermore, IF staining confirmed colocalization of CD38 and CD101 on the majority of TILs in OC tissues. Thus, the present study suggests that coexpression of CD38 and CD101 in peripheral PD-1+CD8+ T cells and TILs may serve as a new indicator for diagnosis and treatment efficacy in patients with epithelial OC.