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|Title:||Cellular immune responses of dengue patients in Singapore||Authors:||BELA-ONG DENNIS||Keywords:||dengue virus, immune responses, NK cell, T cell, dengue patients, Singapore||Issue Date:||15-Jan-2009||Citation:||BELA-ONG DENNIS (2009-01-15). Cellular immune responses of dengue patients in Singapore. ScholarBank@NUS Repository.||Abstract:||Dengue virus (DENV) infection is a major cause of morbidity and mortality in tropical and subtropical areas worldwide. Pathogenesis of dengue is not completely understood. It has been reported that the magnitude of immune responses correlate with severe dengue disease. We therefore characterized DENV-specific T, NK, and CD56+CD3+ T cells in patients with acute dengue infection to find potential targets for intervention and prognostic markers.
As part of the on-going Early DENgue Infection and Outcomes (EDEN) Study in Singapore, fresh whole blood samples were analyzed from patients longitudinally from fever onset to convalescence. We found that dengue patients exhibited significantly lower absolute counts of T, NK, and CD56+ T cells early after infection and significantly higher levels of T cell, NK cell, and CD56+ T cell activation compared to febrile non-dengue controls in the acute phase, which reflects an efficient activation of the immune response early after infection. The percentage of total activated cells did not significantly differ between dengue patients with primary and secondary infection, indicating that naive T cells are as efficiently activated as memory T cells. We also show changes in the T cell compartments of dengue patients potentially leading to the establishment of high numbers of memory cells. These changes did not significantly differ between primary and secondary dengue infection. Activation of naive T cells and reactivation of memory T cells may be protective or disease-enhancing. However, since all our cases were mild, we cannot address the question whether re-activation of T cell memory is potentially immunopathogenic, as suggested by others.
Next, we established methods to detect dengue-specific T cells. PBMCs from dengue patients and healthy DENV-immune individuals were investigated by IFN-gamma T cell ELISPOT and flow cytometry for their DENV-specific responses to peptides spanning conserved regions of the immunodominant NS3 epitope of DENV serotype 2 majority consensus sequence. The specificity of the T cell responses was donor-dependent. IFN-gamma responses of cultured PBMCs (in the absence of irradiated autologous PBMCs) from DENV-immune individuals after restimulation with peptide pools were analyzed by intracellular IFN-gamma staining and flow cytometry. CD4+ and CD8+ T cell responses to peptide pools were observed at 7 and 23 culture days for one out of the three dengue-seropositive donors. ELISPOT analysis of cells from the same donor at day 23 supported intracellular IFN-gamma staining and flow cytometry data, where the NS3 peptide MCHATFTMRLLSPVR(261-275) was found to evoke the strongest response. The HLA restriction for this peptide in the Singaporean population could be further characterized and responses to this epitope could be analyzed to determine the immunodominance of this epitope. As some HLA alleles have been linked to susceptibility to DHF and peptide epitopes have been associated with severe disease, further studies are warranted to increase our understanding of the pathology of severe dengue disease.
Finally, we established T cell clones by antigen re-stimulation ex vivo to be able to characterize DENV-specific T cells. We succeeded in establishing T cell clones from a bulk culture of DENV-stimulated PBMCs from a healthy dengue-immune individual. However, the predominantly CD4+ T cell clones had few IFN-gamma-producing cells and did not expand to numbers sufficient for T cell functional analyses. Our protocol could be further improved and refined to expand CD8+ T cell clones and to make clones expand to numbers sufficient for functional characterization of DENV-specific T cells in order to completely understand the protective or immunopathologic role of T cells and to identify reliable prognostic markers for dengue.
|Appears in Collections:||Master's Theses (Open)|
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