Year: 2008
Type: dissertation
Study design: Observational Study
Abstract: ABSTRACT \nCases of HIV-1 infection are growing in large numbers, and deaths as a result of AIDS \nare escalating in South Africa. Understanding cellular immune responses to HIV-1 by \nexploring general effects and changes occurring at a cellular level, including direct \nengagement of T cells with the virus during exposure or infection will provide \ninformation on possible correlates of viral control. This dissertation focuses on three \ncharacteristics of T cells during HIV infection, dual HIV/TB co-infection and exposure to \nHIV. The characteristics examined are 1) memory and activation status of CD4+ and \nCD8+ T cell subsets; 2) T cell receptor repertoire and 3) HIV-1– specific T cell \nresponses. \nThere are two hypotheses in this dissertation. Firstly, that co-infection with TB leads to \nelevated T cell activation, disruption of the T cell receptor repertoire and altered \npatterns of immunodominance in HIV-1 subtype C-specific T cell responses in infected \nadults. Secondly, that T cell priming occurs in utero in HIV uninfected babies born to \nHIV infected mothers. \nFour cohorts were examined in this dissertation. Three were recruited from a clinic \naround the Welkom area and analysed in a cross-sectional and longitudinal manner. The \ncohorts consisted of HIV-1 infected adults, individuals dually infected with HIV and \nTB and healthy controls. Whole blood samples from the HIV and HIV/TB infected \ngroups were analysed at baseline (before TB chemotherapy), at 2, 8 and 24 weeks. A further cohort consisted of babies born to HIV-1 infected mothers, with some being \nfollowed up at three months after birth. \nThis dissertation consisted of five different methods: 1) the use of four colour flow \ncytometry to measure the frequency of naïve T cells (CD45RA+/CD62L+), memory \n(CD45RA-CD62L-) and activated (CD38) T cell populations in individuals singly \ninfected with TB and dually with HIV and TB. This investigation was aimed at \nobtaining the overall representation of T cells involved in HIV-1 and TB co-infection. \n2) the use of flow cytometry staining with monoclonal antibodies recognizing different \nT cell receptor (TCR) Vβ specificities for quantitation of the percentage of particular \nTCR families in pools of T cells. The aim was to provide an indication of TCR usage in different disease states. 3) the immunoscope assay was used to measure the different \nCDR3 lengths of the TCR and assess Vβ family repertoires in newborn babies. The aim \nwas to show evidence of T cell maturity at birth and whether there was TCR \nengagement in utero by analysing cord blood cells. 4) the use of the IFN-γ ELISPOT to \nmeasure HIV-specific T cell responses in a cohort of HIV and HIV/TB co-infected \nindividuals. The aim was to identify targeted immunodominant regions and to \ndetermine whether TB infection resulted in differing patterns of HIV-1 specific T cell \nimmunity. 5) intracellular cytokine staining (ICS) was used to confirm the responses \nobtained after initial screening with the IFN-γ ELISPOT and was used to delineate CD4+ and CD8+ T cell responsiveness.Whole blood was stained with an array of monoclonal antibodies to measure various T \ncell subsets in HIV-1 and HIV/TB co-infected adults. The CD4+ T cells in HIV-1 \ninfected individuals ranged between 245 - 436, those of HIV/TB co-infected patients \nwere 157-840 and the TB group had CD4+ absolute counts ranging between 583-1757 \ncells/mm3. CD4+ T cells were reduced as a result of HIV-1 infection and HIV/TB coinfection, \nand no loss of these cells was seen as a result of single infection with TB. \nThere was a loss of naïve T cells, with increased memory phenotypes in the presence of \nTB and HIV single infection, which was more pronounced in the presence of HIV and \nTB co-infection. The loss of naïve CD4+ and CD8+ T cells was associated with a high \nHIV-1 plasma RNA load in patients co-infected with HIV and TB. CD8+ T cells in HIV \nsingly infected and HIV/TB co-infected individuals were highly activated when \ncompared to those infected with TB only, which was likely due to the high HIV plasma \nRNA load. The standard course of six months of TB therapy in HIV/TB co-infected \nadults did not lead to recovery of absolute CD4 cells, nor did it stem the loss of naïve \nCD4+ and CD8+ T cells, which remained in a highly activated state: possibly due to \nunchanged HIV-1 RNA loads. \nThe fine specific nature of T cell activation was investigated by examining TCR Vβ \nexpansions in HIV and TB single and co-infected individuals. Whole blood was stained \nwith CD3+, CD4+, CD8+, CD38+ and an array of Vβ-specific antibodies. Polyclonal \nskewing of the TCR Vβ repertoire, showing expansion of various Vβ−CD4+ and -CD8+ \nfamilies was observed in TB and HIV-1 single and dual infection. A more restricted \nusage of the T cell repertoire was observed in both HIV and HIV/TB co-infected patients, \nwhere major and oligoclonal expansions of Vβ11, Vβ16, Vβ20 and Vβ22 were observed. No major expansions were observed in TB single infection. Overall, significantly greater \nuse of TCR Vβ families were found in the CD8+ T cell compartment rather than by \nCD4+ T cells in both HIV-1 and HIV/TB co-infected adults. \nA cohort of neonates born to HIV-infected mothers was used to assess TCR usage using \nimmunoscope analysis to support the hypothesis that the TCR repertoire skewing in cord \nblood cells can be a marker of T cell priming in-utero. The repertoire measured in HIV \nuninfected neonates born to HIV-1 infected mothers displayed a polyclonal skewing of \nvarious TCR families and oligoclonal distribution of Vβ5, Vβ6a, Vβ7, Vβ18, and Vβ23 \nfamilies as compared to the Gaussian distributions seen in healthy controls. This study \nreadily detected perturbations in the TCR repertoire in presumed HIV exposed babies and \nthat newborns possess an intact TCR repertoire. \nMeasurement of HIV-1-specific CD8+ T cells was made to identify which regions of the \nexpressed HIV genome were immunodominant and what impact of co-infection with TB \nmay have. PBMC samples were thawed and cultured in vitro using CD3+/CD4+ bispecific \nantibody to preferentially expand CD8+ T cells and measure IFN-γ producing \ncells in the ELISPOT and confirmed with the ICS. The ELISPOT results were interpreted \nin SFU/million PBMC and as percentages of T cell subsets in ICS assays. HIV-1 subtype \nC-specific CD8+ responses were readily detected in both HIV-1 and HIV/TB co-infected \npatients, however, patterns of peptide targeting were different between the two groups. \nGag was targeted by 85% of HIV-1 infected patients, whereas only 27% of HIV/TB coinfected \npatients targeted Gag. Pol was targeted by 73% in the HIV/TB group. Gag and Nef responses observed in some (n =7) of the patients were confirmed using ICS. These \ndata infer that TB co-infection may change patterns of targeting and in how CD8+ T cells \nrecognize HIV antigens. \nCollectively, this dissertation demonstrated the existence of highly activated CD8+ T \ncells, most probably driven by high HIV-1 plasma RNA loads; restricted TCR usage by \nCD8+ T cells, predominantly in individuals dually infected with HIV and TB; possible \nshifting of immunodominant HIV-specific CD8+ T cell responses as a result of coinfection \nwith TB. Despite successful treatment of TB with chemotherapy, these \nimmunological observations remained unchanged.
Author: Nati Stephina Nyoka
Language: English
Topic: HIV Research and Treatment
Subfield: Virology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
Funder: Fogarty International Center
OpenAlex ID: W2143022612