The characteristics and functional nature of T cells upon HIV-1 infection and exposure
Nyoka, Nati Stephina
ABSTRACT Cases of HIV-1 infection are growing in large numbers, and deaths as a result of AIDS are escalating in South Africa. Understanding cellular immune responses to HIV-1 by exploring general effects and changes occurring at a cellular level, including direct engagement of T cells with the virus during exposure or infection will provide information on possible correlates of viral control. This dissertation focuses on three characteristics of T cells during HIV infection, dual HIV/TB co-infection and exposure to HIV. The characteristics examined are 1) memory and activation status of CD4+ and CD8+ T cell subsets; 2) T cell receptor repertoire and 3) HIV-1– specific T cell responses. There are two hypotheses in this dissertation. Firstly, that co-infection with TB leads to elevated T cell activation, disruption of the T cell receptor repertoire and altered patterns of immunodominance in HIV-1 subtype C-specific T cell responses in infected adults. Secondly, that T cell priming occurs in utero in HIV uninfected babies born to HIV infected mothers. Four cohorts were examined in this dissertation. Three were recruited from a clinic around the Welkom area and analysed in a cross-sectional and longitudinal manner. The cohorts consisted of HIV-1 infected adults, individuals dually infected with HIV and TB and healthy controls. Whole blood samples from the HIV and HIV/TB infected groups 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 followed up at three months after birth. This dissertation consisted of five different methods: 1) the use of four colour flow cytometry to measure the frequency of naïve T cells (CD45RA+/CD62L+), memory (CD45RA-CD62L-) and activated (CD38) T cell populations in individuals singly infected with TB and dually with HIV and TB. This investigation was aimed at obtaining the overall representation of T cells involved in HIV-1 and TB co-infection. 2) the use of flow cytometry staining with monoclonal antibodies recognizing different T cell receptor (TCR) Vβ specificities for quantitation of the percentage of particular TCR 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 CDR3 lengths of the TCR and assess Vβ family repertoires in newborn babies. The aim was to show evidence of T cell maturity at birth and whether there was TCR engagement in utero by analysing cord blood cells. 4) the use of the IFN-γ ELISPOT to measure HIV-specific T cell responses in a cohort of HIV and HIV/TB co-infected individuals. The aim was to identify targeted immunodominant regions and to determine whether TB infection resulted in differing patterns of HIV-1 specific T cell immunity. 5) intracellular cytokine staining (ICS) was used to confirm the responses obtained 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 cell subsets in HIV-1 and HIV/TB co-infected adults. The CD4+ T cells in HIV-1 infected individuals ranged between 245 - 436, those of HIV/TB co-infected patients were 157-840 and the TB group had CD4+ absolute counts ranging between 583-1757 cells/mm3. CD4+ T cells were reduced as a result of HIV-1 infection and HIV/TB coinfection, and no loss of these cells was seen as a result of single infection with TB. There was a loss of naïve T cells, with increased memory phenotypes in the presence of TB and HIV single infection, which was more pronounced in the presence of HIV and TB co-infection. The loss of naïve CD4+ and CD8+ T cells was associated with a high HIV-1 plasma RNA load in patients co-infected with HIV and TB. CD8+ T cells in HIV singly infected and HIV/TB co-infected individuals were highly activated when compared to those infected with TB only, which was likely due to the high HIV plasma RNA load. The standard course of six months of TB therapy in HIV/TB co-infected adults did not lead to recovery of absolute CD4 cells, nor did it stem the loss of naïve CD4+ and CD8+ T cells, which remained in a highly activated state: possibly due to unchanged HIV-1 RNA loads. The fine specific nature of T cell activation was investigated by examining TCR Vβ expansions in HIV and TB single and co-infected individuals. Whole blood was stained with CD3+, CD4+, CD8+, CD38+ and an array of Vβ-specific antibodies. Polyclonal skewing of the TCR Vβ repertoire, showing expansion of various Vβ−CD4+ and -CD8+ families was observed in TB and HIV-1 single and dual infection. A more restricted usage of the T cell repertoire was observed in both HIV and HIV/TB co-infected patients, where major and oligoclonal expansions of Vβ11, Vβ16, Vβ20 and Vβ22 were observed. No major expansions were observed in TB single infection. Overall, significantly greater use of TCR Vβ families were found in the CD8+ T cell compartment rather than by CD4+ T cells in both HIV-1 and HIV/TB co-infected adults. A cohort of neonates born to HIV-infected mothers was used to assess TCR usage using immunoscope analysis to support the hypothesis that the TCR repertoire skewing in cord blood cells can be a marker of T cell priming in-utero. The repertoire measured in HIV uninfected neonates born to HIV-1 infected mothers displayed a polyclonal skewing of various TCR families and oligoclonal distribution of Vβ5, Vβ6a, Vβ7, Vβ18, and Vβ23 families as compared to the Gaussian distributions seen in healthy controls. This study readily detected perturbations in the TCR repertoire in presumed HIV exposed babies and that newborns possess an intact TCR repertoire. Measurement of HIV-1-specific CD8+ T cells was made to identify which regions of the expressed HIV genome were immunodominant and what impact of co-infection with TB may have. PBMC samples were thawed and cultured in vitro using CD3+/CD4+ bispecific antibody to preferentially expand CD8+ T cells and measure IFN-γ producing cells in the ELISPOT and confirmed with the ICS. The ELISPOT results were interpreted in SFU/million PBMC and as percentages of T cell subsets in ICS assays. HIV-1 subtype C-specific CD8+ responses were readily detected in both HIV-1 and HIV/TB co-infected patients, however, patterns of peptide targeting were different between the two groups. Gag was targeted by 85% of HIV-1 infected patients, whereas only 27% of HIV/TB coinfected patients 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 data infer that TB co-infection may change patterns of targeting and in how CD8+ T cells recognize HIV antigens. Collectively, this dissertation demonstrated the existence of highly activated CD8+ T cells, most probably driven by high HIV-1 plasma RNA loads; restricted TCR usage by CD8+ T cells, predominantly in individuals dually infected with HIV and TB; possible shifting of immunodominant HIV-specific CD8+ T cell responses as a result of coinfection with TB. Despite successful treatment of TB with chemotherapy, these immunological observations remained unchanged.
T cells, HIV-1infections