Submitted in fulfillment of the academic requirements for the degree of Doctor of Philosophy in the Discipline of Immunology, School of. The experimental work described in this dissertation was performed at the HIV Pathogenesis Program (HassoPlattner Research Laboratory), Doris Duke Institute for Medical Research, Nelson R. This dissertation contains no text, graphics, or tables copied and pasted from the Internet, except if specifically acknowledged, and the source is detailed in the dissertation and References sections.
I am grateful to the National Research Foundation (NRF), the Howard Hughes Medical Institute (HHMI) and the University of KwaZulu-Natal for funding me during the period of the project. Bingxia Wang, Elena Losina, Musie Ghemremichael and Susan Bryan for their contribution to the statistical analysis in the study. T cell count over the first 12 months of infection Association between HPP Acute Infection cohort haplotypes and baseline.
Sinikithemba chronic infection cohort Association between IL-10 haplotype and IL-2 expression. Sinikithemba chronic infection cohort Association between IL-10 haplotype and IFN-γ expression in Sinikithemba.
ABSTRACT
There was a significant association between the 592AA genotype and a greater breadth of CTL responses compared to the CC and CA (p= 0.002 and 0.004, respectively). The -592AA genotype was significantly associated with reduced CD4 cell loss (p = 0.0496), with -592AA showing the least change in CD4 cells per year. IL-10 promoter variants may influence the rate of HIV-1 disease progression by regulating IL-10 levels, which in turn may influence the breadth of CTL responses.
Furthermore, increased expression of HLA-DR and PD-1 on CD8+ and CD4+ T cells suggests that lower levels of IL-10 are associated with increased immune activation and immune exhaustion. The increased expression of IgG on B cells suggests that in an environment with lower IL-10, a bias towards a Th2 immune response is possible. Further studies to determine if and how the IL-10 pathway can be manipulated for HIV therapeutic or vaccination strategies are warranted.
ETHICAL APPROVAL
IL-10 Genotype and Haplotype Data for all study
- IL-10 AND HIV
IL-10 has been shown to inhibit HIV replication within macrophages and monocytes (Porcheray et al., 2006, Wang and Rice, 2006). Previous genetic studies focused on IL-10 promoter polymorphisms have shown that these SNPs play a role in HIV susceptibility and pathogenesis of infection. In a study by Shin et al in 2000, results showed that IL-10 promoter polymorphisms influence HIV-1 infection and pathogenesis in a cohort comparing individuals of different ethnicity (Shin et al., 2000).
They also found that low-IL-10-producing genotypes were associated with susceptibility to HIV infection and an AIDS-accelerating effect. IL-10 is also reported to enhance the deleterious elimination of dendritic cells by NK cells, thus potentiating immune dysfunction in chronic HIV-1 infection ( Alter et al., 2010 ). The third aim of this study was to determine the relationship between IL-10 variants and different biomarkers of HIV infection.
We also wanted to determine the association between IL-10 variants and the breadth and magnitude of HIV-1-specific CD8+ T cell immune responses. We determined the relationship between CD4+ T cell, CD8+ T cell and B cell activation with IL-10 promoter.
- DNA E XTRACTION
To determine whether there was an association between IL-10 haplotypes and either viral load or CD4+ T cell count, we used the Wilcoxon rank sums test. For this part of the analysis, we analyzed the association between viral load and the -3575 genotype for the CAI cohort. We found no significant association between -3575 genotype and median viral load at any of the three time points measured.
We looked at association between IL-10 haplotype in CAI and viral load after infection. We then analyzed for association between baseline viral load and the IL-10 haplotype in the SK cohort (see Table 2.3.10). We found that there was no significant association between any haplotype and baseline viral load in the SK cohort.
We found no significant association between the −3575 genotype and CD4+ T-cell counts during any of the three time points measured. We found no significant association between haplotype and CD4+ T-cell counts at 0–3 or 3–12 months post-infection. We next investigated the association between IL-10 haplotype and CD4+ T-cell counts in the HAI and SK study groups.
The Wilcoxon rank sum test was used to determine whether there was an association between any haplotype and CD4+ T cell count. We found no significant association between any of the haplotypes and CD4+ T cell counts at baseline. We analyzed the IL-10 haplotype data for association between haplotypes and viral load or CD4+ T cell count longitudinally.
There was also no association between the IL-10 haplotype and the first 12 months of infection. We wanted to determine whether there was an association between biomarkers of HIV-1 infection and IL-10 variants. There was no significant association between any IL-10 haplotype and either viral load or CD4+ T cell count at baseline.
In this cross-sectional analysis, we found no association between any IL-10 variant and either viral load or CD4+ T cell count. We saw no association between the -3575 genotype and either viral load or CD4+ T cell count for the CAI cohort.
Chapter 3: Association between IL-10 variants, cytokine expression and immune
To determine whether there was association between IL-10 haplotype, cytokine production and the breadth and extent of immune responses, we used the Wilcoxon rank sum test. The Pearson's correlation test was used to determine the association between IL-10 concentration and other plasma cytokines. To determine whether there was an association between IL-10 variants and the magnitude or breadth of immune responses, we used the Kruskal-Wallis test.
Plasma IL-10 expression levels were measured from the baseline (entry) time point. To determine whether there was an association between the extended IL-10 haplotype and cytokine expression, we used the Wilcoxon rank sum test. We used Pearson's correlation to determine whether there was any association between IL-10 levels and biomarkers of HIV infection (see Figure 3.3.3).
We therefore investigated the association between IL-10 variants and the magnitude and breadth of CD8+ T-cell immune responses in 409 individuals from the HPP Sinikithemba Chronic Infection cohort. We found no significant association between any IL-10 haplotype and the magnitude of immune responses. We next examined the relationship between IL-10 haplotype and the breadth and extent of immune responses in the chronic infection SK cohort (see Table 3.3.7).
We also found a significant association between the IL-10 haplotype and the breadth of immune responses. Regarding haplotype and IL-10 expression, we found that the CAT haplotype had a strong association. We found no correlation between plasma levels of IL-10 and these biomarkers of HIV-1 infection.
This may explain the lack of correlation between IL-10 expression and biomarkers of HIV-1 infection. We sought to determine the relationship between IL-10 and the predominant Th1 cytokines IFN-γ, IL-2, IL-6 and TNF-α. Individuals with the CAA haplotype had a lower median IL-10 expression, but this was not significant.
Both univariate and multivariate analyzes were performed to assess the association between activation markers and IL-10 genetic variants. In CD4+ T cells and CD8+ T cells, we measured the association between IL-10 genetic variants and the following activation markers: CD38, CD95, Ki67, HLA-DR and PD-1. We next examined the association between IL-10-1082 genotype and markers of CD4+ T cell activation (see Figure 4.3.14).
CFSE assays were used to determine the relationship between the role of IL-10 genetic variants in CD4+ T cell proliferation after IL-10 receptor blockade. We then assessed whether IL-10 genotype played a role in CD4+ T cell proliferation following IL-10 receptor blockade (see Figure 4.3.20). We found no significant association between IL-10 extreme genotype and CD4+ T cell proliferation after IL-10 receptor blockade (p = 0.60).
We also investigated whether the viral load of individuals plays a role in the proliferation of CD4+ T cells after blockade of the IL-10 receptor (see Figure 4.3.21). We next investigated whether IL-10 haplotype was in any way associated with proliferation after IL-10 blockade (see Table 4.3.1). Using the Wilcoxon rank sum test, we found no significant association between any IL-10 haplotype and proliferation after IL-10 blockade.
First, we investigated whether blockade of the IL-10 receptor affected cytokine production (see Figure 4.3.22). Next, we wanted to determine whether viral load plays a role in the production of cytokines after blocking the IL-10 receptor (see Table 4.3.3). Using the Wilcoxon rank sum test, we found that there was a significant association between IL-10 haplotype and IL-2 expression after IL-10 blockade.
We found no significant association between any IL-10 haplotype and IFN-γ expression after IL-10 receptor blockade. Therefore, we wanted to determine whether IL-10 receptor blockade has any effect on cytokine production.
