Doctoral Degree. University of KwaZulu-Natal, Durban.Naturally induced CD8+ T cells do not clear human immunodeficiency virus (HIV) infection, partly because the virus rapidly escapes CD8+ T cell responses and the effector cells are excluded from HIV reservoirs sites. However, optimizing CD8+ T cell
responses could potentially be leveraged in HIV vaccine or cure efforts if epitope escape and barriers to effector CD8+ T cells infiltrating the sites of HIV reservoirs are overcome. In our first study, we described a potential mechanism of HIV-1 control by
CD8+ T cells targeting different variants in individuals infected with HIV-1. Our second study focused on describing the molecular regulation of CXCR5 expression in human CD8+ T cells.
Study 1
HLA-B*81 is associated with control of HIV-1 subtype C infection, while the closely related allele B*42 is not. Interestingly, both alleles present the immunodominant Gag TL9 epitope, and the magnitude of this response correlates negatively with viral load.
To examine the role of T cell receptor (TCR) in this process, we characterized the sequence and function of TL9-specific CD8+ TCR in B*81 and B*42 individuals. TL9-specific CD8+ T cells were identified and isolated using B*81 and/or B*42 TL9
tetramers. TCR beta genes were amplified from single sorted cells and sequenced. Paired alpha genes were identified for selected clones. TCR function was tested using a reporter cell assay where TCR+ Jurkat cells were co-cultured with peptide-pulsed or HIV-1 infected B*81 or B*42 target cells, and signalling quantified by luminescence. TCR recognition was assessed against all single amino acid TL9 variants and results were compared to HIV-1 subtype C sequences.
A population of dual-reactive T cells was detected by both B*81- and B*42-TL9 tetramers in 7/9 (78%) B*81 and 4/11 (36%) B*42 individuals; and this population was associated with lower viremia. Mono- and dual-reactive TCR beta sequences were
collected from six individuals. In B*81 individuals, all TCRs were highly restricted to TRBV12-3. In B*42 individuals, mono-reactive TCRs encoded a variety of V beta genes, while dual-reactive TCRs were restricted to TRBV12-3 and enriched for public clones. Functional analyses indicated that B*81 TCRs (1 mono, 2 dual) and a dualreactive public B*42 TCR displayed similar TL9 cross-reactivity profiles and enhanced capacity to recognize HIV-1 escape mutations compared to mono-reactive B*42 TCRs. This work highlights the impact of TCR promiscuity on T cell-mediated control of HIV-1.
Study 2
HIV-1 infection is difficult to cure even with effective antiretroviral therapy (ART) because of persistent viral replication in immune privileged sites such as the B cell follicles of secondary lymphoid tissues. CD8+ T cells are generally excluded from B
cell follicles, partially due to a lack of expression of the follicular homing receptor CXCR5. Recent murine studies have identified CXCR5+ CD8+ T cells, referred to as follicular CD8+ T cells (fCD8s), that localize in B cell follicles. However, the
mechanisms governing expression of CXCR5 on human CD8+ T cells are not known.
We investigated the epigenetic and transcriptional mechanisms involved in the regulation of CXCR5 expression in human CD8+ T cells. We FACS-sorted CXCR5+CD8+ (fCD8s), CXCR5-CD8+ (non-fCD8s), naïve CD8+ T cells and germinal center T follicular helper cells (GCTfh) from the lymph node of HIV- 1 infected individuals and performed RNA-sequencing (RNA-Seq), DNA methylation assays and the assay for transposase-accessible chromatin using sequencing (ATACSeq).
RNA-Seq was used to quantify the expressed genes in FACS-sorted subsets and to determine transcriptional modules governing CXCR5 expression in CD8+ T cells. ATAC-Seq was used to quantify accessible genes, identify the transcriptional
factors footprinting and determine epigenetic modules governing CXCR5 expression. DNA methylation, a major epigenetic gene silencing mechanism, was used to profile methylation pattern of the CXCR5 gene region in the sorted subsets.
We observed hypermethylation of DNA around the transcriptional start site (TSS) of the CXCR5 gene in non-fCD8s but not in fCD8s. ATAC-Seq analysis revealed a closed chromatin conformation at the TSS in non-fCD8s, but not in fCD8s. Our gene
expression data revealed significant differences in the CXCR5 associated factors between GCTfh and fCD8s. Computational analysis further revealed the presence ofa nucleosome at the TSS of fCD8s, which could be a plausible explanation for lower
expression of CXCR5 in fCD8s as compared to GCTfh. Together, we identified epigenetic regulations involved in CXCR5 expression in human CD8+ T cells and propose that DNA methylation, chromatin structure and nucleosome positioning cooperatively regulate the expression of CXCR5 in CD8+ T cells.
Our data open up the possibility of using epigenetic manipulation as a novel strategy for redirecting CD8+ T cells to B cell follicles where they are needed to eradicate HIV-1 infected cells
