CD8+ T cells located in B cell follicles play an important role in viral and tumor control. However, only a small subset of CD8+ T cells called follicular CD8+ T cells express CXCR5, the chemokine receptor required for cell migration into B cell follicles. We investigated why most LN CD8+ T cells lack CXCR5 expression, and why there is reduced CXCR5 expression on CXCR5+CD8 T cells (fCD8s) relative to GCTfh.
We FACS-sorted CXCR5+CD8+ (fCD8s), CXCR5–CD8+ (non-fCD8s), naïve CD8+ T cells and GCTfh from lymph nodes of HIV-1 infected individuals and performed RNA-sequencing (RNA-Seq). DNA methylation was used to profile methylation pattern of the CXCR5 gene and the Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) was used to quantify accessible genes and to identify epigenetic modules governing CXCR5 expression.
RNA-seq data analysis of fCD8 and non-fCD8s identified 43 gene among the most differentially expressed genes (FDR<0.01) that are associated with epigenetic gene regulation. DNA bisulfite treatment and sequencing showed that 70% of CpG islands in CXCR5 gene were methylated whereas fCD8 had less than 7% methylation levels at equivalent sites. ATAC-Seq analysis revealed a closed chromatin conformation at the CXCR5 TSS in non-fCD8s whereas fCD8s had open chromatin at equivalent sites. Furthermore, analysis of nucleosomal footprinting around the CXCR5 TSS revealed greater nucleosomal occupancy in fCD8s compared to GCTfh, computational simulation indicated that the presence of nucleosomes at the TSS interfered with transcription efficiency resulting in attenuated expression of the CXCR5 gene.
We show that DNA methylation coupled with chromatin compaction at TSS prevent CXCR5 gene expression in non-fCD8s and greater nucleosomal occupancy down-modulate CXCR5 expression levels in fCD8s. Together, these data provide insights into both the underlying molecular mechanisms that repress CXCR5 in non-fCD8s and the molecular mechanisms responsible for the low CXCR5 expression in fCD8s, with implications for HIV cure strategy or eradication of B cell-derived tumors.