Impact of DNA methylation on CXCR5 gene transcription. (A) Non-fCD8s were sorted by FACS and treated for 72 hours with 10 μM Aza, a DNA methyltransferase inhibitor that causes hypomethylation of DNA. Fold change relative to the B2M housekeeping control indicated a significant increase in CXCR5 expression levels after treatment (n = 8). (B) Methylation levels for each subset analyzed across the 15 CpG sites for 3 biological replicates. (C) Representative plot of the quantitative measurement of DNA methylation levels within specific cell subsets (GCTfh’s, fCD8s, non-fCD8s, and naive CD8⁺ T cells), as determined using EpiTYPER DNA Methylation Analysis. Methylation levels were measured from bisulfite-treated genomic DNA, followed by PCR amplification of a 500-bp fragment containing 15 CpG sites (red letters). The naive and non-fCD8s cells show higher levels of methylation within several sites (darker circles), whereas the GCTfh’s and fCD8s show lower levels of methylation (lighter circles) (n = 3). The position of CpG sites are shown relative to the TSS. (D) Analysis of CXCR5 induction using recombinant TGF-β. CXCR5 expression was profiled at baseline and after the sorted cells were cultured in the presence and absence of TGF-β. A significant increase was observed after LNMCs were cultured in the presence of TGF-β (n = 5) for 7 days. Stim/stim, stimulation.