Measurement of HIV-1 reservoirs is usually studied in peripheral blood (PB), but 98% of CD4 T cells are in lymphoid tissues and other organs. Histology has shown characteristic hyperplastic germinal centres (GC) in lymph nodes, recently associated with increases in the T follicular helper cell (Tfh) subset of CD4 T cells, and there is now recognition that GC and Tfh are an important reservoir of HIV-1. We investigated whether ultrasound-guided fine needle biopsies (FNB) from peripheral lymph nodes (LN) provide a minimally invasive means of longitudinally accessing HIV-1 reservoirs in GC.
FNB of inguinal LN were performed on 10 healthy adult controls (HC), 11 HIV+ ART-naive and 10 HIV+ on ART subjects, without adverse events. FNB were analyzed for CD3+CD4+CD45RAnegPD-1hiCXCR5+ICOS+CD127dim Tfh cells and CD19+CD20highCD38highIgDnegKi-67+Bcl-6+ GC B cells, using TruCount tubes on a 4-laser LSR II. CD4 T cells, from FNB and PB, respectively, were purified by cell sorting, from 19 patients, and HIV DNA and cell-associated (CA) HIV RNA were quantified by real-time PCR.
Overall, the median number of CD4 T cells obtained by FNB from HC was 887,000 (IQR: 206,000-1,187,000), and from all 21 HIV+ subjects (519,000 (IQR: 228,000-1,340,000)). However, the median Tfh cell number in FNB from ART-naïve HIV+ subjects was 46,300, significantly higher than in HC (5,600; p=0.04); HIV+ on ART subjects had a median of 15,800 Tfh cells. The median GC B cell number from ART-naïve HIV+ was 153,493 cells, significantly higher than in HC (median: 1,506; p<0.01). For HIV+ on ART, the median GC B cell number was 30,749 cells, so that 4/10 were above HC normal range. Cell sorting from HIV+ FNB yielded a median of 295,000 CD4+ T-cells (purity>96%). HIV DNA was quantified in 19 out of 19, CA HIV RNA in 13 out of 17, samples. HIV DNA copies per 106 CD4+ T cells were higher in CD4+ T cells purified from LN FNB compared to PB in both ART-naïve (3.22 vs 2.56, p=0.02) and ART (3.21 vs 3.08, p=0.13) groups. Unspliced HIV RNA copies per 106 CD4+ T cells were similarly elevated in LN FNB samples from both the ART-naïve (3.97 vs 3.05, p<0.01) and ART (3.66 vs 2.95, p=0.13) groups.
Ultrasound guided FNB of unenlarged LN was well-tolerated, and provided sufficient CD4+ T cells and B cells for high-dimensional flow cytometry and cell sorting for PCR, thereby providing access to Tfh and GC B cells for HIV reservoir, pathogenesis, therapy and vaccine studies.