Background:
HIV-expressing (vRNA+) cells are consistently found in secondary lymphoid tissues (SLT) of people with HIV (PWH) on antiretroviral therapy (ART) and likely drive viral rebound upon ART cessation. Little is known about what affects HIV expression in SLT. We hypothesized that B cells promote HIV expression.
Methods:
Disaggregated tonsil cells from persons without HIV were depleted of CD19+ cells by magnetic beads. CD19- cells were spinoculated with X4 or R5-tropic HIV GFP reporter virus, cultured 3 days with dye-labeled CD19+ or CD19- cells and saquinavir, and %GFP+ of unlabeled CD3+CD8- cells and GFP median fluorescence intensity (MFI) determined by flow cytometry. Tonsil cells were also sorted into CD4+ T follicular helper (TFH; CD3+CD8-CXCR5hiPD-1hi), nonTFH (CD3+CD8-CXCR5+/-PD-1+/-), memory B (IgD-CD38-), pre-germinal center B (pre-GCB; IgD+CD38+), GCB (IgD-CD38+), and naïve B (IgD+CD38-). TFH and nonTFH were infected with X4 HIV, incubated with B cell subsets or control CD4+ T cells, and %GFP+ and GFP MFI determined. Sections of inguinal lymph node (LN) from 6 male PWH on ART for 7-29 years and spleen from 6 male PWH on ART for >5 years were analyzed by in situ hybridization for vRNA and immunofluorescent antibody staining for CD20 to determine follicular (F) regions. Frequencies of vRNA+ cells in F and extrafollicular (EF) regions, EF B cells, and EF B cells adjacent to vRNA+ cells were determined by visual inspection and quantitative image analysis. Data reported are medians. Nonparametric statistical tests were used.
Results:
CD19 depletion reduced %GFP+ and GFP MFI by 32% and 30% in X4 (n=7; p=0.02 and 0.03) and 37% and 29% in R5-HIV infected cells (n=7; both p=0.02) compared to cultures with CD19+ cells. Memory, pre-GCB, GCB, and to a lesser extent naïve B cells elevated HIV expression in both nonTFH (%GFP+ p<0.05; GFP MFI p<0.05; n=6) and TFH (%GFP+ increased by all B cell subsets except naive, p<0.05; n=6; all B cell subsets augmented GFP MFI, p<0.05; n=6). Frequencies of vRNA+ cells were higher in B cell-rich F than EF regions in LN (0.28 vs 0.17 cells/mm2; p=0.03) and spleen (0.07 vs 0.05 cells/mm2; p=ns). In EF areas, vRNA+ cells were adjacent to B cells more often than predicted by chance in all four LN (3.1-fold; range, 3-5.2) and four spleens (1.4-fold, range 1.1-2) evaluated.
Conclusions:
B cells upregulate HIV expression in SLT CD4+ T cells and are associated with vRNA+ cells in SLT of PWH on ART. Mechanisms by which B cells augment HIV expression merit investigation.