Abstract Body

Background: Follicular dendritic cells (FDC) are located within germinal centers (GC) of follicles and harbor large amounts of extracellular virus that are potently infectious to CD4+ T cells in vitro. Although most HIV-producing T cells are located within B cell follicles in secondary lymphoid tissues, it is unknown whether HIV RNA+ cells are concentrated within GC in vivo. The purpose of this study was to quantify HIV RNA+ cells in GC, non-GC follicular (non-GC F) and extra-follicular (EF) compartments of lymph nodes (LN) of HIV-infected individuals. We hypothesized that if FDC are a major source of infectious virus in vivo, then HIV RNA+ cells will be concentrated primarily within GC. Methodology: Frozen LN cross-sections from 22 chronically HIV-infected untreated individuals without AIDS and 10 HIV-seronegative individuals were evaluated. For each LN, in situ hybridization for HIV-1 RNA combined with immunohistochemical (IHC) staining for CD20 and IgD were performed in a minimum of 3 cross-sections located ~60 μm apart. GC was defined as CD20+ IgD- stained areas, non-GC F as CD20+ IgD+, and EF as CD20- IgD-. Visual inspection and quantitative image analysis were used to determine the number of HIV RNA+ cells per mm2. IHC for HIV p24 Ag was used to identify the FDC reservoir of virions in follicles (CD20+). Wilcoxon two-sample t test was used for comparisons. Spearman σ was used to describe correlations. Results: LN cross-sectional area was larger in HIV+ (median, 35.8 mm2) compared to HIV- subjects (median, 15.4 mm2; p=0.06). Percent LN that was GC was 16-fold higher in HIV+ (median, 8%; range 0.8-41%) than HIV- (median, 0.5%; range, 0-1.6%). Frequencies of HIV RNA+ cells in LN were higher in GC (median, 2.95 cells/mm2) than in non-GC F (median, 1.38 cells/mm2; p= 0.0008) and EF (median, 0.13 cells/mm2; p< 0.0001). A median of 55% of HIV RNA+ cells within LN resided in GC, which was 2.5-fold higher than both non-GC F (median, 22%; p=0.0012) and EF (median, 22%; p= 0.0005). Plasma HIV RNA correlated with HIV RNA+ cells/mm2 in GC (σ=0.62, p=0.002), non-GC (σ=0.65, p=0.001) and EF (σ=0.83, p<0.0001). Percent p24 Ag+ area within follicle ranged from 0.1 to 4.1 (median, 0.8), but did not correlate with either plasma HIV RNA (p=0.13) or frequency of HIV RNA+ cells in GC (p=0.56). Conclusions: This study is the first to quantify the distribution and frequency of HIV-1 producing cells within GC of LN. HIV RNA+ cells are concentrated within GC of activated follicles where FDC and their extra-cellular burden of HIV virions are located. Results are consistent with the hypotheses that FDC are major sources of infectious virus in vivo and/or GC T follicular helper cells are highly permissive to HIV.