Germinal Center T Follicular Helper Cells Are Highly Permissive to HIV-1 and Alter Their Phenotype during Virus Replication

Abstract

HIV-1 replication is concentrated within CD4(+) T cells in B cell follicles of secondary lymphoid tissues during asymptomatic disease. Limited data suggest that a subset of T follicular helper cells (TFH) within germinal centers (GC) is highly permissive to HIV-1. Whether GC TFH are the major HIV-1 virus-producing cells in vivo has not been established. In this study, we investigated TFH permissivity to HIV-1 ex vivo by spinoculating and culturing tonsil cells with HIV-1 GFP reporter viruses. Using flow cytometry, higher percentages of GC TFH (CXCR5(high)PD-1(high)) and CXCR5(+)programmed cell death-1 (PD-1)(low) cells were GFP(+) than non-GC TFH (CXCR5(+)PD-1(intermediate)) or extrafollicular (EF) (CXCR5(-)) cells. When sorted prior to spinoculation, however, GC TFH were substantially more permissive than CXCR5(+)PD-1(low) or EF cells, suggesting that many GC TFH transition to a CXCR5(+)PD-1(low) phenotype during productive infection. In situ hybridization on inguinal lymph node sections from untreated HIV-1-infected individuals without AIDS revealed higher frequencies of HIV-1 RNA(+) cells in GC than non-GC regions of follicle or EF regions. Superinfection of HIV-1-infected individuals' lymph node cells with GFP reporter virus confirmed the permissivity of follicular cells ex vivo. Lymph node immunostaining revealed 96% of CXCR5(+)CD4(+) cells were located in follicles. Within sorted lymph node cells from four HIV-infected individuals, CXCR5(+) subsets harbored 11-66-fold more HIV-1 RNA than CXCR5(-) subsets, as determined by RT PCR. Thus, GC TFH are highly permissive to HIV-1, but downregulate PD-1 and, to a lesser extent, CXCR5 during HIV-1 replication. These data further implicate GC TFH as the major HIV-1-producing cells in chronic asymptomatic HIV-1 infection.

FIGURE 1. Follicular cells are highly permissive to HIV-1 following ex vivo infection of disaggregated human tonsil cells with HIV-1 GFP reporter viruses

Disaggregated tonsil cells were spinoculated with R5- (n=6) or X4-tropic HIV-1 GFP reporter viruses (n=8) for 2 hours, cultured for two days, and analyzed by flow cytometry. (A) Representative examples of flow cytometry gating are shown. Cells were stained with a viability dye. Within viable CD3+CD8- cells, GFP expression was determined after gating on CXCR5- and CXCR5+ cells to define extrafollicular (EF) and follicular (F) subsets, respectively. (B) Percentages of GFP+ cells and gMFI of GFP was determined on EF, F, naïve (CD95−), memory (CD95+), central memory (CM; CD95+CD28+CCR7+), effector memory (EM; CD95+CD28-CCR7−), memory EF (CXCR5-CD95+), and memory F cells (CXCR5+CD95+). Statistical analyses were performed using Wilcoxon signed rank test and significance is denoted by asterisks where * = p < 0.05, ** = p < 0.01.

FIGURE 2. GC T_FH are highly permissive to HIV-1 GFP reporter viruses ex vivo

Disaggregated tonsil cells were spinoculated with R5- or X4-tropic HIV-1 GFP reporter viruses for 2 hours, cultured for two days, and analyzed by flow cytometry. (A) Representative examples of flow cytometry gating are shown from cells spinoculated with X4-tropic HIV-1 GFP reporter virus. Within the follicular (F) subset, further subsets were defined as CXCR5+PD-1^low, CXCR5+PD-1^intermediate (non-GC T_FH) and CXCR5^highPD-1^high (GC T_FH). Histograms indicate gMFI of GFP within each subset. (B) Percentages of GFP+ cells and gMFI of GFP within extrafollicular (EF) and F subsets following ex vivo HIV-1 infection with R5- and X4-tropic HIV-1 (n=15). (C) Percentages of CXCR5+PD-1^low and GC T_FH subsets within mock-spinoculated cultures compared to R5- and X4-tropic HIV-1 spinoculated cultures (n=15). (D) gMFI of PD-1 on CXCR5+ cells and gMFI of CXCR5 on total CD3+CD8- cells in mock-spinoculated compared to R5- and X4-tropic HIV-1 spinoculated tonsil cultures (n=15). (E) Percentages and gMFI of GFP+ cells within EF, CXCR5+PD-1^low, and GC T_FH subsets that were sorted prior to spinoculation with R5- or X4-tropic HIV-1 reporter virus from 6 tonsil donors (T282, T290, T294, T165, T169, T171). Statistical analyses were performed using ANOVA (B), or Wilcoxon signed rank test (C,D) and significance is denoted by asterisks where * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.

FIGURE 3. HIV-1 chemokine co-receptor expression and immune activation are not linked to permissivity of GC T_FH ex vivo

(A) Percentages and mean number of molecules per cell of CCR5 and CXCR4 on tonsil cell subsets at baseline prior to spinoculation (n=15). (B) HLA-DR and CD38 coexpression in tonsil cell subsets at baseline prior to infection (n=8). (C) Percentages and gMFI of GFP on DR+38+ cells versus non-DR+38+ cells following spinoculation with R5- and X4-tropic HIV-1 reporter viruses (n=8). Statistical analyses were performed using ANOVA (A,B) and Wilcoxon signed rank tests (C) and significance is denoted by asterisks where ** = p < 0.01 and *** = p < 0.001.

FIGURE 4. HIV-1 RNA+ cells are concentrated within GC of lymph nodes in untreated HIV-1-infected individuals

In situ hybridization for HIV-1 RNA was performed in conjunction with immunostaining for CD20 and IgD in inguinal lymph node tissue sections from 22 HIV-1-infected individuals not receiving antiretroviral therapy. (A) Representative images of a lymph node section demonstrating GC (pink, IgD-CD20+), non-GC follicle (brown, IgD+CD20+), and EF regions (IgD-CD20-). (B) Percentages of total lymph node tissue cross sections that consisted of EF, non-GC follicle, and GC regions. (C) Frequencies of HIV-1 RNA+ cells in lymph node compartments. (D) Proportions of total HIV-1 RNA+ cells found in each lymph node compartment. (E) Representative image of a lymph node tissue section stained with antibodies to CD20 (gray) and HIV-1 p24 antigen (brown) demonstrating that virions demonstrating a typical reticular pattern for virus bound to FDC are localized exclusively in follicles. Statistical analyses were performed using ANOVA (B, C, D) and significance is denoted by asterisks where * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ****p<0.0001.

FIGURE 5. Follicular cells, but not activated cells from lymph nodes of HIV-1-infected individuals are highly permissive to HIV-1 following ex vivo superinfection with HIV-1 X4-tropic GFP reporter virus

Disaggregated lymph node cells from 3 HIV-1-infected individuals were spinoculated with X4-tropic HIV-1 GFP reporter virus, cultured for 3 days, and analyzed by flow cytometry. A subset of cells were CD8 depleted prior to spinoculation. (A) Percentages and gMFI of GFP+ cells in whole and CD8-depleted lymph node cells, and ratios of depleted:whole in follicular (F) and extrafollicular subsets (EF). (B) Percentages and gMFI of GFP+ cells in whole and CD8-depleted lymph node cells, and ratios of depleted:whole in DR+38+ cells compared to all other CD3+CD8− cells (non-DR+38+). Due to the small number of subjects evaluated (n=3), none of the differences shown were statistically significant using a non-parametric Wilcoxon signed rank test.

FIGURE 6. Expression of CXCR5, CD20, and CD4 in a representative lymph node tissue section from an untreated HIV-1-infected individual

Immunofluorescent staining of a representative lymph node tissue section for (A) CXCR5 (green), (B) CD20 (blue), (C) CD4 (red) and (D) composite image revealed that the majority of CXCR5+CD4+ cells (indicated by arrows) were located within B cell follicles. B cell follicles were identified morphologically as a discrete aggregation of CD20+ cells. Images were collected using a 40X objective on a Leica DM5000B fluorescent microscope.