Elizabeth Connick

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.

We previously demonstrated that HIV replication is concentrated in lymph node B cell follicles during chronic infection and that HIV-specific CTL fail to accumulate in large numbers at those sites. It is unknown whether these observations can be generalized to other secondary lymphoid tissues or whether virus compartmentalization occurs in the absence of CTL. We evaluated these questions in SIVmac239-infected rhesus macaques by quantifying SIV RNA(+) cells and SIV-specific CTL in situ in spleen, lymph nodes, and intestinal tissues obtained at several stages of infection. During chronic asymptomatic infection prior to simian AIDS, SIV-producing cells were more concentrated in follicular (F) compared with extrafollicular (EF) regions of secondary lymphoid tissues. At day 14 of infection, when CTL have minimal impact on virus replication, there was no compartmentalization of SIV-producing cells. Virus compartmentalization was diminished in animals with simian AIDS, which often have low-frequency CTL responses. SIV-specific CTL were consistently more concentrated within EF regions of lymph node and spleen in chronically infected animals regardless of epitope specificity. Frequencies of SIV-specific CTL within F and EF compartments predicted SIV RNA(+) cells within these compartments in a mixed model. Few SIV-specific CTL expressed the F homing molecule CXCR5 in the absence of the EF retention molecule CCR7, possibly accounting for the paucity of F CTL. These findings bolster the hypothesis that B cell follicles are immune privileged sites and suggest that strategies to augment CTL in B cell follicles could lead to improved viral control and possibly a functional cure for HIV infection.