Background:
Follicular T helper cells (TFH) are the major viral RNA expressing (vRNA+) cells in secondary lymphoid tissues during chronic HIV/SIV infection prior to AIDS/SAIDS. TFH are variably defined based on location in germinal centers (GC), and/or expression of the canonical transcription factor BCL6 and multiple cell surface markers including PD-1. In some in vitro systems, HIV downregulates PD-1, but whether this occurs in vivo is unknown. We investigated the distribution and expression of BCL-6 and PD-1 by HIV/SIV RNA+ cells in lymph nodes of untreated people living with HIV (PLWH) and SIV-infected rhesus macaques (RM).
Methods:
Inguinal LN frozen in OCT from 7 PLWH (3 female; median age 42y; median viral load 4.35log10; median CD4 706) and 5 RM (4 female; median age 3y; median viral load 6.0log10) was sectioned (10μm) and fixed in paraformaldehyde. In situ hybridization for vRNA and BCL6 mRNA (ACDbio), and immunofluorescent antibody staining for PD1 and CD20 was performed. Slides were imaged (Aperio scanner), vRNA+ cells identified (median vRNA+ cells, 59/92 in PLWH/RM) and phenotypes determined. CD20+ areas defined follicles (F), CD20- areas extrafollicular (EF), and clusters of BCL6 in F defined GC. Nonparametric statistical analyses were performed; data reported are medians.
Results:
In PLWH, most (78%) GC vRNA+ cells were BCL6+ and comparatively fewer nonGCF (60%; p=0.03) and EF (50%; p=0.03). PD-1 was present on 72% of GC, 53% of nonGCF, and 23% of EF vRNA+ cells (p=0.01). Percentages of BCL6+ vRNA+ cells that were PD-1+ were highest in GC (75%), and relatively lower in nonGCF (56%; p=0.2) and EF (11%; p=0.03). In RM, similar trends in BCL6 expression in vRNA+ cells were observed; 90% of GC, 48% of nonGCF and 50% of EF vRNA+ cells were BCL6+. PD-1 expression was substantially lower in RM than in PLWH; PD-1 was found on only 36% of GC, 15% of nonGCF, and 4% of EF vRNA+ cells. Percentages of BCL6+vRNA+ cells that expressed PD-1 were 38% in GC, 25% in nonGCF and 5% in EF.
Conclusions:
PD-1 was not found on 25% of vRNA+ GC TFH in PLWH and 62% in RM, when defining TFH as BCL6+ vRNA+ cells in GC. Whether BCL6+ cells outside of the GC are TFH is unknown, but decreased PD-1 expression on BCL6+ vRNA+ cells outside of the GC is consistent with a model of downregulation of PD-1 on vRNA+ TFH infected in the GC as they migrate into nonGCF and EF. Sorting strategies to quantify vRNA that identify TFH utilizing PD-1 expression likely underestimate TFH contribution to viral burden.