Follicular CTL Accumulate in SIV-Infected Lymph Nodes Due to Immune Activation

Strategies aimed to eradicate HIV/SIV infection require the characterization of cytolytic CD8 T cells that can migrate and recognize infected cells at the sites of active replication. Follicular CD4 T cells (T_FH), located within the germinal centers (GC) of the lymph node (LN) B cell follicles support active HIV/SIV replication even under suppressive combined antiretroviral therapy (cART). Thus, a comprehensive analysis of CD8 T cell dynamics in the LN, particularly in the GC area, will provide valuable information towards HIV/SIV eradication.

Multi-parametric flow cytometry, multiplexed confocal imaging, histocytometry and fluidigm-based analysis were used to characterized PBMC, LN-derived cells and LN tissues from (1) SIV-negative rhesus macaques (RM), (2) acute and chronic SIV-infected RM and (3) chronic SIV-infected African green monkeys (AGM). CM9 (Gag) and TL8 (Tat) tetramers and intracellular cytokine staining after peptide pool stimulation identified virus-specific CD8 T cells while in vitro killing assays using sorted LN CD8 T cell populations and an anti-CD3/VRC07 antibody proved the cytolytic activity of LN-resident CD8 T cells.

Chronic SIV infection in RM was characterized by the accumulation of memory (CD28^hiCD95^hi) and effector (CD28^loCD95^hi) CD8 T cells with a follicular (CCR7^loCXCR5^hi) phenotype (fCD8). Imaging and histocytometry confirmed the accumulation of fCD8 within the GC. fCD8, when compared to CCR7^hiCXCR5^lo non-fCD8, express higher amounts of Granzyme B and exert a higher capacity to mediate in vitro bispecific-mediated redirected killing of infected cells. Chronically infected AGM, on the other hand, did not accumulate fCD8. Fluidigm analysis showed that both memory and effector fCD8 clustered together and were mostly affected by the SIV-infection status. Besides, the frequency of LN CD8 T cells was significantly correlated with LN CD14^hiCD16^hi monocytes. Upon stimulation, monocytes from SIV-infected RM secreted increased amounts of CXCL10, a CXCR3 ligand. Confocal imaging confirmed the proximity of CXCR3-expressing cells and monocytes within the LN.

Cytolytic fCD8 T cells are able of killing infected cells, further justifying the use of the SIV model to develop new eradication strategies. Our results suggest that chronic immune activation is a major force driving the accumulation of fCD8, pointing to specific targets as mediators of the intra-follicular CD8 trafficking.