Background
Lymphoid tissues are major sites of HIV/SIV viral replication and reservoir persistence. The full spectrum of cells that harbor persistent virus and can contribute to viral rebound are not well understood, hindering therapeutic targeting of these cell populations. We used multiplex fluorescent imaging to extensively characterize infected cells in lymphoid tissues of Rhesus macaques (RM).
Methods
Our multiplex in situ imaging approach combines RNAscope and DNAscope to localize infected cells, with highly multiplexed cellular phenotyping of > 50 markers. Using FFPE samples we assessed the phenotype and environment of SIV infected cells in various lymphoid tissues, including male and female RM tissues from all stages of infection (naïve, acute, chronic, on ART, and after ATI). Cell phenotypes and frequencies were evaluated using QuPath, and multiplex phenotyping was corroborated by flow cytometry.
Results
Using multiplex imaging, we identified a non-T cell (CD3-) population of cells harboring SIV genomes that formed unique structures in lymph nodes. These cells were presumptively identified as a plasmacytoid dendritic cell (pDC) population based on expression of CD303, however they lack other major pDC lineage defining markers (CD123). These pDCs are found in tightly packed clusters along the lymphatic ducts, often bordering between the B-cell follicle and T-cell zone. Clusters were most frequently identified in lymph nodes but could also be found in the tonsils and spleen. Clusters ranged in size from 3 cells to > 500 cells/cluster, and their abundance correlated with viral load. Interestingly, a small subset express vRNA, while many harbor vDNA despite expressing the HIV restriction factor SAMHD1, interferon response genes (MxA/ISG15), and cytolytic marker (GZMB).
Conclusions
Here, based off expression of CD303, we provisionally identified a pDC-like population that may constitute an important subset of the persistent viral reservoir and respond to early SIV infection in lymphoid tissues. Similar clusters of pDCs in humans have been observed in cases of reactive lymphadenopathies. While morphologically similar in structure, these cellular clusters in humans expressed the traditional pDC marker CD123, which is lacking in the population we observe in RM, suggesting further work is required to understand the lineage, functional, and species differences observed. We hope to detect this pDC-like population in blood, to better track and understand its role in early SIV infection.