One of the major hurdles for directly phenotyping the in vivo latent HIV reservoir is the need to stimulate these cells ex vivo. As the cells are stimulated, they change expression of many cell surface markers making precise phenotyping of the latent cells difficult. We recently established an approach called PP-SLIDE (Predicted Precursor as determined by SLIDE) that can effectively backtrack in time to establish the cell surface phenotype of a cell before infection (Cavrois et al 2017) using high-dimensional datasets generated by CyTOF phenotyping. This approach was implemented to chart the in vivo latent HIV reservoir and identify biomarkers of these cells.
The ability of PP-SLIDE to trace the phenotype of a reactivated cell to its precursor (non-stimulated) state was first confirmed in an in vitro model of HIV latency. Then, freshly isolated blood and lymph node cells from ART-suppressed, HIV-infected individuals from the SCOPE cohort were stimulated with PMA/ionomycin, and reactivated cells (expressing Gag) were deep-phenotyped using a 39-parameter latency-focused CyTOF panel. PP-SLIDE was used to map the reactivated cells onto a T cell atlas of unstimulated cells created for each patient sample analyzed.
Comparison of latent to non-latent cells revealed unique signatures associated with latency. Receptors preferentially expressed on latently-infected T cells from blood include previously described markers such as PD1, TIGIT, and OX40, as well as novel ones including homing integrin α4β1. Latent cells from lymph nodes also preferentially expressed PD1, TIGIT, and OX40, and differed from latent cells in blood in that they exhibited features of resident memory cells and preferentially expressed the T follicular helper marker CXCR5 and the costimulatory molecules CD28 and ICOS.
By simultaneously detecting the expression of 39 proteins in individual reactivated cells and mapping this information onto an atlas of unstimulated cells, we have begun to establish a high-resolution view of the types of latent cells that persist in HIV-infected individuals. Applying this method to chart the reservoir in blood and tissues of additional donors will provide a more complete picture of the nature of the persistent reservoir. Our findings thus far reveal that latent cells exhibit unique features, some of which differ between blood and tissues consistent with the notion that unique mechanisms of persistence are present in tissues.