Background:
Single cell methods enhance the resolution at which infected cells in blood and tissues can be studied in people with HIV (PWH). Capturing the native state of infected cells obtained from multiple tissue compartments, especially cells in the cerebrospinal fluid (CSF), while minimizing cell loss from cryopreservation remains a challenge, especially in resource limited settings.
Methods:
Fresh blood, CSF, gut, lymph node (LN) and cell-sorted T follicular helper (TFH) cells were collected from participants enrolled in the RV304/SEARCH013 study enrolling people with chronic HIV (PWCH) in Bangkok, Thailand where uptake of optional procedures including leukapheresis, lumbar puncture (LP), gut biopsy, and lymph node (LN) biopsy is high. Longitudinal fresh samples from 4 compartments were collected from one PWH at baseline (ART naïve) and after 32 months of ART. An aliquot of baseline CSF cells were set aside and frozen for separate analysis. We used the 10X genomics platform locally to generate single cell RNA and T-cell/B-cell receptor data from fresh cells within hours of sampling and from frozen CSF cells.
Results:
Single cell data were generated for 18,790 CSF cells, 18,341 gut cells, 37,055 LN cells, 11,561 TFH cells, and 23,349 blood cells. To enhance detection of HIV viral transcripts pre-ART, we generated autologous near full-length patched viral sequences from pre-ART plasma to align single cell sequencing reads and detected high and low HIV transcript containing cells in all compartments pre-ART. Despite 32 months of suppressive ART, transcriptionally active HIV-infected cells were present in all 4 compartments at single cell resolution, including the CNS, predominately in memory CD4 T cells. Notably, single cell comparisons of fresh (9,603 cells) versus frozen CSF cells (9,187 cells) revealed a significant loss of infected HIV cells in CSF after cryopreservation. T cell clonotypes were shared across CSF, gut, LN, and blood compartments; however, the observed HIV transcript containing cell clones were all unique pre-ART.
Conclusions:
Longitudinal multi-compartment studies of fresh cells indicate presence of transcriptionally active HIV in several compartments. In CNS, sensitivity was markedly greater in fresh compared to cryopreserved cells. Future studies should evaluate whether HIV-infected cells from diverse tissue sites may be sensitive to cryopreservation, potentially leading to an inaccurate representation of the viral reservoir.
Logistics and workflow of longitudinal multi- compartment single cell study of fresh cells from RV304 in Bangkok, Thailand. 