Single Cell Transcriptome Sequencing of Human Lymph Node HIV-Infected CD4 Cells

Identification of live HIV-infected CD4 cells allows the characterization of individual cells both by flow cytometry and by transcriptomic analysis.

Cervical lymph nodes were obtained from 3 viremic HIV-infected volunteers, median CD4 count 671(range 247-1,288) and median viral load 17,437(14,075-29,923).  Approximately 90 individual CD45RO⁺ PD1^hi CD4^dim cells were index sorted into individual wells for each patient.  In addition, surface expression of HIV envelope on CD4 T cells was identified using fluorescently labelled PGT121.  Whole transcriptome libraries were generated from the purified RNA of sorted single cells using established methods and deep sequenced on an Illumina HiSeq (Trombetta, JJ et al. (2014) Curr Protoc Mol Biol 4.222.1-422.17).

The median frequency of HIV-RNA containing PD1^hi CD4^dim cells was 8.0 (4.6-8.0)%.  In cells from two of the three lymph nodes the frequency HIV RNA containing cells was higher in cells staining for PGT121, 15 and 18%, compared to 6 and 5%, respectively.  There was no difference in HIV RNA containing cells within PGT121⁺ and PGT121^– populations in the lymph node from the third volunteer.  The percentage of total reads containing HIV RNA varied between 0.02-6.0% in individual HIV+ cells.  In two of the three volunteers the median frequency of HIV reads was higher in PGT121⁺ cells than in PGT121^– cells, 0.15 and 0.6%, compared to 0.04 and 0.03% PGT121^– cells.  In the other sample there was essentially no difference.

These data show that it is possible to identify HIV-infected CD4 T cells at a level which makes whole cell transcriptome analysis of HIV RNA+ and similar HIV RNA- cells possible.