Background
Fully mature SHIV envelope is transported to the CD4 T cell surface. Therefore, expressed envelope represents a means of identifying live SHIV-infected CD4 T cells.
Methods
CD4 T cells from Fiebig-staged SHIVAD8-EO-infected rhesus macaques were stained using 2 fluorescently labelled broadly neutralizing antibodies previously shown to identify in vitro SHIVAD8-EO-infected rhesus macaque CD4 T cells. SHIV-infected cells from 2 rhesus macaques were index sorted between Fiebig stages 1 and 6. For each animal lymph nodes (LNs) were longitudinally sampled 3 times, and blood was longitudinally sampled 5 times. SHIV infection was confirmed by measurement of single cell SHIV gag-RNA by RT PCR. SmartSeq NGS libraries were prepared, then sequenced using an Illumina NovaSeq 6000. Bcl2fastq2, STAR, Picard and HTSeq were used to demultiplex and map reads, sequence and calculate read number and analyze differentially expression for each gene. Benjamini Hochberg correction was used for multiple comparison correction.
Results
The frequency of SHIV-infected cells in both peripheral blood and LN CD4 T cells, intracellular SHIV RNA copy number and blood viral load all peaked between Fiebig stages 2 and 5, and then decreased in these macaques. During this time the frequency of SHIV infected cells increased in PD1 positive and CD28 dim CD4 T cells and then decreased. Intracellular SHIV RNA was inversely associated with CD3 and CD4 fluorescence intensity. Infection of germinal center CD4 T cells was discordant in these animals. One animal showed no germinal center CD4 SHIV infection, in the other most infection occurred in the germinal center.
Envelope staining did not strictly identify SHIV infected CD4 T cells. In some populations the frequency of SHIV RNA in envelope stained CD4 T cells was close to 40%. This allowed the comparison of SHIV-infected and non-infected CD4 T cells in 57 Env+SHIV RNA+ and 80 Env–SHIV RNA– LN CD4 T cells from a single sample. The frequency of SHIV RNA was as high as 5% of the sequenced transcriptome. The frequency of HIV RNA reads increased with CD3 and CD4 down regulation. GSEA analysis suggests G2M arrest and decreased antiviral responses in SHIV-infected CD4 T cells.
Conclusions
Ex vivo isolation of acutely infected SHIV CD4 T cells using surface markers is possible. This enabled the identification of specific subsets of CD4 T cells with greatly increased frequencies of infection, allowing further characterization and transcriptome analysis of SHIV-infected CD4 T cells.