Background: Identification of live HIV-infected CD4 cells would allow the characterization of these cells by flow cytometry and single cell transcriptomic analysis.
Methods: bnAbs were screened for their ability to identify individual BaL-infected primary CD4 T cells as identified by P24 intracellular staining and transcription of Gag, and D1A3 (Tat-associated) and D1A4b (Rev-associated) spliced HIV RNA. CD4 mimic, V1V2 and the V3 glycan bnAbs were found that stained BaL-infected cells. bnAbs used for ex vivo studies were selected based on breadth of neutralization of B clade virus, and sensitivity and intensity of staining of HIV-infected CD4 T cells. Live CD3+CD45RO+CD8–CD14–CD19–TCRδγ– CD4 dim and null cells prepared from lymph nodes were stained for expression of PD-1, CD57, CXCR4, CXCR5, CD27 and PGT121 and then bulk sorted into PD-1+; PD-1–CD57–; and PD-1–CD57+ populations. Individual PD1+ cells were index sorted into individual wells, RNA was extracted, purified, and Gag, and Rev- and Tat-associated RNA copy numbers determined by RT PCR. Measureable Tat- and Rev-associated HIV RNA were assumed to represent active transcription of HIV proviral DNA.
Results: The highest frequency of CD4 T cells transcribing proviral DNA was found in the PD-1+, CD4 dim and null population. PD-1+ cells were index sorted and Gag, Tat- and Rev-associated HIV RNAs measured. Ninety-one of 599 (15%) cells sorted from lymph nodes from six untreated, HIV-infected individuals, were actively transcribing HIV RNA. Eighty-five percent of the cells transcribing Tat- and Rev-associated RNA also contained measurable Gag RNA. Staining of HIV envelope by PGT121 was significantly associated with proviral DNA transcription (P<0.0001). Median percentage of PGT121+ CD4 dim and null T cells actively transcribing virus was 41 (range, 12-64)% compared to 17(3-22)% in PGT121– cells. PGT121 staining was strongly associated with downregulation of CD4 (P<0.0001). A higher percentage of CXCR5+PD1+ cells actively transcribed proviral DNA than did CXCR5–PD-1+ cells (P<0.0001). A lesser proportion of CD57+ CD4 T cells, a marker of germinal center cells, actively transcribed proviral DNA than did CD57– CD4 T cells (P<0.05).
Conclusions: Most CD4 T cells transcribing proviral DNA are Tfh cells. Viral transcription occurs both inside and outside of the germinal center in B cell follicles and is identifiable by bnAbs. bnAbs may be a means of targeting HIV-infected CD4 T cells in lymph nodes.