The role of CD32 during HIV-1 infection

Abstract

Persistence of latent HIV-1 in long-lived resting memory CD4+ T cells is a major barrier to curing HIV-1 infection, and thus a biomarker for latently infected cells would be of great scientific and clinical importance.^,,,, Through an elegant discovery-based approach, Descours et al. reported that CD32a, an Fcγ receptor not normally expressed on T cells, is a potential biomarker for latently infected cells. Using the quantitative viral outgrowth assay, we show that CD32+ CD4+ T cells do not harbor the majority of intact proviruses in the latent reservoir and that the enrichment found by Descours et al. may in part reflect the use of an ultrasensitive ELISA for HIV-1 p24 antigen that does not predict exponential viral outgrowth. Our studies show that CD32 is not a biomarker for the major population of latently infected CD4+ T cells.

Fig. 1.

A) Percentage of CD4+CD32^hi T cells relative to total CD4+ T cells in healthy donors and HIV-1 infected donors. Infected donor values reported by Descours et al. were obtained from Supplementary Table 4 of reference . B) Schematic depicting 3 strategies used to obtain different populations of CD4+ T cells plated in QVOAs. C) Numbers of sorted CD4+CD32^hi and CD3+CD4+CD32^hi T cells from each subject plated in QVOAs. D) Frequencies of latently infected cells among CD4+CD32^hi T cells and CD4+CD32^neg T cells and among total CD4+ T cells from the same subjects previously measured in separate experiments. Cells were isolated using protocol 1 (colors coordinate with subject values from Fig 1c). E) Probability of detecting outgrowth based on measured frequencies of latently infected cells among the CD4+CD32^neg fraction and number of CD4+CD32^hi cells plated assuming various degrees of enrichment of HIV-1 in CD32^hi cells. F) Frequencies of latently infected cells measured in QVOAs using positive or negative selection to obtain total CD4+ cells (protocol 2; positive selection was accomplished using either sorting or CD4 microbead strategies, with similar results). G) Comparison of proviral DNA measurements obtained with Qpcr on total CD4+ cells purified using positive or negative selection (protocol 2). H) Frequencies of latently infected cells among total CD4 cells, and CD3+CD4+CD32^neg and CD3+CD4+CD32^hi populations. Cells were isolated using protocol 3 (colors coordinate with subject values from Fig 1c).

Fig. 2.

Ultrasensitive p24 measurements as a possible factor in overestimation of latent infection. A) p24 values from CD32+ culture wells measured with ELISA versus SIMOA (Lower limit of quantification (LLOQ): 5–10 pg/mL and 0.01 pg/mL, respectively) (data collected from three subjects, for a total of 71 wells). B) Longitudinal p24 values measured with SIMOA in individual culture wells in the QVOA for CD32^neg cells from subject 5, showing wells with and without viral outgrowth (red and blue circles, respectively). C) p24 values in QVOA wells of CD32^hi and CD32^neg cultures. Day 21 values are shown for CD32^neg culture wells with outgrowth as detected by ELISA (red), CD32^neg culture wells with no outgrowth by ELISA but with positive SIMOA values (blue), and CD32^hi culture wells with no outgrowth by ELISA but with positive SIMOA values (orange). Date were collected from subjects 2, 4, and 5. p values were calculated with a non-parametric t-test. D) IUPM calculation based on ELISA versus SIMOA. Symbols in dark grey represent values below the limit of detection. E) Fold enrichment of IUPM in CD32+ cells (subject ID shown on the horizontal axis).