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MAJORITY OF THE LATENT RESERVOIR RESIDES IN CD32A NEGATIVE CD4 T CELLS
Lynn N. Bertagnolli1, Jennifer White1, Subul A. Beg1, Gregory Laird1, Francesco R. Simonetti1, Jun Lai1, Costin Tomescu2, Alexandra J. Murray1, Annukka Antar1, Hao Zhang1, Joseph B. Margolick1, Luis Montaner2, Robert Siliciano1, Janet Siliciano1
1The Johns Hopkins University, Baltimore, MD, USA,2Wistar Institute, Philadelphia, PA, USA
The persistence of HIV-1 in a stable reservoir in resting CD4+ T cells is the major barrier to curing HIV-1 infection. Cell surface biomarkers that could distinguish cells comprising this latent reservoir from uninfected cells have been lacking. If identified, these biomarkers could significantly enhance the progress towards an HIV cure. A recent study (Descours et al., 2017) identified the cell-surface protein CD32a, a low-affinity Fc receptor for IgG that is commonly found on B cells and cells of the myeloid lineage, as a potential marker for the latent reservoir.
To explore this biomarker, CD4+ T cells were isolated from 6 HIV-1 infected patients virally suppressed on cART for at least 6 months and sorted for the expression of CD32. CD4+CD32- and CD4+CD32+ T cells were plated and tested for the presence of infectious HIV-1 using the quantitative viral outgrowth assay (QVOA). Additional studies compared viral outgrowth and HIV-1 proviral DNA levels in CD4+ T cells isolated using differing selection methods in order to investigate the possibility that CD32+ CD4 T cells were being removed using the negative depletion method for purifying CD4+ T cells.
In cultures from 6 aviremic patients in which CD32 sorting was performed, no viral outgrowth was detected in CD4+ T cells expressing CD32 using the standard ELISA assay for HIV-1 p24 antigen. In contrast, CD4+CD32- cultures showed viral outgrowth that was comparable in frequencies previously measured from the same patients as well as to historical controls. Since an ultrasensitive p24 assay was used in the original report, we also analyzed culture supernatants with this method. Using this assay, low levels of p24 slightly above the limit of detection were seen in CD32+ cultures, but levels did not increase exponentially over time. Studies using different modes of total CD4+ T cell isolation, including positive selection or negative depletion, showed no difference in viral outgrowth.
We conclude that an enrichment of HIV-1 infected cells is not observed in viral outgrowth cultures of CD32+ CD4+ T cells while CD32- CD4+ T cells from the same donors had expected levels of infected cells. Detection of p24 antigen using ultrasensitive methods may represent defective virus or assay artifacts. Our results demonstrate that the cell-surface molecule CD32a does not specifically mark the latent reservoir, and that additional efforts are needed to identify biomarkers for latently infected cells.