Naive CD4+ T Cells Harbor a Large Inducible Reservoir of Latent, Replication-competent Human Immunodeficiency Virus Type 1

Abstract

Background: The latent human immunodeficiency virus type 1 (HIV-1) reservoir represents a major barrier to a cure. Based on the levels of HIV-1 DNA in naive (TN) vs resting memory CD4+ T cells, it is widely hypothesized that this reservoir resides primarily within memory cells. Here, we compared virus production from TN and central memory (TCM) CD4+ T cells isolated from HIV-1-infected individuals on suppressive therapy.

Methods: CD4+ TN and TCM cells were purified from the blood of 7 HIV-1-infected individuals. We quantified total HIV-1 DNA in the CD4+ TN and TCM cells. Extracellular virion-associated HIV-1 RNA or viral outgrowth assays were used to assess latency reversal following treatment with anti-CD3/CD28 monoclonal antibodies (mAbs), phytohaemagglutinin/interleukin-2, phorbol 12-myristate 13-acetate/ionomycin, prostratin, panobinostat, or romidepsin.

Results: HIV-1 DNA was significantly higher in TCM compared to TN cells (2179 vs 684 copies/106 cells, respectively). Following exposure to anti-CD3/CD28 mAbs, virion-associated HIV-1 RNA levels were similar between TCM and TN cells (15 135 vs 18 290 copies/mL, respectively). In 4/7 donors, virus production was higher for TN cells independent of the latency reversing agent used. Replication-competent virus was recovered from both TN and TCM cells.

Conclusions: Although the frequency of HIV-1 infection is lower in TN compared to TCM cells, as much virus is produced from the TN population after latency reversal. This finding suggests that quantifying HIV-1 DNA alone may not predict the size of the inducible latent reservoir and that TN cells may be an important reservoir of latent HIV-1.

Keywords: HIV-1; latent; memory; naive; reservoir.

Figure 1.

Quantification of the total human immunodeficiency virus type 1 (HIV-1) DNA reservoir in CD4+ T naive (T_N), T central memory (T_CM), transitional memory (T_TM)+effector memory (T_EM) cells purified from HIV-infected individuals on antiretroviral therapy. A, Quantification of total HIV-1 DNA in CD4+ T_N, T_CM, and T_TM+T_EM cells. Total HIV-1 DNA was normalized to the cell number (determined by quantification of the CCR5 gene). B, The frequency of different resting CD4 T-cell subsets in the peripheral blood of HIV-1–infected individuals. C, Contribution (%) of the CD4+ T_N, T_CM, and T_TM+T_EM cells to the total HIV-1 DNA reservoir, taking into account both the frequency of the CD4+ T-cell subset and the frequency of HIV-1 DNA in that subset. In the plots, each colored dot represents a unique donor. All P values were determined using a Mann–Whitney test. Abbreviation: PBMC, peripheral blood mononuclear cell.

Figure 2.

Total virus recovery from CD4+ T naive (T_N) and T central memory (T_CM) cells from human immunodeficiency virus type 1 (HIV-1)–infected individuals on antiretroviral therapy following latency reversal. A, Total copies of extracellular virion-associated HIV-1 RNA produced from T_N or T_CM cells after exposure to the latency reversing agents indicated. B, Copies of HIV-1 RNA produced per infected CD4+ T_N or T_CM cell. Each colored dot represents a unique donor. Solid dots represent data from CD4+ T_N cells, while open dots represent data from CD4+ T_CM cells. N = 7; except for phorbol-myristate-acetate +Iono, prostratin, and panobinostat, where N = 6. Abbreviations: HIV-1, human immunodeficiency virus type 1; lono, ionomycin; LOQ, limit of quantification; PHA, phytohaemagglutinin; PMA, phorbol-myristate-acetate; T_EM, T effector memory cell.

Figure 3.

Impact of the latency reversing agent (LRA) on global T-cell activation and cell viability on CD4+ T naive (T_N) and T central memory (T_CM) cells purified from human immunodeficiency virus type 1 (HIV-1)–infected individuals on antiretroviral therapy. A, Expression of the T-cell activation markers CD25, CD69, and HLA-DR on CD4+ T_N and T_CM cells 7 days post-LRA exposure. All data are shown as the mean ± standard error of the mean (SEM; N = 7). B, CD4+ T_N or T_CM cell viability assessed 7 days post-LRA exposure. All data are shown as the mean ± SEM (N = 4). No significant differences were noted between the 2 cell types, as measured by a Mann–Whitney test. Abbreviations: lono, ionomycin; PHA, phytohaemagglutinin; PMA, phorbol-myristate-acetate; T_EM, T effector memory cell.

Figure 4.

Quantification of replication-competent human immunodeficiency virus type 1 (HIV-1) in CD4+ T naive (T_N), T central memory (T_CM), transitional memory (T_TM)+effector memory (T_EM) cells from HIV-1–infected donors on antiretroviral therapy. A, Infectious units per million cells (IUPM) values determined from T_N, T_CM, and T_TM+T_EM cells. B, IUPM values determined for T_N cells with (T_N cells) and without (T_NCD95-) T stem cell-like memory cells. Each colored dot represents a unique donor. Data are shown as the mean ± standard error of the mean. P values were determined using an unpaired t test.