Fig 1.
Resting primary CD4+ T cells support both productive and latent HIV infection.
(A) Diagram of the HIV Duo-Fluo I virus, in which eGFP has replaced the nef gene, and a whole transcription unit—consisting of an EF1α promoter driving the expression of an mCherry fluorescent marker—has been inserted downstream. Upon infection with the HIV Duo-Fluo I virus, cells that express GFP alone or GFP and mCherry are considered productively infected; cells that express only mCherry are considered latently infected; cells that lack expression of either fluorescent marker are considered uninfected. (B) Expression of the activation markers CD69 and CD25 in resting primary CD4+ T cells either left untreated or stimulated with CCL19, IL-7, or αCD3/αCD28 activating beads for 72 h. Mean Fluorescence Intensity (MFI) for CD25 expression is also shown. (C) Infection profiles of untreated or stimulated primary CD4+ T cells 6 days after infection via flow cytometry. Untreated resting CD4+ T cells were infected with either HIV Duo-Fluo I virus alone or the Vpx-containing HIV Duo-Fluo I virus. Stimulated cells were infected with HIV Duo-Fluo I alone. Productive infection (GFP+ and GFP/mCherry double-positive) and latent infection (mCherry+) were analyzed by flow cytometry. Data shown are from a single donor but are representative of three separate donors. (D) Quantified values of latent infection and productive infection from panel C. Data represents the average of three donors. (E) Ratios of latent infection to productive infection were calculated using data from panel D. Data represent the average of three donors. (F) Quantified values for reactivation of pre-integration latent virus and post-integration provirus calculated from the isolated uninfected populations (GFP/mCherry double-negative) of untreated and stimulated primary CD4+ T cells via flow cytometry (S4 Fig). Six days after infection, uninfected cells were isolated via fluorescence-activated cell sorting (FACS) and were either left unstimulated or stimulated with αCD3/αCD28 activating beads alone or αCD3/αCD28 activating beads in the presence of raltegravir for 48 h. Reactivatable pre-integration latent virus was calculated by subtracting the amount of productive infection from cells treated with αCD3/αCD28 activating beads alone and cells treated with αCD3/αCD28 activating beads in the presence of raltegravir. Reactivatable post-integration latent provirus was calculated by subtracting the amount of productive infection from unstimulated cells and cells treated with αCD3/αCD28 activating beads in the presence of raltegravir. Data represent the average of three donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant.
Fig 2.
Primary CD4+ T cells transitioning from an activated state back to a resting state are more likely to become latently infected.
(A) Schematic of experimental procedure. Primary CD4+ T cells were isolated from uninfected donor blood and stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 72 h and were then allowed to return to a resting state over 20 days in the presence of IL-2. Cells were infected at peak activation (day 4) and every 5 days thereafter as they returned to a resting state. (B) Expression of activation markers CD69 and CD25 as the cells transition from an activated state to a resting state. Flow cytometry was performed 72 h post activation and every 5 days after the activation beads were removed. Data shown are from a single donor, but representative of three separate donors. Percentage of live cells is calculated from the live gate (forward vs side scatter plots) in the FACS analysis and represents the average of three donors. (C) Quantified values of the cells’ activation status from panel B. Data represents the average of three donors. (D) Infection profiles of primary CD4+ T cells as they transition back to a resting state. Cells were spinoculated with HIV Duo-Fluo I 72 h after activation and every 5 days after the activation beads were removed. Infection was analyzed by flow cytometry 72 h post-infection. Data shown are from a single donor, but representative of three separate donors. (E) Quantified values of latent infection and productive infection from panel D. Data represents the average of three donors. (F) Ratios of latent infection to productive infection were calculated using data from panel E. Data represents the average of three donors. *, P < 0.05; **, P < 0.01; ns, non-significant.
Fig 3.
Productively infected and latently infected primary CD4+ T cells isolated by FACS return to a resting state.
(A) Schematic of experimental procedure. Primary CD4+ T cells were isolated from uninfected donor blood and stimulated with αCD3/αCD28 activating beads in the presence of IL-2 for 72 h and then infected with HIV Duo-Fluo I virus. Productive, latent, and uninfected cell populations were isolated via FACS 4 days after infection and were allowed to return to a resting state for 11 days in the presence of IL-2. (B) Expression of activation markers CD69 and CD25 in each isolated population as they return to a resting state. (C) Infection profiles of each isolated population as they return to a resting state, as analyzed by flow cytometry. (D) Reactivation of isolated cell populations after returning to a resting state. Cells were split in half and either left unstimulated or stimulated with αCD3/αCD28 activating beads for 48 h. All data shown are from a single donor, but representative of three separate donors.
Fig 4.
Primary CD4+ T cells and total lymphoid cell populations isolated from peripheral blood and tonsillar and splenic tissues are more likely to become latently infected.
(A) Expression of activation markers CD69 and CD25 on CD4+ T cells and total lymphoid cell populations either left untreated or stimulated with αCD3/αCD28 activating beads for 72 h. Data shown are from a single donor, but are representative of three separate donors. (B) Infection profiles of CD4+ T cells and total lymphoid cell populations from panel A. Cells were infected with HIV Duo-Fluo I and analyzed for productive and latent infection 72 h after infection. Data shown are from a single donor, but are representative of three separate donors. (C) Quantification of latent infection and productive infection from panel B. Data represent the average of three donors. (D) Ratios of latent infection to productive infection were calculated using data from panel C. Data represent the average of three donors. *, P < 0.05; ***, P < 0.001; ns, nonsignificant.