Latent HIV-1 infection occurs in multiple subsets of hematopoietic progenitor cells and is reversed by NF-κB activation

Abstract

The ability of HIV-1 to establish a latent infection presents a barrier to curing HIV. The best-studied reservoir of latent virus in vivo is resting memory CD4(+) T cells, but it has recently been shown that CD34(+) hematopoietic progenitor cells (HPCs) can also become latently infected by HIV-1 in vitro and in vivo. CD34(+) cells are not homogenous, however, and it is not yet known which types of CD34(+) cells support a latent infection. Furthermore, the mechanisms through which latency is established in this cell type are not yet known. Here we report the development of a primary cell model for latent HIV-1 infection in HPCs. We demonstrate that in this model, latent infection can be established in all subsets of HPCs examined, including HPCs with cell surface markers consistent with immature hematopoietic stem and progenitor cells. We further show that the establishment of latent infection in these cells can be reversed by tumor necrosis factor alpha (TNF-α) through an NF-κB-dependent mechanism. In contrast, we do not find evidence for a role of positive transcription elongation factor b (P-TEFb) in the establishment of latent infection in HPCs. Finally, we demonstrate that prostratin and suberoylanilide hydroxamic acid (SAHA), but not hexamethylene bisacetamide (HMBA) or 5-aza-2'-deoxycytidine (Aza-CdR), reactivate latent HIV-1 in HPCs. These findings illuminate the mechanisms through which latent infection can be established in HPCs and suggest common pathways through which latent virus could be reactivated in both HPCs and resting memory T cells to eliminate latent reservoirs of HIV-1.

Figures

Fig 1

(A to D) GM-CSF and TNF-α reactivate latent virus in a primary cell model for HIV-1 infection of CD34+ HPCs. (A) Time course for latency experiments. (B) Schematic diagrams of the HIV-1 reporter viruses used for these studies. Viruses were pseudotyped with the VSV-G envelope (center) or the CXCR4-tropic HIV-1 HXB envelope (right). The shading of vpr and vpu in HXB-ePLAP indicates that these genes are defective in full-length HXB. Filled rectangles represent genes that have been added to or deleted from the wild-type viral clone. (C) Flow cytometric analysis of cells sorted for reactivation protocols. (Top) Cord blood-derived HPCs infected with HXB-ePLAP/VSV-G and PLAP− cells isolated by magnetic sorting; (bottom) cells infected with NL4-3-ΔGPE-GFP/VSV-G and GFP− cells isolated by flow sorting. The percentages of live cells that are PLAP+ or GFP+ are given in each panel. Live cells were gated using forward scatter (FSC) and side scatter (SSC) parameters. (D) Flow cytometric analysis of cells for which results are shown in panel C after overnight incubation with STIF medium or GM-CSF (100 ng/ml) and TNF-α (2.5 ng/ml). CD34+ cells are gated on at the left; then PLAP or GFP expression in uninfected, STIF-treated, or GM-CSF- and TNF-α-treated cells is shown on the right. Numbers indicate the percentages of cells within the labeled gate. Live cells were gated based on FSC, SSC, and 7-AAD. (E) CD3+ T cells are not present in the CD34+ HPC population. Cord blood-derived HPCs were sorted and infected with NL4-3-ΔGPE-GFP/VSV-G as outlined in panel A. CD3 and CD34 staining was assessed on day 6 following flow sorting and overnight incubation in STIF medium. Live cells were gated using FSC, SSC, and 7-AAD. Data are representative of two independent experiments, one each using cord blood- or bone marrow-derived HPCs. (F to H) Latent infection that can be reactivated with GM-CSF and TNF-α occurs in immature CD133+ HPCs. (F) Flow cytometric analysis of CD133+ cells treated with HXB-ePLAP/VSV-G and sorted to remove actively infected (PLAP+) cells. The percentages of live cells are given in each quadrant. Live cells were gated using forward scatter and side scatter. (G) Flow cytometric analysis of cells sorted as in panel F and then stimulated overnight with STIF medium or GM-CSF and TNF-α. Live cells were defined by FSC, SSC, and 7-AAD. Numbers indicate the percentages of cells falling within the indicated gate. (H) Quantitation of reactivation in CD133+ cells (cells sorted for CD133 prior to stimulation) relative to that in cells incubated in STIF medium. Means and standard errors for 4 independent experiments are shown. The asterisk indicates a significant difference (P = 0.01) from the expected fold increase of 1 by a 1-sample t test.

Fig 2

Latent infection occurs in all subsets of CD34+ HPCs defined by CD38 and CD45RA expression. (A) Diagram of hematopoiesis showing CD34, CD133, CD38, and CD45RA expression on each cell subset. HSC, hematopoietic stem cell; MPP, multipotent progenitor; MLP, multilymphoid progenitor; CMP, common myeloid progenitor; MEP, megakaryocyte/erythrocyte progenitor; GMP, granulocyte/monocyte progenitor; B-NK, B and NK cell progenitor. (B) Flow cytometric analysis of CD133+ cells treated with HXB-ePLAP/HXB Env, sorted to remove actively infected (PLAP+) cells, and stimulated overnight with STIF medium or GM-CSF and TNF-α. Live cells were defined using forward scatter (FSC), side scatter (SSC), and 7-AAD; then they were analyzed for CD34, CD38, CD45RA, and PLAP expression. The percentage of total cells is given in each quadrant. (C) Summary of experiments similar to those shown in panel B but using the HXB-ePLAP/VSV-G envelope to infect cells. Shown is the fold induction of active infection in cells treated with GM-CSF and TNF-α or with TNF-α alone relative to cells incubated in medium. Labels are colored to match cell types in panel A. Means and standard errors for 6 independent experiments are given. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001) by a 1-sample t test from the expected fold increase of 1,. Differences in the fold induction of active infection between cell subsets are not significant (P, 0.26 by 1-way analysis of variance). (D) Summary of experiments for which results are shown in panel B. Shown is the fold induction of active infection in cells treated with GM-CSF and TNF-α or with TNF-α alone relative to unstimulated cells. Means and standard errors for 2 independent experiments are shown. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) by a 1-sample t test from the expected fold increase of 1. Differences in the fold induction of active infection between cell subsets are not significant (P, 0.53 by 1-way analysis of variance). (E) Summary of experiments similar to those shown in panel B but using the NL4-3-ΔGPE-GFP/VSV-G envelope to infect bone marrow cells. Shown is the fold induction of active infection in cells treated with TNF-α relative to unstimulated cells. Means and standard errors for 2 independent experiments are given. Asterisk indicates a significant difference (*, P < 0.05) by a 1-sample t test from the expected fold increase of 1. Differences in the fold induction of active infection between cell subsets are not significant (P, 0.11 by 1-way analysis of variance).

Fig 3

GM-CSF- and TNF-α-treated HPCs have higher nuclear NF-κB DNA binding activity than STIF medium-treated cells. (A) Quantitation of the results of a transcription factor ELISA measuring NF-κB DNA-binding activity in nuclear lysates from HPCs incubated with STIF medium or with GM-CSF plus TNF-α. Absorbance was normalized to that of the positive control on each plate; then nonspecific activity (defined by the activity obtained using cytoplasmic extracts) was subtracted from the nuclear absorbance, and the difference was graphed as arbitrary units (AU). The value for the positive control, a Raji nuclear cell extract, was set to 1 AU. Means and standard errors for 3 independent experiments are shown. Asterisk indicates a significant difference (*, P < 0.05) by a paired t test. (B) Quantitation of the results of a transcription factor ELISA measuring AP-1 DNA-binding activity in nuclear lysates from HPCs incubated with STIF medium or with GM-CSF plus TNF-α overnight. Data were analyzed as described for panel A except that nuclear extracts of K-562 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate were used as the positive control. Means and standard errors for 2 independent experiments are shown. AP-1 nuclear activity was not significantly elevated by GM-CSF and TNF-α treatment (P, >0.2 for all subunits by a paired t test); however, JunB expression in STIF medium-treated cells (P < 0.05) and FosB (P < 0.01) and Fra-1 (P < 0.05) expression in GM-CSF- and TNF-α-treated cells were significantly different from zero by a 1-sample t test.

Fig 4

TNF-α reactivates latent infection in HPCs via an NF-κB-dependent pathway. (A) TNF-α is sufficient to reactivate latent virus. CD133+ cells treated with NL4-3-ΔGPE-GFP/VSV-G, sorted to remove actively infected cells, and incubated overnight with the indicated cytokines were analyzed by flow cytometry. Numbers show the percentages of cells falling within the indicated gates. Live cells were gated using forward scatter (FSC), side scatter (SSC), and 7-AAD. (B) Quantitative analysis of reactivation in cells treated as described for panel A and incubated with 10-fold dilutions of TNF-α in the presence or absence of an antibody to TNF-α. The mean fold increase in the percentage of cells expressing GFP over that for cells incubated in STIF medium alone is shown. Error bars represent standard errors for two independent experiments. (C) Quantitation of reactivation in cells treated as described for panel B except that cells were treated with HXB-ePLAP/VSV-G and were reactivated with or without the classical NF-κB pathway inhibitor IKK2 VI (10 μM). The mean fold increase in the percentage of cells expressing PLAP over that for cells incubated in STIF medium plus DMSO is shown. Error bars represent standard errors for two independent experiments.

Fig 5

The NF-κB inhibitor IKK2 VI counteracts TNF-α-induced reactivation of latent virus in HPCs. (A, C, and E) Flow cytometric analysis of CD133+ cells infected using the indicated envelope, sorted to remove actively infected cells, and stimulated overnight as indicated. Live cells were gated using forward scatter (FSC), side scatter (SSC), and 7-AAD; numbers indicate the percentage of cells within each gate. Results are representative of 4 (A) or 2 (C and E) independent experiments. (B, D, and F) Summary of reactivation for panels A, C, and E, respectively. The fold increase in the percentage of cells expressing PLAP over that for cells incubated in STIF medium plus DMSO was calculated. Means and standard errors for 4 (B) or 2 (D or F) independent experiments are shown. Asterisk indicates a significant difference (P, <0.01 [B], <0.02 [D], or <0.03 [F]) by a paired t test. (G) Quantitation of nuclear NF-κB p50 DNA binding activity for cells treated as for panel A and analyzed as described for Fig 4A. Means and standard errors for two experiments are shown.

Fig 6

Nuclear P-TEFb in HPCs is not increased by stimulation with GM-CSF and TNF-α. Western blot analysis was carried out for cyclin T1 and pCDK9 in nuclear extracts from CD133+ HPCs (CB) incubated for 5 days in STIF medium and then treated with STIF medium or GM-CSF (100 ng/ml GM-CSF) plus TNF-α (2.5 ng/ml). Controls were nuclear extracts from resting memory T (RM T) cells isolated from peripheral blood by magnetic selection for CD4+ CD45RO+ HLA-DR− CD25− CD69− cells and lysed immediately or after stimulation with anti-CD3/anti-CD28 beads overnight, as well as nuclear extracts from Jurkat cells incubated overnight in medium or 3 ng/ml TNF-α. Results are representative of two independent experiments using cells from separate donors (T cells) or separate donor pools (cord blood).

Fig 7

Prostratin and SAHA reactivate latent virus in CD34+ HPCs. (A) Quantitation of viral reactivation in CD133+ cells treated with prostratin, SAHA, or HMBA for 13 h. Reactivation was measured using the latency reactivation assay described for Fig 1 after infection with NL4-3-ΔGPE-GFP/VSV-G or HXB-ePLAP/VSV-G. The mean fold increase in the percentage of live CD34+ cells expressing PLAP or GFP over that with the solvent control is shown. Error bars represent standard errors for 3 (prostratin, SAHA) or 2 (HMBA) independent experiments. Asterisk indicates a significant difference (*, P < 0.05) by a 1-sample t test from the expected fold increase of 1. (B) Quantitation of reactivation by cells treated with prostratin (5 μM) or SAHA (10 μM) for 13 h by using the reactivation assay described for Fig 1. Means and standard errors for 7 independent experiments in cord blood (6 experiments) or bone marrow (1 experiment) are shown. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) by a paired t test. (C) Quantitation of reactivation in U1 cells stimulated for 48 h with 5 mM HMBA or a solvent control (H2O) and analyzed by flow cytometry to assess induction of HIV-1 Gag expression. Means and standard errors for three replicates are shown. (D) Quantitation of reactivation by cells treated with TNF-α, prostratin, SAHA, or HMBA for 24 h using the reactivation assay described for Fig. 1. Means and standard errors for 6 (TNF-α), 5 (HMBA), or 4 (prostratin or SAHA) independent experiments are shown. Asterisk indicates a significant difference (*, P < 0.03) by a 1-sample t test from the expected fold increase of 1. Induction of active infection with prostratin, SAHA, and HMBA is not significantly different from induction with TNF-α stimulation. (E) Quantitation of the results of a transcription factor ELISA measuring NF-κB DNA binding activity in nuclear extracts from HPCs infected with NL4-3-ΔGPE-GFP/VSV-G and stimulated for 24 h with the indicated compounds. ELISA data were analyzed as described for Fig 4A. Means and standard deviations for two replicates are shown. The fold increase in the percentage of cells expressing GFP over that with the solvent control is given under the bar graph. Results are representative of two independent experiments using cells from separate donor pools. (F) Time course of survival of HPCs cultured with TNF-α (3 ng/ml), prostratin (5 μM), or SAHA (10 μM). CD133+ HPCs were isolated from cord blood and were cultured in STIF medium for 5 days; then they were split into a solvent control or subjected to the conditions shown for an additional 3 days. The number of treated cells remaining is displayed as a percentage of the number of cells remaining in the solvent control (taken as 100%), as a function of time. Means and standard errors for 3 independent experiments are shown. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001) by a 1-sample t test from the expected cell count of 100% of the cell count in the solvent. (G) Images, obtained with light microscopy at ×400 magnification, of the cells analyzed in panel F. Images were collected at the indicated times poststimulation. Data are representative of 3 independent experiments.

Fig 8

Aza-CdR does not reactivate latent virus in CD34+ HPCs. (A) Quantitation of reactivation in J-Lat cells treated with the indicated compounds and assayed for GFP expression by flow cytometry. J-Lat cells treated with 30 ng/ml TNF-α and/or 1 μM Aza-CdR for 24 h were assayed for GFP expression (reporter for HIV-1 LTR activity) by flow cytometry after an additional 48 h. Means and standard errors of results from three J-Lat clones (J-Lat 8.4, J-Lat 9.2, and J-Lat 6.3) are shown. Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) by a paired t test. (B) Quantitation of reactivation of viral gene expression in CD133+ HPCs using the reactivation assay shown in Fig 1 following treatment with the indicated concentrations of Aza-CdR. The mean fold increase in the percentage of cells expressing GFP over that with the solvent control is shown. Error bars represent standard errors for 2 independent experiments. (C) Quantitation of reactivation of viral gene expression in CD133+ HPCs using the reactivation assay shown in Fig 1 following treatment with TNF-α and either Aza-CdR or the DMSO solvent control. The fold increase in the percentage of cells expressing GFP over that with the solvent control was calculated. Means and standard errors for 2 independent experiments are shown.