Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size

Abstract

Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCMdespite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIVex vivo Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not.

Importance: HIV infection is incurable due to a long-lived reservoir of HIV(+)memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients.

FIG 1

Central memory CD4 T cells express CASP8 and Casp8p41 in HIV infection. (A) Cytosolic extracts from T_CM and T_EM were assessed for expression of Casp8 and BCL-2 by Western blotting and densitometry. (B) PBMCs from five HIV-infected patients were assessed for intracellular Casp8p41 expression in CD4⁺ T cell subsets and in CD3 negative cells. (C) Depicted is representative flow cytometry data showing the gating strategy to determine intracellular Casp8p41 expression in CD4⁺ T cell subsets: naive (T_N), central memory (T_CM), effector memory (T_EM), and total CD4 T cells. CD3⁻ cells, which are not infected by HIV and thus do not express HIV protease, were used as negative gating controls.

FIG 2

Central memory CD4 T cells are more resistant to apoptosis than effector memory CD4 T cells. (A and B) PBMCs from three donors were treated overnight with CH11, cycloheximide (CHX), etoposide (ETP), camptothecin (CPT), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), or hydrogen peroxide (H₂O₂). Cell death was assessed by flow cytometry using light scatter (A) and Live/Dead stains (B); mean (SEM) results of treatment specific killing (treatment-control) from three independent donors. (C) T_CM and T_EM were isolated from two HIV-infected donors by magnetic bead separation and gene expression measured by RNA sequencing. Depicted are differentially expressed genes associated with cell proliferation and cell death. The genes are sorted by log₂-fold change. At the top are the cell proliferation genes. Below are the cell death genes. (D) Gene expression differences in T_CM versus T_EM were validated in a separate publicly available data set of six uninfected donors.

FIG 3

BCL-2 overexpression decreases cell death induced by Casp8p41 and increases viral replication. (A) Parental Jurkat T cells or Jurkat T cells stably overexpressing BCL-2 (Jurkat-BCL-2) were transfected with GFP-Casp8p41 or GFP alone and assessed for cell death via TUNEL. (B) Mean (standard deviation [SD]) results from three independent experiments as in panel A. (C) The relative GFP-Casp8p41 expression in GFP-positive cells was compared between Jurkat and Jurkat-BCL-2 cells. (D and E) Parental Jurkat cells or Jurkat-BCL-2 cells were transfected with GFP-Casp8p41 or GFP alone and assessed for active BAK expression in GFP-positive cells, using a conformational specific antibody which detects only active BAK. Depicted are the individual mean fluorescence intensities (MFI) of active BAK (D) and representative dot plots (E) from two independent experiments. (F) Parental Jurkat cells or Jurkat-BCL-2 cells were infected with HIVIIIb or mock infected and then assessed for HIV p24 production in culture supernatant at day 9 postinfection. Depicted are the mean (SD) values of three independent experiments.

FIG 4

Casp8p41 binds to the BH3 binding groove of antiapoptotic BCL-2 family members. (A) 293T cells transfected with HA-empty vector, HA-Casp8p41, or HA-Casp8p41 EK were immunoprecipitated with HA-matrix or agarose-conjugated antibody (IgG or BCL-2) and immunoblotted as indicated. (B) Binding of 12.5 to 200 nM GST-BCL-2 to immobilized GST-Casp8p41, as assessed by SPR. (C) K_ds for GST-BCL-2, GST-BCL-X_L, and GST-MCL-1 binding to GST-Casp8p41. (D) The multiple low-mass molecular dynamics simulation-refined model of the BH3-like domain of Casp8p41 (green) bound at the human BCL-2 BH3-binding groove. (E) Binding of 800 nM GST-BCL-2 or GST-BCL-2 R146A to immobilized GST-Casp8p41. (F) K_ds determined as in panel E. (G) Binding of 200 nM GST-Casp8p41 or GST-Casp8p41 Val150Glu/Leu157Glu to immobilized GST-BCL-2. (H) K_ds determined as in panel G. Bars in panels C, F, and H: means ± the SD from three independent experiments. (I) Binding of 800 nM GST-BCL-2 or GST-BCL-2 Arg146Ala to immobilized Casp8p41 activator BH3-like peptide. GST, glutathione S-transferase.

FIG 5

Venetoclax increases Casp8p41-induced apoptosis but spares cells that do not express Casp8p41. (A) Jurkat T cells pretreated with increasing concentrations of venetoclax or diluent dimethyl sulfoxide (DMSO) were transfected with EGFP-Casp8p41, vector control, or no DNA and assayed for cell death by TUNEL. Data are representative of three independent experiments. (B) Primary CD4 T cells from seven uninfected donors were treated with venetoclax (1 μM) or diluent and assayed for cell viability by flow cytometry over 5 days. (C) Primary CD4 T cells from four uninfected donors were stimulated with phytohemagglutinin (2 μg/ml) in the presence of venetoclax (1 μM) or diluent and assayed for proliferation by CFSE staining. (D) Primary CD4 T cells from seven ART-suppressed HIV-positive patients were treated with venetoclax (1 μM) or DMSO control for 24 h and assessed for viability by trypan blue exclusion.

FIG 6

Apoptotic cell death is associated with loss of protein and mRNA markers. Jurkat T cells stably expressing eGFP were treated with DMSO or CPT and assessed for cell viability by ATP content (A) and light scatter (B) over time. (C) Expression of eGFP was measured by flow cytometry. (D) Actin mRNA expression was assessed by qRT-PCR and expressed as mean C_T values normalized to baseline DMSO control. (E to G) Cell death was assessed by active-caspase 3 expression (E), TUNEL (F), and Live/Dead viability stain (G) and compared between eGFP-positive and eGFP-negative cells. Values in panels A to D represent the mean (range) of two independent experiments. The results the panels E to G are representative of two independent experiments.

FIG 7

Selective BCL-2 inhibition reduces cell associated DNA during HIV reactivation ex vivo. (A) Primary CD4 T cells isolated by negative selection from cryopreserved PBMCs of long-term virologically suppressed HIV-infected patients (n = 4) were treated for 16 h with venetoclax or DMSO vehicle in the presence of tenofovir and raltegravir to prevent spreading infection and then exposed to plate-bound αCD3 and soluble αCD28 antibody to induce HIV reactivation. After 72 h, cell-associated HIV DNA was measured. (B) Freshly obtained peripheral CD4 T cells from an additional 16 suppressed HIV-infected patients were treated with venetoclax (1 μM) or DMSO in the presence of tenofovir/raltegravir, followed by αCD3/αCD28 for 72 h, and cell associated HIV DNA was measured. Depicted is the ratio of HIV DNA in venetoclax-treated versus diluent-treated samples for each patient, with measurable HIV DNA in the diluent sample (n = 11). (C) Primary CD4 T cells from four HIV-infected subjects were treated with venetoclax or diluent and induced to reactivate HIV using CD3/CD28, and cell death was measured in HIV P24-positive or -negative cells using activated caspase 3 staining. (D) HIV RNA was measured in cell culture supernatant from 6 of the 11 patient experiments from panel B.

FIG 8

BCL-2 antagonism as a model of “prime, shock, and kill” for decreasing the HIV reservoir. (A) Reactivation of latent HIV does not lead to death of the reactivated cell due to inhibition of Casp8p41 by direct binding to BCL-2. Therefore, the reactivated cell survives and produces progeny virus. (B) Antagonism of BCL-2 activity (as with venetoclax treatment) allows Casp8p41 to bind proapoptotic BAK, leading to apoptotic death of the reactivated cell and to decreased total HIV DNA.