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. 2019 Jan 4;93(2):e01073-18.
doi: 10.1128/JVI.01073-18. Print 2019 Jan 15.

BCL6 Inhibitor-Mediated Downregulation of Phosphorylated SAMHD1 and T Cell Activation Are Associated with Decreased HIV Infection and Reactivation

Yanhui Cai  1 , Mohamed Abdel-Mohsen  1 , Costin Tomescu  1 , Fengtian Xue  2 , Guoxin Wu  3 , Bonnie J Howell  3 , Yong Ai  2 , Jie Sun  4 , Livio Azzoni  1 , Carole Le Coz  5 , Neil Romberg  5 , Luis J Montaner  6
Affiliations

BCL6 Inhibitor-Mediated Downregulation of Phosphorylated SAMHD1 and T Cell Activation Are Associated with Decreased HIV Infection and Reactivation

Yanhui Cai et al. J Virol. .

Abstract

Clearance of HIV-infected germinal center (GC) CD4+ follicular helper T cells (Tfh) after combination antiretroviral therapy (ART) is essential to an HIV cure. Blocking B cell lymphoma 6 (BCL6; the master transcription factor for Tfh cells) represses HIV infection of tonsillar CD4+ Tfh ex vivo, reduces GC formation, and limits immune activation in vivo We assessed the anti-HIV activity of a novel BCL6 inhibitor, FX1, in Tfh/non-Tfh CD4+ T cells and its impact on T cell activation and SAMHD1 phosphorylation (Thr592). FX1 repressed HIV-1 infection of peripheral CD4+ T cells and tonsillar Tfh/non-Tfh CD4+ T cells (P < 0.05) and total elongated and multispliced HIV-1 RNA production during the first round of viral life cycle (P < 0.01). Using purified circulating CD4+ T cells from uninfected donors, we demonstrate that FX1 treatment resulted in downregulation pSAMHD1 expression (P < 0.05) and T cell activation (HLA-DR, CD25, and Ki67; P < 0.01) ex vivo corresponding with inhibition of HIV-1 and HIV-2 replication. Ex vivo HIV-1 reactivation using purified peripheral CD4+ T cells from HIV-infected ART-suppressed donors was also blocked by FX1 treatment (P < 0.01). Our results indicate that BCL6 function contributes to Tfh/non-Tfh CD4+ T cell activation and cellular susceptibility to HIV infection. BCL6 inhibition represents a novel therapeutic strategy to potentiate HIV suppression in Tfh/non-Tfh CD4+ T cells without reactivation of latent virus.IMPORTANCE The expansion and accumulation of HIV-infected BCL6+ Tfh CD4+ T cells are thought to contribute to the persistence of viral reservoirs in infected subjects undergoing ART. Two mechanisms have been raised for the preferential retention of HIV within Tfh CD4+ T cells: (i) antiretroviral drugs have limited tissue distribution, resulting in insufficient tissue concentration and lower efficacy in controlling HIV replication in lymphoid tissues, and (ii) cytotoxic CD8+ T cells within lymphoid tissues express low levels of chemokine receptor (CXCR5), thus limiting their ability to enter the GCs to control/eliminate HIV-infected Tfh cells. Our results indicate that the BCL6 inhibitor FX1 can not only repress HIV infection of tonsillar Tfh ex vivo but also suppress HIV infection and reactivation in primary, non-Tfh CD4+ T cells. Our study provides a rationale for targeting BCL6 protein to extend ART-mediated reduction of persistent HIV and/or support strategies toward HIV remission beyond ART cessation.

Keywords: BCL6; HIV infection; SAMHD1; T cell activation; Tfh.

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Figures

FIG 1
FIG 1
Phenotypic analysis and immune response protein profiles among different CD4+ T subsets. (A) Flow cytometry gating strategy, phenotype analysis, and BCL6 expression in five tonsillar CD4+ T cell subsets (n = 5). (B) Gating strategy for sorting human tonsil Tfh and non-Tfh cells after enrichment of CD4 subsets by a negative-selection method. (C) Relative expression levels of BCL6 proteins in naive cells (n = 3), resting cells (n = 3), anti-CD3/CD28 activated CD4+ T cells (n = 4), tonsillar non-Tfh cells (CD4+ CD45RA– CXCR5– PD1-/dim, n = 3), and Tfh CD4+ T cells (n = 3) were quantified with a NanoString assay. (D) Heat maps of protein arrays for naive cells (n = 3), resting cells (n = 2), anti-CD3/CD28 activated CD4+ T cells (n = 3), tonsillar non-Tfh cells (n = 2), and Tfh CD4+ T cells (n = 2) were obtained using a NanoString assay with an nCounter Vantage 3D protein immune cell signaling panel.
FIG 2
FIG 2
BCL6 inhibition via FX1 represses ex vivo HIV infection of tonsillar Tfh/non-Tfh CD4+ T cells. Productive HIV-infected CD4+ T cells (p24+ CD4–) were determined by quantification of intracellular HIV p24 by a flow cytometry assay at 4 days after infection. (A) A representative flow image shows productive HIV-infected T cells in uninfected, HIVNL4-3-infected, and HIVNL4-3-infected/FX1-treated and FX1-treated naive, non-Tfh, and Tfh CD4+ T cells. (B) The percentages of productively infected CD4+ T cells (HIVp24+ CD4–) were plotted, and Wilcoxon matched-pair signed-rank tests were performed to compare the differences in the productively infected CD4+ T cells between FX1 treatment and DMSO treatment in naive (n = 6), non-Tfh (n = 6), and Tfh CD4+ (n = 3) T cells. (C) The percentages of productively infected T cells in the HIVNL4-3- or HIVBaL-infected pantonsillar CD4+ T cells (CD8/CD56–) treated with FX1 or DMSO (n = 6) were plotted and compared to Wilcoxon matched-pair signed-rank tests. (D) The HIV p24 levels in the cell culture supernatant of HIVNL4-3- or HIVBaL-infected pantonsillar mononuclear cells were measured by a standard ELISA (n = 6). Wilcoxon matched-pair signed-rank tests were performed to compare the results between two groups (*, P < 0.05). (E) The viability of PBMCs in the presence of different FX1 doses was analyzed with an XTT assay and deduced by comparing the absorbance at 490 nm to that in cells without FX1 (n = 3).
FIG 3
FIG 3
FX1 treatment of peripheral CD4+ T cell reduces ex vivo HIV infection by lowering the transcription of total elongated and multispliced HIV RNA. (A) Flow cytometry analysis of intracellular HIV p24 expression indicated viral infection rate in uninfected, HIVNL4-3-infected, and HIVNL4-3-infected/FX1-treated peripheral CD4+ T cells (n = 9, left panel). The percentages of infected T cells in uninfected, HIVBaL-infected, and HIVBaL-infected/FX1-treated peripheral CD4+ T cells (n = 6, right panel) are presented. (B) The percentages of HIVNL4-3-infected CD4+ T cells (p24+ CD4–) in the presence of different doses of FX1. (C) Flow cytometry analysis indicated the expression level of HIV receptor (CXCR4 and CCR5) on activated CD4+ T cells in the presence or absence of 50 μM FX1 for 48 h. The isotype antibody was used to stain the control cells and show the background of staining. (D) The relative RNA expression level (RLU) of HIV early reverse transcription (RT) and late RT in infected CD4+ T cells treated with DMSO (control) or 50 μM FX1 for 12 h postinfection (n = 9) was quantified by using real-time qPCR. Efavirenz at 100 nM (a reverse transcription inhibitor) was used as a suppression control. (E) The levels of total, integrated, and 2-LTR circular HIV DNA-infected CD4+ T cells of DMSO-treated (negative control) or FX1-treated cells was measured at 12 and 48 h postinfection (n = 6). HIV DNA in HIVNL4-3-infected CD4+ cells treated with DMSO or 50 μM FX1 were harvested at 12 h or 48 h postinfection (n = 6) and then underwent preamplification prior to quantative real-time PCR (see Table 1 for the primers). The level of HIV DNA was determined by extrapolation against a five-point standard curve (6 to 60,000 copies) performed in parallel. The cellular input was deduced by comparing the signal using CD3 primers and probe to a standard curve (6 to 60,000 copies). (F) The relative expression levels of unspliced, total elongated, polyadenylated, and multispliced HIV RNA were measured at 22 and 48 h postinfection (n = 9). The cells from three donors were treated with 10 ng/ml bicyclam JM-2987 (ADM3100; NIH-AIDS) 3 h after spin infection (noted in red color). The nucleic acid input was normalized by the cell number deduced by quantification of Tert (the telomerase reverse transcriptase, one copy per chromosome in a normal cell). A Wilcoxon test was performed to compare the HIV DNA/RNA between groups at each time points (*, P < 0.05; **, P < 0.01).
FIG 4
FIG 4
FX1 treatment downregulates the phosphorylation of SAMHD1 and the activation of CD4+ T cells ex vivo. Peripheral CD4+ T cells were activated by stimulation with anti-CD3/CD28 microbeads for 3 days and then treated with DMSO (control) or 50 μM FX1 for additional 48 h (n = 8). The cells were harvested and analyzed either by flow cytometry or Western blotting. (A) A Wilcoxon test was performed to compare the percentages of activated cells (HLA-DR+ CD4+, CD25+ CD4+, or Ki67+ CD4+) between control and FX1-treated groups. (B) Representative images of the blots from SDS-PAGE Western blotting analyses exhibited the protein levels of p-SAMHD1, SAMHD1, or β-actin in resting, anti-CD3/CD28-activated, and anti-CD3/CD28-activated and FX1-treated peripheral CD4+ T cells. (C) Quantification of p-SAMHD1, SAMHD1, or β-actin expression was performed by analyzing the intensity of their specific bands on blots using Image Studio Lite software. The intensity of the target band of p-SAMHD1 or SAMHD1 was normalized to that of β-actin to obtain the relative protein level in CD4+ T cells. The relative expression levels were used to calculate the ratio of p-SAMHD1 to SAMHD1. The fold change was calculated by comparing the relative protein level of p-SAMHD1 in resting, anti-CD3/CD28-activated, or anti-CD3/CD28-activated CD4+ T cells and in FX1-treated CD4+ T cells (n = 7). (D) Flow cytometry analysis of HIV-1 (NL4-3)- and HIV-2 (7312a)-infected resting CD4+ T cells in the presence or absence of 50 μM FX1 (n = 3). We used a Wilcoxon test to compare the results between two groups (*, P < 0.05; **, P < 0.01).
FIG 5
FIG 5
FX1 treatment represses HIV reactivation ex vivo. The CD4+ T cells of the HIV-infected ART-suppressed subjects were isolated by a negative selection method (see Materials and Methods). About four million CD4+ T cells were treated with DMSO (and isotype antibodies) as controls, as well as anti-CD3/CD28, anti-CD3/CD28 and 50 μM FX1, or with 50 μM FX1 alone for 24 h. The DNA and RNA were prepared from the cell pellets for a real-time qPCR assay to analyze HIV RNA and DNA directly. The level of RPLP0 (a housekeeping gene) was used to normalize and calculate the relative expression levels of HIV RNA transcripts. The level of HIV DNA was measured by determined by extrapolation against a five-point standard curve (6 to 60,000 copies) performed in parallel. The cellular input was deduced by comparing the signal using CD3 primers and probe to a standard curve (6 to 60,000 copies). The levels of HIV RNA (A) or total HIV DNA (B) in control, anti-CD3/CD28-treated, anti-CD3/CD28- and FX1-treated, or FX1-treated CD4+ T cells of HIV-infected cART-suppressed subjects were compared and are presented (n = 8). (C) Fold changes in HIV RNA production were calculated by comparing the relative RNA amounts in the anti-CD3/CD28-treated, anti-CD3/CD28- and FX1-treated, or FX1-treated CD4+ T cells to that of the CD4+ T cells in the control group (n = 8). (D) HIV production in the cell culture supernatants and cell lysates of two donors was measured by a Simoa-based ultrasensitive HIV p24 assay. The fold change was obtained by comparing the p24 level in the cell culture supernatant or cell lysate of anti-CD3/CD28-treated, anti-CD3/CD28- and FX1-treated, or FX1-treated CD4+ T cells to that of control CD4+ T cells from the same donor (n = 2). (E) The levels of HIV p24 in the cell culture supernatant from PMA/IO-stimulated, PMA/IO-stimulated and FX1-treated, or FX1-treated PBMCs were compared to those of DMSO-treated PBMCs from the same donor (n = 2). (F) Flow cytometry analysis of the green fluorescent protein (GFP) expression level in PMA/IO-stimulated, PMA/IO-stimulated and FX1-treated, or FX1-treated J-Lat GFP cells (Jurkat cells contain a single integration site of an HIV retroviral vector expressing LTR-driven GFP expression) (n = 3). A Wilcoxon test was performed to compare the results between two groups (*, P < 0.05; **, P < 0.01).
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