In-vivo administration of histone deacetylase inhibitors does not impair natural killer cell function in HIV+ individuals

Abstract

Objective: Histone deacetylase inhibitors (HDACi) have proven to induce HIV-RNA and antigen expression in resting CD4 T cells of antiretroviral therapy (ART)-treated HIV-infected individuals. However, to achieve viral eradication, immune clearance must follow latency reversal, and thus it is essential to understand the impact of latency reversal agents on immune function.

Design: Here we evaluate the impact of in-vivo administration of vorinostat (VOR) and panobinostat (PNB) during clinical trials on natural killer (NK) cell function and phenotype.

Methods: Cryopreserved peripheral blood mononuclear cells from HIV-positive participants receiving VOR (NCT01319383) or PNB (NCT01680094) were selected to assess the impact of the drugs on cell composition, activation, NK cell phenotype (CD16, NKG2D, NKp30, NKp46 and DNAM-1), cytotoxic activity (CD107a), and interferon (IFN)-γ production.

Results: No impairment of NK cell function was observed during treatment with either VOR or PNB. An increase in the frequency of CD3CD56 NK cells was consistently observed. Interestingly, after VOR administration, NK cells increased expression of NKp46 and CD16, and showed improved degranulation and IFN-γ production capacity. Moreover, taking together VOR and PNB samples, HIV DNA levels in CD4 cells were negatively correlated with NK cell frequency and NK cell expression of CD16.

Conclusions: In-vivo treatment with HDACi does not have measurable negative effects on NK cell function, with some evidence of improved function in vitro. These results have important implications for potential combinatorial approaches to target HIV reservoirs, suggesting that the use of HDACis as a latency reversal agent could be paired with interventions to enhance NK cell activity or recruitment.

Conflict of interest statement

CONFLICT OF INTEREST

None declared.

Figures

Fig. 1.. Frequency of cell populations, proliferation and activation during HDAC inhibitor treatment.

PBMC were stained with a panel of monoclonal antibodies to analyze cell populations and expression of proliferation and activation markers. A. Representative plot of cell gating strategy. B. VOR treatment: The proportion of CD4 and CD8+T cells did not change during VOR treatment, but frequency of NK cells increased after 11 doses of VOR, with increases maintained after 22 doses. Expression of the proliferation marker Ki67 and the activation marker CD69 decreased during VOR treatment. C. PNB treatment: CD4 and CD8+T cells remained stable during PNB treatment, but NK cells showed an increase, that was lost after the end of treatment. A decrease in CD69 was observed during PNB treatment. Each symbol represents cells from a different study participant. Wilcoxon matched-pairs signed rank test.

Fig. 2.. NK cell cytotoxicity and cytokine production.

NK cells were negatively isolated from thawed PBMCs and cultured with the cell line K562 to measure degranulation through the marker CD107a and IFN-γ production by intracellular staining. A. Representative plots of cells from one participant. B. VOR treatment: An increase in both degranulation and IFN-γ production was observed after VOR exposure. C. PNB treatment: Cytotoxic and IFN-γ potential remained stable during PNB treatment. Each symbol represents cells from a different study participant. Wilcoxon matched-pairs signed rank test.

Fig. 3.. Expression of activating receptors on NK cells.

NK cells were negatively isolated from thawed PBMC and stained with a panel of monoclonal antibodies to analyze the expression of activating receptors. A. VOR treatment: NKp30, DNAM-1 and NKG2D expression did not change during treatment, but expression of CD16 and NKp46 was increased to some extent after VOR administration. B. PNB treatment: No changes were observed during PNB treatment. Each symbol represents cells from a different study participant. Wilcoxon matched-pairs signed rank test.

Fig. 4.. Correlation with the HIV reservoir size.

Size of the HIV reservoir was measured as total DNA per million CD4 cells or as IUPMs after quantitative outgrowth assay. HIV DNA correlated negatively with NK cell frequency, CD16 expression and IFN-γ production, while it correlated positively with NKG2D expression. Circles correspond to samples from the VOR study and squares to the PNB study. Green represent samples before HDACi treatment, purple during treatment and red after treatment.