In vivo Effects of Romidepsin on T-Cell Activation, Apoptosis and Function in the BCN02 HIV-1 Kick&Kill Clinical Trial

Abstract

Romidepsin (RMD) is a well-characterized histone deacetylase inhibitor approved for the treatment of cutaneous T-cell lymphoma. in vitro and in vivo studies have demonstrated that it is able to induce HIV-1 gene expression in latently infected CD4⁺ T cells from HIV-1⁺ individuals on suppressive antiretroviral therapy. However, in vitro experiments suggested that RMD could also impair T-cell functionality, particularly of activated T cells. Thus, the usefulness of RMD in HIV-1 kick&kill strategies, that aim to enhance the immune system elimination of infected cells after inducing HIV-1 viral reactivation, may be limited. In order to address whether the in vitro observations are replicated in vivo, we determined the effects of RMD on the total and HIV-1-specific T-cell populations in longitudinal samples from the BCN02 kick&kill clinical trial (NCT02616874). BCN02 was a proof-of-concept study in 15 early treated HIV-1⁺ individuals that combined MVA.HIVconsv vaccination with three weekly infusions of RMD given as a latency reversing agent. Our results show that RMD induced a transient increase in the frequency of apoptotic T cells and an enhanced activation of vaccine-induced T cells. Although RMD reduced the number of vaccine-elicited T cells secreting multiple cytokines, viral suppressive capacity of CD8⁺ T cells was preserved over the RMD treatment. These observations have important implications for the design of effective kick&kill strategies for the HIV-1 cure.

Keywords: HDAC inhibitor; kick&kill strategy; latency reversing agent (LRA); romidepsin; therapeutic vaccine.

Figure 1

Study design. The BCN02 study was a single arm, open label, proof-of-concept study to address safety and effect on the viral reservoir of a kick&kill strategy combining MVA.HIVconsv vaccines with the HDACi RMD. Timepoints used for the analysis presented here are indicated for each assay by filled circles.

Figure 2

Effect of RMD on the viability on T cells. Apoptotic cells (Annexin V⁺/7AAD⁺ and Annexin V⁺/7AAD⁻) percentages are shown for CD8⁺ (A) and CD4⁺ T cells (B). Changes relative to baseline in the percentage of apoptosis in HLA-DR⁺ (in red) and HLA-DR⁻ cells (in purple) are shown for CD8⁺ (C) and CD4⁺ (D) T cells. RMD administration cycles are indicated by gray bars. Sampling time points at 4 h, 8 h, 3 days, and 7 days after each RMD administration are indicated. Median with interquartile range is shown. P-values (*p < 0.05, **p < 0.01, ***p <0.001) are indicative for the corresponding timepoint compared to pre-RMD₁.

Figure 3

Evolution of T-cell subsets over the course of the intervention. Averages of relative frequency of CD8⁺ (A) and CD4⁺ (B) T-cell subsets based on CCR7 and CD45RA expression are shown. TEMRA: highly differentiated effector cells expressing CD45RA (CCR7⁻/CD45RA⁺), EM: effector memory cells (CCR7⁻/CD45RA⁻), CM: central memory (CCR7⁺/CD45RA⁻), and naïve (CCR7⁺/CD45RA⁺). P-values (*p < 0.05, **p < 0.01, ***p < 0.001) are indicative for the corresponding timepoint compared with values from baseline samples prior to MVA₁.

Figure 4

CD4⁺ and CD8⁺ T cell activation status over the course of the intervention. Percentage of HLA-DR expression is shown for CD8⁺ in orange and CD4⁺ T cells in purple (A). Levels of HLA-DR are shown for CD8⁺ (B) and CD4⁺ T cells (C) in HIVconsv specific and total T cells. HIVconsv-specific T cells were defined upon in vitro antigen-specific stimulation and detection based on their ability to produce cytokines (IL-2, MIP1-β, TNF-α, IFN-γ) in response to stimulation. Percentage of PD-1 is shown for CD8⁺ in orange and CD4⁺ T cells in purple (D). Median with interquartile range is shown. P-values (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) are indicative for the corresponding timepoint compared with baseline.

Figure 5

Longitudinal assessment of the T-cell cytokine production over the course of the intervention. Polyfunctionality of CD8⁺ and CD4⁺ T cells was analyzed by Boolean gating. Pie charts illustrate relative proportion of each of the different subsets (cells producing 1, 2, 3, or 4 cytokines, respectively).

Figure 6

In vitro viral replication inhibition capacity. Levels of CD8⁺ viral inhibitory capacity are shown for HIV-1_BaL (A) and HIV-1_IIIB (B) at two E/T ratios (effector/target ratio) 1:1 and E/T 1:10 in individuals that underwent the intervention (n = 14 for HIV-1_BaL and n = 15 for HIV-1_IIIB). P-values (*p < 0.05) are indicative for the corresponding timepoint compared with respective bsl.