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. 2018 Jun 29:9:1514.
doi: 10.3389/fimmu.2018.01514. eCollection 2018.

In Vitro Exposure to Prostratin but Not Bryostatin-1 Improves Natural Killer Cell Functions Including Killing of CD4+ T Cells Harboring Reactivated Human Immunodeficiency Virus

Maria Giovanna Desimio  1 , Erica Giuliani  1 , Angelo Salvatore Ferraro  2 , Gaspare Adorno  2   3 , Margherita Doria  1
Affiliations

In Vitro Exposure to Prostratin but Not Bryostatin-1 Improves Natural Killer Cell Functions Including Killing of CD4+ T Cells Harboring Reactivated Human Immunodeficiency Virus

Maria Giovanna Desimio et al. Front Immunol. .

Abstract

In the attempt of purging the HIV-1 reservoir through the "shock-and-kill" strategy, it is important to select latency-reversing agents (LRAs) devoid of deleterious effects on the antiviral function of immune effector cells. Here, we investigated two LRAs with PKC agonist activity, prostratin (PRO) and bryostatin-1 (BRY), for their impact on the function of natural killer (NK) cells, the major effectors of innate immunity whose potential in HIV-1 eradication has emerged in recent clinical trials. Using NK cells of healthy donors, we found that exposure to either PRO or BRY potently activated NK cells, resulting in upmodulation of NKG2D and NKp44 activating receptors and matrix metalloprotease-mediated shedding of CD16 receptor. Despite PRO and BRY affected NK cell phenotype in the same manner, their impact on NK cell function was diverse and showed considerable donor-to-donor variation. Altogether, in most tested donors, the natural cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells were either improved or maintained by PRO, while both activities were impaired by BRY. Moreover, we analyzed the effect of these drugs on the capacity of treated NK cells to kill autologous latently infected CD4+ T cells reactivated via the same treatment. First, we found that PRO but not BRY increased upmodulation of the ULBP2 ligand for NKG2D on reactivated p24+ cells. Importantly, we showed that clearance of reactivated p24+ cells by NK cells was enhanced when both targets and effectors were exposed to PRO but not to BRY. Overall, PRO had a superior potential compared with BRY as to the impact on key NK cell functions and on NK-cell-mediated clearance of the HIV-1 reservoir. Our results emphasize the importance of considering the effects on NK cells of candidate "shock-and-kill" interventions. With respect to combinative approaches, the impact on NK cells of each LRA should be re-evaluated upon combination with a second LRA, which may have analogous or opposite effects, or with immunotherapy targeting NK cells. In addition, avoiding co-administration of LRAs that negatively impact ADCC activity by NK cells might be essential for successful application of antibodies or vaccination to "shock-and-kill" strategies.

Keywords: HIV; NKG2D; antibody-dependent cellular cytotoxicity; bryostatin-1; latency-reversing agents; natural killer cell function; prostratin; protein kinase C agonists.

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Figures

Figure 1
Figure 1
Prostratin (PRO) and bryostatin-1 (BRY) activate natural killer (NK) cells and modulate activating receptors. (A) The purity of NK cells isolated from PBMCs was examined by flow cytometry measuring the frequency of CD3−CD56+CD16+/− cells. (B) NK cells were cultured for 18, 46, and 76 h in medium alone [not treated (nt)] or supplemented with 1 or 10 µM PRO, or with 5 or 10 nM BRY. The viability of gated NK cells was examined by LIVE/DEAD staining and expressed relatively to nt cells set at 100%. Bars represent mean ± SEM (n = 3). (C–F) NK cells treated or not with 1 µM PRO, 10 nM BRY, or 12.5 ng/ml of IL-15 were examined at various time points as indicated (C,D,F) or after 18 h only (E) for the expression of various markers: (C,D) the frequency of CD69+ and CD107a+ NK cells among nt (filled gray histograms, gray percentages) and treated cells (open histograms, black percentages) is shown together with control IgG signal (dashed line) for a representative experiment (C) and as mean ± SEM (D); (E,F) mean ± SEM of both percentage of positive cells and mean fluorescence intensity (MFI) for NKG2D, DNAM-1, NKp46, NKp44, NKp30, and CD16 is shown. Experiments were performed with at least three independent donors [up to eight in panel (E)]. *P < 0.05, **P < 0.01, and ***P < 0.001 by paired t test.
Figure 2
Figure 2
CD16 is shed from natural killer (NK) cells upon exposure to prostratin (PRO) or bryostatin-1 (BRY). (A) Soluble CD16 (sCD16) was measured in the media of NK cells after 18 h culture without [not treated (nt)] or with the addition of 1 µM PRO or 10 nM BRY. Each symbol represents one donor (n = 4). The mean ± SEM is reported. (B,C) NK cells were pre-exposed to 25 µM MMPI-III inhibitor or equivalent amounts of DMSO for 1 h, then 1 µM PRO or 10 nM BRY were added or not for 18 h and finally analyzed for cell-surface CD16 expression. (B) Histograms show CD16 signal on nt, PRO- and BRY-treated cells either exposed to MMPI-III (filled gray histograms) or to DMSO (solid line) in one representative experiment. Signal of control IgG (dashed line) and the frequency of CD16+ cells (gray or black when MMPI-III or DMSO are present, respectively) are also shown. (C) Bars represent mean ± SEM (n = 3). (D) The catalytic 80 kDa form of ADAM17 (top) and GAPDH as internal control (bottom) were detected by western blotting analysis of total protein extracts of nt, PRO- and BRY-treated NK cells. Molecular mass standards are indicated (kDa). (E) Quantification of ADA17 induction by PRO and BRY relative to nt condition. The ADA17 signal normalized for GAPDH signal was quantified and divided for the value obtained in the nt sample. Data show mean ± SEM of three independent experiments like the one shown in (D). *P < 0.05 and **P < 0.01 by paired t test.
Figure 3
Figure 3
Impact of prostratin (PRO) and bryostatin-1 (BRY) on natural killer (NK)-cell cytotoxicity. (A) NK cells cultivated for 18 h without stimuli [not treated (nt)], with 12.5 ng/ml of IL-15, 10 µM PRO or 5 nM BRY were tested for cytotoxicity against K562 cell targets at the indicated E:T ratio. The percent specific lysis of two representative experiments is shown. (B) The NK cell-mediated lysis measured in four independent experiments like the one shown in panel (A) was converted in lytic units (LU, mean ± SEM). Each symbol represents one donor. (C,D) NK cells treated for 18 h with 1 µM PRO or 10 nM BRY or nt were tested against Raji cells coated or not with rituximab (Rtx) monoclonal antibody. The efficiency of overall lysis (C) and antibody-dependent cellular cytotoxicity (D) were measured in five donors. The mean ± SEM is reported. *P < 0.05 by paired t test.
Figure 4
Figure 4
ULBP2 is induced on latently infected CD4+ T cells upon HIV-1 reactivation by prostratin (PRO) and, to a lesser extent, by bryostatin-1 (BRY). To establish viral latency, freshly isolated CD4+ T cells were cultivated in the presence of CCL19 for 1–3 days, infected or not with HIV-1, and further cultured for 3 days, as described in details in Section “Materials and Methods.” Then, cells were stimulated with 10 µg/ml phytohemagglutinin (PHA), 1 µM PRO, or 10 nM BRY, or not stimulated (ns), further cultivated for 3 days and finally analyzed by two-color flow cytometry for the expression of intracellular p24 and cell-surface MICA/B and ULBP2. (A) Representative dot plots show the frequency of reactivated p24+ cells gated on non-infected PHA-stimulated control cells. (B) Percentage of p24+ cells was determined as shown in panel (A) in four independent experiments and normalized to HIV-infected PHA-treated cultures (mean ± SEM). (C) Histograms show MICA/B and ULBP2 fluorescence in the gated p24− (top panels) and p24+ (bottom panels) cell populations measured in a representative experiment on ns, PRO- and BRY-stimulated cell samples. Signals obtained with control IgG (filled histograms) and the percentage of ligand-positive cells are shown. (D) ULBP2 expression (mean ± SEM), both% of positive cells and mean fluorescence intensity (MFI), was determined as shown in panel (C) in four independent experiments. (E) RT-qPCR was used to assess ULBP2 mRNA levels in latently infected and in control non-infected CD4+ T cells cultivated for 24 h in ns, PRO, and BRY conditions. Results are expressed relative to ns non-infected sample (set to 1). Mean ± SEM values obtained in three independent experiments performed in duplicate are shown. *P < 0.05 and **P < 0.01 by paired t test. Values that differ significantly from ns non-infected samples in panel (E) are also indicated (#P < 0.05 by paired t test).
Figure 5
Figure 5
Prostratin (PRO) enhances the capacity of natural killer (NK) cells to suppress HIV-infected CD4+ T cells that exit from latency. A co-culture assay of latently infected CD4+ T cells at day 2 post-stimulation with 1 µM PRO or 10 nM bryostatin-1 (BRY) and autologous NK cells at a 1:1 E:T ratio was performed overnight (18 h) in the presence of the same drug. Then, cells were analyzed to measure the frequency of p24+ cells among gated CD3+ targets and calculate % reduction of 24+ targets by NK cells. (A) The CD3+ cell gate was set on cultures of targets alone, so that effectors cells were excluded, as shown in control cultures of effectors alone as well as in co-cultures of targets and effectors in a representative experiment. (B) A representative set of results with PRO- and BRY-exposed cultures as well as control not treated (nt) cultures is shown. (C) The NK-cell-mediated p24+ cell killing was measured in seven independent experiments and expressed as % 24+ killing (mean ± SEM). (D) NK cells were preincubated with anti-NKG2D monoclonal antibody (mAb) or isotype control IgG1 immediately before the overnight co-culture with targets in nt, PRO, and BRY conditions, then the % 24+ killing was calculated in three independent experiments. *P < 0.05 by paired t test.
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