Defining the Effects of PKC Modulator HIV Latency-Reversing Agents on Natural Killer Cells

Abstract

Background: Latency reversing agents (LRAs) such as protein kinase C (PKC) modulators can reduce rebound-competent HIV reservoirs in small animal models. Furthermore, administration of natural killer (NK) cells following LRA treatment improves this reservoir reduction. It is currently unknown why the combination of a PKC modulator and NK cells is so potent and whether exposure to PKC modulators may augment NK cell function in some way.

Methods: Primary human NK cells were treated with PKC modulators (bryostatin-1, prostratin, or the designed, synthetic bryostatin-1 analog SUW133), and evaluated by examining expression of activation markers by flow cytometry, analyzing transcriptomic profiles by RNA sequencing, measuring cytotoxicity by co-culturing with K562 cells, assessing cytokine production by Luminex assay, and examining the ability of cytokines and secreted factors to independently reverse HIV latency by co-culturing with Jurkat-Latency (J-Lat) cells.

Results: PKC modulators increased expression of proteins involved in NK cell activation. Transcriptomic profiles from PKC-treated NK cells displayed signatures of cellular activation and enrichment of genes associated with the NFκB pathway. NK cell cytotoxicity was unaffected by prostratin but significantly decreased by bryostatin-1 and SUW133. Cytokines from PKC-stimulated NK cells did not induce latency reversal in J-Lat cell lines.

Conclusions: Although PKC modulators have some significant effects on NK cells, their contribution in "kick and kill" strategies is likely due to upregulating HIV expression in CD4⁺ T cells, not directly enhancing the effector functions of NK cells. This suggests that PKC modulators are primarily augmenting the "kick" rather than the "kill" arm of this HIV cure approach.

Keywords: Acquired Immunodeficiency Syndrome; HIV-1; Immunity; Killer Cells, Natural; Protein Kinase C; Virus Latency.

Figure 1.

Viability and cell-surface expression of activation and degranulation markers on NK cells treated with PKC modulators. NK cells were cultured for 24 hours untreated (DMSO only), with 10nM bryostatin-1, 1μM prostratin, or 10nM SUW133 and analyzed for viability (A), CD69 (B), NKG2D (C), and CD107a (D) via flow cytometry. Data from 4 healthy human donors are shown, with each color and shape representing results from a different donor. Horizontal lines indicate the mean. Error bars indicate the standard error of the mean (SEM). An unpaired, unequal variance Student's t-test was performed, with (***) indicating P < 0.001 and (****) indicating P < 0.0001.

Figure 2.

Cytokine induction in NK cells by PKC modulators. Supernatant from NK cells treated with 10nM bryostatin-1, 1µM prostratin, or 10nM SUW133 was filtered and analyzed using a Luminex 38-plex human cytokine immunoassay for cytokine composition. (A) Heatmap showing the mean fold change relative to the untreated (DMSO only) control from 5 different biological donors. (B) Example cytokine profiles from data shown in panel A, with each color and shape representing results from a different human donor (n=5). Horizontal lines indicate the mean. Error bars indicate the standard error of the mean (SEM). Dashed red lines indicate the lower limit of detection for the assay, which varies between different cytokines. A 2-tailed, unpaired, unequal variance Student's t-test was performed, with (*) indicating P < 0.05 and (**) indicating P < 0.01

Figure 3.

Transcriptomic profiles of NK and CD4⁺ T cells treated with PKC modulators. RNA-seq was performed on NK and CD4⁺ T cells cultured for 24 hours untreated (DMSO only), with 10nM bryostatin-1, 1μM prostratin, or 10nM SUW133. (A) Principal component analysis (PCA) plot of data from 5 healthy human donors is shown, with each point representing results from a different donor and each color representing a different treatment group. Venn diagram illustrating the overlap of upregulated (B) and downregulated (C) genes between the 3 different PKC modulators in NK and CD4⁺ T cells. Areas shown are proportional to the numbers of genes within each category.

Figure 4.

Differentially expressed genes induced by SUW133 in NK and CD4⁺ T cells. RNA-seq was performed on NK and CD4⁺ T cells cultured for 24 hours untreated (DMSO only) or with 10nM SUW133. Volcano plot of the distribution of all differentially expressed genes in NK cells (A) and CD4⁺ T cells (C). The red and blue dots represent the upregulated and downregulated genes (q-value < 0.01 and |log₂FC| > 2), respectively. The 15 most differentially expressed genes are labeled on each plot and shown in tables (B) & (D).

Figure 5.

Impact of PKC modulators on NK cell cytotoxicity. NK cells were cultured for 24 hours untreated (DMSO only), with 10nM bryostatin-1, 1μM prostratin, or 10nM SUW133 and tested for cytotoxicity against K562 cells at the indicated effector-to target (E:T) ratios. The mean percentage specific lysis from 5 independent biological replicates was measured. Error bars indicate the standard error of the mean (SEM). An unpaired, unequal variance Student's t-test was performed, with (***) indicating P <0.001 and (****) indicating P < 0.0001.

Figure 6.

Evaluation of secreted factors from NK cells treated with SUW133 and their effect on latency reversal in select J-Lat clones. Conditioned media (CM) generated with NK cells from 3 different healthy human donors were analyzed using a Luminex 38-plex human cytokine immunoassay. (A) Heatmap showing the mean fold change relative to the untreated (DMSO only) control. (B) Example cytokine profiles from data shown in panel A, with each color and shape representing results from a different human donor. (C - F) Jurkat-Latency (J-Lat) clones (10.6 and A2) were treated with direct LRA or CM for 48 hours and assessed for viability (C, D) and GFP+ expression (E, F). All control conditions (media only and direct stimulations) were technical singlets in 3 independent biological replicates, resulting in an n = 3; For CM samples, technical triplicates in 3 independent biological replicates per donor (3 donors each) resulted in n = 9. A 2-tailed, unpaired, unequal variance Student's t-test was performed, with (*) indicating P <0.01 and (**) indicating P < 0.001. The single asterisk above the 10nM SUW133 CM condition in panel D corresponds to a comparison with direct stimulation with media only.