HIV can establish a long-lived latent infection in cells harboring integrated non-expressing proviruses. Latency reversing agents (LRAs), including protein kinase C (PKC) modulators, can induce expression of latent HIV, thereby reducing the latent reservoir in animal models. However, PKC modulators such as bryostatin-1 also cause cytokine upregulation in peripheral blood mononuclear cells (PBMCs), including cytokines that might independently reverse HIV latency. To determine whether cytokines induced by PKC modulators contribute to latency reversal, primary human PBMCs were treated with bryostatin-1 or the bryostatin analog SUW133, a superior LRA, and supernatant was collected. As anticipated, LRA-treated cell supernatant contained increased levels of cytokines compared to untreated cell supernatant. However, exposure of latently-infected cells with this supernatant did not result in latency reactivation. These results indicate that PKC modulators do not have significant indirect effects on HIV latency reversal in vitro and thus are targeted in their latency reversing ability.

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.