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.
Background: Insulin-degrading enzyme (IDE) is a highly conserved metallopeptidase initially described because of its ability to degrade insulin. Results: (i) IDE expression is stress-inducible; (ii) IDE concentration is up-regulated in some CNS tumors; (iii) IDE downregulation impairs SHSY5Y cell proliferation/viability. Conclusion: IDE is a multifunctional protein.Significance: IDE is a novel HSP with important implications in cell growth regulation.
In pilot HIV-1 eradication studies, patients' immune responses were ineffective at killing viral reservoirs reactivated through latency reversing agents (LRAs) like suberoylanilide hydroxamic acid (SAHA). We hypothesized that T cells harboring reactivated HIV-1 express MIC and ULBP ligands for the activating NKG2D receptor of natural killer (NK) cells. Here, we demonstrated that MICA/B and ULBP2 are induced by SAHA on primary T cells harboring reactivated virus. Using latently HIV-1-infected J-Lat 6.3/8.4/9.2 and J1.1 cell lines, we showed that SAHA reverts latency and, simultaneously, up-regulates MICA/B and ULBP2 acting at the transcriptional level and through ATR activation, thus sensitizing T cells with reactivated virus to NKG2D-mediated killing by NK cells. Moreover, IL-2 and IL-15 potently boosted NKG2D expression and cytotoxicity of NK cells against SAHA-reactivated p24 target cells. Therefore, immunotherapy with cytokines enhancing NKG2D-mediated NK-cell cytotoxicity combined with administration of LRAs up-modulating NKG2D ligands, represents a promising approach towards HIV-1 eradication.
In the present study, we demonstrate that minimal concentrations (≤ 1 nM) of retinoic acid (RA), equivalent to the quantity contaminating serum-containing culture medium, are sufficient to promote meiotic entry and progression through meiotic prophase I (MPI) stages in isolated 12.5-days postcoitum (dpc) XX and XY mouse primordial germ cells (PGCs) in culture. Similarly, we found that the same low RA concentration up-regulated or induced stimulation by retinoic acid 8 (Stra8) in such cells, both at mRNA and protein level. In preleptotene/leptotene germ cells, STRA8 was localized in nuclear dots that disappeared at later MPI stages. In addition to Stra8, other meiotic genes such as Dmc1 and Rec8 appeared stimulated by RA directly in PGCs with similar concentration-dependent trends. Finally, we found that RA induced Stra8, Sycp3, Dmc1, and Rec8 transcripts, promoting meiotic entry in culture also in pregonadal 10.5-dpc PGCs of both sexes. When cultured isolated from somatic cells, such PGCs, however, were unable to progress through MPI stages, while after entering meiosis, they progressed through MPI when cultured within aorta/gonad/mesonephros tissues. We conclude that besides RA, germ cell intrinsic factors and other exogenous signals from the surrounding somatic cells are probably necessary for meiotic entry and progression in mouse PGCs.
The ‘shock-and-kill’ strategy to purge the latent HIV reservoir relies on latency-reversing agents (LRAs) to reactivate the provirus and subsequent immune-mediated killing of HIV-expressing cells. Yet, clinical trials employing histone deacetylase inhibitors (HDACis; Vorinostat, Romidepsin, Panobinostat) as LRAs failed to reduce the HIV reservoir size, stressing the need for more effective latency reversal strategies, such as 2-LRA combinations, and enhancement of the immune responses. Interestingly, several LRAs are employed to treat cancer because they up-modulate ligands for the NKG2D NK-cell activating receptor on tumor cells. Therefore, using in vitro T cell models of HIV latency and NK cells, we investigated the capacity of HDACis, either alone or combined with a distinct LRA, to potentiate the NKG2D/NKG2D ligands axis. While Bortezomib proteasome inhibitor was toxic for both T and NK cells, the GS-9620 TLR-7 agonist antagonized HIV reactivation and NKG2D ligand expression by HDACis. Conversely, co-administration of the Prostratin PKC agonist attenuated HDACi toxicity and, when combined with Romidepsin, stimulated HIV reactivation and further up-modulated NKG2D ligands on HIV+ T cells and NKG2D on NK cells, ultimately boosting NKG2D-mediated viral suppression by NK cells. These findings disclose limitations of LRA candidates and provide evidence that NK cell suppression of reactivated HIV may be modulated by specific 2-LRA combinations.
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