Human immunodeficiency virus type 1 (HIV-1) can be controlled by combined antiretroviral therapy (cART) to the point at which viral loads in the peripheral blood are undetectable. However, the main obstacle to total HIV eradication and a functional cure is the presence of latent HIV-1 reservoirs, which exist in the peripheral blood and lymphoid tissues. To functionally cure HIV-1, several strategies have been proposed, including gene editing technologies based on CCR5, broadly neutralizing antibodies (bNAbs), the “shock and kill” strategy for eradication of shallow reservoirs, and the “block and lock” or “permanent silencing” approach for eradication of deep reservoirs (1). Many latency-reversing agents (LRAs) have been researched as part of the “shock and kill” approach, but only a small number of candidates have been evaluated in clinical trials. Although the majority of LRAs can activate viral transcription, they are unable to induce “killing” effectively. While preventative medications and treatments exist to reduce the disease burden of acquired immunodeficiency syndrome (AIDS), there is no effective cure for HIV; therefore, it is critical to develop useful approaches for HIV immunotherapy.

HIV infects humans through immune cells, including macrophages (2, 3), dendritic cells (DCs) (4), and especially CD4⁺ T cells; this can destroy immune function and result in severe infection, malignant tumors, autoimmune illnesses, and other complications (5). In the past years, the innate immune system has been shown to play a significant role in fighting HIV. The innate immune system is the body’s initial line of defense against foreign pathogens, and it includes a variety of cells and cytokines that fight off pathogens in a nonspecific manner. Among innate immune cells, natural killer (NK) cells play important roles in antiviral and anti-tumor immune responses. Advances in NK cell technology have been spurred by the development of cancer immunotherapy, and these advances have the potential to enhance HIV immunotherapy and to overcome some of the problems with strategies targeting T cells. Numerous data and literature demonstrate the efficacy of NK immunotherapy against tumors (6–9) and liver inflammation (10) have been discussed elsewhere (6–9). In the present paper, we will review recent advances in NK cell technology and the potential of NK cells for use in HIV immunotherapy.

Natural killer cells are components of the innate immune system (11) that are phenotypically comparable to T cells, particularly CD8⁺ T cells (2). Human NK cells are derived from CD34⁺ lymphoid lineage cells (HPCs) (12, 13) and found in the liver, peritoneum, placenta, and other organs; they account for 5-15% of peripheral blood mononuclear cells (PBMCs). Immature NK cells in secondary lymph nodes are stimulated by cytokines such as IL-15 and IL-12 from DCs or other antigen presenting cells (APCs) (14) and are subsequently transferred to the peripheral blood, where they gradually acquire cytotoxicity during differentiation and development. NK cells are divided into three or five subgroups according to their expression of CD56 and CD16: CD3⁻CD56⁻CD16⁺, CD3⁻CD56^dimCD16⁺, and CD3⁻CD56^briCD16^+/−, and the total population of NK cells included these three subsets (9, 15), with the CD56^dim NK cells being the most numerous in peripheral blood and the CD56^bright NK cells are largely enriched in the liver (16). CD56^bright liver-resident NK cells (lrNKs) can be further defined based on the expression of CD69, CD49a, CCR5 and CXCR6 (17). Moreover, technological advances in mass cytometry revealed peripheral blood NK cell diversity with at least 6000 phenotypic populations in an individual assessed by more than 30 parameters simultaneously, which can be used for immunotherapeutic strategies for infection, reproduction, and transplantation (18).

NK cells are multifunctional natural effector cells that have antiviral and antitumor properties. They participate in killing and lysing target cells by recognizing virus-infected cells or tumor cells through their surface receptors. NK cells have diverse mechanisms of killing, including release of cytotoxic granules and apoptosis mediated by the Fas/FasL pathway or TNF-related apoptosis-inducing ligand (TRAIL) (19, 20). NK cells can also engage in antibody-dependent cell cytotoxicity (ADCC) leading to death of antibody-coated cells (21). Finally, NK cells can secrete cytokines and chemokines that contribute to their antiviral effects, including IFN-γ, GM-CSF, TNF-α, CCL3, CCL4, CCL5, XCL1, and XCL2 (14, 17, 18). In addition to their natural killing function, NK cells have significant immunomodulatory roles; for example, they regulate adaptive immune cells such as T and B cells and innate immune cells such as DCs and macrophages to impact the outcome of infections (22, 23). Multiple features of NK cells indicate that they play dynamic roles in immune-mediated protection and homeostasis (24).

NK cells play significant roles in HIV-1 infection. In the early stages of viral infection, NK cells act more quickly than adaptive immune cells, and cytotoxic CD56^low NK cells expand faster than CD8⁺ T cells (25). HIV infection changes the distribution and functions of NK cell subpopulations (26–28) even after they are partially restored by antiretroviral therapy (ART) (29). Another study found that CD56^lowCD16⁺ NK cells decreased significantly during HIV-1 infection, whereas CD56^highCD16⁺ NK cells populations were unchanged (26, 30), and this impacted the rapid and early progression of AIDS (31). In addition, The CD56^–CD16⁺ NK cell subpopulation reduces spontaneous NK cytotoxicity (26). Furthermore, NK cells in HIV-1-exposed seronegative intravenous drug users (HESN-IDU) produce higher levels of IFN-γ and TNF-α (32, 33) compared to NK cells in healthy controls. Another study demonstrated that NK cells can dramatically inhibit viral entry into CD4⁺ T cells and prevent the spread of HIV-1 by producing β-chemokines following stimulation with IL-2 and IL-15 (34). NK cells are also associated with the development of bNAbs during HIV-1 infection (35). Immunogenetics, viral development, and immune escape studies showed that enhanced NK cell activity supported by cytotoxic cytokines and chemokines production are involved in delaying AIDS progression and controlling of HIV infection (36).

“Shock and kill” therapy strategies attempt to reactivate the latent HIV-1 in lymphocytes and tissues by LRAs, and this is followed by killing of the infected cells by the immune system and ART (37). Some classic LRAs have been reported to influence the function and fate of NK cells. For example, the protein kinase C (PKC) and NF-κB activator prostratin induced non-specific NK cell activation and a strong antiviral response, whereas panobinostat decreased NK cell viability, antiviral activity, and cytotoxicity; these factors should be carefully considered when evaluating strategies to eliminate HIV-1 infection (38). In a clinical trial of people living with HIV (PLWH), following analytical treatment interruption (ATI), those treated with panobinostat had decreased proviral HIV-1 DNA levels and increased viral rebound times, and this was correlated with higher frequencies of immunomodulatory CD56⁺ NK cells, CD56^low NK cells, and plasmacytoid dendritic cells (39). The impact of panobinostat on NK cell populations may be due to LRA-mediated alterations in germline-encoded NK receptor ligand expression on HIV-infected CD4⁺ T cells. A study of HIV-1 and Hepatitis C Virus (HCV) coinfected individuals receiving cART showed that pegylated interferon-α (PEG-IFNα) induced NK cell activation, and levels of cell-associated proviral DNA were negatively correlated with NK cell quantities (40). Peg-IFN-α2a-mediated HIV-1 suppression was also correlated with NK cell cytotoxicity and innate immune activity (41). These findings indicate that the reduction in HIV-1 reservoirs following treatment with PEG-IFNα occurs mainly due to the antiviral properties of NK cells. It is also worth noting that targeting NK cell subsets to restore any residual dysfunction could enhance their antiviral properties and reduce related comorbidities.

Immune checkpoint inhibition can restore the function of exhausted T cells during chronic infection and in the tumor microenvironment; this approach is widely used to treat cancer and could be an appealing adjuvant therapeutic approach for HIV. In addition to iNKRs, NK cells express immune checkpoint receptors previously found on T cells, such as programmed death receptor 1 (PD-1), lymphocyte activation gene-3 (LAG3)/CD223, cytotoxic T lymphocyte associateprotein-4 (CTLA-4), T cell immunoglobulin-3 (TIM-3), T cell immune receptor with Ig and ITIM domains (TIGIT) and the recently identified CD276 (B7-H3) (89). Checkpoint inhibitors may strengthen NK cell activation and cytolytic effectiveness (90). Initial studies of immune checkpoint inhibitors in HIV mainly focused on CD4⁺ T cells. Although checkpoint inhibition has been shown to significantly improve NK cell activity in cancer patients, the benefits of checkpoint inhibition in PLWH on ART have yet to be determined. NK cells, like T cells, can develop an exhausted phenotype during HIV or simian immunodeficiency virus (SIV) infections (91). NK cell exhaustion can cause various problems including poor proliferative capacity, decreased expression of activating receptors (56, 92, 93), and increased expression of inhibitory receptors (26). Furthermore, exhausted NK cells are unable to degranulate, produce cytokines, and promote ADCC (93). PD-1, an key marker of T cell exhaustion during HIV-1 infection, was upregulated on NK cells from HIV-1 positive individuals following ART. Increased PD-1 expression was associated with limited NK cell proliferation, which may impact NK cell maintenance during HIV-1 infection (94). HIV infection and treatment may alter invariant natural killer T cell (iNKT) induced IFN-γ levels. iNKT dysfunction is uniquely associated with the expression of LAG-3, an immune checkpoint marker similar to PD-1, and persistent LAG-3 expression may result in deficient immune reconstitution in PLWH during ART (95).

Multiple activating and inhibitory interactions between T cells and APCs can regulate the immune response. Immune checkpoints promote suppressive interactions between immune cells to maintain homeostasis. Immune checkpoint inhibition has been consistently demonstrated to enhance not only T cell function, but also NK cell function during HIV infection. One group demonstrated that in both untreated and ART-suppressed PLWH, combined PD-1 and IL-10 blockade could enhance NK cell cytokine secretion, degranulation, and killing capacity as well as restore CD4⁺ T cell function. This implies that using immune checkpoint inhibition to improve the cooperation between T and NK cells is a potential therapeutic approach for HIV (96). Therefore, immunotherapeutic interventions targeting immune checkpoint could augment NK cell activity and function against HIV and PLWH with cancers.

Toll-like receptors (TLRs) are pathogen recognition receptors (PRRs) that detect and modulate signaling responses based on molecular motifs that are conserved across pathogenic microorganisms. TLRs are mainly found on APCs and epithelial and endothelial cells (97), and they are localizated in cell surface (TLR1, 2, 4, 5, 6, 10, 11) and endosomal (TLR3, 7, 8, 9, 12, 13) (98). TLR2 heterodimerizes with TLR1 or TLR6 to function. TLR agonists have been studied as LRAs with the potential to reverse latent HIV-1 and enhance antiviral responses through immunomodulation. TLR agonists enhance immune activation, adaptive responses, and innate antiviral responses in vitro and in vivo (99). Because of unique immunomodulatory properties of TLR agonists have, further research into the approach to combine latency reversal and viral clearance strategies is undergoing in vivo (99). Different TLRs are expressed in NK cells, according to reports, TLR1-TLR9 mRNA was expressed in human NK cells, with TLR1 expression being the highest, followed by moderate levels of TLR2, TLR3, TLR5, and TLR6, and low or undetectable expression of TLR9 levels (100, 101), (reviewed in (102). TLR ligands can activate NK cells directly or indirectly, for example, a new synthetic TLR1/2 ligand named XS15 (103) can stimulate NK cells by monocytes and TLR-7/8 agonist Clo97 can activate NK cells by polymorphonuclear neutrophils (104) indirectly; NK cells also were activated and the cytotoxicity of them were enhanced by autologous DC cultured with poly (I:C) (105). In addition, TLR ligands such as TLR1/2 (Pam3CSK4 or SMU-Z1) or TLR2/6 (MALP-2) and TLR3 (poly I:C) can directly activate human NK cells (106–109). Although poly I:C treatment had no significant effect on CD4⁺ T cells and plasma viral control, the data suggested that poly I:C can induce innate immune responses in PLWH, indicating that it could be a promising adjuvant for HIV therapeutic vaccines (NCT02071095) (110). In SIV-infected rhesus monkeys, TLR7 agonist GS-9620 combined with bNAb PGT121 effectively activated NK cells and CD4⁺ T cells and delayed viral rebound after ART interruption, indicating the potential of NK cell stimulation in conjunction with bNAbs for targeting HIV reservoirs (111). GS-9620 has already been studied in clinical trials NCT03060447 and NCT02858401 in PLWH receiving ART. TLR-7 and/or TLR-8 agonists are being tested for their ability to directly activate NK cells. Although purified NK cells do not express TLR-7 or -8, TLR-7/TLR-8 agonists (R-848) enhance NK-cell cytotoxicity in vivo and control NK cell activation indirectly through immune modifiers, and TLR-8 ligands result in indirect NK-cell activation by IL-18 and IL-12p70 and possibly by other cytokines (112). Furthermore, the TLR9 agonist MGN1703 has been shown in clinical studies to activate NK cells in PLWH (113). In summary, TLR agonists have been shown to target cellular and tissue HIV reservoirs in a variety of in vivo studies (114). Combined with these results, it is reasonable to expect that TLR agonists will soon become a safe and multipotent clinical drug designed to reduce the HIV reservoir.

Many interleukins such as IL-2, -12, -15, -18 and -21 have been found to boost NK cytotoxicity and proliferation both in vitro and in vivo (7). In particular, IL-15 is known to promote the proliferation, differentiation, and maturation of NK cells and induces NK cell receptor expression, ADCC, cytotoxicity, and IFN-γ production. After exposure to LRAs, NK cells stimulated with IL-15 were able to kill latent HIV-infected cells (115). Remarkably, the IL-15 superagonist ALT-803/N-803 has been identified as a LRA and can induce HIV elimination in a cooperative manner with CD8⁺ T cells (116). Moreover, in ART-treated macaques, ALT-803 could consistently reactivate SIV in cases of CD8⁺ T cell depletion (117). Furthermore, ALT-803 could be used as an immunomodulator to effectively prevent the formation HIV/SIV: it transiently increased NK cell, CD8⁺ T and memory T cell populations as well as decreased viral loads in SIV-positive rhesus macaques (118). ALT-803 has been evaluated in clinical trials for lymphoma (119), solid and hematological tumors (120, 121), and for synergy with other immunotherapies (NCT01885897). ALT-803 is also undergoing clinical trials to assess the possibility of HIV reservoir elimination (NCT02191098). A phase II, randomized, unblinded, controlled trial will be conducted to investigate the safety, tolerability, and immunomodulatory effect of combining N-803 with ART during acute HIV infection (NCT04505501). The efficacy of N-803 with or without bNAbs is being evaluated for curing HIV in PLWH receiving analytic treatment interruption (ATI) (NCT04340596). There will be more good news to report in the future. ALT-803 has shown significant results in the treatment of bladder cancer and initial success in HIV infection, however, only one drug is not enough to eliminate AIDS and more combinations need to be explored.

In addition to T cells, NK cells are an efficient part of the immune response to HIV and act as a bridge between the adaptive and innate immune systems. Several NK cell-based therapeutics are currently used to treat cancer, and it is likely that they will be re-deployed to treat HIV. Some researches has focused on natural killer cell-based HIV immunotherapeutics (53, 74, 88, 160, 161). Herein, we outline several approaches and highlight ongoing studies for treating HIV by enhancement of NK cell activation or cytotoxicity ( Figure 2 ). NK cell super-agonists may also be useful for HIV viral suppression and reservoir clearance, and NK cell transfer is another powerful weapon for fighting HIV. CAR-NK cells are advantageous because they do not require HLA-matching and are abundantly available; therefore, they have the potential to be made into off-the-shelf products that can be used immediately and extensively in the clinic. Donor-related variability in NK cell expansion makes the utilization of NK cell lines an efficient and attractive substitute for cell therapy. Strategies that combine NK cell-based immunotherapy with other therapeutic approaches have shown success in numerous of clinical trials. In conclusion, the inherent nature of NK cells will likely make them a crucial tool for future multimodal strategies to clear HIV-1.

SL contributed to the conception of the review. SD and SL wrote and revised the manuscript. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (grant 81773787 to SL), the National Science and Technology Major Project (grant 2018ZX10301101 to SL), and the Guangdong Natural Science Foundation Research Team Project (grant 2018030312010 to SL).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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