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TRIGGERED: could refocused cell signaling be key to natural killer cell-based HIV immunotherapeutics?

Sugawara, Shoa; Manickam, Cordeliaa; Reeves, R. Keitha,b

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doi: 10.1097/QAD.0000000000002743
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Although antiretroviral therapy (ART) revolutionized the treatment of individuals living with HIV-1 infection [1], complete eradication of HIV-1 remains a high bar. People living with HIV-1 (PLWH) suffer from adverse health outcomes including higher contraction rate of cancer and cardiovascular diseases [2–4]. Immunotherapies for PLWH could potentially improve the control of HIV-1 infection and alleviate their health problems. Among the potential targets for the therapy, natural killer (NK) cells are considered major targets because of their multifaceted role(s) in immunity. The significance of NK cell responses has been demonstrated in multiple diseases. NK cell responses are readily associated with slower progression of solid tumors and elimination of other neoplastic cells [5–7]. In addition to their responses against cancers, NK cells confer protection against a wide range of viral infections including influenza and cytomegalovirus (CMV) infections [8,9]. NK cells exert their cytotoxic functions by directly recognizing virally infected cells through multiple mechanisms [10], or indirectly through antibodies, known as antibody-dependent cellular cytotoxicity (ADCC) [11]. In addition to their cytolytic responses, NK cells are well appreciated as major producers of numerous cytokines, such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and macrophage inflammatory protein (MIP)-1α [12]. Moreover, NK cells interact with other immune cell subsets to modulate their functions including dendritic cells, CD4+ and CD8+ T cells [13–15]. Although NK cells are generally categorized as innate effector cells, recent studies suggest that a certain subset of NK cells, collectively referred to as adaptive NK cells, can exert functions similar to adaptive immune cells [16]. Adaptive NK cells are typically characterized by lower expression of CD7, CD161, and the Fc receptor gamma chain and increased CD57 expression [17], and respond in an antigen-specific or memory-like fashion [16]. Moreover, adaptive NK cells seem to exhibit recall responses to secondary infections and vaccine antigens [16,17] For instance, adaptive NK cells from HIV-1 envelope (env)-vaccinated persons specifically kill target cells presenting HIV-1 env antigen [16]. Similarly, adaptive NK cells from healthy donors who experienced varicella zoster virus (VZV) infection exhibited VZV-specific responses even years after exposure [16]. To regulate these diverse adaptive and innate functions, NK cells integrate the signals from activating and inhibitory receptors [18,19]. As NK cell signaling strongly influences the nature of immune responses leading to their clearance of cancer and viral infection, NK cells are increasingly targeted for therapeutic development. Some of the NK cell-based therapeutics include checkpoint blockade and chimeric antigen receptor (CAR)-NK cells, and small molecule-based alteration of intracellular signaling [20–23]. Therefore, a better understanding of NK cell intracellular signaling events is highly critical. In this review, we will describe the general NK cell receptor signaling cascades, alteration of NK cell signaling in the context of multiple diseases, particularly in HIV/simian immunodeficiency virus (SIV) infection, and the potential application of NK cell signaling for therapeutics.

Overview of natural killer cell receptor signaling in humans

Receptors involved in NK cell signaling include CD16 (Fcγ receptor III), NKG2 family members, natural cytotoxicity receptors (NCRs), killer-cell immunoglobulin-like receptors (KIRs), and co-receptors, such as 2B4, DNAM-1, and CD2 (Fig. 1). The activating and inhibitory signals from these receptors regulate NK cell activation by modulating hundreds of genes critical for cytolytic responses [24]. These transcriptional changes then regulate NK cell functions including degranulation, target cell killing by release of lytic granules, and cytokine production [19]. Among the signals from receptors, CD16 signaling is by far the most studied signaling pathway in NK cells. CD16 plays a primary role in ADCC mediated by NK cells [11,25]. CD16 is constituted of an α surface subunit for ligand recognition, and an intracellular γ chain (FcRγ) for signal transduction [26]. In addition to FcRγ, CD3ζ can interact with CD16 to trigger signaling [27]. The cytoplasmic tails of FcRγ and CD3ζ have Immunoreceptor Tyrosine-based Activation Motifs (ITAM; YxxL/Ix6–8YxxL/I domain; where any amino acid can be x) [18,19], which relay activating signals. Upon ligation of the Fc portions of antibodies, Src family kinases including Lck and Fyn are activated and phosphorylate the ITAM domain of FcRγ and/or CD3ζ. The phosphorylated ITAMs allow protein kinases including Syk and ZAP70 to get recruited and phosphorylated [28]. Subsequently, Syk and ZAP70 phosphorylate transmembrane or cytosolic adaptor molecules, such as SLP76 and LAT [18]. On the basis of the knowledge from T-cell signaling and direct stimulation of CD16, downstream signaling events, including activation of JNK, ERK, phospholipase Cγ (PLCγ), and Vav-2 and Vav-3 are triggered after phosphorylation of these adaptor molecules [17,18,29–37]. Eventually, the activation of these numerous signaling molecules induces ADCC by NK cells, as well as cytokine secretion, such as IFN- γ and TNF-α [11,25,37].

Fig. 1:
Diagram of natural killer cell receptor signaling.

NKG2 family members are another class of receptors, which can have activating or inhibitory role in NK cell signaling in both humans and mice. Specifically, NKG2A, NKG2C, and NKG2D are the most studied molecules of NKG2 family proteins in NK cells. Both NKG2A and NKG2C form a heterodimer with CD94+[38,39], and recognize HLA-E [40]. The NKG2C/CD94 heterodimer is associated with another ITAM-bearing molecule DAP12. Upon stimulation of NKG2C/CD94, DAP12 is phosphorylated, which leads to subsequent signaling events including activation of Syk [41], JNK, and ERK [32]. In response to NKG2C signals, NK cells exhibit enhanced cytotoxicity and IFN-γ production [42,43]. Conversely, NKG2A has an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM; I/V/YxxL motif) in its cytoplasmic tail [44]. When the NKG2A/CD94 heterodimer binds to HLA-E, its ITIM domain gets phosphorylated, which allows the recruitment of the phosphatase SHP-1 [44,45]. SHP-1 then dephosphorylates signaling molecules including CD3ζ and DAP12 [28,44,45] and prevents cytolytic function and cytokine secretion of NK cells [42,46].

NKG2D utilizes signaling molecules distinct from the NKG2A/C-mediated pathways. Human NKG2D signals through DAP10 [47,48], which does not bear ITAMs, but has an YxxM motif [49]. DAP10 can activate distinct kinases including PI3K, PLCγ2, Vav-1, and NFκB, and its signaling is independent of signaling molecules in ITAM-based pathway, such as Syk, LAT, and ZAP70 [48,50]. NKG2D-mediated signaling eventually initiates the cytolytic function of NK cells against tumors [51] but does not trigger IFN-γ secretion [48].

Similar to CD16 and NKG2 family proteins, natural cytotoxicity receptors (NCRs) utilize ITAM-dependent signaling cascades. The well known NCRs include NKp46, NKp44, and NKp30, which transduce activating signals to NK cells [18,19]. NKp46 and NKp30 are generally expressed on all NK cells [18,52], whereas NKp44 expression is observed only on activated NK cells [53]. NKp46 and NKp30 are associated with FcRγ or CD3ζ, whereas NKp44 triggers signaling through DAP12 [53,54]. Similar to CD16 and NKG2C/CD94 signaling, recognition of ligands by NCRs triggers phosphorylation of FcRγ, CD3ζ, and DAP12, thereby inducing the activation of downstream signaling molecules including Syk, ZAP70, ERK, PLCγ, and Vav proteins [18,29–33,55]. These signaling events result in killing of target cells, such as tumors and virus-infected cells, [56,57] and secretion of IFN-γ [58,59].

In addition to NCRs, killer immunoglobulin-like receptors (KIRs) are also important receptors involved in NK cell signaling. KIRs are transmembrane glycoproteins bearing extracellular immunoglobulin (Ig)-like domains and expressed on primate NK cells [60]. KIRs are highly polymorphic [60,61], and recognize MHC class I molecules [60,62]. KIRs are named based on the number of its immunoglobulin (Ig)-like domains (characterized by ‘2D’ for instance), and the length of its cytoplasmic tail (written as ‘L’ for long or ‘S’ for short) [63]. For example, KIR2DL2 has 2 Ig-like domains along with a long cytoplasmic tail, and KIR3DS1 bears 3 Ig-like domains and a short intracellular domain. Depending on the length of their cytosolic tail, KIRs can trigger either activating or inhibitory signals. KIRs with short cytoplasmic tail (KIR-S) interact with DAP12, which transduces activating signals to NK cells [60,64]. In contrast, KIRs with long cytoplasmic tail (KIR-L) contain an ITIM domain in their intracellular region, which can recruit SHP-1 upon activation and inhibit NK cell signaling [60]. For instance, crosslinking of KIR3DS1 induces target cell killing and production of IFN-γ [65,66], whereas KIR3DL1 engagement diminishes cytotoxicity and IFN-γ and TNF-α secretion [65,67]. KIR2DL4 is the only KIR with a long intracellular domain, which is coupled to FcRγ [68] and thereby provides activating NK cell signals [60]. Interestingly, signals from KIR2DL4 induces both cytolytic function and IFN-γ production of activated NK cells but resting NK cells initiate only cytokine secretion in response to KIR2DL4 signaling [69].

Other co-activating receptors including 2B4, DNAM1, and CD2 augment NK cell signaling. 2B4 is a member of the SLAM family of receptors, which contains an intracellular Immunoreceptor Tyrosine-based Switch Domain (ITSM; S/TxYxxL/I motif). Upon stimulation, ITSMs get phosphorylated and allow the binding of SLAM-associated protein (SAP) [70]. Then the Src family kinase, Fyn, is recruited to SAP [71–73], initiating activating signaling cascades that include phosphorylation of Vav-1 and inhibition of phosphatase recruitment [73]. 2B4 signaling also activates NFκB, which synergizes with NKG2D signaling, and enhances NKp46-mediated degranulation of NK cells [50,74]. In the absence of SAP, phosphatases and inhibitory kinases including SHIP-1, SHIP-2, and Csk can bind to 2B4 [70], which in turn relays inhibitory signals to NK cells. In addition to 2B4, DNAM-1 exhibits an important function on NK cells, especially in the context of antitumor responses [75]. DNAM-1 stimulation activates Akt, ERK, Vav1, PI3K, and PLCγ1, but DNAM-1 does not utilize Fyn for its signal transduction [76]. DNAM-1 signaling also augments other activating signals including signals from NKp46 [74]. CD2 is another important receptor in NK cell function, particularly for mediating ADCC and adaptive functions [17,77]. Rolle et al.[77] illustrated that blocking of CD2 or one of its ligands CD58 impairs the cytokine production from adaptive NK cells. CD2 is also known to participate in CD16+-mediated signaling by increasing the phosphorylation of ZAP70, Syk, CD3ζ, and ERK [17,78]. NKp46 signaling is also enhanced by CD2 crosslinking [74]. In summary, NK cell activation is regulated by numerous receptors but these signals converge to the common signaling cascades, such as ERK, PLCγ, and PI3K to orchestrate NK cell functions including lysis of target cells and cytokine secretion.

Natural killer cell signaling and HIV/simian immunodeficiency virus infection

HIV-1 and SIV infections are known to modulate innate and adaptive immune cells through evasive strategies including alteration of cytokine milieu. In people living with HIV-1 (PLWH), numerous cytokines and chemokines, including IL-15, IL-18, and IFNγ are elevated in plasma compared with HIV-1 uninfected individuals [79]. Type 1 IFN signaling is also enhanced in PLWH, acutely and chronically SIV-infected rhesus macaques [80,81]. As IL-15, IL-18, and type 1 IFN are known to alter NK cell functions [82–84], one can expect the modulation of NK cell responses in HIV/SIV infection. Several groups reported the over activation of NK cells in PLWH [85,86], but it could be misleading because NK cell activation is typically assessed by the levels of HLA-DR, CD38, and CD69, which are commonly used markers of CD8+ T-cell activation [85,86]. However, recent single cell transcriptomic analysis of NK cells from PLWH revealed that unstimulated PLWH NK cells upregulated a number of genes similar to in-vitro stimulated NK cells from healthy controls but lacked the expression of cytotoxicity-related genes and cytokine receptor genes [24]. This indicates downregulated NK cell responses in HIV-1 infection may be independent of activation markers. Indeed, NK cells from HIV-1 infection became less functional compared with healthy controls, which correlated with altered expression of several activating and inhibitory receptors [87]. Furthermore, in PLWH with controlled viremia, reduced expression of CD3ζ, DAP12, and FcRγ was observed in CD56dimCD16+ NK cells. The phosphorylation of ZAP70/Syk by ex-vivo CD16 stimulation was dampened in the same population [88]. The reduction in NK cell activity could also be attributed to the accumulation of unique CD56CD16+ NK cell subset in PLWH [89–93]. These NK cells are characterized by low expression of Siglec-7, NKG2A, and NCR, and high NKG2C and SHIP-1 expression [90,93,94], and exerted lower cytokine expression and cytolytic activity [89–93].

HIV-1 infection can also alter NK cell function by changing the expression of MHC molecules, the ligand for NKG2A/C and KIRs, on infected target cells. In-vitro infection of primary HIV-1 isolates demonstrated the modulation of HLA-A, B, C and E expression on CD4+ T cells [95,96], which could potentially regulate KIR-mediated and NKG2A/C-mediated signaling. In fact, PLWH-expressing KIR3DS1 and HLA-Bw4-I80 are known to have stronger NK cell responses and slower AIDS progression [97,98]. Additionally, the KIR3DL1 haplotype is associated with better protection of HIV-1 in HLA-B∗57+ individuals [99]. Conversely, HIV-1 accumulates mutation in the HIV-1 gag protein, which improves binding of KIR2DL3 with HLA-C03 : 04 and suppresses NK cell degranulation [100]. Although numerous groups have reported the dysfunction of NK cells in PLWH [101], the effects on intracellular signaling status, especially by stimulation of combinatorial NK cell receptors has not been critically investigated. As the available data on intracellular signaling is only from CD16 crosslinking studies, it is challenging to assess whether HIV-1 specifically perturbs CD16 signaling or globally impairs signaling from multiple activating receptors. Therefore, it is critical to evaluate the differential activation and/or suppression of intracellular NK cell-signaling pathways in HIV-1 infection.

Similar to NK cells in PLWH, SIV infection dampens NK cell activity through modulation of surface receptor expression. Overall, NK cells from SIV-infected animals diminish activating receptor expression and cytokine secretion [102]. Ries et al.[103] demonstrated KIR3DL05+ NK cells increased during acute SIV infection in rhesus macaques, whereas KIR3DL01+CD16+ NK cells decreased during chronic infection. They also observed delayed degranulation and cytokine secretion by KIR3DL01+ and KIR3DL05+ NK cells compared with KIR3DL01KIR3DL05 NK cells [103]. NK cell malfunction is caused by not only the expression of inhibitory signaling molecules but also its trafficking to LNs [104]. Huot et al.[104] characterized the upregulated CXCR5 expression on NK cells from SIV-infected African Green Monkeys (AGM)s that do not develop SIV pathogenesis, but not on NK cells from rhesus macaques with pathogenic SIV infection. As a consequence, lack of NK cell trafficking into LN follicles was described in rhesus macaques but not in AGMs [104]. However, the connection between the change in intracellular signaling activation and NK cell trafficking is still unclear. Though one of the advantages using SIV infection models is to analyze NK cells in multiple anatomical locations, the change in intracellular signaling is poorly understood similar to PLWH studies. It would be beneficial to comprehend the signaling perturbation in NK cells in circulation and tissues from SIV-1-infected macaques as dysregulated NK cell signaling could also influence NK cell trafficking.

In order to address the gap in knowledge between the surface receptor expression profile and downstream signaling, our lab investigated the induction of NK cell signaling in SIV-infected RMs by CD16 crosslinking studies. Along with the lower protein expression of ZAP70, and DAP12, reduced CD3ζ, Syk, and ZAP70 phosphorylation was observed in NK cells from SIV-infected rhesus macaques when they were stimulated with anti-CD16 antibody ex vivo[36]. The decrease in CD3ζ, Syk, and ZAP70 phosphorylation was also demonstrated in FcRγSyk adaptive NK cells. Recently, our group performed RNA-seq analyses to characterize NK cell gene signatures in rhesus macaques that have never been described before [105]. We observed a significant overlap of NK cell-specific genes between rhesus macaques and humans including major pathways of TLR, KIR, and cytokine signaling, RNA-seq analyses also revealed the modulated expression of hundreds of NK cell signature genes in SIV infection [105].

Modulation of natural killer cell signaling in other diseases

Similar to HIV-1/SIV infections, NK cell signaling in other pathogenic infections and cancers is perturbed by modulation of surface receptor and intracellular signaling molecule expression (Table 1). The levels of surface receptors on NK cells including CD11a, CD16, CD69, NKp46, NKp30, NKG2D, and DNAM-1 are altered in multiple bacterial and viral infections as well as cancers [106–110]. In many cases, the modulated surface receptor expression is associated with impaired T-cell responses [107] and disease progression [108]. Specifically, many cancers and virus-infected cells, such as hepatitis C virus upregulate HLA-E, which enhances inhibitory signals through NKG2A [20,46,114]. In addition, viruses and cancers also downregulate the levels of intracellular signaling molecules in NK cells, such as CD3ζ [55] T-bet and Eomes [110], which leads to impaired NK cell activity. Several studies suggest the link between the dysfunctional NK cell responses and immunosuppressive cytokines, such as IL-10 and TGF-β1 [106,108] but the direct contribution of these cytokines still needs to be evaluated.

Table 1 - Alteration of natural killer cell signaling by pathogens and diseases.
Pathogen/disease NK cell modulation
Tuberculosis [106] Reduced CD11a, CD16, CD69 expression
LCMV [107] Expansion of NKG2D+ NK cells
Gastric cancer [108], cervical cancer [109] Reduced NKp46, NKp30, DNAM-1, NKG2D expression
HCV [46], SCCHN [20] NKG2A-mediated inhibition
Influenza [55] CD3ζ degradation
B/T cell lymphoma [110] Reduced NKG2D, NK1.1, T-bet, Eomes expression
HCMV [111,112], rhCMV [36,113] Expansion of NKG2C+ and Δg adaptive NK cells
rhCMV [36,113] Increased CD3ζ usage
HIV [88–93], SIV [36] Expansion of defective CD56CD16+ NK cellsSuppressed global, Δg and CD56dimCD16+ NK cell signaling
The mechanisms by which specific pathogens and disease states may modulate NK cell signaling is summarized. HCMV, human cytomegalovirus; HCV, hepatitis C virus; LCMV, lymphocytic choriomeningitis virus; rhCMV, rhesus cytomegalovirus; SCCHN, squamous cell carcinoma of the head and neck; SIV, simian immunodeficiency virus.

In addition to the modulation of NK cells in general, adaptive NK cell signaling is also altered specifically by CMV infection. A unique subset of adaptive NK cells deficient in FcRγ expression (Δg NK cells) accumulated in HCMV-infected individuals [111,112]. Macaques with rhesus CMV (rhCMV) infection also exhibited increased numbers of adaptive Δg NK cells and NKG2C+ NK cells [36,113]. Δg NK cells are characterized by distinct surface receptor expression including lower Siglec-7, CD7, and TIM-3, and higher CD2 and Fas expression [111]. This subset exerts stronger responses by CD16 stimulation than conventional NK cells [111]. The distinct function of Δg NK cells can be attributed to its intracellular signaling molecule expression, such as loss of Syk and FcRγ [111], Taken together, dysregulated NK cell signaling is induced in many diseases, highlighting the potential importance for both pathogenesis and therapeutics.

Potential application of novel signaling assays to natural killer cells

Due to limits on multiplex evaluation, comprehensive studies of intracellular NK cell signaling are challenging despite its potential significance. The activation of NK cell signaling is currently most commonly assessed by transcriptomic analysis, western blot, and phospho-flow. Costanzo et al. [24] evaluated the activation of NK cell signaling by gene chip-based transcriptome analysis followed by gene set enrichment analysis. This approach could identify potential elevation of signaling based on gene expression profile [24], but it could not measure the levels of posttranslational modifications of proteins including phosphorylation and ubiquitination. To evaluate the levels of posttranslational modification, western blot is widely used for many cell types including NK cells [48,115]. However, western blot has limited quantitative analysis for some proteins as its sensitivity varies depending on molecular weight and endogenous protein expression [115]. Although the quantification of posttranslational modifications is difficult by western blot, phospho-flow can quantitatively measure the levels of phosphorylated protein by mean-fluorescent intensity [36,88]. Another strength of phospho-flow is that the levels of protein phosphorylation can be quantified in multiple cell types at the same time. By using phospho-flow, Shah et al.[36] was able to monitor phosphorylation of signaling proteins in both conventional and adaptive NK cells simultaneously. Nonetheless, phospho-flow is still not ubiquitous for exhaustive analysis of signaling activation because of the limited availability of antibodies with fluorophores for signaling molecules [116].

For comprehensive screening of NK cell signaling activation, multiplex signaling assay or proteomics approach as used in T-cell biology would be beneficial. Karlsson et al.[117] tested the activation of kinases in CAR-T cells by measuring the phosphorylation of a library of peptides bearing known phosphorylation sites. They further confirmed a similar trend in the activation of seven signaling molecules in T-cell receptor (TCR) signaling by bead-based multiplex signaling assay [117]. The elevation of several canonical signaling pathways including NFκB, STAT3, and JNK signaling, has also been analyzed by the bead-based multiplex assay in myeloid leukemia cells, pediatric leukemia, and human odontoblast-like cells [118–120]. Unlike phospho-flow or western blot, multiplex signaling assay can monitor the activation of several signaling molecules simultaneously in one experiment [117–120]. However, multiplex assays have disadvantages, such as reduced sensitivity to each signaling molecule and optimal timepoint selection for multiple analytes.

Global phosphoproteomics approaches can also be useful to discover novel signaling events in NK cells. Phosphoproteomics approach is the technique to diagnostically evaluate phosphorylation of cellular proteins in combination with mass spectrometry [121]. Using this technique, TCR signaling activation has been exhaustively analyzed in murine T cells [122] and human peripheral blood mononuclear cells [123]. This strategy was also implemented to analyze primary CD4+ T cells susceptible to HIV-1 entry and revealed new phosphorylated proteins in response to HIV-1 infection [124]. In addition, Awasthi et al.[125] screened the difference in phosphorylation events between Raji cells (B-cell lymphoma cell line) that are sensitive or resistant to NK cell-mediated ADCC using phosphoproteomics. They discovered differential phosphorylation events in ERK pathway including MAPK1, and elucidated the evasion of ADCC by Raji cells is inducible by MAPK1 inhibition [125]. Although phosphoproteomics approaches have challenges including detection of many irrelevant phosphorylation events and large amount of input materials [121], they are successful in describing uncharacterized signaling pathways. Thus, it is worthwhile to apply proteomics strategies to NK cells from PLWH and SIV-infected macaques so that the key signaling events in NK cell dysfunction can be identified.

Therapies targeting natural killer cell signaling

Targeting NK cell signaling for immunotherapy, including checkpoint blockade, CAR-NK cells, and intracellular signaling modulation by small molecules, has demonstrated a promising result in treatment for cancer and HCV infection. Currently, blocking KIRs and NKG2A has been implemented to improve NK cell responses towards cancers and viral infections. Lirilumab, which blocks KIR2DL1, 2, and 3, was used for acute myeloid lymphoma (AML) and multiple myeloma therapy [126,127]. In both studies, anti-KIR therapy induced improved responses to tumors [126,127] with good tolerability in phase I clinical trial, but not in phase II trial ( ID: NCT02399917). NKG2A blockade is also being tested as many tumors upregulate HLA-E [20]. Although the number of studies is limited, blocking NKG2A along with anti-EGFR therapy decreased the size of squamous cell carcinoma of the head and neck presumably by NK cell-mediated ADCC [20]. NKG2A blockade also enhanced not only NK cell but also HCV-specific CD8+ T-cell responses in mice model [46]. As HIV-1 infection modulates HLA-A, B, C, and E expression on CD4+ T cells in vitro[95,96], therapies targeting NKG2A/C or KIRs could be a reasonable approach for HIV-1 treatment.

In addition to checkpoint blockades, CAR-NK cell therapy could be another effective strategy to treat multiple diseases. CAR is an artificial immune receptor that was originally developed for T-cell therapeutics [128]. Most CARs consist of single chain fragments of the variable region of antibody (scFv) for target recognition, and signaling molecules including CD3ζ or FcRγ conjugated to scFvs [128]. CAR was also demonstrated to function in cord blood NK cells and iPSC-derived NK cells [21,22]. Recently, anti-CD19 CAR-NK cell therapy was performed on individuals with lymphoid tumors. CAR-NK cells expanded in patients and survived at least 12 months after infusion. Liu et al.[129] illustrated CAR-NK cell therapy reduced tumors in a number of patients, and did not develop severe side effects unlike CAR-T cell treatment [129]. CAR-NK cell-based clinical trial is also being tested for COVID-19-infected people ( ID: NCT04324996) owing to its effectiveness and safety in cancer treatment.

CAR-NK cell treatment could also improve immune responses to HIV-1 infection based on the promising data from CAR-T-cell studies in HIV-1/SIV. A number of groups reported that CAR T cells can kill HIV-1/SIV-infected cells in vitro[130,131]. Haran et al.[130] also demonstrated that co-expression of CXCR5 on T cells along with CAR enables them to migrate to lymph nodes, implying the possibility that CAR cells can target HIV/SIV-infected cells in tissues. Similar to CAR T cells, CD4-TCRζ CAR-NK cells were capable of killing HIV-1-infected CEM cells in vitro[132]. Currently, adoptive NK cell transfer is the only NK cell-based clinical trial performed for HIV-1 treatment ( ID: NCT03346499) but CAR-NK cell therapy could be an alternative approach for PLWH. However, results from CAR-T-cell strategy suggest CAR-NK cells may require multiple signaling domains to overcome lack of activating signals in vivo[133].

In order to optimize CAR-NK cell functions, co-stimulatory molecules, and NK cell-specific signaling molecules are incorporated to CAR development. Imai et al.[134] elucidated that incorporation of 4–1BB into CAR-NK cells enhanced their cytolytic responses to tumor cell lines. Furthermore, NKG2D-DAP10-TCRζ CAR NK cells exerted not only cytotoxicity against tumors but also secreted a number of cytokines [135], which cannot be induced by NKG2D crosslinking alone [48]. Thus, one strategy to maximize CAR-NK cell activity is to test a library of CARs with any possible combination of surface and intracellular signaling molecules, as tested in CAR-T cell [133,136]. Nevertheless, it is still unclear whether CAR-NK cells become dysfunctional regardless of the components of CARs by immunosuppressive environment generated by HIV-1 infection.

Another strategy to enhance NK cell activity is to modulate intracellular NK cell signaling by small molecules. Several studies reported the elevation of specific signaling pathways in cancer, such as Notch and hedgehog (Hh) signaling, and antitumor therapy has attempted to incorporate inhibition of these signaling cascades [137]. Although the effect varies depending on tumor types, Notch and Hh signaling inhibition demonstrated antitumor activity in patients with AML [138] and desmoid tumors [139]. Similarly, the responses from NK cells could be enhanced by targeting intracellular signaling. Recently, Hideshima et al.[140] demonstrated the surface receptor-independent NK cell activation and increased NK cell cytotoxicity by ZAP70 activator. Considering the reduced phosphorylation of ZAP70 in NK cells from PLWH and SIV-infected animals [36,88], surface receptor-independent ZAP70 activation by small molecules could restore NK cell functions in PLWH. As, defective CD56CD16+ NK cells in PLWH exhibit SHIP-1 upregulation [94], NK cell responses could also be improved by a SHIP-1 inhibitor. Indeed, transient SHIP-1 inhibition by small molecules increased antitumor NK cell responses in a mouse model [23]. Giordano-Attianese et al.[141] engineered novel CAR-T cells whose function can be regulated by small molecule-based inhibition of Bcl-XL protein interaction. This suggests the potential improvement of CAR-NK cell responses by small molecules even if CAR-NK cells become dysfunctional in the course of treatment. However, similar to Notch and Hh inhibition in cancer [137], it is important to evaluate the potential off-target effect by ZAP70 activators or SHIP-1 inhibitors as ZAP70 and SHIP-1 are also expressed in other immune cells [18]. The toxicity of ZAP70 activators or SHIP-1 inhibitors also needs to be investigated as several side effects have been reported in cancer patients with Notch or Hh inhibitor therapy [138,139]. Regardless, extensive analysis of intracellular signaling is still required to determine the target signaling molecules.


In this review, we have highlighted the distinct and common pathways in NK cell signaling, overview pathogen and disease modulation of NK cell receptor signaling, particularly in HIV/SIV infection, and discuss how recent NK cell-based therapies may be beneficial in infectious diseases. As NK cells play varied roles in many diseases, therapies targeting NK cells could enhance not only cytolytic activities but also regulate other direct and indirect immune functions of other immune cells. Particularly, applications for PLWH treatment would be a valid strategy based on the impaired NK cell responses observed in multiple studies. However, many studies have traditionally focused on the modulation of surface receptor expression without carefully investigating the changes in intracellular signaling. Furthermore, despite the strength of SIV-infection model to understand NK cell signaling in numerous tissues, only few groups have ever investigated NK cell signaling in SIV infection. Due to the incomplete understanding of intracellular NK cell signaling, NK cell therapy is limited to the intervention of one or two signals from surface receptors. Further, it is still unclear, which intracellular signaling molecules are crucial to restore NK cell functions that may be impaired by HIV-1 infection. By determining the key cytosolic signaling molecules using multiplex signaling and proteomics analysis, NK cell therapy could be complemented by modulation of the key signaling molecules using small molecule inhibitors, knockdown of phosphatases, and overexpression of kinases. Therefore, extensive study of intracellular signaling in NK cells in PLWH and SIV-infected animals is critical to both understand disorders that could be potentially reversed and immunotherapies that could be enhanced.


Funding: This work was supported by National Institutes of Health grants R01 AI120828 and P01 AI120756 (both to R.K.R.).

Author contributions: S.S. and C.M. generated the idea prepared the manuscript. RKR edited the final version. All authors contributed to the writing.

Conflicts of interest

There are no conflicts of interest.


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cell signaling; HIV; natural killer cells; simian immunodeficiency virus

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