key: cord-0877703-5uwizpl0 authors: Alves, Eric; McLeish, Emily; Blancafort, Pilar; Coudert, Jerome D.; Gaudieri, Silvana title: Manipulating the NKG2D Receptor-Ligand Axis Using CRISPR: Novel Technologies for Improved Host Immunity date: 2021-08-12 journal: Front Immunol DOI: 10.3389/fimmu.2021.712722 sha: 195c6c1f15db5455d1f6f035fb02f57207f552cf doc_id: 877703 cord_uid: 5uwizpl0 The activating immune receptor natural killer group member D (NKG2D) and its cognate ligands represent a fundamental surveillance system of cellular distress, damage or transformation. Signaling through the NKG2D receptor-ligand axis is critical for early detection of viral infection or oncogenic transformation and the presence of functional NKG2D ligands (NKG2D-L) is associated with tumor rejection and viral clearance. Many viruses and tumors have developed mechanisms to evade NKG2D recognition via transcriptional, post-transcriptional or post-translational interference with NKG2D-L, supporting the concept that circumventing immune evasion of the NKG2D receptor-ligand axis may be an attractive therapeutic avenue for antiviral therapy or cancer immunotherapy. To date, the complexity of the NKG2D receptor-ligand axis and the lack of specificity of current NKG2D-targeting therapies has not allowed for the precise manipulation required to optimally harness NKG2D-mediated immunity. However, with the discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins, novel opportunities have arisen in the realm of locus-specific gene editing and regulation. Here, we give a brief overview of the NKG2D receptor-ligand axis in humans and discuss the levels at which NKG2D-L are regulated and dysregulated during viral infection and oncogenesis. Moreover, we explore the potential for CRISPR-based technologies to provide novel therapeutic avenues to improve and maximize NKG2D-mediated immunity. Interactions between hosts and pathogens are constantly evolving, and in this ongoing arms race each side aims to outsmart the other. Host and pathogen genetics form a key part of this competitive evolutionary relationship, with variation in their respective genomes having a considerable impact on hostpathogen dynamics. Many pathogens, such as viruses, generate this variation in the form of mutations as a by-product of their rapid, error-prone replication. Some of these mutations may confer a selective advantage to the pathogen, and via the process of natural selection are retained within the variant "pool", termed quasispecies (1) . Interestingly, this process of pathogen evolution bears an uncanny resemblance to what is seen during oncogenesis. During oncogenic transformation, genomic instability gives rise to tumor variants, which undergo a selective process to similarly maintain "fitter" variants within the tumor quasispecies (2) . In both the tumor and pathogen contexts, host immune pressure constitutes a major selective force of pathogen/tumor evolution. In humans, the immune response relies on the strategic orchestration of innate and adaptive immunity, which comprises a variety of cell types and soluble molecules. This is regulated by the interaction of multiple receptors and ligands expressed at the cell membrane or released as soluble proteins. As such, pressure exerted via receptor-ligand mediated immune responses inadvertently selects for viral or oncogenic mutations that dysregulate receptor/ligand expression (3) (4) (5) . Consequently, in order to compete against pathogen diversification and oncogenic transformation in this way, humans have over the course of this perennial host-pathogen battle developed in their arsenal a high level of polymorphism at loci encoding receptors/ligands responsible for immune recognition (6) (7) (8) . One such receptorligand axis is the type II lectin-like transmembrane natural killer group 2 member D (NKG2D) receptor and its cognate ligands (NKG2D-L). Herein, we briefly overview the NKG2D receptorligand axis in humans, explore the levels at which NKG2D-L regulation/dysregulation occurs, and discuss how clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies are poised to harness NKG2D-mediated immunity in the analogous contexts of oncogenic transformation and viral infection. The NKG2D Receptor NKG2D is the most versatile and widely distributed activating/ co-stimulatory natural killer (NK)-related receptor. First identified in human NK cells in 1991 (9) , NKG2D has since been discovered on numerous cell subsets including, activated (ab and gd) T cells, natural killer T (NKT) cells, and mucosalassociated invariant T (MAIT) cells (10) (11) (12) (13) . Increasingly, NKG2D expression is also being identified on tissue-resident innate lymphoid cells (ILC), such as certain ILC1 (14, 15) , ILC2 (16) and ILC3 (17) subsets. In humans, NKG2D is encoded by the KLRK1 gene and is located within the NK gene complex (NKC) on chromosome 12p. Moreover, NKG2D is highly conserved across multiple vertebrate species (18, 19) . To date, two major human haplotype alleles of NKG2D have been identified, termed LNK1 (low activity) and HNK1 (high activity) alleles (20, 21) , with surface expression of NKG2D lower in carriers of the low activity LNK1/LNK1 genotype (20, 22) . When expressed at the cell surface, the 42 kDa homodimeric NKG2D receptor combines with the DNAX-activating protein 10 (DAP10) homodimer and following its engagement with cognate ligands initiates a cytotoxic cellular response and/or the secretion of pro-inflammatory cytokines ( Figure 1A ) (23) (24) (25) . NKG2D is also capable of facilitating the function of other activating receptors, depending on the inflammatory milieu and/ or expressing cell type. For example, in activated NK cells primed by pro-inflammatory cytokines (e.g. interleukin (IL)-2 and IL-15), NKG2D provides direct stimulatory signals (26) (27) (28) (29) , whereas in resting NK cells, it synergizes as a co-activator with other receptors, such as NKp46 and 2B4 (30, 31) . In ab T cells, NKG2D typically provides a co-stimulatory signal, acting to promote T cell receptor (TCR)-dependent cytotoxicity, production of pro-inflammatory cytokines and memory differentiation (32) (33) (34) (35) (36) (37) . Interestingly, prolonged exposure to IL-15 has been shown to increase expression of NKG2D in CD8 + T cell subsets, potentiating TCR-independent activation (38, 39) . Similarly in gd T cells, NKG2D can function as a costimulatory molecule (40) , but may also directly trigger cytotoxicity in a TCR-independent fashion (41) . Alternatively, some gd T cells have been shown to bypass the NKG2D receptor and recognize NKG2D-L, such as ULBP4 or MICA/B, directly via their TCR, implying a TCR agonistic role (42) (43) (44) . In innatelike T cells, such as invariant NKT cells, NKG2D is restricted to the CD4subsets, and functions to mediate direct lysis of target cells and co-stimulatory activation (12) . Whereas, in MAIT cells, NKG2D is more prominent on CD8 + subsets, and functions as a co-stimulatory molecule (45) or, if in the presence of IL-15, exerts NKG2D-dependent innate-like cytotoxicity (13, 46) . Lastly, in ILC subsets, early studies highlight that expression of NKG2D may aid in the production of pro-or anti-inflammatory mediators depending on the surrounding microenvironment (15, 16) . However, further investigation is required to completely elucidate the impact of NKG2D-mediated signaling on ILC function. Altogether, although NKG2D expression, regulation and function differ across the above cell types, it undoubtedly plays a central regulatory role in the immune response and is vital for immunological surveillance against tumorigenic transformation and viral infection. (ULBP) family (ULBP1-ULBP6) ( Figure 1B ) (47) (48) (49) (50) . NKG2D-L are, for the most part, not constitutively expressed, but instead are selectively induced upon cellular stress, damage or transformation, as is caused by events such as viral infection or oncogenesis (32, 51) . Moreover, as reviewed by Lanier (52) , essentially all cell types are capable of expressing one or more types of NKG2D-L if given the appropriate stimulus. For example, Fujita and colleagues (53) identified two distinct ligand expression profiles in non-neoplastic epithelial tissues: ULBP5-ULBP3-MICA/B and ULBP2/6-ULBP1-ULBP4. Moreover, in cells undergoing tumorigenic transformation, high heterogeneity in NKG2D-L expression has been reported. Notably, expression of two or more NKG2D-Ls (often MICA and The natural killer group 2 member D (NKG2D) receptor consists of a disulphidelinked homodimer that associates with the DNAX-activating protein 10 (DAP10) disulphide-linked homodimer for cellular signaling. DAP10 harbors a Tyr-X-X-Met (YxxM) motif, which binds the p85 subunit of phosphatidylinositol-3 kinase following phosphorylation. (B) NKG2D ligands encompass the MHC class-I polypeptiderelated sequence A (MICA), MICB and six UL16-binding proteins (ULBP1-6). MICA/B (also termed PERB11.1/11.2) share similar structural and functional properties, with both containing three extracellular domains (a1, a2 and a3) and a transmembrane domain for binding to the cell surface. In comparison with full-length MICA alleles, MICA*008 differs by encoding a truncated protein due to a nucleotide insertion in the transmembrane domain and is known to acquire a glycolsylphosphatidylinositol (GPI) lipid anchor for cell surface expression. ULBP1-6 (also termed RAET1I/H/N/E/G/L) lack the a3 extracellular domain and are either bound to the cell surface by a GPI-anchor (ULBP1-3, 6) or transmembrane domain (ULBP4, 5). (C) Various functional MICA, ULBP4 and ULBP5 splice variants have been identified. MICA-A, -B1, -B2, -C and -D are known isoforms lacking the extracellular a3 domain and the a2 domain in the majority of isoforms (A, B1, C and D). Moreover, MICA-A and MICA-C both lack a transmembrane and cytoplasmic domain, which impairs their expression at the cell surface. RAET1E1/ULBP4-I, RAET1E2/ULBP4-II and RAET1E3/ULBP4-III are membrane-spanning splice variants with an extended a1 domain, reduced a1 domain and reduced a2 domain, respectively. RAET1G2/ULBP5-II and RAET1G3/ULBP5-III are truncated soluble splice variants resulting from two alternative premature stop codons before the transmembrane domain. The surface-expressed splice variants have been shown to bind NKG2D to a similar degree as compared to their wildtype isoforms, except for MICA-B2 and -D that bind NKG2D with a significantly weaker affinity. MICB) is more common in solid tumors, compared to hematological tumors (shown to predominantly express MICB) (54) . Furthermore, co-expression of multiple allelic forms of the same ligand has also previously been identified at the surface of cells undergoing stress, which is strongly suggestive of functional redundancy in these molecules. It is also worth noting that NKG2D-L differ in their affinity (K D ) and avidity for NKG2D, such that ULBP1 (the only ULBP member tested to date) has the highest affinity (1.1 mM), followed by MICA (0.9-1 mM) and MICB (800 nM) (55) (56) (57) (58) (59) . Therefore, it is speculated that NKG2D may transduce different signals or activate separate downstream pathways based on which ligand or allelic variant is bound (60) , which supports the various functions of NKG2D discussed prior. Surprisingly, the role of NKG2D-L extends beyond providing a signal for cellular stress. In cells of the myeloid lineage, these ligands can mediate lymphocyte activation leading to cytotoxicity, cytokine production and proliferation. For instance, expression of MICA/B on dendritic cell-derived exosomes plays an important role in promoting NK cell differentiation and proliferation (61) . Furthermore, ULBP2/3 expression levels are increased during CD34 + hematopoietic progenitor commitment to the granulomonocyte lineage, suggesting that NKG2D-L play a role in promoting myeloid differentiation (62) . Alternatively, NKG2D-L expression on myeloid cells can lead to lymphocyte inactivation and maintenance of immune homeostasis. For example, persistent expression of membrane bound ULBP1 and MICB on myeloid cells induces NKG2D internalization and desensitization of NK cells (63) . Moreover, overexpression of MICA on activated CD8 + T cells makes them susceptible to NK cell lysis, indicating that NKG2D-L may participate in immune homeostasis during ongoing immune responses (64) . During infection with Mycobacterium tuberculosis, heightened ULBP1 expression on expanded T regulatory cells (Tregs) facilitates NK cell-mediated killing of these cells, thereby enhancing the overall immune response (65) . Ultimately, NKG2D-L expression is associated with both cytotoxic and regulatory processes, as is reflected by the diverse roles played by these molecules in host immunity. The MICA and MICB genes were originally described as stressinduced MHC class I polypeptide-related sequences and are located in the MHC region on the short arm of chromosome 6 (47, 66) . These genes are highly polymorphic (67) , with, to date, over 100 described alleles (allelefrequencies.net). The MIC alleles have variations that are, to a certain extent, concentrated in the extracellular domains as well as truncated forms due to coding frame-shifts (e.g. MICA 5.1). Specific alleles have been associated with disease outcomes (68) (69) (70) , influence the amount of soluble protein and impact binding affinity to its cognate receptor, NKG2D (71) . Moreover, numerous MICA splice variants have been documented thus far ( Figure 1C) , with the majority binding NKG2D similarly as their wildtype counterparts, highlighting that lack of a domain (e.g. a3 domain) does not necessarily reduce binding affinity (60) . Interestingly, there is a naturally occurring MICA-MICB null combination (deletion of MICA and premature stop codon for MICB) that exists on the HLA-B48 haplotype found in East Asian and South American populations (72) (73) (74) . Furthermore, the MICA-MICB genes are merged in chimpanzees resulting in a hybrid form (75) , while they are absent in mice (76) . The presence of the null haplotype without obvious phenotypic consequences suggests redundancy in the NKG2D receptor-ligand axis but its overall importance is highlighted by the overlapping mechanisms exhibited by cancers and viruses to evade it (77). Although the ULBPs are distantly related to MICA/B in sequence, they differ in their location, mapping instead to the opposite (long) arm of chromosome 6 (78, 79) . Emerging data suggests that the extensive diversity seen in the ULBP family may be due to the functional or locational specialties of each ligand, as is evidenced with ULBP4 and its predominant expression in skin (80) (81) (82) . Splice variants exist (ULBP4 (83) , and ULBP5 (83, 84); Figure 1C ) providing significant within locus diversity. Furthermore, as reviewed by Carapito & Bahram (19) , clear differences in allele frequencies between geographically distinct populations exist for the ULBP family, which suggest that polymorphisms in ULBP may be a consequence of divergent selective pressures. Moreover, the possession of a large ULBP family in humans and other species is thought to provide a selective host advantage in the evasion of viruses and tumors. However, overall, the ULBP family appears to be less polymorphic than the MIC genes, albeit studies of ULBP gene polymorphisms and haplotypes remain limited (50, (85) (86) (87) (88) . The appropriate regulation of NKG2D-L is integral to the effective detection and elimination of virally infected or neoplastically-transformed cells. Many reviews to date have discussed the various levels of regulation involved in the control of NKG2D-L expression. For a comprehensive overview of these mechanisms in health and disease, we refer to previously published reviews (59, 89) . In this section, however, we focus on convergent regulatory mechanisms exploited by both tumors and viruses to evade NKG2D-mediated immunity. NKG2D-L expression is regulated at the level of transcription, post-transcription and post-translation through numerous pathways and molecules intrinsically linked to cellular stress ( Table 1) . As such, it is unsurprising that both viruses and tumors harbor various mechanisms that work in combination to hijack and dysregulate NKG2D-L at multiple levels. At the transcriptional level, viral proteins (e.g. HBV's HBx and HBc) have been shown to directly suppress MICA/B (154) . A similar strategy is achieved by tumors (e.g. melanoma), whereby cells with highly methylated NKG2D-L loci are selected for, given the resultant suppression of transcription (54) . Although mechanistically different, both viral proteins and tumormediated methylation converge at the DNA level to hinder transcription of NKG2D-L and facilitate immune evasion. At the post-transcriptional level, viruses and tumor cells exhibit convergence in their use of microRNAs (miRNAs) to inhibit NKG2D-L transcript translation. For instance, viral microRNAs have been shown to directly bind to the 3' untranslated region of MICB (HCMV-miR-UL112, EBV-pri-miR-BART2-5p, KSHV-miR-K12-7) (128, 131) and ULBP3 (JCV-miR-J1-3p) (155) , to trigger transcript destabilization and degradation. In addition, viral miRNAs can act indirectly by preventing translation of key components of the GPI-anchoring machinery (HSV1-miR-H8) (156) or blocking translation of surface shedding inhibitors (HCMV-miR-US25-2-3p) (157) . Interestingly, host oncogenic miRNAs (124, (126) (127) (128) 158 ) also bind to the 3' untranslated region of MICA (miR-520b) (127), MICB (miR-17-5p, miR-20a, miR-93, miR-106b, miR-372, miR-373, miR-520c) (128) and ULBP2 (miR-32a/c) (126) , in a similar fashion to viral miRNAs, with some binding sites identified by Bauman & Mandelboim (155) as overlapping with those targeted by the viral miRNAs referenced above. Altogether, the shared binding sites and action of oncogenic and viral miRNAs indicate the convergent evolution of tumorigenic and viral immune evasion mechanisms at the post-transcriptional level. Interference with NKG2D-L expression at the protein level is often a major target of both viruses and tumors. The two major mechanisms of reducing NKG2D-L surface expression have been reviewed by others (77, 89, 159, 160) , and are understood to be (1), intracellular retention and degradation, and (2), surface shedding. In the context of viruses, key viral proteins (e.g. HCMV's UL16, UL142) have been found to reside in the endoplasmic reticulum and cis-Golgi apparatus of cells, and cause intracellular retention and degradation of MICA/B and ULBP1-3 (161) (162) (163) (164) . Alternatively, viral infection with HIV or HCMV has also been shown to activate the shedding molecules ADAM10/17, which are otherwise essential for development and homeostasis, resulting in cleavage of MICA/B and ULBP2 (4, 157, 165) . Manipulation at the protein level in a similar fashion is also extensively seen in cancer. For example, NKG2D-L have previously been shown to be retained in the endoplasmic reticulum or cytoplasm in a variety of cancer types, including melanoma, breast, colorectal, lung and gastric cancers (51, 166) , resulting in reduced cell surface expression. Similarly, NKG2D-L shedding from tumor cells via enhanced expression of ADAM10/17, is understood to contribute significantly to the poor immunogenicity of many cancers (143) (144) (145) . Again, the similarities between viruses and tumors in targeting NKG2D-L at the DNA, RNA and protein level is strongly suggestive of convergent evolution and highlights fundamental immune evasion mechanisms. The ability to modify loci at the genetic and epigenetic level in a specific manner using CRISPR-associated (Cas) proteins, such as Cas9 (167, 168) , has greatly expanded our knowledge of diseases, their genetic components and the development of targeted therapies. When combined with a short guide RNA (sgRNA), consisting of a non-coding trans-activating RNA annealed to a target-specific 20 nucleotide RNA, Cas9 is able to base pair with any target DNA located adjacent to a conserved protospaceradjacent motif (5'-NGG-3' in the frequently used Streptococcus pyogenes Cas9) and induce specific DNA cleavage. This process allows efficient and precise DNA editing. Beyond the CRISPR-Cas9 system, the synthesis of engineered variants, such as nuclease-deactivated Cas9 (dCas9), has provided new avenues for gene editing and regulation. The CRISPR-dCas9 system harbors two mutations (D10A and H840A), which deactivate Cas9's cleavage capability (169) . In doing so, the RNA-guided DNA-binding specificity of Cas9 can be harnessed to precisely direct effector domains that mediate transcriptional activation (170) (171) (172) or repression (173) (174) (175) . Despite the success of wild-type Cas9, its ability to introduce irreversible genetic changes, particularly at off-target sites, has raised safety concerns. Therefore, to date, clinical trials utilizing the CRISPR-Cas9 system have been performed ex vivo, where extensive off-target checks can be conducted (176, 177) . Although clinical use of Cas9 remains limited due to these trepidations, the application of dCas9 in gene therapy is becoming increasingly likely, given its transient nature and inability to permanently alter the genetic code (178) . Moreover, unlike other methods of gene therapy, dCas9-based methods are highly scalable and versatile, with the capability to target multiple loci simultaneously, termed multiplexed editing (179) . Furthermore, as seen in combinatorial Cas9 screening systems (180) , multiple orthologues of Cas9 can be used concurrently, allowing for synchronized activation and repression of separate loci. In the context of viral infection and cancer, significant potential lies in the use of CRISPR-Cas9 [including other Cas proteins, such as the alternative DNA nuclease Cas12 (181) , and RNA nuclease Cas13 (182) ] and dCas9-based methods to target NKG2D and its ligands for enhanced immune recognition and elimination. The ease of use, high specificity and multiplexable nature of CRISPR-Cas9/dCas9-based genetic and epigenetic editing has clear applications in the development and improvement of NKG2D-directed therapies, as discussed below. To date, adoptive cell transfer (ACT) therapies, whereby peripheral blood mononuclear cells are collected, edited, expanded and reinfused back into patients, have successfully been developed to express NKG2D (largely on T cells) and shown considerable anti-tumoral and anti-viral potential in vitro (183) (184) (185) . However, in vivo responses to NKG2Dfocused chimeric antigen receptor (CAR) ACT therapy to date do not appear to be as robust, suggesting that inducing NKG2D alone may not be sufficient (186) . To our knowledge, transduction of NKG2D in combination with other receptors within the same cell for improved CAR therapy has only recently been applied clinically, with one (1/9) active registered trial (as of 27 July 2021 on ClinicalTrials.gov) combining transduction of NKG2D and ACE2 for treatment of SARS-CoV-2 (NCT04324996). Most currently active trials (8/9) aim to solely target the NKG2D receptor, and this is partially to reduce the risk of insertional mutagenesis and gene dysregulation (187, 188) . CRISPR-Cas9, however, provides a novel avenue for ACT therapy by providing a means to conduct targeted gene insertion in a multiplexed fashion to maximize host immunity (176, 177) . Strong evidence suggests that CRISPR-based knockout of receptors responsive to immunosuppressive mediators, such as transforming growth factor beta receptor 2 (TGFbR2), or immune checkpoints (dampeners of cellular activation), such as programmed cell death 1 (PD-1), which play key roles in physiological immune homeostasis, are likely to improve NKG2D-mediated cellular cytotoxicity (189) (190) (191) . Simultaneous editing of multiple loci, particularly immune checkpoints, in autologous or allogenic cells in this way is also likely to complement immune checkpoint inhibitor (ICI) therapy, such as anti-PD-L1. Moreover, CRISPR-Cas9 expands the potential of ACT therapeutics by facilitating targeted insertion of gene sequences (192) , such as KLRK1, in cell subsets that otherwise have no or low expression of the NKG2D receptor. Similarly, knockout of inhibitory receptors within the same effector cell using CRISPRbased therapies is predicted to be an excellent starting point in improving ACT therapy outcomes via CRISPR-based methods. Notably, enhancement of NKG2D-dependent immune responses in this way requires careful consideration and evaluation of the potential for collateral adverse autoimmune reactions. Indeed, given the central role of the NKG2D receptor-ligand axis in autoimmune conditions, such as Crohn's disease (36) , coeliac disease (193) , and rheumatoid arthritis (194) , care needs to be taken to not generate autoreactive lymphocytes. Apart from direct gene editing to induce or improve NKG2Dmediated immunity, enhancing NKG2D-L on tumor or virusinfected cells represents both an alternative and complementary strategy to augment ACT therapy, ICIs and overall host immunity. In this approach, CRISPR-dCas9-based transcriptional activation or repression may be used to directly activate NKG2D-L loci and ensure their surface expression. Recently, Sekiba et al. (195) , have shown this to be possible in vitro by applying CRISPR-dCas9 to transcriptionally activate MICA in Huh7 and HepG2 human hepatocellular carcinoma cell lines. Although no other NKG2D-L have been targeted in this way to date, it is predicted that multiplexed activation of several types of NKG2D-L is likely to be most effective in promoting NKG2D-mediated immunity and may resolve the low response rate of NKG2D-ACT therapy in vivo. However, our group and others (178, 196, 197) have extensively reviewed that multiplexed CRISPR-based editing, particularly in vivo within the tumor or virally infected cell, is best conducted with an optimized set of targets, rather than a large panel, so as to avoid reduced editing efficiency (retroactivity) and extensive off-target effects. Therefore, an accurate understanding of individual ligands and outcome of their binding to NKG2D is strongly recommended. For instance, given the locational and functional specialties within the ULBP family (80, 81) , we suggest that activation of these ligands, either individually or in combination, is needed to elucidate their contribution to the NKG2D pathway in different tumoral/viral contexts. Moreover, it is likely that only a subset of ULBP or MIC members need to be targeted, with some unable to engage NKG2D effectively, as previously reported (81) . Notably, given the higher affinity of ULBP1 for the NKG2D ligand, compared to MICA and MICB (59) , it may serve as a better therapeutic target. However, further investigation is needed to elucidate the affinities of the remaining ULBP family members, and whether higher affinity to the NKG2D ligand directly translates to improved cytotoxicity. CRISPR-dCas9 can also theoretically be applied to other levels of NKG2D-L regulation ( Table 1) for improved expression and immunity. An obvious example of this is in the transcriptional repression of genes involved in NKG2D-L shedding, such as ADAM10/17 or MMP9/14, which are commonly hijacked during viral infection and tumorigenesis, and are known to be targetable using CRISPR-based technologies ( Table 1 ) (143, 165) . Targeting the molecules responsible for proteolytic shedding of NKG2D-L in this way is likely to be beneficial both in the clinic and in furthering our basic biological understanding of the mechanisms driving proteolytic shedding. Although CRISPR-dCas9 epigenetic editing is transient and does not induce permanent genetic alterations, significant care needs to be taken to deliver this technology specifically to the target tissue or cell using precise delivery systems, so as to avoid inducing a severe systemic inflammatory state, particularly if used in combination with NKG2D-ACT therapeutics. Manipulation of the NKG2D receptor-ligand axis to improve host immunity is emerging as a novel therapeutic avenue in the era of CRISPR-based technologies. In addition to the direct enhancement of NKG2D on cell subsets to generate potent cytotoxic effector cells using Cas9 genetic editing, potential exists to use dCas9-based epigenetic editing methods to activate and lock NKG2D-L expression on tumor or virusinfected cells to promote their recognition and elimination. Although gaps remain in understanding how to optimize NKG2D-mediated immunity in different contexts, CRISPRbased multiplexed editing of NKG2D jointly with other genes on effector cells, or epigenetic activation of NKG2D-L in combination with one another on tumor or virally infected cells is likely to provide important insights to novel therapeutic approaches. The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author. 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Activation Pathways Synergy Among Receptors on Resting NK Cells for the Activation of Natural Cytotoxicity and Cytokine Secretion Minimal Requirement for Induction of Natural Cytotoxicity and Intersection of Activation Signals by Inhibitory Receptors Costimulation of CD8ab T Cells by NKG2D via Engagement by MIC Induced on Virus-Infected Cells NKG2D Is a Costimulatory Receptor for Human Naive CD8+ T Cells NKG2D Receptor Regulates Human Effector T-Cell Cytokine Production NKG2D Signaling Certifies Effector CD8 T Cells for Memory Formation CD4 +NKG2D+ T Cells in Crohn's Disease Mediate Inflammatory and Cytotoxic Responses Through MICA Interactions Activating NK-Cell Receptors Co-Stimulate CD4+CD28-T Cells in Patients With Rheumatoid Arthritis Coordinated Induction by IL15 of a TCR-Independent NKG2D Signaling Pathway Converts CTL Into Lymphokine-Activated Killer Cells in Celiac Disease IL-15 Induces CD8+ T Cells to Acquire Functional NK Receptors Capable of Modulating Cytotoxicity and Cytokine Secretion Activation of Vg9vd2 T Cells by NKG2D MICA Engagement by Human Vg2vd2 T Cells Enhances Their Antigen-Dependent Effector Function Recognition of Stress-Induced MHC Molecules by Intestinal Epithelial gd T Cells Vd1 T Lymphocytes From B-CLL Patients Recognize ULBP3 Expressed on Leukemic B Cells and Up-Regulated by Trans-Retinoic Acid The NKG2D Ligand ULBP4 Binds to Tcrg9/d2 and Induces Cytotoxicity to Tumor Cells Through Both Tcrgd and NKG2D MAIT Cells Detect and Efficiently Lyse Bacterially-Infected Epithelial Cells The CD4-CD8-MAIT Cell Subpopulation Is a Functionally Distinct Subset Developmentally Related to the Main CD8+ MAIT Cell Pool A New Polymorphic and Multicopy MHC Gene Family Related to Nonmammalian Class I Genomic Characterization of the Region Between HLA-B and TNF: Implications for the Evolution of Multicopy Gene Families Allelic and Interlocus Comparison of the PERB11 Multigene Family in the MHC ULBP6/RAET1L Is an Additional Human NKG2D Ligand MHC Class I Chain-Related Protein A and B (MICA and MICB) Are Predominantly Expressed Intracellularly in Tumour and Normal Tissue NKG2D Receptor and Its Ligands in Host Defense Immunohistochemical Validation and Expression Profiling of NKG2D Ligands in a Wide Spectrum of Human Epithelial Neoplasms Methylation of NKG2D Ligands Contributes to Immune System Evasion in Acute Myeloid Leukemia The MICA-129 Dimorphism Affects NKG2D Signaling and Outcome of Hematopoietic Stem Cell Transplantation CRISPR-Mediated Manipulation of NKG2D/NKG2D-L Complex Structure of the Activating Immunoreceptor NKG2D and Its MHC Class Ilike Ligand MICA Conformational Plasticity Revealed by the Cocrystal Structure of NKG2D and Its Class I MHC-Like Ligand ULBP3 Interactions of Human NKG2D With its Ligands MICA, MICB, and Homologs of the Mouse RAE-1 Protein Family Regulation of Ligands for the NKG2D Activating Receptor Alternative Splice Transcripts for MHC Class I-Like MICA Encode Novel NKG2D Ligands With Agonist or Antagonist Functions Dendritic Cell-Derived Exosomes Promote Natural Killer Cell Activation and Proliferation: A Role for NKG2D Ligands and IL-15ra Tnfa Stimulates NKG2D-Mediated Lytic Activity of Acute Myeloid Leukemic Cells Immune Evasion Mediated by Tumor-Derived Lactate Dehydrogenase Induction of NKG2D Ligands on Myeloid Cells in Glioblastoma Patients Activation-Induced Expression of MICA on T Lymphocytes Involves Engagement of CD3 and CD28 NK Cells Lyse T Regulatory Cells That Expand in Response to an Intracellular Pathogen A Second Lineage of Mammalian Major Histocompatibility Complex Class I Genes Different Evolutionary Histories in Two Subgenomic Regions of the Major Histocompatibility Complex Association Study Identifies a Susceptibility Locus for HCV-Induced Hepatocellular Carcinoma Hepatitis B Virus-Induced Hepatocellular Carcinoma: Functional Roles of MICA Variants Association of MICA-129met/Val Polymorphism With Clinical Outcome of Anti-TNF Therapy and MICA Serum Levels in Patients With Rheumatoid Arthritis The MICA-129met/Val Dimorphism Affects Plasma Membrane Expression and Shedding of the NKG2D Ligand MICA Wide Distribution of the MICA-MICB Null Haplotype in East Asians High Frequency of MIC Null Haplotype the Angaite Amerindian Community in Paraguay Comparative Sequencing of Human and Chimpanzee MHC Class I Regions Unveils Insertions/Deletions as the Major Path to Genomic Divergence MICAgen Mice Recapitulate the Highly Restricted But Activation-Inducible Expression of the Paradigmatic Human NKG2D Ligand MICA Convergent Evolution by Cancer and Viruses in Evading the NKG2D Immune Response Novel MHC Class I-Related Molecules, Bind to CMV Glycoprotein UL16 and Stimulate NK Cytotoxicity Through the NKG2D Receptor UL16-Binding Proteins, Novel MHC Class I-Related Proteins, Bind to NKG2D and Activate Multiple Signaling Pathways in Primary NK Cells ULBP4 Is a Novel Ligand for Human NKG2D Human Ligands of the NKG2D Receptor, Stimulate Tumor Immunity With Enhancement by IL-15 Transfer of the Human NKG2D Ligands UL16 Binding Proteins (ULBP) 1-3 Is Related to Lytic Granule Release and Leads to Ligand Retransfer and Killing of ULBP-Recipient Natural Killer Cells Four Novel ULBP Splice Variants Are Ligands for Human NKG2D Cellular Expression, Trafficking, and Function of Two Isoforms of Human ULBP5/RAET1G Polymorphisms of NKG2D Ligands: Diverse RAET1/ULBP Genes in Northeastern Thais RAET1/ULBP Alleles and Haplotypes Among Kolla South American Indians Single Nucleotide Polymorphism Analysis of the NKG2D Ligand Cluster on the Long Arm of Chromosome 6: Extensive Polymorphisms and Evidence of Diversity Between Human Populations Characterization of 5′ Promoter and Exon 1-3 Polymorphism of the RAET1E Gene One for All, All for One Cell Stress-Regulated Human Major Histocompatibility Complex Class I Gene Expressed in Gastrointestinal Epithelium Regulation of NKG2D Ligand Gene Expression Human NK Cells Are Alerted to Induction of P53 in Cancer Cells by Upregulation of the NKG2D Ligands ULBP1 and ULBP2 P53 Gene Repair With Zinc Finger Nucleases Optimised by Yeast 1-Hybrid and Validated by Solexa Sequencing TALEN-Mediated Targeting of HPV Oncogenes Ameliorates HPV-Related Cervical Malignancy Synthesizing a Genetic Sensor Based on CRISPR-Cas9 for Specifically Killing P53-Deficient Cancer Cells Oxidative Stress Increases MICA and MICB Gene Expression in the Human Colon Carcinoma Cell Line (CaCo-2) NKG2D Ligands Are Expressed on Stressed Human Airway Epithelial Cells Promoter Region Architecture and Transcriptional Regulation of the Genes for the MHC Class I-Related Chain A and B Ligands of NKG2D Oxidative Stress Mediates a Reduced Expression of the Activating Receptor NKG2D in NK Cells From End-Stage Renal Disease Patients Thermaland Oxidative Stress Causes Enhanced Release of NKG2D Ligand-Bearing Immunosuppressive Exosomes in Leukemia/Lymphoma T and B Cells Reactive Oxygen Species-and DNA Damage Response-Dependent NK Cell Activating Ligand Upregulation Occurs at Transcriptional Levels and Requires the Transcriptional Factor E2F1 Novel Role for STAT3 in Transcriptional Regulation of NK Immune Cell Targeting Receptor MICA on Cancer Cells Dual Blockage of STAT3 and ERK1/2 Eliminates Radioresistant GBM Cells NF-kb Regulates MICA Gene Transcription in Endothelial Cell Through a Genetically Inhibitable Control Site NF-kb Regulates Expression of the MHC Class I-Related Chain A Gene in Activated T Lymphocytes BCR/ABL Oncogene Directly Controls MHC Class I Chain-Related Molecule A Expression in Chronic Myelogenous Leukemia The Decreased Susceptibility of Bcr/Abl Targets to NK Cell-Mediated Lysis in Response to Imatinib Mesylate Involves Modulation of NKG2D GM1 Expression, and Synapse Formation Efficient Disruption of Bcr-Abl Gene by CRISPR RNA-Guided FokI Nucleases Depresses the Oncogenesis of Chronic Myeloid Leukemia Cells CRISPR/ Cas9-Mediated Gene Deletion Efficiently Retards the Progression of Philadelphia-Positive Acute Lymphoblastic Leukemia in a P210 BCR-ABL1T315I Mutation Mouse Model HER2/HER3 Signaling Regulates NK Cell-Mediated Cytotoxicity via MHC Class I Chain-Related Molecule A and B Expression in Human Breast Cancer Cell Lines The PI3K Subunits, P110a and P110b Are Potential Targets for Overcoming P-Gp and BCRP-Mediated MDR in Cancer CRISPR-Mediated Targeting of HER2 Inhibits Cell Proliferation Through a Dominant Negative Mutation CRISPR-Cas9 Epigenome Editing Enables High-Throughput Screening for Functional Regulatory Elements in the Human Genome P38 MAPK Differentially Controls NK Activating Ligands at Transcriptional and Post-Transcriptional Level on Multiple Myeloma Cells Up-Regulated Expression of MICA on Activated T Lymphocytes Involves Lck and Fyn Kinases and Signaling Through MEK1/ERK, P38 MAP Kinase, and Calcineurin Regulates Expression of NKG2D Ligands ULBP1/2/3 in AML and Modulates Their Susceptibility to NK-Mediated Lysis Upregulation of Myc Promotes the Evasion of NK Cell−Mediated Immunity Through Suppression of NKG2D Ligands in K562 Cells Identification of Focally Amplified Lineage-Specific Super-Enhancers in Human Epithelial Cancers Dcas9-Based Epigenome Editing Suggests Acquisition of Histone Methylation Is Not Sufficient for Target Gene Repression Multiple Mechanisms Downstream of TLR-4 Stimulation Allow Expression of NKG2D Ligands to Facilitate Macrophage/NK Cell Crosstalk CRISPR-Cas9-Based Gene Editing of Human Mesenchymal Stromal Cells to Improve the Outcome of Cell Therapy A Forward Genetic Screen Reveals Novel Independent Regulators of ULBP1, an Activating Ligand for Natural Killer Cells ATF4 Regulates MYB to Increase g-Globin in Response to Loss of b-Globin MiR-10b Downregulates the Stress-Induced Cell Surface Molecule MICB, A Critical Ligand for Cancer Cell Recognition by Natural Killer Cells Genome Editing Reveals Glioblastoma Addiction to microRNA-10b Tumor Suppressive microRNAs miR-34a/C Control Cancer Cell Expression of ULBP2, A Stress-Induced Ligand of the Natural Killer Cell Receptor NKG2D Cutting Edge: Down-Regulation of MHC Class I-Related Chain A on Tumor Cells by IFNg-Induced microRNA Human microRNAs Regulate Stress-Induced Immune Responses Mediated by the Receptor NKG2D Small Indels Induced by CRISPR/Cas9 in the 5′ Region of microRNA Lead to Its Depletion and Drosha Processing Retardance Sequence-Specific Inhibition of microRNA via CRISPR/CRISPRi System Diverse Herpesvirus microRNAs Target the Stress-Induced Immune Ligand MICB to Escape Recognition by Natural Killer Cells Host Immune System Gene Targeting by a Viral miRNA An Identical miRNA of the Human JC and BK Polyoma Viruses Targets the Stress-Induced Ligand ULBP3 to Escape Immune Elimination RNA-Binding Proteins Regulate the Expression of the Immune Activating Ligand MICB Vigilin Regulates the Expression of the Stress-Induced Ligand MICB by Interacting With its 5′ Untranslated Region The RNA Binding Protein IMP3 Facilitates Tumor Immune Escape by Downregulating the Stress-Induced Ligands ULPB2 and MICB. eLife Combinatorial Recognition of Clustered RNA Elements by the Multidomain RNA-Binding Protein IMP3 Cutting Edge: Down-Regulation of MICA on Human Tumors by Proteolytic Shedding Cutting Edge: The Membrane Type Matrix Metalloproteinase MMP14 Mediates Constitutive Shedding of MHC Class I Chain-Related Molecule A Independent of a Disintegrin and Metalloproteinases MMP9 Mediates MICA Shedding in Human Osteosarcomas Inhibition of MMP Activity can Restore NKG2D Ligand Expression in Gastric Cancer, Leading to Improved NK Cell Susceptibility MMP9 Mediates Acute Hyperglycemia-Induced Human Cardiac Stem Cell Death by Upregulating Apoptosis and Pyroptosis In Vitro Shedding of Endogenous MHC Class I-Related Chain Molecules A and B From Different Human Tumor Entities: Heterogeneous Involvement of the "A Disintegrin and Metalloproteases" 10 and 17 A Disintegrin and Metalloproteinases 10 and 17 Modulate the Immunogenicity of Glioblastoma-Initiating Cells Tumor-Associated MICA Is Shed by ADAM Proteases Cutting Edge: The Metalloproteinase ADAM17/TNF-a-Converting Enzyme Regulates Proteolytic Shedding of the MHC Class I-Related Chain B Protein Cleavage Site Localization Differentially Controls Interleukin-6 Receptor Proteolysis by ADAM10 and ADAM17. Sci Rep Sorafenib Inhibits the Shedding of Major Histocompatibility Complex Class I-related Chain A on Hepatocellular Carcinoma Cells by Down-Regulating a Disintegrin and Metalloproteinase 9 Predominance of Regorafenib Over Sorafenib: Restoration of Membrane-Bound MICA in Hepatocellular Carcinoma Cells A Disintegrin and Metalloproteinase 9 Domain (ADAM9) Is a Major Susceptibility Factor in the Early Stages of Encephalomyocarditis Virus Infection Disulphide-Isomerase-Enabled Shedding of Tumour-Associated NKG2D Ligands Histamine Reduces Susceptibility to Natural Killer Cells via Down-Regulation of NKG2D Ligands on Down-Regulation of NKG2D and NKp80 Ligands by Kaposi's Sarcoma-Associated Herpesvirus K5 Protects Against NK Cell Cytotoxicity HBV Suppresses Expression of MICA/B on Hepatoma Cells Through Up-Regulation of Transcription Factors GATA2 and GATA3 to Escape From NK Cell Surveillance MicroRNA Based Immunoevasion Mechanism of Human Polyomaviruses HSV1 microRNA Modulation of GPI Anchoring and Downstream Immune Evasion Altered microRNA Expression After Infection With Human Cytomegalovirus Leads to TIMP3 Downregulation and Increased Shedding of Metalloprotease Substrates, Including MICA Silencing NKG2D Ligand-Targeting miRNAs Enhances Natural Killer Cell-Mediated Cytotoxicity in Breast Cancer NKG2D Ligands-Critical Targets for Cancer Immune Escape and Therapy The Role of the NKG2D Receptor for Tumor Immunity CRISPR-Mediated Manipulation of NKG2D/NKG2D-L Human Cytomegalovirus Glycoprotein UL16 Causes Intracellular Sequestration of NKG2D Ligands, Protecting Against Natural Killer Cell Cytotoxicity Selective Intracellular Retention of Virally Induced NKG2D Ligands by the Human Cytomegalovirus UL16 Glycoprotein NKG2D Ligand MICA Is Retained in the Cis-Golgi Apparatus by Human Cytomegalovirus Protein UL142 Intracellular Sequestration of the NKG2D Ligand ULBP3 by Human Cytomegalovirus HIV Nef, Paxillin, and Pak1/2 Regulate Activation and Secretion of TACE/ ADAM10 Proteases Intracellular Retention of the NKG2D Ligand MHC Class I Chain-Related Gene A in Human Melanomas Confers Immune Privilege and Prevents NK Cell-Mediated Cytotoxicity A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity Classification and Evolution of Type II CRISPR-Cas Systems Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression Waking Up Dormant Tumor Suppressor Genes With Zinc Fingers, TALEs and the CRISPR/dCas9 System Activating PTEN Tumor Suppressor Expression With the CRISPR/dCas9 Tumour Suppression by Targeted Intravenous non-Viral CRISPRa Using Dendritic Polymers Highly Specific Epigenome Editing by CRISPR-Cas9 Repressors for Silencing of Distal Regulatory Elements RNA-Guided Transcriptional Silencing In Vivo With S. Aureus CRISPR-Cas9 Repressors Transcriptional Repression of PTEN in Neural Cells Using CRISPR/dCas9 CRISPR-Engineered T Cells in Patients With Refractory Cancer Safety and Feasibility of CRISPR-Edited T Cells in Patients With Refractory Non-Small-Cell Lung Cancer Reprogramming the Anti-Tumor Immune Response via CRISPR Genetic and Epigenetic Editing Multiplexed Activation of Endogenous Genes by CRISPRa Elicits Potent Antitumor Immunity Orthologous CRISPR-Cas9 Enzymes for Combinatorial Genetic Screens Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System C2c2 Is a Single-Component Programmable RNA-Guided RNA-Targeting CRISPR Effector Chimeric NK-Receptor-Bearing T Cells Mediate Antitumor Immunotherapy A Chimeric Receptor With NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells Attacking Latent HIV With convertibleCAR-T Cells, a Highly Adaptable Killing Platform Phase I Trial of Autologous CAR T Cells Targeting NKG2D Ligands in Patients With AML/MDS and Multiple Myeloma Insertional Oncogenesis in 4 Patients After Retrovirus-Mediated Gene Therapy of SCID-X1 Generation of Knock-Out Primary and Expanded Human NK Cells Using Cas9 Ribonucleoproteins Elevated TGF-b1 Secretion and Down-Modulation of NKG2D Underlies Impaired NK Cytotoxicity in Cancer Patients A Genetically Engineered Primary Human Natural Killer Cell Platform for Cancer Immunotherapy CRISPR-Mediated Integration of Large Gene Cassettes Using AAV Donor Vectors A Direct Role for NKG2D/MICA Interaction in Villous Atrophy During Celiac Disease Stimulation of T Cell Autoreactivity by Anomalous Expression of NKG2D and Its MIC Ligands in Rheumatoid Arthritis Transcriptional Activation of the MICA Gene With an Engineered CRISPR-Cas9 System Multiplexed CRISPR Technologies for Gene Editing and Transcriptional Regulation CRISPR-Based Gene Expression Control for Synthetic Gene Circuits 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.Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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