NKG2D Receptor Research Articles

Effects of homo‐ and heterozygous TREM2 mutations on natural killer cell activity

AbstractBackgroundNasu‐Hakola disease (NHD) is a rare genetic neurodegenerative disorder characterised by progressive presenile dementia and bone cysts. Dysfunctions in myeloid‐derived innate immune cells such as microglia due to loss‐of‐function mutations in the TREM2 (trigger receptor 2 expressed on myeloid cells) gene cause the clinical manifestations. Heterozygous carriers have strongly increased risk of developing Alzheimer’s Disease but do not present with NHD symptoms. TREM2 is associated with DAP12 protein, which contains an immunoreceptor tyrosine‐based activation motif that is structurally similar to natural killer (NK) cell receptors. In this study, we aimed to analyse the effects of TREM2 mutation and its effect on the peripheral immune system represented by NK cell functions.MethodSix patients with NHD with a defined homozygous TREM2 mutation, 7 carriers with heterozygous TREM2 mutations and 10 age‐ and gender‐matched controls were included. NK cell subsets, KIR expressions (NKG2A, NKG2D, NKp46), cytotoxic activity (CD107a, Perforin, Granzyme A) and cytokine secretions (IL‐4, IFN‐γ, TNF‐α, IL‐10, IL‐17, TGF‐β) were analysed by flow cytometry.ResultCD3−CD56dimCD16− (p = 0.003) subsets were found to be increased in the NHD group compared to carriers. Activating C‐type lectin receptor NKG2D expression was increased in CD8+ (p = 0.003) NK cell subsets in the NHD group. Inhibiting C‐type lectin receptor NKG2A expression was increased in total (p = 0.011) as well as CD8+ (p = 0.007) NK cell subsets in carriers compared to controls. In total NK cells, intracellular TNF‐α (p = 0.005) and TGF‐β (p = 0.013) levels were decreased, IL‐17 (p = 0.05) and IL‐4 (p = 0.005) levels were increased in the NHD group. When NK cells were grouped due to their CD8 expression, TNF‐α levels were decreased, and IL‐17 levels were increased in both subsets in the NHD group. Cytotoxic activity showed no differentiation in both unstimulated and K562 stimulated conditions between groups.ConclusionUnlike previous case series, NK cell ratios did not differ between groups. Over‐expression of inhibiting receptors in the carriers group, unlike high levels of activating receptors in the NHD, might explain the lack of symptoms in the carriers. The increased ratio of CD3−CD56dimCD16− NK cell subsets that overexpress inhibitory checkpoints might explain the low levels of pro‐inflammatory cytokines in the NHD group.

Preclinical Characterization of the Anti-Leukemia Activity of the CD33/CD16/NKG2D Immune Modulating TriNKET ® BMS-986357 (CC-96191)

CD33 is widely expressed by myeloid cells and is a validated drug target in acute myeloid leukemia (AML), as shown by the benefit of some patients from the CD33 antibody-drug conjugate gemtuzumab ozogamicin (GO). However, GO is often ineffective and toxic, prompting interest in more potent CD33-directed therapies. Success of T cell-engaging bispecific antibodies in lymphoid malignancies has raised enthusiasm for similar agents targeting CD33, but toxicities from activated T cells cause significant challenges for their use and provide a strong rationale to explore drugs engaging natural killer (NK) cells. Here, we investigated BMS-986357, a TriNKET ® that activates NK cells via CD16 and NKG2D receptors while targeting CD33. This format was chosen because simultaneous engagement of multiple activating NK cell receptors yields greater cytotoxic activity against neoplastic cells than engagement via a single activating receptor. Surface plasmon resonance analyses showed BMS-986357 simultaneously binds CD33, NKG2D, and CD16a, and co-engagement of NKG2D and CD16a increases the avidity. In the presence of primary human NK cells from healthy donors or a human NK cell line, the TriNKET ® demonstrated broad cytolytic anti-leukemia activity in a strictly CD33-dependent manner using a panel of CD33-expressing or CD33-deficient/deleted acute leukemia cells. Consistent with this, removing the CD33 binding domain rendered BMS-986357 ineffective against CD33+ AML cells. Drug-induced cytotoxicity was greater with BMS-986357 than with monoclonal CD33 antibodies, including lintuzumab and an afucosylated antibody with enhanced NK function, demonstrating the benefit of the TriNKET ® format. Compared to BMS-986357, substantially less cytotoxicity was observed with an Fc-mutant variant of BMS-986357 that abrogates binding to CD16a or an NKG2D dead-arm TriNKET ®, and cytolytic effects were reduced with a CD16-deficient NK cell line vs. a CD16-expressing subline, indicating maximal efficacy of BMS-986357 required co-engagement of CD16a and NKG2D. Using sublines of OCI-AML3 and KG-1 cells (both cell lines with low endogenous CD33 expression) to overexpress wild-type CD33 at increasing levels, we found low abundance of CD33 curtailed BMS-986357-induced cytotoxicity. Flow cytometric binding studies identified BMS-986357 to recognize the membrane-distal V-set domain of CD33. Unlike GO, BMS-986357-mediated cytolysis was not affected by over-expression of ABC transporter proteins by AML cells. The potency but not maximal activity of BMS-986357 was reduced by super-physiologic concentrations of soluble CD33; in contrast, the soluble form of the NKG2D ligand MICA did not impact TriNKET ® activity. In the presence of CD33+ AML cells, BMS-986357 activated NK cells but not T cells from healthy donor peripheral blood mononuclear cells and produced similar levels of maximum cytolysis as a CD33/CD3 bispecific antibody. Despite this, BMS-986357 elicited 10 to >100-fold lower soluble cytokine levels including IFN-γ, TNF, IL-6, and GM-CSF than a CD33/CD3 bispecific antibody, suggesting BMS-986357 has the potential for CD33/CD3 bispecific-like efficacy without the associated risk of cytokine release syndrome. Finally, in bone marrow specimens from patients with AML, BMS-986357 killed AML cells but not normal monocytes, suggesting selectivity of drug-induced cytotoxicity toward neoplastic cells, consistent with the notion NK cells are capable to differentiate between healthy and leukemic CD33+ cells. This selectivity suggests BMS-986357 may produce less myelosuppressive toxicity than what has been observed with CD33 targeting antibody-drug conjugates. In summary, our findings demonstrate that BMS-986357 TriNKET ® has potent CD33-dependent cytolytic activity in vitro against human AML cells, supporting the drug's exploration in early phase clinical trials. One such trial (NCT04789655) has opened accrual for adults with relapsed/refractory AML.

Utilizing Chemotherapy to Enhance the Anti-Tumor Properties of Ex Vivo Expanded Gamma Delta (γδ) T Cells Against Acute Myeloid Leukemia

Background: Acute myeloid leukemia (AML) is the second most common type of blood cancer in children with a high rate of treatment failure and aggressive relapse. While allogeneic HSCT offers a cure, limitations arise in achieving remission prior to transplant. Immunotherapy-based approaches can induce remission, gaining interest in AML. Here, we explore the use of gamma delta (γδ) T cells as a promising tool to fulfill this unmet need. Unlike predominant alpha beta (αβ) T cells, γδ T cells do not require antigen presentation and identify their targets in a major histocompatibility complex (MHC) independent manner. Allogeneic γδ T cells thus have minimal risk of causing graft-versus-host disease (GvHD), creating ‘off-the-shelf’ cellular therapeutics. This would be ideal to target AML, especially given the aggressive nature of relapsed disease. Cytotoxic mechanisms used by γδ T cells include recognition of cellular stress molecules such as NKG2D and DNAM-1 receptor ligands on target cells, activation of death receptor pathway through Fas-FasL and TRAIL-R interactions, as well as γδ ΤCR mediated killing through recognition of butyrophilins (BTNs) on target cells. These stress molecules are expressed on AML cells and can be further upregulated through chemotherapeutic agents such as azacitidine (a hypomethylating agent) and venetoclax (BCL-2 antagonist), drugs currently in use to treat relapsed AML. Here, we tested these drugs in combination with γδ T cells to target AML. Methods Our studies explored four different AML cell lines (Kasumi-1, Nomo-1, MV4-11 and MOLM13). Each one was respectively treated at their corresponding inhibitory concentration 25 (IC 25) and 50 (IC 50) of azacitidine and/or venetoclax for 24 hours. After treatment, AML cells were assessed by flow cytometry for expression of NKG2D and DNAM-1 ligands, death receptor pathway molecules (CD95, TRAIL-R1/R2) as well as the butyrophillins (BTN2A and BTN3A1) to elucidate which pathways are over-expressed, aiding in γδ T cell targeting and cytotoxicity. Next, we co-cultured AML cells pre-treated with azacitidine and/or venetoclax with γδ T cells and measured cytotoxicity using a flow cytometry-based assay and a bioluminescence assay. Results: Each cell line expressed a unique pattern of markers at baseline. Cell lines treated with azacitidine had a robust expression of the NKG2D ligands ULBP1, ULBP2/5/6 and ULBP3. Azacitidine also increased CD155 and TRAIL-R2 in Nomo-1 while CD112 increased in MOLM13. Most venetoclax-treated AML cells over-expressed BTN2A and BTN3A1 along with CD95 in MOLM13. Cell lines were then treated with a combination of both drugs. Dual treatment of cells resulted in a significant increase in expression of most NKG2D markers as well the death receptor Fas (CD95). Additionally, increase in CD155 as well as BTNs was seen in several cell lines. Importantly, azacitidine and venetoclax had minimal effects on the viability of γδ Τ cells when treated for 24 hours. In vitro cytotoxicity studies indicate increased cell death across multiple AML cell lines when γδ T cells were co-cultured with cells pretreated with azacitidine and venetoclax. Conclusions: Azacitidine and venetoclax treatment resulted in upregulation of several markers involved in γδ Τ cell-mediated cytotoxicity. Ex vivo expanded γδ Τ cells combined with chemotherapy-treated AML cells resulted in increased overall cytotoxicity, which is likely both additive and synergistic. Our next steps include preclinical validation of this combinatorial approach in an in-vivo mouse study.

Selinexor Enhances Natural Killer Cell Function Against Multiple Myeloma Cells

Introduction: Natural killer (NK) cells are innate immune effector cells which can mediate antibody dependent cellular cytotoxicity (ADCC) and lyse cancer cells. Harnessing NK cell function against multiple myeloma (MM) is of high interest and multiple strategies are currently under development. Exportin-1 (XPO1) is a nuclear export protein which inhibits the function of tumour suppressor proteins and facilitates oncogene mRNA translation. Selinexor is a first-in-class XPO1 inhibitor approved for the treatment of relapsed or refractory MM that induces cancer cell apoptosis. In addition, selinexor has recently been shown to promote NK cell cytotoxicity against B cell lymphoma cells. This study aimed to determine whether selinexor augments NK cell activation against MM cells both alone, and in combination with the anti-CD38 antibody daratumumab. Methods: A panel of MM cell lines (MM.1S, U266 and RPMI-8226) were incubated with clinically relevant concentrations of selinexor for 24 hours, then either assessed for expression of ligands for NK cell receptors (KIR, NKG2A, NKG2D and NKp46), or co-cultured with primary human NK cells ± daratumumab. Following co-culture, NK-mediated lysis of MM cells was assessed by propidium iodide staining and NK cell degranulation assessed by CD107a expression. Results: Selinexor pre-treatment of MM cells significantly increased NK cell degranulation (p<0.05) and enhanced NK specific lysis of MM targets (p<0.05). Furthermore, selinexor pre-treatment significantly (p<0.05) enhanced NK cell activation in combination with daratumumab compared to daratumumab alone. Importantly, selinexor did not alter surface expression of CD38 on MM cell lines. To investigate the mechanism behind this enhanced NK cell function, we assessed the effect of selinexor on the expression of NK cell ligands by MM cells. Selinexor increased the expression of ULBP1/2/5/6 (2-fold, p<0.001) which bind to the NK cell activating receptor NKG2D. In addition, selinexor decreased MM cell expression of HLA-E (30% decrease, p<0.05) which binds to the inhibitory NK receptor NKG2A. Conclusions: These data suggest that selinexor may promote the efficacy of NK targeted therapeutics in multiple myeloma.

CD19-Specific CAR NK Cells Coexpressing IL-21 Exhibit Superior Expansion and Antitumor Activity Against CD19-Postive Lymphoma

Background Chimeric antigen receptor (CAR) expressing NK cells induce robust antitumor responses in patients with hematologic malignancies but have limited persistent without cytokines supplement. IL-15 and IL-21 promote NK cell expansion, survival, and possess both overlapping and distinctive functions. CAR NK cells engineered to express IL-15 have shown long-term persistence and potent antitumor activity, however, the pleiotropic effects of IL-21 on CAR NK cell-mediated tumor rejection are lacking. Therefore, we compared the therapeutic potency of CD19-specific human NK cells that constitutively express IL-15 or IL-21. Methods The codon-optimized minigene encoding cytokine IL-21 or IL-15, linked with CD19-CAR containing 4-1BB costimulatory domain by a P2A sequence were synthesized and sub-cloned into the pSFG retroviral vector yielding the BBz.IL21 or BBz.IL15 retroviral construct. To produce CD19-CAR retrovirus, 293T/17 cells were transfected with a combination of plasmids containing CAR constructs, RDF plasmid and PegPam3 plasmid. Human peripheral blood NK cells isolated from healthy volunteer donors were stimulated with CD16/CD137 mAb and expanded with 500 IU/ml IL-2 (PeproTech) for 5 days before transduced with CAR retrovirus in plates coated with RetroNectin. The expression of CAR, phenotype, degranulation, cytokine production, and cytotoxicity of CAR NK cells were analyzed on day 3 post transduction. To measure IL-15 and IL-21 production, supernatants were collected from CAR NK cells cultured in the absence of IL-2 for 24h and evaluated by ELISA. Results After transduction, all constructs were stably expressed by NK cells, with construct containing IL-21 (71.23 ± 4.05 %, n = 3) demonstrating higher transduction efficiency compared with the BBz.IL15 (61.13 ± 3.43 %), but slightly lower than the BBz construct (79.63 ± 3.83 %). High levels of IL-21 were found in the supernatant of BBz.IL21 NK cells after resting 24h (3464 ± 199 pg/ml), while no detectable IL-21 was observed on BBz and BBz.IL15 NK cells. NK cells expressing BBz.IL15 produced significantly more IL-15 than BBz.IL21 and BBz NK cells as baseline (BBz.IL15: 213 ± 14 pg/ml, BBz.IL21: 61.86± 4 pg/ml, BBz: 60.56 ± 11 pg/ml). Given that IL-15 or IL-21 could potentially induce antigen-independent NK cell proliferation, we compared the ability of each NK-cell group to maintain growth in the presence of IL-2. The expansion of CAR NK cells expressing IL-21 was significantly higher than that of CAR NK cells expressing IL-15, while the number of BBz.IL15 NK cells and BBz NK cells were comparable (fold expansion in 5 days: BBz.IL21: 26.6 fold, BBz.IL15: 15.9 fold, BBz: 17.2 fold), suggesting a synergistic effect of IL-21 and IL-2 on CAR NK cell expansion. BBz.IL21 NK cells shared a similar immunophenotypic profile to BBz.IL15 NK cells, as the expression of inhibitory receptors PD1, LAG3, TIM3 and NKG2A, as well as activation receptors NKG2D and CD69 were comparable. CAR engagement by CD19-postive Raji cells specifically induced cytokines production by CD19-CAR NK cells but not by control NK cells. IL-21 coexpression significantly enhanced IFN-γ and TNF-α production compared with CAR NK cells as well as CAR NK cells expressing IL-15 (BBz.IL21 vs. BBz.IL15, P < 0.05; BBz.IL21 vs. BBz, P < 0.05; BBz.IL15 vs. BBz, P > 0.05). Moreover, a higher percentage of degranulated NK cells was observed on BBz.IL21 NK cells compared to BBz.IL15 NK cells and BBz NK cells. To confirm this observation, NK cells were cocultured with Raji cells at various E:T ratios for 6h. CAR NK cells expressing IL-21 showed superior cytotoxicity, compared to CAR NK cells expressing IL-15. Conclusion CD19-CAR NK cells engineered to express IL-21 gene produced high levels of IL-21 during cultivation and enabled greater ex vivo expansion of CAR NK cells compared with CAR NK cells expressing IL-15. IL-21 coexpression induced a significant increase in IFN-γ, TNF-α production and degranulation compared with IL-15 coexpression, resulting in enhanced antitumor activity of CD19-CAR NK cells against CD19-postive lymphoma.

Autophagy-mediated NKG2D internalization impairs NK cell function and exacerbates radiation pneumonitis.

Radiation pneumonitis is a critical complication that constrains the use of radiation therapy for thoracic malignancies, leading to substantial morbidity via respiratory distress and lung function impairment. The role of Natural killer (NK) cells in inflammatory diseases is well-documented; however, their involvement in radiation pneumonitis is not fully understood. To explore the involvement of NK cells in radiation pneumonitis, we analyzed tissue samples for NK cell presence and function. The study utilized immunofluorescence staining, western blotting, and immunoprecipitation to investigate CXCL10 and ROS levels, autophagy activity, and NKG2D receptor dynamics in NK cells derived from patients and animal models subjected to radiation. In this study, we observed an augmented infiltration of NK cells in tissues affected by radiation pneumonitis, although their function was markedly diminished. In animal models, enhancing NK cell activity appeared to decelerate the disease progression. Concomitant with the disease course, there was a notable upsurge in CXCL10 and ROS levels. CXCL10 was found to facilitate NK cell migration through CXCR3 receptor activation. Furthermore, evidence of excessive autophagy in patient NK cells was linked to ROS accumulation, as indicated by immunofluorescence and Western blot analyses. The association between the NKG2D receptor and its adaptor proteins (AP2 subunits AP2A1 and AP2M1), LC3, and lysosomes was intensified after radiation exposure, as demonstrated by immunoprecipitation. This interaction led to NKG2D receptor endocytosis and subsequent lysosomal degradation. Our findings delineate a mechanism by which radiation-induced lung injury may suppress NK cell function through an autophagy-dependent pathway. The dysregulation observed suggests potential therapeutic targets; hence, modulating autophagy and enhancing NK cell activity could represent novel strategies for mitigating radiation pneumonitis.

High population frequencies of MICA copy number variations originate from independent recombination events.

MICA is a stress-induced ligand of the NKG2D receptor that stimulates NK and T cell responses and was identified as a key determinant of anti-tumor immunity. The MICA gene is located inside the MHC complex and is in strong linkage disequilibrium with HLA-B. While an HLA-B*48-linked MICA deletion-haplotype was previously described in Asian populations, little is known about other MICA copy number variations. Here, we report the genotyping of more than two million individuals revealing high frequencies of MICA duplications (1%) and MICA deletions (0.4%). Their prevalence differs between ethnic groups and can rise to 2.8% (Croatia) and 9.2% (Mexico), respectively. Targeted sequencing of more than 70 samples indicates that these copy number variations originate from independent nonallelic homologous recombination events between segmental duplications upstream of MICA and MICB. Overall, our data warrant further investigation of disease associations and consideration of MICA copy number data in oncological study protocols.

Porcine UL-16 Binding Protein 1 Is Not a Functional Ligand for the Human Natural Killer Cell Activating Receptor NKG2D.

Natural killer (NK) cells play a vital role in xenotransplantation rejection. One approach to induce NK cell immune tolerance is to prevent the NK cell-mediated direct killing of porcine cells by targeting the interaction of the activating receptor NKG2D and its ligands. However, the identity of porcine ligands for the human NKG2D receptor has remained elusive. Previous studies on porcine UL-16 binding protein 1 (pULBP-1) as a ligand for human NKG2D have yielded contradictory results. The goal of the present study was to clarify the role of pULBP-1 in the immune response and its interaction with human NKG2D receptor. To accomplish this, the CRISPR/Cas9 gene editing tool was employed to disrupt the porcine ULBP-1 gene in a 5-gene knockout porcine endothelial cell line (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin, 5GKO). A colony with two allele mutations in pULBP-1 was established as a 6-gene knockout pig cell line (6GKO). We found that pULBP-1-deficient pig cells exhibited a reduced binding capacity to human NKG2D-Fc, a recombinant chimera protein. However, the removal of ULBP-1 from porcine endothelial cells did not significantly impact human NK cell degranulation or cytotoxicity upon stimulation with the pig cells. These findings conclusively demonstrate that pULBP-1 is not a crucial ligand for initiating xenogeneic human NK cell activation.

NKX101, an Allogeneic Off-the-Shelf CAR NK Cell Therapy Targeting NKG2D-Ls, Has Potent Anti-Leukemic Activity Alone or in Combination with Ara-C

Introduction NKG2D-Ligands (NKG2D-Ls) are a family of immunomodulatory proteins that are upregulated on damaged or transformed cells but have limited expression on normal tissues. Natural killer (NK) cells target and eliminate NKG2D-L + cells through NKG2D receptor binding and subsequent activation. NKX101 is an allogeneic off-the-shelf healthy donor-derived chimeric antigen receptor (CAR) NK cell therapy candidate engineered to express an NKG2D-L targeting CAR and membrane-bound IL-15 for extended persistence. In a clinical trial, NKX101 treatment combined with modified lymphodepletion incorporating cytarabine (Ara-C) has shown promise for the treatment of relapsed/refractory (r/r) AML, including generating complete responses (CR) with MRD negativity (MRD -). To support the rationale of NKX101 combined with modified lymphodepletion, we developed assays to (i) investigate the cytotoxic activity of NKX101 against individual NKG2D-Ls, (ii) determine the effect of Ara-C pre-treatment on NKG2D-L expression on AML cell lines, and (iii) evaluate the combined anti-leukemic activity of NKX101 and Ara-C. Methods NKX101 cells were generated from peripheral blood leukopaks from healthy donors. CHO-K1 cells were engineered to stably express individual NKG2D-Ls or empty vector controls and were combined with NKX101 cells for cytotoxicity experiments. AML cell lines were treated with increasing levels of Ara-C and evaluated for NKG2D-L expression by flow cytometry. To measure NKX101 cytotoxicity against AML cell lines, NucRed™-labeled target cells were pre-treated with Ara-C using IC 50 and IC 90 concentrations and co-cultured with NKX101. Specific cell killing of target cells was measured using an Incucyte® instrument for five days. Assessment of the contribution of each agent was determined using a dose-response matrix of increasing concentrations of NKX101 cells and Ara-C to generate specific cytotoxicity values. Combination effects were analyzed using the SynergyFinder Plus web application. Results Expression of a single NKG2D-L was sufficient to trigger NKX101 cytotoxicity against target cell lines. Flow cytometry analysis revealed that pre-treatment with Ara-C upregulated NKG2D-Ls on AML cell lines in a dose-dependent manner. NKX101 showed enhanced anti-leukemic activity against AML cells pre-treated with Ara-C compared to untreated controls, demonstrating that the combination can enhance NKX101 potency. Assessment of the combination of NKX101 and Ara-C using a dose-response matrix revealed that the two modalities combine to potently kill AML cells. Conclusions In summary, we show that NKX101 potently targets and eliminates AML cells expressing NKG2D-Ls further validating NKG2D-Ls as therapeutic targets. Additionally, we show that the combination of Ara-C and NKX101 leads to increased anti-leukemic activity, supporting continued investigation of NKX101 CAR NK cell therapy after lymphodepletion incorporating Ara-C for the treatment of r/r AML.

MICa/b-dependent activation of natural killer cells by CD64+ inflammatory type 2 dendritic cells contributes to autoimmunity.

Primary Sjögren's syndrome (pSS) is an inflammatory autoimmune disorder largely mediated by type I and II interferon (IFN). The potential contribution of innate immune cells, such as natural killer (NK) cells and dendritic cells (DC), to the pSS pathology remains understudied. Here, we identified an enriched CD16+ CD56hi NK cell subset associated with higher cytotoxic function, as well as elevated proportions of inflammatory CD64+ conventional dendritic cell (cDC2) subtype that expresses increased levels of MICa/b, the ligand for the activating receptor NKG2D, in pSS individuals. Circulating cDC2 from pSS patients efficiently induced activation of cytotoxic NK cells ex vivo and were found in proximity to CD56+ NK cells in salivary glands (SG) from pSS patients. Interestingly, transcriptional activation of IFN signatures associated with the RIG-I/DDX60 pathway, IFN I receptor, and its target genes regulate the expression of NKG2D ligands on cDC2 from pSS patients. Finally, increased proportions of CD64hi RAE-1+ cDC2 and NKG2D+ CD11b+ CD27+ NK cells were present in vivo in the SG after poly I:C injection. Our study provides novel insight into the contribution and interplay of NK and cDC2 in pSS pathology and identifies new potential therapy targets.

Efficacy of Human iPSC-Derived CAR-NK Cells Targeting Multiple Myeloma Cells

NK cells armed with chimeric antigen receptors (CAR) enable NK cells specifically to target cancer cells by recognizing tumor associated antigens. Previous studies have proven that human iPSC-derived CAR-NK cells have enhanced anti-tumor activity. Targeted B-cell maturation antigen (BCMA) therapy for relapsed refractory MM (rrMM) has been widely used with antibody-drug conjugate (ADC), bispecific antibodies, and CART cells, which has produced a rapid response, but relapse is common. Recent work has revealed GPRC5D as an alternative target in MM for immunotherapy and patients who have previously received anti-BCMA therapy have responded to GPRC5D CART cells. To assess target GPRC5D therapy with CAR-NK cells, which may in future serve as allogeneic immunotherapy, we transduced anti-GPRC5D CAR into an iPSC line derived from a healthy donor using a piggybac transposon system. More than 97% of CAR inserted-iPSCs (CAR-iPSCs) expressed anti-GPRC5D scFV with a similar expression level identified in CAR-iPSC-derived-NK cells (CAR-iNK, 92.7%). The anti-GPRC5D CAR-iNK demonstrated high purity (>99.9% for CD45 + and CD56 +) and expressed a high level of CD16 (63.6%), the NK cell activating receptor NKG2D (96.3%) and NKp30 (98.7%), and the co-stimulatory receptors CD244 (99.6%) and CD226 (97.8%). Nonetheless expression of TCR⍺β and TCRƳ- was relatively low (<2%). Cytotoxicity assay revealed that anti-GPRC5D CAR-iNK had similar cytotoxicity against K562 cells (No GPRC5D antigen) to cord blood-derived NK cells (CB-NK), wide type (WT) iNK and anti-BCMA CAR-iNK. Cytotoxicity was dose-dependent indicating that these NK cells had similar innate anti-cancer cytotoxicity. When incubated at a ratio of 4:1 with NCI-H929 cells (MM) for 4 hours, which express a high level of both BCMA and GPRC5D, anti-GPRC5D CAR-iNK demonstrated an overwhelming advantage (approximately 90% killing rate) over the CB-NK (no killing) and WT iNK (around 10% killing rate). Furthermore, the anti-GPRC5D CAR-iNK also showed superior cytotoxicity to anti-BCMA CAR-iNK (Figure 1), indicating that GPRC5D may be a more efficient target for CAR-NK-based immunotherapy in MM. We further confirmed that anti-GPRC5D CAR-NK have acceptable cytotoxicity to NCI-H929 and OPM-2 cells, even when transported over a long distance (more than 2000 km) and following cryopreservation. The lower expression by OPM-2 cells of BCMA and GPRC5D compared with NCI-H929 indicates that both fresh and cryopreserved anti-GPRC5D CAR-iNK can specifically target MM with a higher and lower expression of GPRC5D. In addition, cryopreserved anti-GPRC5D CAR-iNK cells maintain comparable antigen-specific cytotoxicity in vitro, and demonstrate the ability to reduce tumor burden in an antigen-specific manner in an OPM-2 xenograft model. Our results demonstrate that genome-engineered human iPSCs can provide an unlimited source for manufacturing scalable, off-the-shelf, and cost-effective CAR-NK cells, and GPRC5D is a potent immunotherapeutic target in multiple myeloma.

Bispecific CAR-iNKT Immunotherapy for High Risk MLL-Rearranged Acute Lymphoblastic Leukemia

MLL-rearranged CD19+ acute lymphoblastic leukemia ( MLLr-ALL), the commonest infant leukemia, has a high mortality rate (<40% leukemia-free survival). While stem cell transplantation has not improved outcomes, CD19-directed immunotherapeutic approaches, including CAR-T cells, have shown promise. However, as patients can still relapse with CD19-, or lineage-switched disease, alternative strategies are needed. To enhance efficacy and mitigate against immune escape, we aimed to develop iNKT cell-based bispecific CAR immunotherapy. iNKT are rare, CD1d-restricted, glycolipid-reactive T cells that bridge adaptive and innate immunity. Furthermore, allogeneic iNKT protect from aGVHD making them ideal candidates for ‘off-the-shelf’ immunotherapy without need for TCR deletion. To target both CD19 and CD133, which is often co-expressed with CD19 on MLLr blasts and possibly also on CD19-CD133+ leukemia-initiating cells, we developed a CD19-28z/CD133-41BBz bispecific as well as CD19-28z and CD133-4-1BBz monospecific CARs and compared their anti-leukemic activity. In short- and long-term cytotoxicity assays against MLLr leukemic cell lines (SEM, RS4;11, KOPN8) we show that bispecific CAR-iNKT are more potent than their monospecific counterparts. In assays involving single or dual knock-out of CD19 and CD133 in SEM cells, both the CD19 and CD133 moieties of the bispecific CAR were reactive and specific against their respective targets. While bispecific CAR-iNKT retained killing activity against either single target-expressing leukemia cells, monospecific CAR-iNKT only killed leukemia cells expressing their cognate target. In line with the in vitro findings, in xenograft assays, a low dose of 10 6 bispecific CAR-iNKT cells significantly prolonged survival of luc-SEM leukemia-bearing mice as compared to monospecific CAR-iNKT-treated and untreated control animals (Fig 1A). Of note, in vivo as well as in vitro, bispecific CAR-iNKT very effectively eliminated high and low CD19/CD133 co-expressing, but not, as expected, CD19-CD133- disease. Next, we compared bispecific CAR-iNKT vs their CAR-T counterparts. In vitro, CAR-iNKT from three donors demonstrated higher killing activity than CAR-T, while in vivo, compared to 10 6 CAR-T, CAR-iNKT cells induced significantly improved survival of leukemia-bearing mice (Fig 1B). Of note, while CAR-T cells were not detectable in the BM and spleen at sacrifice, CAR-iNKT were readily detectable in the organs of CAR-iNKT cell-treated animals, suggesting they persist longer. Remarkably, mice treated with 5x10 6 bispecific CAR-iNKT cells survived >60 days without any evidence of leukemia by serial bioluminescence assay, or in the bone marrow, spleen (flow-cytometry) and leptomeninges (immunohistochemistry) upon culling. Since SEM cells do not express CD1d, we explored alternative mechanism(s) that would account for the more potent anti-leukemic activity of CAR-iNKT cells. We found that CAR-iNKT express significantly higher baseline levels of the activating receptor NKG2D than CAR-T cells. Upon co-culture with SEM cells, nearly all CAR-iNKT but only a fraction of CAR-T express NKG2D, and a blocking NKG2D Ab significantly abrogated the superior cytotoxic effect of CAR-iNKT cells. Consistent with an NKG2D-dependent mechanism, mRNA of one or more NKG2D ligands is expressed by primary MLLr blasts as well as leukemia cell lines. Since CD133 is expressed on hematopoietic stem and progenitor cells (HSPC), the potential hematopoietic toxicity of bispecific CAR-iNKT cells was tested in a series of experiments involving cord blood humanisation of NSGS mouse hematopoiesis. We found that transfer of 10 7 bispecific CAR-iNKT cells significantly but transiently depleted engrafted CB CD19+ B cells. However, phenotypic analysis showed that long term engraftment levels were similar for all mature cell lineages, CD34+ HSPC and specifically for Lin-CD34+CD38- HSC. We conclude that bispecific CAR-iNKT cell immunotherapy is a very effective treatment for pre-clinical aggressive MLLr-ALL, it outperforms CAR-T cell immunotherapy in an NKG2D-dependent manner and has the potential to protect from immune escape, leptomeningeal disease and lineage switch without discernible hematological toxicity. These findings provide the basis for clinical development of bispecific CD19/CD133 CAR-iNKT cells as an ‘off-the-shelf’ treatment for MLLr-ALL.

NKG2D-mediated detection of metabolically stressed hepatocytes by innate-like T cells is essential for initiation of NASH and fibrosis.

Metabolic-associated fatty liver disease (MAFLD) is a spectrum of clinical manifestations ranging from benign steatosis to cirrhosis. A key event in the pathophysiology of MAFLD is the development of nonalcoholic steatohepatitis (NASH), which can potentially lead to fibrosis and hepatocellular carcinoma, but the triggers of MAFLD-associated inflammation are not well understood. We have observed that lipid accumulation in hepatocytes induces expression of ligands specific to the activating immune receptor NKG2D. Tissue-resident innate-like T cells, most notably γδ T cells, are activated through NKG2D and secrete IL-17A. IL-17A licenses hepatocytes to produce chemokines that recruit proinflammatory cells into the liver, which causes NASH and fibrosis. NKG2D-deficient mice did not develop fibrosis in dietary models of NASH and had a decreased incidence of hepatic tumors. The frequency of IL-17A+ γδ T cells in the blood of patients with MAFLD correlated directly with liver pathology. Our findings identify a key molecular mechanism through which stressed hepatocytes trigger inflammation in the context of MAFLD.

Upregulation of the NKG2D Ligand ULBP2 by JC Polyomavirus Infection Promotes Immune Recognition by Natural Killer Cells.

JC polyomavirus (JCPyV) causes progressive multifocal leukoencephalopathy (PML), a potentially fatal complication of severe immune suppression with no effective treatment. Natural killer (NK) cells play critical roles in defense against viral infections; however, NK-cell response to JCPyV infection remains unexplored. NK- and T-cell responses against the JCPyV VP1 were compared using intracellular cytokine staining upon stimulation with peptide pools. A novel flow cytometry-based assay was developed to determine NK-cell killing efficiency of JCPyV-infected astrocyte-derived SVG-A cells. Blocking antibodies were used to evaluate the contribution of NK-cell receptors in immune recognition of JCPyV-infected cells. In about 40% of healthy donors, we detected robust CD107a upregulation and IFN-γ production by NK cells, extending beyond T-cell responses. Next, using the NK-cell-mediated killing assay, we showed that coculture of NK cells and JCPyV-infected SVG-A cells leads to a 60% reduction in infection, on average. JCPyV-infected cells had enhanced expression of ULBP2-a ligand for the activating NK-cell receptor NKG2D, and addition of NKG2D blocking antibodies decreased NK-cell degranulation. NKG2D-mediated activation of NK cells plays a key role in controlling JCPyV replication and may be a promising immunotherapeutic target to boost NK-cell anti-JCPyV activity.

NKG2D receptor regulates CD4+T cell differentiation via interaction with dendritic cells in patients with juvenile idiopathic arthritis

NKG2D provides a costimulatory signal for activation of CD4+ T cells. We explored its role in interactions of CD4+ T cells and dendritic cells (DCs) in juvenile idiopathic arthritis (JIA) patients by using NKG2D genetically modified CD4+ T cells. We found active JIA patients had significantly higher content of CD4 + NKG2D+ T cells than healthy controls. Expression of NKG2D on CD4+ T cells, and MICA and MICB on DCs were significantly greater in articular JIA than systemic JIA. NKG2D induced IL- 12 and suppressed IL-10 and TGF-β from CD4+ T cells, increased IFN-γ + CD4+ T and IL-17+ CD4+ T cells, RORc and T-bet, but reduced CD25+ Foxp3+ CD4+ T cells, IL-4+ CD4+ T cells, Foxp3, and GATA3 in JIA patients. NKG2D decreased IL-10 and increased CD83, MICA, and MICB of DCs in JIA and controls. So NKG2D regulates differentiation of CD4+ T cells directly and the maturation of DCs indirectly.

MICA and NLRP3 gene polymorphisms interact synergistically affecting the risk of ankylosing spondylitis.

Ankylosing spondylitis (AS) is an autoinflammatory disease that affects the sacroiliac joints, causing stiffness and pain in the back. MICA is a ligand of the NKG2D receptor, and an increase in its expression affects the immune response in various diseases. NLRP3 is a multiprotein complex that promotes the release of IL-1β, but its role in AS has been minimally explored. The objective of this study was to analyze the association and interaction of polymorphic variants of the MICA and NLRP3 genes in patients with AS. In this case-control study, patients with AS were included and compared with healthy controls of Mexican origin. The polymorphisms rs4349859 and rs116488202 of MICA and rs3806268 and rs10754558 of NLRP3 were genotyped using TaqMan probes. Associations were determined using logistic regression models, while interactions were analyzed by the multifactorial dimensionality reduction (MDR) method. A P value < 0.05 was considered statistically significant. The minor allele of rs4349859 (A) and rs116488202 (T) of MICA polymorphisms showed risk associations with AS (OR = 9.22, 95% CI = 4.26-20.0, P < 0.001; OR = 9.36, 95% CI = 4.17-21.0, P < 0.001), while the minor allele of the rs3806268 (A) polymorphism of NLRP3 was associated with protection (OR = 0.55, 95% CI = 0.33-0.91, P = 0.019). MDR analysis revealed synergistic interactions between the MICA and NLRP3 polymorphisms (P = 0.012). In addition, high- and low-risk genotypes were identified among these variants. The study findings suggest that the MICA rs4349859 A allele and rs116488202 T allele are associated with AS risk. An interaction between MICA and NLRP3 was observed which could increase the genetic risk in AS.

Association of MICA Gene Polymorphism in Opisthorchis viverrini-Induced Periductal Fibrosis in Northeastern Thais.

Chronic Opisthorchis viverrini (OV) infection is the cause of advanced periductal fibrosis (APF), subsequently leading to cholangiocarcinoma (CCA). Natural killer (NK) cells can kill hepatic stellate cells (HSCs), the initiating cells for fibrosis formation, by using the interaction between the natural killer group 2 member D (NKG2D) receptor and its ligand on the HSCs. This can inhibit the fibrosis formation. Major histocompatibility complex class I chain-related A (MICA) is the ligand of the NKG2D receptor and has highly polymorphic characteristics that are involved in NKG2D binding and NK cell activation. This study aimed to investigate the polymorphism of MICA in OV-induced fibrosis. MICA typing was performed by polymerase chain reaction- sequence specific primer (PCR-SSP) and sequencing in two groups: OV infection without fibrosis (N = 99) and with fibrosis (N = 290). Six alleles were identified and the MICA*010 allele had the highest frequency in both groups. The MICA*00201-02 allele was a protective factor for fibrosis (OR= 0.508, 95%CI= 0.34-0.76, Pc <0.05), while the MICA*019 allele was suggested to be a risk allele for fibrosis (OR=1.95, 95%CI=1.25-3.03, Pc<0.005). In addition, two motifs, glycine (G) at position 14 and glutamine (Q) at position 251, were negatively associated with fibrosis (G14: OR=0.508, 95%CI=0.34-0.76, Pc <0.05 and Q251: OR=0.586, 95%CI=0.41-0.84, Pc <0.05). Moreover, the distribution of the MICA-129 genotype also showed the protective genotype (Pc<0.05, OR=0.319, 95%CI= 0.12-0.54) for fibrosis. The MICA*00201-02 allele encoded all these motifs, and this suggested that it might lead to strong NK cell activation to kill HSCs, subsequently preventing fibrosis formation. This study described initial evidence suggesting that the polymorphism of the MICA gene might be a marker for OV-derived periductal fibrosis.

SOLUBLE FORMS OF THE NKG2D RECEPTOR AND LIGAND NKG2DL1 IN PATIENTS WITH COLORECTAL CANCER

Relevance. Natural killer (NK) cells are considered to be effective cytotoxic cells of the tumor microenvironment, whose function is to effectively eliminate tumor cells. Their activation can occur when the NKG2D receptor interacts with the NKG2DL ligand expressed, for example, on tumor cells, which leads to the death of the latter. The aim of the study was a comparative study of the content of soluble forms of the sNKG2D receptor and its ligand sNKG2DL1 in the blood serum of healthy donors and patients with colorectal cancer (CRC), taking into account the clinical and morphological characteristics of the disease and prog-nosis. Material and methods. We examined 65 patients with CRC (32 women and 33 men), who were treated at the “National Medical Research Center of Oncology named after N.N. Blokhin” of the Ministry of Health of Russia aged (median 60 years) and 30 healthy donors (20 women and 10 men) aged (median 47 years). The clinical diagnosis in all patients was confirmed by the data of the morphological study of the tumor according to the Interna-tional Histological Classification of Tumors of the Digestive System (WHO, 2019), all had adenocarcinoma. The concentration of NKG2D and NKG2DL proteins was determined by ELISA in blood serum before treatment with Human B7-H3 Quantikine ELISA Kit reagents firm R&amp;D (USA) in accordance with the manufacturer's instructions. The measurements were carried out on an automatic enzyme immunoassay analyzer BEP 2000 Advance (Siemens Healthcare Diagnostics, Germany). Statistical analysis of the obtained results was carried out using GraphPad Prizm v. 9. Overall survival analysis was performed by constructing survival curves using the Kaplan-Meier method. Results. In patients with colorectal cancer, a significant increase in the content of sNKG2DL in blood serum was found compared to the control group. sNKG2D concentrations did not differ between controls and CRC patients. No significant associations were found between the content of sNKG2D and sNKG2DL1 proteins in the blood serum of CRC patients and the main clinical and morphological characteristics of the disease. A decrease in the con-centration of sNKG2D was noted with the progression of the disease, and the highest content of sNKG2D was found in the localization of the tumor in the caecum. For the ligand sNKG2DL1, the opposite pattern was observed, namely, its lowest concentration in blood serum in tumors located in the caecum. For the ligand sNKG2DL1, the opposite pattern was observed, namely, its lowest concentration in blood serum in tumors located in the cae-cum. At the same time, the levels of sNKG2D and sNKG2DL1 in the control group directly and significantly correlated with each other (r=0.453; p=0.014), while no such pattern was found in the group of CRC patients (r=0.014; p=0.935). sNKG2DL1 concentrations are not a prognostic factor in CRC, while high levels of sNKG2D tended to lead to a favorable prognosis of the disease, which indirectly confirms the positive significance of tumor in-filtration by NK cells. Conclusions. The mechanisms of the relationship between CRC avoidance of immune surveillance and the expression of the sNKG2D receptor and the sNKG2DL ligand are discussed as one of the directions in the study of immune checkpoints. We believe that the effect of CRC immunotherapy can be achieved by increasing the expression of NKG2D on immune cells and inducing the expression of NKG2DL in tumor cells, which is the basis of the con-cept of this line of research.

Significant increase in MIC-A and MIC-B and soluble MIC-A and MIC-B in canine lymphomas

Non-Hodkin's lymphoma (NHL) is the most frequent hematologic malignancy in humans and dogs. NKG2D is one of the most critical receptors on NK cells, recognizing their natural ligands on malignant cells such as A and B major histocompatibility complex-related proteins (MIC-A and MIC-B). Soluble molecules (sMIC-A and sMIC-B) can interfere with immune synapsis between NK cells and tumor cells, impeding NK cytotoxicity. The main objectives of this study were to analyze, in dogs with diffuse large B cell lymphoma, NK cell lymphoma, and reactive lymphadenopathies, the role of NK cells, their activating receptors NKG2D and NKp46, and their ligands MIC-A and MIC-B, as well as soluble molecules sMIC-A and sMIC-B. Thirty-six dogs with a possible diagnosis of NHL and eight healthy dogs were studied. NHL was diagnosed in 28 (78 %) dogs; in the other 8 (22 %), reactive lymphadenopathies were present. Most of the lymphomas corresponded to B cell NHL (82 %). The most predominant subtype was diffuse large B cell lymphoma (21, 71.5 %), followed by five cases (18 %) that were Non-B Non-T lymphomas (presumably NK cell lymphomas) and other B cell lymphomas (3, 10.5%). There were no cases of T cell NHL. MIC-A was positive in 7 of 27 (26 %) cases of NHL, and MIC-B in 20 of 27 (74 %) NHL. In non-malignant lymphadenopathies, three (37.5 %) dogs were positive for MIC-A, and five (62.5 %) expressed MIC-B. Dogs with lymphoma had higher numbers of NK cells than eight healthy dogs. In 15 dogs (12 cases with NHL and three cases with reactive adenopathies) and eight controls, there were no differences in the number of NK cells expressing NKP46 and NKG2D. NHL dogs had higher values of sMIC-A and sMIC-B. B-cell and NK cell lymphomas correspond to 86 % and 14 % of all canine lymphomas. MIC-A, MIC-B, and sMIC-A and sMIC-B were increased in canine lymphomas.

Targeting NKG2DL with Bispecific NKG2D-CD16 and NKG2D-CD3 Fusion Proteins on Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a particularly aggressive subtype of breast cancer with a poor response rate to conventional systemic treatment and high relapse rates. Members of the natural killer group 2D ligand (NKG2DL) family are expressed on cancer cells but are typically absent from healthy tissues; thus, they are promising tumor antigens for novel immunotherapeutic approaches. We developed bispecific fusion proteins (BFPs) consisting of the NKG2D receptor domain targeting multiple NKG2DLs, fused to either anti-CD3 (NKG2D-CD3) or anti-CD16 (NKG2D-CD16) Fab fragments. First, we characterized the expression of the NKG2DLs (MICA, MICB, ULBP1-4) on TNBC cell lines and observed the highest surface expression for MICA and ULBP2. Targeting TNBC cells with NKG2D-CD3/CD16 efficiently activated both NK and T cells, leading to their degranulation and cytokine release and lysis of TNBC cells. Furthermore, PBMCs from TNBC patients currently undergoing chemotherapy showed significantly higher NK and T cell activation and tumor cell lysis when stimulated with NKG2D-CD3/CD16. In conclusions, BFPs activate and direct the NK and T cells of healthy and TNBC patients against TNBC cells, leading to efficient eradication of tumor cells. Therefore, NKG2D-based NK and T cell engagers could be a valuable addition to the treatment options for TNBC patients.