Results of a phase I trial with Haploidentical mbIL-21 ex vivo expanded NK cells for patients with multiply relapsed and refractory AML.

Natural killer (NK)-cells have potent anti-tumor effects, yet it remains unclear if they are effective for patients with relapsed acute myeloid leukemia (AML). In a phase I clinical trial, we treated 12 patients (median age 60 years) with refractory AML (median 5 lines of prior therapy, median bone marrow blast count of 47%) with fludarabine/cytarabine followed by 6 infusions of NK-cells expanded from haploidentical donors using K562 feeder cells expressing membrane-bound IL21 and 4-1BBL. Patients received 106-107/kg/dose. No toxicity or graft-versus-host disease (GVHD) was observed and MTD was not reached. Seven patients (58.3%) responded and achieved a complete remission (CR) with/without count recovery. Median time to best response was 48 days. Five responding patients proceeded to a haploidentical transplant from the same donor. After a median follow-up of 52 months, 1-year overall survival (OS) for the entire group was 41.7%, better for patients who responded with CR/CRi (57.14%), and for patients who responded and underwent transplantation (60%). Persistence and expansion of donor-derived NK-cells were identified in patients' blood, and serum IFNγ levels rose concurrently with NK cell infusions. A higher count-functional inhibitory KIR was associated with higher likelihood of achieving CR/CRi. In conclusion, we observed a significant response to ex vivo expanded NK-cell administration in refractory AML patients without adverse effects.

Trabectedin Enhances Oncolytic Virotherapy by Reducing Barriers to Virus Spread and Cytotoxic Immunity in Preclinical Pediatric Bone Sarcoma.

We previously reported that the DNA alkylator and transcriptional-blocking chemotherapeutic agent trabectedin enhances oncolytic herpes simplex viroimmunotherapy in human sarcoma xenograft models, though the mechanism remained to be elucidated. Here we report trabectedin disrupts the intrinsic cellular anti-viral response which increases viral transcript spread throughout the human tumor cells. We also extended our synergy findings to syngeneic murine sarcoma models, which are poorly susceptible to virus infection. In the absence of robust virus replication, we found trabectedin enhanced viroimmunotherapy efficacy by reducing immunosuppressive macrophages and stimulating granzyme expression in infiltrating T and NK cells to cause immune-mediated tumor regressions. Thus, trabectedin enhances both the direct virus-mediated killing of tumor cells and the viral-induced activation of cytotoxic effector lymphocytes to cause tumor regressions across models. Our data provide a strong rationale for clinical translation as both mechanisms should be simultaneously active in human patients.

Single-cell functional genomics reveals determinants of sensitivity and resistance to natural killer cells in blood cancers.

Cancer cells can evade natural killer (NK) cell activity, thereby limiting anti-tumor immunity. To reveal genetic determinants of susceptibility to NK cell activity, we examined interacting NK cells and blood cancer cells using single-cell and genome-scale functional genomics screens. Interaction of NK and cancer cells induced distinct activation and type I interferon (IFN) states in both cell types depending on the cancer cell lineage and molecular phenotype, ranging from more sensitive myeloid to less sensitive B-lymphoid cancers. CRISPR screens in cancer cells uncovered genes regulating sensitivity and resistance to NK cell-mediated killing, including adhesion-related glycoproteins, protein fucosylation genes, and transcriptional regulators, in addition to confirming the importance of antigen presentation and death receptor signaling pathways. CRISPR screens with a single-cell transcriptomic readout provided insight into underlying mechanisms, including regulation of IFN-γ signaling in cancer cells and NK cell activation states. Our findings highlight the diversity of mechanisms influencing NK cell susceptibility across different cancers and provide a resource for NK cell-based therapies.

Knockout of the inhibitory receptor TIGIT enhances the antitumor response of ex vivo expanded NK cells and prevents fratricide with therapeutic Fc-active TIGIT antibodies.

BACKGROUND: Inhibitory receptor T-cell Immunoreceptor with Ig and ITIM domains (TIGIT) expressed by Natural Killer (NK) and T cells regulates cancer immunity and has been touted as the next frontier in the development of cancer immunotherapeutics. Although early results of anti-TIGIT and its combinations with antiprogrammed death-ligand 1 were highly exciting, results from an interim analysis of phase III trials are disappointing. With mixed results, there is a need to understand the effects of therapeutic anti-TIGIT on the TIGIT+ immune cells to support its clinical use. Most of the TIGIT antibodies in development have an Fc-active domain, which binds to Fc receptors on effector cells. In mouse models, Fc-active anti-TIGIT induced superior immunity, while Fc receptor engagement was required for its efficacy. NK-cell depletion compromised the antitumor immunity of anti-TIGIT indicating the essential role of NK cells in the efficacy of anti-TIGIT. Since NK cells express TIGIT and Fc-receptor CD16, Fc-active anti-TIGIT may deplete NK cells via fratricide, which has not been studied. METHODS: CRISPR-Cas9-based TIGIT knockout (KO) was performed in expanded NK cells. Phenotypic and transcriptomic properties of TIGIT KO and wild-type (WT) NK cells were compared with flow cytometry, CyTOF, and RNA sequencing. The effect of TIGIT KO on NK-cell cytotoxicity was determined by calcein-AM release and live cell imaging-based cytotoxicity assays. The metabolic properties of TIGIT KO and WT NK cells were compared with a Seahorse analyzer. The effect of the Fc-component of anti-TIGIT on NK-cell fratricide was determined by co-culturing WT and TIGIT KO NK cells with Fc-active and Fc-inactive anti-TIGIT. RESULTS: TIGIT KO increased the cytotoxicity of NK cells against multiple cancer cell lines including spheroids. TIGIT KO NK cells upregulated mTOR complex 1 (mTORC1) signaling and had better metabolic fitness with an increased basal glycolytic rate when co-cultured with cancer cells compared with WT NK cells. Importantly, TIGIT KO prevented NK-cell fratricide when combined with Fc-active anti-TIGIT. CONCLUSIONS: TIGIT KO in ex vivo expanded NK cells increased their cytotoxicity and metabolic fitness and prevented NK-cell fratricide when combined with Fc-active anti-TIGIT antibodies. These fratricide-resistant TIGIT KO NK cells have therapeutic potential alone or in combination with Fc-active anti-TIGIT antibodies to enhance their efficacy.

Opportunities and challenges of combining adoptive cellular therapy with oncolytic virotherapy.

The use of oncolytic viruses (OVs) and adoptive cell therapies (ACT) have independently emerged as promising approaches for cancer immunotherapy. More recently, the combination of such agents to obtain a synergistic anticancer effect has gained attention, particularly in solid tumors, where immune-suppressive barriers of the microenvironment remain a challenge for desirable therapeutic efficacy. While adoptive cell monotherapies may be restricted by an immunologically cold or suppressive tumor microenvironment (TME), OVs can serve to prime the TME by eliciting a wave of cancer-specific immunogenic cell death and inducing enhanced antitumor immunity. While OV/ACT synergy is an attractive approach, immune-suppressive barriers remain, and methods should be considered to optimize approaches for such combination therapy. In this review, we summarize current approaches that aim to overcome these barriers to enable optimal synergistic antitumor effects.

Deletion of Glycogen Synthase Kinase 3 Beta Reprograms NK Cell Metabolism.

Loss of cytotoxicity and defective metabolism are linked to glycogen synthase kinase 3 beta (GSK3ß) overexpression in natural killer (NK) cells from patients with acute myeloid leukemia or from healthy donors after expansion ex vivo with IL-15. Drug inhibition of GSK3ß in these NK cells improves their maturation and cytotoxic activity, but the mechanisms of GSK3ß-mediated dysfunction have not been well studied. Here, we show that expansion of NK cells with feeder cells expressing membrane-bound IL-21 maintained normal GSK3ß levels, allowing us to study GSK3ß function using CRISPR gene editing. We deleted GSK3B and expanded paired-donor knockout and wild-type (WT) NK cells and then assessed transcriptional and functional alterations induced by loss of GSK3ß. Surprisingly, our data showed that deletion of GSK3B did not alter cytotoxicity, cytokine production, or maturation (as determined by CD57 expression). However, GSK3B-KO cells demonstrated significant changes in expression of genes related to rRNA processing, cell proliferation, and metabolic function, suggesting possible metabolic reprogramming. Next, we found that key genes downregulated in GSK3B-KO NK cells were upregulated in GSK3ß-overexpressing NK cells from AML patients, confirming this correlation in a clinical setting. Lastly, we measured cellular energetics and observed that GSK3B-KO NK cells exhibited 150% higher spare respiratory capacity, a marker of metabolic fitness. These findings suggest a role for GSK3ß in regulating NK cell metabolism.

Cerebral organoids containing an AUTS2 missense variant model microcephaly.

Variants in the AUTS2 gene are associated with a broad spectrum of neurological conditions characterized by intellectual disability, microcephaly, and congenital brain malformations. Here, we use a human cerebral organoid model to investigate the pathophysiology of a heterozygous de novo missense AUTS2 variant identified in a patient with multiple neurological impairments including primary microcephaly and profound intellectual disability. Proband cerebral organoids exhibit reduced growth, deficits in neural progenitor cell (NPC) proliferation and disrupted NPC polarity within ventricular zone-like regions compared to control cerebral organoids. We used CRISPR-Cas9-mediated gene editing to correct this variant and demonstrate rescue of impaired organoid growth and NPC proliferative deficits. Single-cell RNA sequencing revealed a marked reduction of G1/S transition gene expression and alterations in WNT-ß-catenin signalling within proband NPCs, uncovering a novel role for AUTS2 in NPCs during human cortical development. Collectively, these results underscore the value of cerebral organoids to investigate molecular mechanisms underlying AUTS2 syndrome.

Pediatric versus adult high grade glioma: Immunotherapeutic and genomic considerations.

High grade gliomas are identified as malignant central nervous tumors that spread rapidly and have a universally poor prognosis. Historically high grade gliomas in the pediatric population have been treated similarly to adult high grade gliomas. For the first time, the most recent classification of central nervous system tumors by World Health Organization has divided adult from pediatric type diffuse high grade gliomas, underscoring the biologic differences between these tumors in different age groups. The objective of our review is to compare high grade gliomas in the adult versus pediatric patient populations, highlighting similarities and differences in epidemiology, etiology, pathogenesis and therapeutic approaches. High grade gliomas in adults versus children have varying clinical presentations, molecular biology background, and response to chemotherapy, as well as unique molecular targets. However, increasing evidence show that they both respond to recently developed immunotherapies. This review summarizes the distinctions and commonalities between the two in disease pathogenesis and response to therapeutic interventions with a focus on immunotherapy.

Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV.

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.

Natural Killer Cell Recognition and Control of Epithelial Cancers.

ABSTRACT: Natural killer (NK) cells possess an innate ability to recognize cancer and are key mediators of cytotoxic efficacy for anticancer antibodies. Recent advances in the ability to generate, qualify, and safely infuse NK cells have led to a wide variety of clinical trials in oncology. Although their efficacy is best established for liquid cancers, their potential application in solid cancers has received increased attention. Here, we provide general background across a disparate group of exemplary solid tumors for which there is evidence for an NK cell role, discuss NK cell recognition motifs specific to each and murine and human studies of each that are supportive of NK cell adoptive immunotherapy, and end with special considerations relevant to the solid tumor microenvironment.

CRISPR Gene Editing of Human Primary NK and T Cells for Cancer Immunotherapy.

Antitumor activity of immune cells such as T cells and NK cells has made them auspicious therapeutic regimens for adaptive cancer immunotherapy. Enhancing their cytotoxic effects against malignancies and overcoming their suppression in tumor microenvironment (TME) may improve their efficacy to treat cancers. Clustered, regularly interspaced short palindromic repeats (CRISPR) genome editing has become one of the most popular tools to enhance immune cell antitumor activity. In this review we highlight applications and practicability of CRISPR/Cas9 gene editing and engineering strategies for cancer immunotherapy. In addition, we have reviewed several approaches to study CRISPR off-target effects.

Evaluation of allogeneic and autologous membrane-bound IL-21-expanded NK cells for chronic lymphocytic leukemia therapy.

Successes with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered vs unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated NK cells have shown promise as a cellular therapy, but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound interleukin-21 (mbIL-21) and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells, whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in 2 xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21-expanded NK cells. Collectively, these data support development of mbIL-21-expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.

The Future of Natural Killer Cell Immunotherapy for B Cell Non-Hodgkin Lymphoma (B Cell NHL).

OPINION STATEMENT: Natural killer (NK) cells have played a critical-if largely unrecognized or ignored-role in the treatment of B cell non-Hodgkin lymphoma (NHL) since the introduction of CD20-directed immunotherapy with rituximab as a cornerstone of therapy over 25 years ago. Engagement with NK cells leading to lysis of NHL targets through antibody-dependent cellular cytotoxicity (ADCC) is a critical component of rituximab's mechanism of action. Despite this important role, the only aspect of B cell NHL therapy that has been adopted as standard therapy that even indirectly augments or restores NK cell function is the introduction of obinutuzumab, a CD20 antibody with enhanced ability to engage with NK cells. However, over the last 5 years, adoptive immunotherapy with effector lymphocytes of B cell NHL has experienced tremendous growth, with five different CAR T cell products now licensed by the FDA, four of which target CD19 and have approved indications for some subtype of B cell NHL-axicabtagene ciloleucel, brexucabtagene autoleucel, lisocabtagene maraleucel, and tisagenlecleucel. These T cell-based immunotherapies essentially mimic the recognition, activation pathway, and cytotoxic machinery of a CD19 antibody engaging NK cells and lymphoma targets. Despite their efficacy, these T cell-based immunotherapies have been difficult to implement because they require 4-6 weeks of manufacture, are costly, and have significant toxicities. This renewed interest in the potential of cellular immunity-and the manufacturing, supply chain, and administration logistics that have been addressed with these new agents-have ignited a new wave of enthusiasm for NK cell-directed therapies in NHL. With high safety profiles and proven anti-lymphoma efficacy, one or more new NK cell-directed modalities are certain to be introduced into the standard toolbox of NHL therapy within the next few years, be it function-enhancing cytokine muteins, multi-domain NK cell engagers, or adoptive therapy with expanded or genetically modified NK cells.

Assessment of Beta-2 Microglobulin Gene Edited Airway Epithelial Stem Cells as a treatment for Sulfur Mustard Inhalation.

Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient's immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD50 on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously (n = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.

Decrease post-transplant relapse using donor-derived expanded NK-cells.

In this phase I/II clinical trial, we investigated the safety and efficacy of high doses of mb-IL21 ex vivo expanded donor-derived NK cells to decrease relapse in 25 patients with myeloid malignancies receiving haploidentical stem-cell transplantation (HSCT). Three doses of donor NK cells (1 × 105-1 × 108 cells/kg/dose) were administered on days -2, +7, and +28. Results were compared with an independent contemporaneously treated case-matched cohort of 160 patients from the CIBMTR database.After a median follow-up of 24 months, the 2-year relapse rate was 4% vs. 38% (p = 0.014), and disease-free survival (DFS) was 66% vs. 44% (p = 0.1) in the cases and controls, respectively. Only one relapse occurred in the study group, in a patient with the high level of donor-specific anti-HLA antibodies (DSA) presented before transplantation. The 2-year relapse and DFS in patients without DSA was 0% vs. 40% and 72% vs. 44%, respectively with HR for DFS in controls of 2.64 (p = 0.029). NK cells in recipient blood were increased at day +30 in a dose-dependent manner compared with historical controls, and had a proliferating, mature, highly cytotoxic, NKG2C+/KIR+ phenotype.Administration of donor-derived expanded NK cells after haploidentical transplantation was safe, associated with NK cell-dominant immune reconstitution early post-transplant, preserved T-cell reconstitution, and improved relapse and DFS. TRIAL REGISTRATION: NCT01904136 ( https://clinicaltrials.gov/ct2/show/NCT01904136 ).

Adoptive immunotherapy with double-bright (CD56bright /CD16bright ) expanded natural killer cells in patients with relapsed or refractory acute myeloid leukaemia: a proof-of-concept study.

Patients with acute myeloid leukaemia (AML) have a five-year survival rate of 28·7%. Natural killer (NK)-cell have anti-leukaemic activity. Here, we report on a series of 13 patients with high-risk R/R AML, treated with repeated infusions of double-bright (CD56bright /CD16bright ) expanded NK cells at an academic centre in Brazil. NK cells from HLA-haploidentical donors were expanded using K562 feeder cells, modified to express membrane-bound interleukin-21. Patients received FLAG, after which cryopreserved NK cells were thawed and infused thrice weekly for six infusions in three dose cohorts (106 -107 cells/kg/infusion). Primary objectives were safety and feasibility. Secondary endpoints included overall response (OR) and complete response (CR) rates at 28-30 days after the first infusion. Patients received a median of five prior lines of therapy, seven with intermediate or adverse cytogenetics, three with concurrent central nervous system (CNS) leukaemia, and one with concurrent CNS mycetoma. No dose-limiting toxicities, infusion-related fever, or cytokine release syndrome were observed. An OR of 78·6% and CR of 50·0% were observed, including responses in three patients with CNS disease and clearance of a CNS mycetoma. Multiple infusions of expanded, cryopreserved NK cells were safely administered after intensive chemotherapy in high-risk patients with R/R AML and demonstrated encouraging outcomes.

Defining the AHR-regulated transcriptome in NK cells reveals gene expression programs relevant to development and function.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates cellular processes in cancer and immunity, including innate immune cell development and effector function. However, the transcriptional repertoire through which AHR mediates these effects remains largely unexplored. To elucidate the transcriptional elements directly regulated by AHR in natural killer (NK) cells, we performed RNA and chromatin immunoprecipitation sequencing on NK cells exposed to AHR agonist or antagonist. We show that mature peripheral blood NK cells lack AHR, but its expression is induced by Stat3 during interleukin-21-driven activation and proliferation, coincident with increased NCAM1 (CD56) expression resulting in a CD56bright phenotype. Compared with control conditions, NK cells expanded in the presence of the AHR antagonist, StemRegenin-1, were unaffected in proliferation or cytotoxicity, had no increase in NCAM1 transcription, and maintained the CD56dim phenotype. However, it showed altered expression of 1004 genes including those strongly associated with signaling pathways. In contrast, NK cells expanded in the presence of the AHR agonist, kynurenine, showed decreased cytotoxicity and altered expression of 97 genes including those strongly associated with oxidative stress and cellular metabolism. By overlaying these differentially expressed genes with AHR chromatin binding, we identified 160 genes directly regulated by AHR, including hallmark AHR targets AHRR and CYP1B1 and known regulators of phenotype, development, metabolism, and function such as NCAM1, KIT, NQO1, and TXN. In summary, we define the AHR transcriptome in NK cells, propose a model of AHR and Stat3 coregulation, and identify potential pathways that may be targeted to overcome AHR-mediated immune suppression.

NKG2D-CAR-transduced natural killer cells efficiently target multiple myeloma.

CAR-T-cell therapy against MM currently shows promising results, but usually with serious toxicities. CAR-NK cells may exert less toxicity when redirected against resistant myeloma cells. CARs can be designed through the use of receptors, such as NKG2D, which recognizes a wide range of ligands to provide broad target specificity. Here, we test this approach by analyzing the antitumor activity of activated and expanded NK cells (NKAE) and CD45RA- T cells from MM patients that were engineered to express an NKG2D-based CAR. NKAE cells were cultured with irradiated Clone9.mbIL21 cells. Then, cells were transduced with an NKG2D-4-1BB-CD3z-CAR. CAR-NKAE cells exhibited no evidence of genetic abnormalities. Although memory T cells were more stably transduced, CAR-NKAE cells exhibited greater in vitro cytotoxicity against MM cells, while showing minimal activity against healthy cells. In vivo, CAR-NKAE cells mediated highly efficient abrogation of MM growth, and 25% of the treated mice remained disease free. Overall, these results demonstrate that it is feasible to modify autologous NKAE cells from MM patients to safely express a NKG2D-CAR. Additionally, autologous CAR-NKAE cells display enhanced antimyeloma activity demonstrating that they could be an effective strategy against MM supporting the development of NKG2D-CAR-NK-cell therapy for MM.

Donor Killer Immunoglobulin Receptor Gene Content and Ligand Matching and Outcomes of Pediatric Patients with Juvenile Myelomonocytic Leukemia Following Unrelated Donor Transplantation.

Natural killer (NK) cell determinants predict relapse-free survival after allogeneic hematopoietic cell transplantation (HCT) for acute myelogenous leukemia, and previous studies have shown a beneficial graft-versus-leukemia effect in patients with juvenile myelomonocytic leukemia (JMML). However, whether NK cell determinants predict protection against relapse for JMML patients undergoing HCT is unknown. Therefore, we investigated NK cell-related donor and recipient immunogenetics as determinants of HCT outcomes in patients with JMML. Patients with JMML (age 0 to <19 years) who underwent a first allogeneic HCT from an unrelated donor between 2000 and 2017 and had available donor samples from the Center for International Blood and Marrow Transplant Research Repository were included. Donor killer immunoglobulin receptor (KIR) typing was performed on pre-HCT samples. The primary endpoint was disease-free survival (DFS); secondary endpoints included relapse, grade II-IV acute graft versus-host-disease (aGVHD), chronic GVHD (cGVHD), GVHD-free relapse-free survival, transplantation-related mortality, and overall survival (OS). Donor KIR models tested included KIR genotype (AA versus Bx), B content (0-1 versus ≥2), centromeric and telomeric region score (AA versus AB versus BB), B content score (best, better, or neutral), composite score (2 versus 3 versus 4), activating KIR content, and the presence of KIR2DS4. Ligand-ligand and KIR-ligand mismatch effects on outcomes were analyzed in HLA-mismatched donors (≤7/8; n = 74) only. Univariate analyses were performed for primary and secondary outcomes of interest, with a P value <.05 considered significant. One hundred sixty-five patients (113 males), with a median follow-up of 85 months (range, 6 to 216 months) met the study criteria. Of these, 111 underwent an unrelated donor HCT and 54 underwent a UCB HCT. Almost all (n = 161; 98%) received a myeloablative conditioning regimen. After exclusion of recipients of reduced-intensity/nonmyeloablative conditioning regimens and ex vivo T cell-depleted grafts (n = 8), there were 42 AA donors and 115 Bx donors, respectively. Three-year DFS, OS, relapse, and GRFS for the entire cohort were 58% (95% confidence interval [CI], 50% to 66%), 67% (95% CI, 59% to 74%), 26% (95% CI, 19% to 33%), and 27% (95% CI, 19% to 35%), respectively. The cumulative incidence of grade II-IV aGVHD at 100 days was 36% (95% CI, 27% to 44%), and that of cGVHD at 1 year was 23% (95% CI, 17% to 30%). There were no differences between AA donors and Bx donors for any recipient survival outcomes. The risk of grade II-IV aGVHD was lower in patients with donors with a B content score of ≥2 (hazard ratio [HR], 0.46; 95% CI, 0.26 to 0.83; P = .01), an activating KIR content score of >3 (HR, 0.52; 95% CI, 0.29 to 0.95; P = .032), centromeric A/B score (HR, 0.57; 95% CI, 033 to 0.98; P = .041), and telomeric A/B score (HR, 0.58; 95% CI, 0.34 to 1.00; P = .048). To our knowledge, this is the first study analyzing the association of NK cell determinants and outcomes in JMML HCT recipients. This study identifies potential benefits of donor KIR-B genotypes in reducing aGVHD. Our findings warrant further study of the role of NK cells in enhancing the graft-versus-leukemia effect via recognition of JMML blasts.