Natural Killer Effector Function Research Articles

Abstract 1241: Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE)

Abstract With an annual incidence rate exceeding 660,000 cases, and a death toll surpassing 325,000 per year, head and neck cancer (HNC) ranks as the seventh most common cancer in the world. Surgery, radiation and chemotherapy are used to treat HNC patients with modest and variable clinical success. However, these treatments prove less effective for human papillomavirus negative (HPV-) HNC patients, a subset of HNC patients with markedly worse prognosis. While significant advancements have been made in cancer immunotherapy over the past decade, its success also remains elusive for HNC due to factors such as the hypoxic solid tumor microenvironment (TME). To address the critical need for an improved therapeutic intervention, we leveraged the ability of natural killer (NK) cells in killing cancer cells without prior sensitization by developing a novel tri-specific killer engager (TriKE) that can direct NK cell killing of tumor within the hypoxic solid TME. The TriKE is composed of three domains: a humanized nanobody arm binding the activating receptor CD16 on NK cells, an interleukin (IL)-15 moiety that can drive expansion of NK cells, and a nanobody arm binding B7H3, a protein which high expression can be negatively correlated with overall survival of HPV- HNC patients. B7H3 is a prime target candidate because it is highly expressed on HPV- HNC cells, but minimally expressed on healthy tissues. In vitro testing using HPV- HNC patient blood samples revealed that B7H3 TriKE enhances activation (measured by NK cell degranulation and interferon-gamma production) and expansion of NK cells from these patients at levels equivalent to those observed in healthy controls. Furthermore, B7H3 TriKE is efficacious in its ability to drive high NK cell cytotoxicity in both acute (<48-hours) and prolonged (7-days) hypoxic (1% oxygen) models of HNC. Under acute hypoxia, B7H3 TriKE induces significantly more killing of targets by NK cells compared to IL-15 treatment, where NK cell cytotoxicity is impaired. In addition, B7H3 TriKE can boost the killing efficacy of NK cells exposed to prolonged hypoxia, surpassing limitations seen with IL-15 treatment. These findings strongly suggest that the B7H3 TriKE can bypass hypoxic suppression of NK effector functions in the solid TME. In vivo studies using immunocompromised mice engrafted with HPV- HNC cells revealed that B7H3 TriKE treatment significantly extends the survival of mice, compared to IL-15 treatment. Moreover, NK cells persisted in the blood of B7H3 TriKE-treated mice 28 days post-NK cell injection, highlighting the promising clinical translation of this immunotherapy. More in-depth characterization of NK cells from HPV- HNC patients are underway but altogether, these robust pre-clinical data present a novel avenue for the management of HNC for these patients. We plan to translate results from these studies to clinical trials in fall 2024. Citation Format: Melissa Khaw, Nicholas A. Zorko, Carly Selleck, Laura Bendzick, Zachary Davis, Peter Hinderlie, Madison Shackelford, Ann Lu, James Lim, Naomi Fujioka, Margaret MacMillan, John Wagner, Martin Felices, Jeffrey S. Miller. Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1241.

Metabolic regulation of NK cell function: implications for immunotherapy.

Natural killer (NK) cells are innate immune lymphocytes capable of rapidly responding to tumors and infection without prior sensitization. There is increasing interest and success in harnessing NK cell function for the treatment of disease, in particular cancers. NK cell activation is dependent on integration of signals through cytokine and germline-encoded activating and inhibitory receptors. The availability of metabolic fuels and pathways is required for NK effector functions including proliferation, killing, and production of interferon gamma (IFN-γ). An understanding of NK cell immunometabolism is thus essential for developing immunotherapy approaches that will allow for optimal effector functions in patients. Studies in mice and humans have demonstrated stimulation-dependent metabolic changes that are required for NK cell function. Here we review the most recent findings in NK cell immunometabolism relevant to disease models and translation to therapy of patients.

Identification of a novel mechanism for LFA-1 organization during NK cytolytic response.

The elimination of transformed and viral infected cells by natural killer (NK) cells requires a specialized junction between NK and target cells, denominated immunological synapse (IS). After initial recognition, the IS enables the directed secretion of lytic granules content into the susceptible target cell. The lymphocyte function-associated antigen (LFA)-1 regulates NK effector function by enabling NK-IS assembly and maturation. The pathways underlying LFA-1 accumulation at the IS in NK cells remained uncharacterized. A kinase anchoring protein 350 (AKAP350) is a centrosome/Golgi-associated protein, which, in T cells, participates in LFA-1 activation by mechanisms that have not been elucidated. We first evaluated AKAP350 participation in NK cytolytic activity. Our results showed that the decrease in AKAP350 levels by RNA interference (AKAP350KD) inhibited NK-YTS cytolytic activity, without affecting conjugate formation. The impairment of NK effector function in AKAP350KD cells correlated with decreased LFA-1 clustering and defective IS maturation. AKAP350KD cells that were exclusively activated via LFA-1 showed impaired LFA-1 organization and deficient lytic granule translocation as well. In NK AKAP350KD cells, activation signaling through Vav1 was preserved up to 10 min of interaction with target cells, but significantly decreased afterwards. Experiments in YTS and in ex vivo NK cells identified an intracellular pool of LFA-1, which partially associated with the Golgi apparatus and, upon NK activation, redistributed to the IS in an AKAP350-dependent manner. The analysis of Golgi organization indicated that the decrease in AKAP350 expression led to the disruption of the Golgi integrity in NK cells. Alteration of Golgi function by BFA treatment or AKAP350 delocalization from this organelle also led to impaired LFA-1 localization at the IS. Therefore, this study characterizes AKAP350 participation in the modulation of NK effector function, revealing the existence of a Golgi-dependent trafficking pathway for LFA-1, which is relevant for LFA-1 organization at NK-lytic IS.

Allele imputation for the killer cell immunoglobulin-like receptor KIR3DL1/S1.

Highly polymorphic interaction of KIR3DL1 and KIR3DS1 with HLA class I ligands modulates the effector functions of natural killer (NK) cells and some T cells. This genetically determined diversity affects severity of infections, immune-mediated diseases, and some cancers, and impacts the course of immunotherapies, including transplantation. KIR3DL1 is an inhibitory receptor, and KIR3DS1 is an activating receptor encoded by the KIR3DL1/S1 gene that has more than 200 diverse and divergent alleles. Determination of KIR3DL1/S1 genotypes for medical application is hampered by complex sequence and structural variation, requiring targeted approaches to generate and analyze high-resolution allele data. To overcome these obstacles, we developed and optimized a model for imputing KIR3DL1/S1 alleles at high-resolution from whole-genome SNP data. We designed the model to represent a substantial component of human genetic diversity. Our Global imputation model is effective at genotyping KIR3DL1/S1 alleles with an accuracy ranging from 88% in Africans to 97% in East Asians, with mean specificity of 99% and sensitivity of 95% for alleles >1% frequency. We used the established algorithm of the HIBAG program, in a modification named Pulling Out Natural killer cell Genomics (PONG). Because HIBAG was designed to impute HLA alleles also from whole-genome SNP data, PONG allows combinatorial diversity of KIR3DL1/S1 with HLA-A and -B to be analyzed using complementary techniques on a single data source. The use of PONG thus negates the need for targeted sequencing data in very large-scale association studies where such methods might not be tractable.

Filamin A Is Required for NK Cell Cytotoxicity at the Expense of Cytokine Production via Synaptic Filamentous Actin Modulation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions.

Modulation of Siglec-7 Signaling Via In Situ-Created High-Affinity cis-Ligands.

Sialic acid-binding immunoglobulin-like lectins, also known as Siglecs, have recently been designated as glyco-immune checkpoints. Through their interactions with sialylated glycan ligands overexpressed on tumor cells, inhibitory Siglecs on innate and adaptive immune cells modulate signaling cascades to restrain anti-tumor immune responses. However, the elucidation of the mechanisms underlying these processes is just beginning. We find that when human natural killer (NK) cells attack tumor cells, glycan remodeling occurs on the target cells at the immunological synapse. This remodeling occurs through both the transfer of sialylated glycans from NK cells to target tumor cells and the accumulation of de novo synthesized sialosides on the tumor cells. The functionalization of NK cells with a high-affinity ligand of Siglec-7 leads to multifaceted consequences in modulating a Siglec-7-regulated NK-activation. At high levels of ligand, an enzymatically added Siglec-7 ligand suppresses NK cytotoxicity through the recruitment of Siglec-7 to an immune synapse, whereas at low levels of ligand an enzymatically added Siglec-7 ligand triggers the release of Siglec-7 from the cell surface into the culture medium, preventing a Siglec-7-mediated inhibition of NK cytotoxicity. These results suggest that a glycan engineering of NK cells may provide a means to boost NK effector functions for related applications.

Therapeutic Approaches Targeting the Natural Killer-Myeloid Cell Axis in the Tumor Microenvironment.

Immunotherapy has transformed cancer treatment by promoting durable clinical responses in a proportion of patients; however, treatment still fails in many patients. Innate immune cells play a key role in the response to immunotherapy. Crosstalk between innate and adaptive immune systems drives T-cell activation but also limits immunotherapy response, as myeloid cells are commonly associated with resistance. Hence, innate cells have both negative and positive effects within the tumor microenvironment (TME), and despite investment in early clinical trials targeting innate cells, they have seen limited success. Suppressive myeloid cells facilitate metastasis and immunotherapy resistance through TME remodeling and inhibition of adaptive immune cells. Natural killer (NK) cells, in contrast, secrete inflammatory cytokines and directly kill transformed cells, playing a key immunosurveillance role in early tumor development. Myeloid and NK cells show reciprocal crosstalk, influencing myeloid cell functional status or antigen presentation and NK effector function, respectively. Crosstalk between myeloid cells and the NK immune network in the TME is especially important in the context of therapeutic intervention. Here we discuss how myeloid and NK cell interactions shape anti-tumor responses by influencing an immunosuppressive TME and how this may influence outcomes of treatment strategies involving drugs that target myeloid and NK cells.

Selection of allogeneic hematopoietic cell transplant donors to optimize natural killer cell alloreactivity

Natural killer (NK) cells are potent mediators of the graft versus leukemia phenomenon critical to the success of allogeneic hematopoietic cell transplantation. Central to calibrating NK effector function via their interaction with class I human leukocyte antigens are the numerous inhibitory killer Ig-like receptors (KIR). The KIR receptors are encoded by a family of polymorphic genes, whose expression is largely stochastic and uninfluenced by human leukocyte antigens genotype. These features provide the opportunity to select hematopoietic cell donors with favorable KIR genotypes that confer enhanced protection from relapse via NK-mediated graft versus leukemia. Over the last 2 decades, a large body of work has emerged examining the use of KIR genotyping to stratify potential donors based on anticipated NK alloreactivity. Overall, these results support KIR-based donor selection for patients undergoing allogeneic hematopoietic cell transplantation for a diagnosis of acute myelogenous leukemia. Despite this, the underlying factors that control NK cell responsiveness are not completely understood, and opportunities remain to refine donor selection using NK cell receptor genotyping. In this review, we will summarize the relevant findings with respect to KIR genotyping as a selection parameter for allogeneic hematopoietic cell donors and address practical considerations with respect to KIR-based selection of donors for patients with myeloid neoplasia.

Purinergic Receptor P2Y <sub>6</sub> Inhibits the Maturation and Function of NK Cells by Suppressing mTOR Signaling Pathway

Natural killer (NK) cells are a critical immune cells of innate immunity that can kill tumor cells, cancer-initiating cells and clear virus infections by producing cytokines and cytotoxic granules. However, there are few reports that elucidate the role of purinergic receptor in regulating NK cells. In this study, we discovered the expression of purinergic receptor P2Y6 was decreased after the activation of NK cells. In P2Y6-deficient mice, we found that P2Y6 deficiency promoted the development of NKp cells into iNK cells and the maturation of NK cells. We also found P2Y6 deficiency increased the production of IFN-γ in NK cells after stimulated with Poly (I:C), IL-15, the combination of IL-12 and IL-15 or PMA. P2Y6 receptor agonist UDP or UDP analogue 5-OMe-UDP inhibited the production of IFN-γ in NK cells after stimulation. In addition, we demonstrated P2Y6 deficiency increased the cytotoxicty function of NK cells in vitro and anti-metastatic activity in vivo. Mechanistically, we found P2Y6 deletion increased the expression of T-bet with or without IL-15 stimulation. Additionally, P2Y6 deficiency increased the level of p-AKT and pS6 after IL-15 stimulation, respectively. Collectively, we identify P2Y6 receptor, as a negative regulation of of NK cells function, inhibits the expression of T-bet by suppressing mTOR signaling pathway activation, eventually prevents NK cells maturation and effector function.

Abstract B044: Engineering CAR NK cells for antigen-dependent autocrine expansion

Abstract Given promising results with chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies attention has turned to whether CAR natural killer (NK) cells might have utility in cancer immunotherapy. NK cells are known to have potent cytolytic function and the ability to secrete interferons and cytokines, including GM-CSF and interferon-γ (IFNγ) upon stimulation. In pre-clinical models CAR NK cells have been shown to have efficacy and possibly some advantages over CAR T-cells. Several biotechs and pharmas are currently exploring this approach, but a major limitation of NK cells is their inability to produce autocrine growth factors to promote their own expansion and activation. In the context of CAR NK cell therapy, a major limitation is the inability of the infused CAR NK cells to expand in the absence of cytokine signals to initiate their proliferation and enhance their effector functions. IL-12 and IL-15 are essential for the clonal expansion of NK cells; however, recombinant IL-12 and IL-15 are quite toxic when administered systemically. Additionally, engineering NK cells to constitutively secrete IL-12 or IL-15 is not an attractive strategy as this may also result in off-target toxicity. Our goal is to engineer chimeric receptors with the intracellular signaling domains of IL-12 or IL-15 receptors fused to the extracellular domains of cytokine receptors. Consequently, self-secreted cytokines by activated NK cells or recognition of tumor cells will provide autocrine growth signal, permit NK clonal expansion and enhance NK effector functions. Citation Format: Avishai Shemesh, Kole T. Roybal, Lewis L. Lanier. Engineering CAR NK cells for antigen-dependent autocrine expansion [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B044.

Cord Blood Derived Natural Killer Cells Loaded with a Tetravalent Bispecific Antibody Construct (AFM13) As Off-the-Shelf Cell Therapy for CD30+ Malignancies

Chimeric antigen receptors to redirect T cell specificity against tumor antigens have shown remarkable clinical responses against CD19+ malignancies. However, the manufacture of an engineered autologous T cell product is expensive and cumbersome. Natural killer (NK) cells provide an alternative source of immune effectors for the treatment of cancer. NK cell cytolytic function can be directed towards specific targets by exploiting their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) through the NK cell Fc receptor, CD16 (FcγRIIIa). AFM13 is a tetravalent bispecific antibody construct based on Affimed's ROCK™ platform. AFM13 is bispecific for CD30 and CD16A, designed for the treatment of CD30 expressing malignancies. It binds CD16A on the surface of NK cells, thus activating and recruiting them to CD30 expressing tumor cells and mediating subsequent tumor cell killing. Since autologous NK effector function is impaired in many patients with malignancies, we propose to overcome this by the use of allogeneic NK cells in combination with AFM13. Cord blood (CB) is a readily available (“off-the-shelf”) source of allogeneic NK cells that can be expanded to large, highly functional therapeutic doses. The feasibility and safety of therapy with allogeneic ex vivo expanded CB-derived NK cells have been shown by our group and others.In this study, we hypothesized that we can redirect the specificity of NK cells against CD30+ malignancies by preloading ex vivo activated and expanded CB-derived NK cells with AFM13 prior to adoptive infusion. Briefly, mononuclear cells were isolated from fresh or frozen CB units by ficoll density gradient centrifugation. CD56+ NK cells were cultured with rhIL-12, rhIL-18 and rhIL-15 for 16 hrs, followed by ex vivo expansion with rhIL-2 and irradiated (100 Gy) K562-based feeder cells expressing membrane-bound IL-21 and CD137-ligand (2:1 feeder cell:NK ratio). After 14 days, NK cells were loaded with serial dilutions of AFM13 (0.1, 1, 10 and 100 mg/ml). After washing twice with PBS, we tested the effector function of AFM13-loaded NK-cells (AFM13-NK) compared to expanded CB-NK cells without AFM13 against Karpas-299 (CD30 positive) and Daudi (CD30 negative) lymphoma cell lines by 51Cr release and intracellular cytokine production assays. AFM13-NK cells killed Karpas-299 cells more effectively at all effector:target ratios tested than unloaded NK cells (Figure 1) and produced statistically more INFγ and CD107a (P=0.0034; P=0.0031 respectively, n=4). In contrast, AFM13-NK cells and unloaded NK cells exerted similar cytotoxicity against Daudi cells. Next, we established the optimal concentration of AFM13 for loading (determined to be 100 μg/ml) and the optimal incubation time to obtain maximal activity (1 h) in a series of in vitro experiments. We also confirmed that the activity of AFM13-NK cells against Karpas-299 cells remains stable for at least 72h post-wash (Figure 2).Additionally, we characterized the phenotype of AFM13-NK vs. unloaded NK cells by flow cytometry using monoclonal antibodies against 22 markers, including markers of activation, inhibitory receptors, exhaustion markers and transcription factors. Compared to unloaded NK cells, AFM13-NK cells expressed higher levels of CD25, CD69, TRAIL, NKp44, granzyme B and CD57, consistent with an activated phenotype.We next tested the in vivo anti-tumor efficacy of AFM13-NK cells in an immunodeficient mouse model of FFluc-Karpas-299. Briefly, six groups of NOD/SCID/IL2Rγc null mice (n=5 per group) were transplanted by tail-vein injection with 1 x 10e5 FFluc-transduced Karpas cells. Group 1 and 6 received tumor alone or tumor + AFM13 and served as a control. Groups 2-4 receive Karpas FFLuc with either expanded NK cells or AFM13-NK cells (NK cells loaded with AFM13) or expanded NK cells and AFM13 injected separately. Group 5 received AFM13-NK cells without tumor. Initial studies confirm the antitumor activity of AFM13-NK cells.In summary, we have developed a novel premixed product, comprised of expanded CB-NK cells loaded with AFM13 to ‘redirect’ their specificity against CD30+ malignancies. The encouraging in vitro and in vivo data observed in this study, provide a strong rationale for a clinical trial to test the strategy of an off-the-shelf adoptive immunotherapy with AFM13-loaded CB-NK cells in patients with relapsed/refractory CD30+ malignancies. [Display omitted] DisclosuresChamplin:Sanofi: Research Funding; Otsuka: Research Funding. Koch:Affimed GmbH: Employment. Treder:Affimed GmbH: Employment. Shpall:Affirmed GmbH: Research Funding. Rezvani:Affirmed GmbH: Research Funding.

Peripheral Immunotype Correlates with Minimal Residual Disease Status and Is Modulated by Immunomodulatory Drugs in Multiple Myeloma

Data indicate reversal of immune dysfunction with active treatment; however, the precise contribution of specific immune effector and immune suppressor components to achieve a minimal residual disease (MRD) state and immunomodulatory drug–mediated immunomodulatory effects in multiple myeloma (MM) patients remains poorly understood. In this prospective proof-of-principle study we sought to determine the dynamic alterations in natural killer (NK), NK-T, and T cells, including maturation and activating/inhibitory repertoire associated with MRDpos versus MRDneg status after autologous stem cell transplantation (ASCT) and during lenalidomide-based maintenance therapy. Of the 46MM patients enrolled, 36 had bone marrow MRD assessment 60+ days post-ASCT, 30 had longitudinal blood immunotyping during maintenance (pretherapy and after cycles 1, 3, and 6), and 20 had both MRD assessment and longitudinal immunotyping. Multicolor flow cytometry was used for MRD and immunotyping. Although the absolute number of NK cells was significantly lower in patients with MRDpos response, phenotypically NK cells in these patients displayed higher expression of activating receptors KIRDS4 and decreased expression of inhibitory molecules NKG2A compared with the MRDneg group. Furthermore, we observed significantly lower frequencies of T cells displaying KIR3DL1 in MRDpos versus MRDneg patients. Longitudinal immunotyping during lenalidomide maintenance showed loss of mature NK effector function, augmentation of NK-T effector function, and acquisition of PD1 independent anergic state. Our findings also suggest skewing of T cells toward an exhausted state during the maintenance phase in MRDpos patients. Put together, these observations provide a distinctive signature for MRDneg and MRDpos groups. These data support exploration of immune profiling in prospective clinical trials according to MRD-defined responses to identify patients that may benefit from maintenance intensification/modification or maintenance withdrawal.

Crosstalks between mTORC1 and mTORC2 variagate cytokine signaling to control NK maturation and effector function

The metabolic checkpoint kinase mechanistic/mammalian target of rapamycin (mTOR) regulates natural killer (NK) cell development and function, but the exact underlying mechanisms remain unclear. Here, we show, via conditional deletion of Raptor (mTORC1) or Rictor (mTORC2), that mTORC1 and mTORC2 promote NK cell maturation in a cooperative and non-redundant manner, mainly by controlling the expression of Tbx21 and Eomes. Intriguingly, mTORC1 and mTORC2 regulate cytolytic function in an opposing way, exhibiting promoting and inhibitory effects on the anti-tumor ability and metabolism, respectively. mTORC1 sustains mTORC2 activity by maintaining CD122-mediated IL-15 signaling, whereas mTORC2 represses mTORC1-modulated NK cell effector functions by restraining STAT5-mediated SLC7A5 expression. These positive and negative crosstalks between mTORC1 and mTORC2 signaling thus variegate the magnitudes and kinetics of NK cell activation, and help define a paradigm for the modulation of NK maturation and effector functions.

Abstract 4743: Inhibition of the Hif1a-mediated checkpoint refuels NK activation in cancer

Abstract Solid tumors remain a major challenge for immunotherapy of cancer due to their immunosuppressive microenvironment. Hypoxia is a common feature of solid tumors and might affect immune cell activation within the tumor tissue. Upon hypoxia the transcription factor Hypoxia inducible factor 1a (HIF-1α) that affects many cellular processes including cell proliferation, survival and effector function becomes induced. Its impact on innate immune cells such as Natural Killer (NK) cells, however, is still incompletely understood. Natural Killer (NK) cells are considered as highly potent effector cells against tumors. Within the tumor tissue, however, NK effector function is often impaired. Our microarray analyses of tumor-infiltrated NK cells revealed upregulated levels of Hif1a. To determine the role of the HIF-1α in NK cell function, we generated the mouse strain Hif1aΔ/ΔNcr1-iCreTg in which HIF-1α is deficient in the NKp46+ lineage. Hif1a deficient NK cells showed greatly enhanced anti-tumor activity compared to wild-type NK cells, leading to a pronounced reduction of tumor growth. Tumor-infiltrated Hif1a deficient NK cells expressed lower levels of checkpoint molecules and show enhanced responses to tumor cells indicating a less exhausted phenotype. Hif1a deficient NK cells preferentially utilize oxidative phosphorylation (OXPHOS) than glycolysis to fuel their energy resulting in enhanced IFN-γ production in the hypoxic environment. In addition, using HIF-1α inhibitors we were able to greatly enhance human NK cell anti-tumor activity. Accordingly, our data reveal HIF-1α as a negative metabolic checkpoint for NK cells. Moreover, inhibition of HIF-1α holds high promise to refuel NK cell activation in cancer with a high potential for improving current strategies of NK cell-based cancer immunotherapy. Citation Format: Jing Ni, Lea Bühler, Ana Stojanovic, Annette Arnold, Veronika Sexl, Adelheid Cerwenka. Inhibition of the Hif1a-mediated checkpoint refuels NK activation in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4743.

Abstract 3696: Tumor-intrinsic PD-L1 alters tumor chemokines, NK cell trafficking and function, and renders distant PD-L1 null tumors responsive to αPD-L1

Abstract Programmed death ligand (PD-L) 1 is expressed on many tumors and inhibits anti-tumor T cells through programmed death (PD)-1. Tumor PD-L1 predicts αPD-L1 treatment effects, but mechanism(s) for PD-L1- tumor response to αPD-L1 are unclear. Our studies suggest tumor-intrinsic PD-L1 signals and spatially varied PD-L1 expression may contribute to response deviation. We used PD-L1+ B16 melanoma (ctrl) and made PD-L1KO by CRISPR. αPD-L1 slowed ctrl but not PD-L1KO B16 growth in mice as expected, but PD-L1KO also responded to αPD-L1 if ctrl B16 was on the trans flank. αPD-L1 elicited similar CD3+ T cell infiltration into ctrl vs. PD-L1KO tumors, but without detectable B16-specific T cell increase. CD11b+ cell infiltration was similar in ctrl and PD-L1KO. Strikingly, NKp46+ and NK1.1+ natural killer (NK) cells infiltrated PD-L1KO > ctrl. NK cells increased significantly (~2-fold) after αPD-L1 in PD-L1KO vs. ctrl, along with NK effector functions (e.g., IFN-γ). Tumor PD-L1 altered tumor chemokines (e.g., CXCL12, CCL2) that could explain trafficking. CD4+ and CD8+ T cells chemotaxed to PD-L1KO slightly > ctrl (with slightly > PD-1+ T cells) in transwells, but NK cells migrated ~2-fold more to PD-L1KO vs. ctrl (with increased activation (e.g., PD-1, CD69), also seen in vivo). We observed that tumor β2 microglobulin (B2M) expression was suppressed by tumor PD-L1. Tumor PD-L1 alteration of immune infiltrates by altering chemokines is a novel mechanism for PD-L1 TIL control. These data also indicate that tumor PD-L1 blunts NK cell infiltration in B16 and reduces B2M, which could boost NK cell anti-tumor activity. These changes correlate with rescued αPD-L1 response of PD-L1KO B16. As αPD-L1 did not increase B16-specific T cells in PD-L1low, we hypothesize that rescue of αPD-L1 response is in part NK cell-mediated. Preliminary in vivo NK cell depletion + αPD-L1 experiments implicate a role for NK cells in PD-L1KO tumor response, but more work is needed to identify mechanisms. Effects extend beyond B16 as ctrl (PD-L1+) MB49 bladder cancer cells in trans elicited αPD-L1 response of PD-L1KO MB49 tumors. Mouse breast and ovarian cancers, and human ovarian and bladder cells exhibit similar PD-L1-intrinsic effects. Finally, αPD-L1 directly altered tumor cell chemokine production in vitro, suggesting additional, novel αPD-L1 treatment mechanisms requiring more study. Our models are useful to understand αPD-L1 (and likely αPD-1) responses based on tumor PD-L1 expression and will help define strategies to improve responses in PD-L1low tumors, and possibly poorly responsive PD-L1+ tumors. Citation Format: Curtis A. Clark, Harshita B. Gupta, Alvaro Padron, Deyi Zhang, Vincent Hurez, Mary Jo Turk, Rong Li, Tyler Curiel. Tumor-intrinsic PD-L1 alters tumor chemokines, NK cell trafficking and function, and renders distant PD-L1 null tumors responsive to αPD-L1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3696. doi:10.1158/1538-7445.AM2017-3696

Regulation of NK Cell Activation and Effector Functions by the IL-12 Family of Cytokines: The Case of IL-27

Natural killer (NK) cells are characterized by their ability to detect and induce apoptosis of susceptible target cells and by secretion of immunoregulatory cytokines such as IFN-γ. Activation of these effector functions is triggered upon recognition of tumor and pathogen (mostly virus)-infected cells and because of a bidirectional cross talk that NK cells establish with other cells of myeloid origin such as dendritic cells (DC) and macrophages. A common characteristic of these myeloid cells is their ability to secrete different members of the IL-12 family of cytokines such as IL-12, IL-23, and IL-27 and cytokines such as IL-15 and IL-18. Although the effect of IL-12, IL-15, and IL-18 has been characterized, the effect of IL-23 and IL-27 on NK cells (especially human) remains ill-defined. Particularly, IL-27 is a cytokine with dual functions as it has been described as pro- and as anti-inflammatory in different experimental settings. Recent evidence indicates that this cytokine indeed promotes human NK cell activation, IFN-γ secretion, NKp46-dependent NK cell-mediated cytotoxicity, and antibody (Ab)-dependent NK cell-mediated cytotoxicity (ADCC) against monoclonal Ab-coated tumor cells. Remarkably, IL-27 also primes NK cells for IL-18 responsiveness, enhancing these functional responses. Consequently, IL-27 acts as a pro-inflammatory cytokine that, in concert with other DC-derived cytokines, hierarchically contributes to NK cells activation and effector functions, which likely contributes to foster the adaptive immune response in different physiopathological conditions.

Selection of Unrelated Allogeneic Hematopoietic Cell Donors Based on KIR3DL1 Allotypes Is Feasible and Results in Improved Disease-Free Survival in Transplant Recipients with MDS and AML

Donor natural killer (NK) cells impart a potent anti-leukemia effect after allogeneic hematopoietic cell transplantation (allo HCT). The killer Ig-like receptors (KIR) play a central role in modulating NK effector function. Chief among them, the inhibitory KIR3DL1 exhibits a high degree of allelic polymorphism. We previously demonstrated that highly expressed KIR3DL1 alleles (KIR3DL1 allele groups *001 and *002) bind preferentially to HLA-Bw4 with I80 to confer a high degree of inhibitory signaling. Similarly, poorly expressed KIR3DL1 alleles (KIR3DL1 allele groups *005 and *007) bind to HLA-Bw4 with T80 to confer a strong inhibitory signal. In a retrospective cohort of 299 patients undergoing allo HCT for AML, we demonstrated that these highly inhibitory donor KIR3DL1/recipient HLA-Bw combinations (KIR3DL1-HIGH) resulted in a greater incidence of recipient relapse when compared to other donor/recipient KIR3DL1/HLA-Bw pairings with low or absent inhibitory signal (KIR3DL1-LOW/NO). (Giglio F et al, ASH Annual Meeting Abstracts, 2012) Here, we evaluated whether it was feasible to prospectively type and use KIR3DL1 allotypes in unrelated allo HCT donor selection, and whether this intervention would result in improved outcomes in patients undergoing transplant for AML and MDS. We performed PCR-SSP based intermediate resolution KIR3DL1 allele typing on all unrelated adult donors evaluated for patients with MDS and AML at our center from 2013 to present as previously described. (Boudreau J et al, PLoS One, 2014) KIR3DL1 status was provided to the treating physicians, who made the final donor selection.A total of 941 prospective allo HCT donors underwent high resolution HLA typing and KIR3DL1 allotyping for 252 patients (median per patient = 4, range 1-12). Among all donors evaluated, 27% were found to be KIR3DL1-HIGH when considering the respective recipient HLA-Bw. Having multiple donors evaluated improved the likelihood of avoiding a KIR3DL1-HIGH donor: Among recipients with one donor evaluated, 27% had only KIR3DL1-HIGH donors available compared to 12% in recipients with 2-3 donors evaluated and 4% for recipients with >3 donors evaluated (P < 0.0001). Among all evaluated patients, 41% had both KIR3DL1-HIGH and KIR3DL1-LOW/NO donors available, indicating a potential selection based on KIR3DL1 was possible.Among 252 patients evaluated, 115 proceeded to allo HCT (48 with MDS, 67 with AML). The median age at transplant was 62 years (range 3.6-78). Donors were HLA matched in 105 transplants and were single loci mismatched in 10 transplants. Conditioning was myeloablative in 73 (11 with total body irradiation). GVHD prophylaxis was with ex vivo CD34+ selection in 65 patients and with tacrolimus/methotrexate in the remaining patients. The median follow-up in transplanted patients was 13.1 months (range 0.5-43.3 months). On univariate analysis, the 2-year disease-free survival was 64% (95% confidence interval: 54-76%) in those with KIR3DL1-LOW/NO donors versus 39% (22-68%) in recipients with KIR3DL1-HIGH donors (P = 0.05). The incidence of relapse was similar but favored patients with KIR3DL1-LOW/NO donors (26% [15-37%] versus 35% [13-57%], P = 0.5). 2-year overall survival was similar between those with KIR3DL1-LOW/NO versus KIR3DL1-HIGH donors (75% [65-86%] versus 69% [52-91%], P = 0.43).The time from the initiation of a formalized donor search to transplant did not differ between recipients with KIR3DL1-LOW/NO versus KIR3DL1-HIGH donors (median 81 v. 83 days, respectively, P = 0.97). Recipients with KIR3DL1-LOW/NO donors were CIBMTR Transplant Risk Score low = 53, intermediate = 6, and high = 33; whereas recipients of KIR3DL1-HIGH donors were low = 17, intermediate = 2, and high = 4 (P = 0.15). These results indicate that disease severity and transplant urgency did not influence whether a KIR3DL1-LOW/NO donor was able to be selected.In summary, these prospective data on 115 patients from a single center support improved outcomes in patients with AML and MDS undergoing unrelated donor allo HCT using a donor with low or absent KIR3DL1 inhibition. A multi-center, prospective study to evaluate the prospective use of HLA and KIR genotype based selection of URDs for patients undergoing allo HCT for AML is underway (NCT02450708). [Display omitted] [Display omitted] DisclosuresKernan:Gentium: Research Funding; The National Cancer Institute of the National Institutes of Health: Research Funding.

Axl signaling induces development of natural killer cells in vitro and in vivo.

Natural killer (NK) cells have been well known to play a critical role in innate immunity, but they are also capable of regulating adaptive immunity through the induction of T cell-mediated memory response and B cell-mediated autoimmune response. NK cells are differentiated from hematopoietic stem cells (HSCs) in the bone marrow (BM), and a series of surface molecules are expressed on NK cells in a differentiation stage-specific manner. Axl receptor tyrosine kinase is originally identified as homeostatic regulators for antigen-presenting cells, and its ligand, growth-arrest-specific gene 6 (Gas6), has been reported to promote cell survival, proliferation, and migration, but their regulatory role in the development and effector function of NK cells is not yet fully understood. In this study, to investigate whether Axl is required for the regulation of NK cell development, the expression of mature NK (mNK) cell-specific receptors and NK cell-associated genes was analyzed in the differentiated HSCs-derived NK cells in vitro and the NK cells harvested from Axl-/- mice. We found that agonistic anti-Axlantibody or recombinant Gas6 specifically upregulated the expression of mNK cell-specific receptors, such as LY49A, Ly49G2, Ly49C/F/I, NKG2A/C/E (1.5- to 3.5-fold increase), and NK cell-associated genes, such as IL-2Rβ (2.3- or 2.4-fold increase), Perforin (4.1- or 2.1-fold increase), IL-15Rα (2.14- or 2.04-fold increase), and IFN-γ (3.3- or 2.8-fold increase) compared to each isotype control, whereas it was abrogated by treatment ofAxl-Ig. Anti-Axlantibody or rGas6 also induced a 2.5- or 1.9-fold increase in the proliferation of developing NK cells compared to each control, respectively. mNK cell populations expressing mNK cell-specific receptors were reduced about twofold in NK cells differentiated from HSCs of Axl-/- mice compared with those of wild-type mice. Furthermore, the triggering of Axl signaling by agonistic anti-Axlantibody promoted the cytolytic activity (1.5- to 1.9-fold increase) against target tumor cells. In B16F10 melanoma-bearing mice, the number of metastatic colonies was decreased by 83% by the administration of mNK cells treated with anti-Axlantibody compared to control Ig. These data suggest that Axl plays an essential role in the regulation of NK cell development as well as NK effector function.

Specific combinations of donor and recipient KIR-HLA genotypes predict for large differences in outcome after cord blood transplantation

AbstractThe ability of cord blood transplantation (CBT) to prevent relapse depends partly on donor natural killer (NK) cell alloreactivity. NK effector function depends on specific killer-cell immunoglobulin-like receptors (KIR) and HLA interactions. Thus, it is important to identify optimal combinations of KIR-HLA genotypes in donors and recipients that could improve CBT outcome. We studied clinical data, KIR and HLA genotypes, and NK-cell reconstitution in CBT patients (n = 110). Results were validated in an independent cohort (n = 94). HLA-KIR genotyping of recipient germline and transplanted cord blood (CB) grafts predicted for large differences in outcome. Patients homozygous for HLA-C2 group alleles had higher 1-year relapse rate and worse survival after CBT than did HLA-C1/C1 or HLA-C1/C2 (HLA-C1/x) patients: 67.8% vs 26.0% and 15.0% vs 52.9%, respectively. This inferior outcome was associated with delayed posttransplant recovery of NK cells expressing the HLA-C2-specific KIR2DL1/S1 receptors. HLA-C1/x patients receiving a CB graft with the combined HLA-C1-KIR2DL2/L3/S2 genotype had lower 1-year relapse rate (6.7% vs 40.1%) and superior survival (74.2% vs 41.3%) compared with recipients of grafts lacking KIR2DS2 or HLA-C1. HLA-C2/C2 patients had lower relapse rate (44.7% vs 93.4%) and better survival (30.1% vs 0%) if they received a graft with the combined HLA-C2-KIR2DL1/S1 genotype. Relapsed/refractory disease at CBT, recipient HLA-C2/C2 genotype, and donor HLA-KIR genotype were independent predictors of outcome. Thus, we propose the inclusion of KIR genotyping in graft selection criteria for CBT. HLA-C1/x patients should receive an HLA-C1-KIR2DL2/L3/S2 CB graft, while HLA-C2/C2 patients may benefit from an HLA-C2-KIR2DL1/S1 graft.

Let-7 microRNAs target the lineage-specific transcription factor PLZF to regulate terminal NKT cell differentiation and effector function.

Lethal-7 (let-7) microRNAs are the most abundant in the genome but their role in developing thymocytes is unclear. We now report that let-7 miRNAs target Zbtb16 mRNA, which encodes the lineage-specific transcription factor PLZF, to post-transcriptionally regulate PLZF expression and NKT cell effector function. Dynamic up-regulation of let-7 miRNAs during NKT thymocyte development down-regulates PLZF expression and directs terminal differentiation into interferon-γ-producing NKT1 cells. Without let-7 up-regulation, NKT thymocytes maintain high PLZF expression and terminally differentiate into IL-4-producing NKT2 and IL-17-producing NKT17 cells. Let-7 up-regulation in developing NKT thymocytes can be signaled by IL-15, vitamin D and retinoic acid. Such miRNA targeting of a lineage-specific transcription factor constitutes a new level of developmental regulation in the thymus.