Conventional Natural Killer Cells Research Articles

Diversity of group 1 innate lymphoid cells in human tissues.

Group 1 innate lymphoid cells (ILC1s) are cytotoxic and interferon gamma-producing lymphocytes lacking antigen-specific receptors, which include ILC1s and natural killer (NK) cells. In mice, ILC1s differ from NK cells, as they develop independently of the NK-specifying transcription factor EOMES, while requiring the repressor ZFP683 (ZNF683 in humans) for tissue residency. Here we identify highly variable ILC1 subtypes across tissues through investigation of human ILC1 diversity by single-cell RNA sequencing and flow cytometry. The intestinal epithelium contained abundant mature EOMES- ILC1s expressing PRDM1 rather than ZNF683, alongside a few immature TCF7+PRDM1- ILC1s. Other tissues harbored NK cells expressing ZNF683 and EOMES transcripts; however, EOMES protein content was variable. These ZNF683+ NK cells are tissue-imprinted NK cells phenotypically resembling ILC1s. The tissue ILC1-NK spectrum also encompassed conventional NK cells and NK cells distinguished by PTGDS expression. These findings establish a foundation for evaluating phenotypic and functional changes within the NK-ILC1 spectrum in diseases.

DNA demethylase Tet2 promotes the terminal maturation of natural killer cells.

The cytotoxicity feature to eliminate malignant cells makes natural killer (NK) cells a candidate for tumor immunotherapy. However, this scenario is currently hampered by inadequate understanding of the regulatory mechanisms of NK cell development. Ten-Eleven-Translocation 2 (Tet2) is a demethylase whose mutation was recently shown to cause phenotypic defects in NK cells. However, the role of Tet2 in the development and maturation of NK cells is not entirely clear. Here we studied the modulatory role of Tet2 in NK cell development and maturation by generating hematopoietic Tet2 knockout mice and mice with Tet2 conditional deletion in NKp46+ NK cells. The results showed that both hematopoietic and NK cell conditional deletion of Tet2 had no effect on the early steps of NK cell development, but impaired the terminal maturation of NK cells defined by CD11b, CD43, and KLRG1 expression. In the liver, Tet2 deletion not only prevented the terminal maturation of NK cells, but also increased the proportion of type 1 innate lymphoid cells (ILC1s) and reduced the proportion of conventional NK cells (cNK). Moreover, hematopoietic deletion of Tet2 lowered the protein levels of perforin in NK cells. Furthermore, hematopoietic deletion of Tet2 downregulated the protein levels of Eomesodermin (Eomes), but not T-bet, in NK cells. In conclusion, our results demonstrate that Tet2 plays an important role in the terminal maturation of NK cells, and the Eomes transcription factor may be involved.

Natural Killer Cell‐Derived Extracellular Vesicles as Potential Anti‐Viral Nanomaterials

In viral infections, natural killer (NK) cells exhibit anti-viral activity by inducing apoptosis in infected host cells and impeding viral replication through heightened cytokine release. Extracellular vesicles derived from NK cells (NK-EVs) also contain the membrane composition, homing capabilities, and cargo that enable anti-viral activity. These characteristics, and their biocompatibility and low immunogenicity, give NK-EVs the potential to be a viable therapeutic platform. This study characterizes the size, EV-specific protein expression, cell internalization, biocompatibility, and anti-viral miRNA cargo to evaluate the anti-viral properties of NK-EVs. After 48 h of NK-EV incubation in inflamed A549 lung epithelial cells, or conditions that mimic lung viral infections such as during COVID-19, cells treated with NK-EVs exhibit upregulated anti-viral miRNA cargo (miR-27a, miR-27b, miR-369-3p, miR-491-5p) compared to the non-treated controls and cells treated with control EVs derived from lung epithelial cells. Additionally, NK-EVs effectively reduce expression of viral RNA and pro-inflammatory cytokine (TNF-α, IL-8) levels in SARS-CoV-2 infected Vero E6 kidney epithelial cells and in infected mice without causing tissue damage while significantly decreasing pro-inflammatory cytokine compared to non-treated controls. Herein, this work elucidates the potential of NK-EVs as safe, anti-viral nanomaterials, offering a promising alternative to conventional NK cell and anti-viral therapies.

Natural killer cell subsets and their functional molecules in peripheral blood of the patients with breast cancer.

Natural killer (NK) cells, CD3- lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3- NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). Thirty untreated females with BC and 20 age-matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome-conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four-color flow cytometer. Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3-CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3-CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3-CD56brightCD16- /+) NK cells and CD3-CD56-CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis-related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3-CD56-CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

Innate lymphoid cells: New players in osteoimmunology.

Innate lymphoid cells (ILCs) are the most recently identified immune cell types existing in lymphoid and nonlymphoid organs. Albeit they lack the expression of antigen receptors, ILCs play vital roles in innate immune responses by producing multiple effector cytokines. The ILC family includes conventional natural killer cells and cytokine-producing ILCs, which are divided into group 1, group 2, and group 3 ILCs based on their effector cytokines and developmental requirements. Emerging evidence has indicated that ILCs are essential immune regulators of bone homeostasis, playing a critical role in osteoimmunology. In this mini-review, we discuss recent advances in the understanding of ILC functions in bone homeostasis under physiological and pathological conditions, with an emphasis on the communication between ILCs and bone cells including osteoclasts and osteoblasts, as well as the underlying immunoregulatory networks involving ILC-derived cytokines and growth factors. This review also discusses future research directions and the potential of targeting ILCs for the treatment of inflammation-associated bone disorders.

Circulating NK cells establish tissue residency upon acute infection of skin and mediate accelerated effector responses to secondary infection

Natural killer (NK) cells are present in the circulation and can also be found residing in tissues, and these populations exhibit distinct developmental requirements and are thought to differ in terms of ontogeny. Here, we investigate whether circulating conventional NK (cNK) cells can develop into long-lived tissue-resident NK (trNK) cells following acute infections. We found that viral and bacterial infections of the skin triggered the recruitment of cNK cells and their differentiation into Tcf1hiCD69hi trNK cells that share transcriptional similarity with CD56brightTCF1hi NK cells in human tissues. Skin trNK cells arose from interferon (IFN)-γ-producing effector cells and required restricted expression of the transcriptional regulator Blimp1 to optimize Tcf1-dependent trNK cell formation. Upon secondary infection, trNK cells rapidly gained effector function and mediated an accelerated NK cell response. Thus, cNK cells redistribute and permanently position at sites of previous infection via a mechanism promoting tissue residency that is distinct from Hobit-dependent developmental paths of NK cells and ILC1 seeding tissues during ontogeny.

C–C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model

BackgroundPrevious studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure.AimThis study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism.ResultsBy gene array analysis, chemokine (C–C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1β or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model.ConclusionThe CCR5-MIP-1β/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.

Adoptively Infused Memory-like Natural Killer Cells Impact Adaptive Immune Responses in Patients with Acute Myeloid Leukemia

Natural killer (NK) therapies have been challenging owing to antigen escape, restricted trafficking, and limited tumor infiltration, translating into modest clinical benefit. We have shown that memory-like (ML)-NK cells acquire an in vivo differentiated phenotype distinct from conventional NK cells and are well-tolerated without cytokine release syndrome, GVHD, or neurotoxicity. WU-NK-101 (W-NK) is an allogeneic cytokine-reprogrammed, expanded, cryopreserved ML-NK cell product derived from healthy human peripheral blood mononuclear cells, with scalable manufacturability, that is currently in clinical development in relapsed/refractory (R/R) AML (NCT# 05470140). We conducted a multi-modal analysis of the immune tumor microenvironment (TME) in 13 patients with R/R AML receiving ML-NK cells (NCT#01898793) with the aim to determine whether TME modifications induced by ML-NK cells correlate with clinical benefit. NK cells were purified from leukapheresate, and activated with IL-12, IL-15, and IL-18 for 12-16 hours. Patients were lymphodepleted with fludarabine and cyclophosphamide. NK cells were infused in escalating doses: 0.5 × 10 6/kg, 1.0 × 10 6/kg, and all NK cells from a single leukapheresis, capped at 10 × 10 6/kg. Response was defined as <5% BM blasts on d+28. BM trephines for immune gene expression profiling (nCounter IO360®) and spatial proteomics (GeoMx™ DSP; n=53 immuno-oncology [IO] targets; NanoString Technologies, Seattle) were collected at baseline (BL) and on treatment (OT). Immune infiltration, NK and T cell number from BM samples was quantified manually on immunofluorescent imaging. Oncomine™ TCR Pan-Clonality assay (Thermo Fisher) was used to profile TCRb and g repertoires by NGS. The Shannon index was used to describe TCR repertoire diversity, and clonal evenness was used as a measure of the similarity of clone sizes. BM infiltration with CD8 + effectors, enhanced glycolysis and cell cycling was higher in BM samples from responders (CR) to ML-NK cells. By contrast, OXPHOS RNA scores and gene signatures of immune senescence and terminal T-cell exhaustion were higher in non-responders (NR). We employed machine learning to construct an 8-gene predictor of response which encompassed T/NK markers, such as CD7, and IFN regulatory factors and TLRs. This score was significantly higher at baseline in CR and positively correlated with response duration. Principal component analysis (PCA) of spatially resolved IO proteins separated CR from NR, particularly in OT specimens, where T-cell markers CD3/CD8 and inhibitory receptors CTLA4/LAG3 were among the top contributing features. CD34, type I IFN signaling molecule STING1 and PARP1 were significantly higher at baseline compared with OT, with OT showing higher expression of cytotoxicity markers and inhibitory receptors (e.g., Tim-3 and CD276). This observation strongly suggests modulation of the immune TME after the infusion of ML-NK cells. Response to ML-NK cells was associated with coordinated up-regulation of T cell, NK cell and B cell protein markers. To further substantiate the co-localization of T cells and NK cells in the BM FFPE sections (Fig. A), and its correlation with clinical efficacy we annotated the 93 ROIs in our spatial proteomics dataset based on a median split of CD3 and CD56 protein barcodes. ROIs from CR were largely CD3 highCD56 high, both at BL and OT. Conversely, ROIs from BM sections of NR to ML-NK cells had a predominantly CD3 lowCD56 low phenotype. Quantitative analysis of T-cell infiltration post ML-MK dosing showed a significant association with response, 0.9 (PD) vs. 5.3 (CR)-fold-increase ( P=0.01). Interestingly, data demonstrate selected intra-BM clonal expansion as evidenced by decreased TCR diversity (mean ± SEM, 4.6±0.3 vs. 3.3±0.4, pre vs. post; P=0.013)and increased clonality (Fig. B). Thus, pre-existing T-cell responses appear to be co-opted in the AML TME of CR, suggesting the potential for durable effectiveness of ML-NK cells. Multi-modal analyses of WU-NK-101 highlight unique transcriptional and functional states, which could underpin in vivo potency and persistence. WU-NK-101 are currently being evaluated in a phase 1 study of R/R AML (#NCT05470140).

G-NK cells from umbilical cord blood are phenotypically and functionally different than g-NK cells from peripheral blood

ABSTRACT FcRγ-deficient natural killer (NK) cells, designated as g-NK cells, exhibit enhanced antibody-dependent cellular cytotoxicity (ADCC) capacity and increased IFN-γ and TNF-α production, rendering them promising for antiviral and antitumor responses. g-NK cells from peripheral blood (PB) are often associated with prior human cytomegalovirus (HCMV) infection. However, the prevalence, phenotype, and function of g-NK cells in umbilical cord blood (UCB-g-NK) remain unclear. Here, we demonstrate significant phenotypical differences between UCB-g-NK and PB-g-NK cells. Unlike PB-g-NK cells, UCB-g-NK cells did not show heightened cytokine production upon CD16 engagement, in contrast to the conventional NK (c-NK) cell counterparts. Interestingly, following in vitro activation, UCB-g-NK cells also exhibited elevated levels of IFN-γ production, particularly when co-cultured with HCMV and plasma from g-NK+ adults. Furthermore, g-NK+ plasma from PB even facilitated the in vitro expansion of UCB-g-NK cells. These findings underscore the phenotypic and functional heterogeneity of g-NK cells based on their origin and demonstrate that components within g-NK+ plasma may directly contribute to the acquisition of an adult phenotype by the “immature” UCB-g-NK cells.

CTLA-4 Blockade Results in the Enrichment of Proliferative CD56 dimCD16 + NK Cells Following Infusion of Haploidentical Donor Memory-like Natural Killer Cells Plus IL-15 Superagonist in a Phase 1 Trial

Introduction Regulatory T cells (Tregs) blunt anti-tumor responses of major immune effector cells including CD8 T cells and natural killer (NK) cells. Tregs constitutively express CTLA-4 and thus the use of ipilimumab, a CTLA-4 blocker can potentially overcome the Treg-mediated immune suppression. We (and others) have shown that a brief activation of conventional NK cells with the cytokine combination IL-12, IL-15, and IL-18 generates cytokine-induced memory-like (CIML) NK cells which exhibit enhanced anti-tumor responses, have a longer half-life (up to 90 vs ~12 days of conventional NK cells), and show promising activity in several early phase clinical trials. N-803 is an IL-15 super-agonist with a long half-life that preferentially activates and expands NK and CD8+ T cells without affecting Tregs, making it an ideal cytokine for combining with adoptive immunotherapy. In the current study, we hypothesized the use of ipilimumab (IPI) will abrogate Treg-mediated inhibition and thus allow enhanced proliferation, activation, and anti-tumor responses of the adoptively transferred CIML NK cells.To address this, we conducted a phase 1 trial of HLA-haploidentical CIML NK cells in combination with IPI and N-803 in patients with advanced head and neck cancer (HNC). Using flow cytometry and CITE-Seq on the banked peripheral blood samples, we performed an in-depth characterization of NK cells expanded following IPI pre-exposure and compared it to those who did not receive IPI. Methods We enrolled 10 patients with refractory HNC regardless of human papillomavirus (HPV) status who had prior platinum and immunotherapy (NCT04290546). All patients received lymphodepletion with fludarabine (25 mg/m 2 x 5 days) and cyclophosphamide (60 mg/kg x 2 days) during days -6 to -2 prior to haploidentical CIML NK cell infusion on day 0 (5-10 x 10 6 cells/kg=dose level 0) followed by N-803 (15 mcg/kg subcutaneously) starting on day +1 every 21-days for 4-doses. In cohort 2, patients received IPI (3 mg/kg) on day -7, no ipilimumab was given to the first 6 patients treated on the lead-in safety cohort 1. Flow cytometry using a custom NK cell panel was performed on samples collected on days +7, +14, +21, +28, +42, and +60 after CIML NK cell infusion. FlowSOM clustering was performed on NK cell markers. UMAP clustering was applied to CITE sequencing data acquired from samples collected on day +7 and day +28, and well-defined markers genes for each cluster were used to identify cell populations. NK cell function (cytotoxicity, IFNg, and CD107a) was assessed using flow-cytometry based assays. Results The median age of the patients was 58, 90% were men and 80% had oropharyngeal primaries. Mild CRS (grade 2 or lower) was observed in 60% of patients. No neurotoxicity or graft-versus-host disease was observed. Maximal expansion of NK cells occurred on day +7 following CIML NK cell infusion in 9/10 of patients. The single patient without NK cell expansion was found to have a donor-specific anti-HLA antibody. Day +7 NK cells exhibited upregulation of IFNg expression following cytokine restimulation as compared to resting NK cells. Tumor regression was observed in 50% of treated patients at day +30 based on RECIST v1.1 criteria. Patients who had tumor regression had an expansion of CD56 dimCD16 + NK cell clusters by day +14. IPI treated patients were noted to have a relative enrichment of CD16 + NK cell metaclusters at day +14 compared to no IPI patients (Figure 1A). At the time of maximal NK cell expansion, CITEseq defined NK cell clusters were dominant in both IPI and no IPI patients, but the proliferative gene sets were enriched in the IPI treated patient (Figure 1B). IPI exposure had no effect on the distribution of regulatory T cells or CD8 + T cell populations at day +28 compared to the no IPI patients. Conclusions The use of donor-derived CIML NK cells is safe and associated with tumor regression in patients with advanced HNC. The use of IPI was associated with the preferential expansion of CD16 + NK cell clusters that had enrichment of proliferative gene sets. Tumor regression was associated with CD56 dimCD16 + NK cell expansion. Further work is required to elucidate the mechanism by which IPI exposure is associated with the expansion of specific subsets of CIML NK cells.

Memory-like Differentiation, Tumor-Targeting mAbs, and Chimeric Antigen Receptors Enhance Natural Killer Cell Responses to Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell therapy-refractory cancers, but are frequently dysfunctional in patients with HNSCC. Strategies are needed to enhance NK cell responses against HNSCC. We hypothesized that memory-like (ML) NK cell differentiation, tumor targeting with cetuximab, and engineering with an anti-EphA2 (Erythropoietin-producing hepatocellular receptor A2) chimeric antigen receptor (CAR) enhance NK cell responses against HNSCC. We generated ML NK and conventional (c)NK cells from healthy donors, then evaluated their ability to produce IFNγ, TNF, degranulate, and kill HNSCC cell lines and primary HNSCC cells, alone or in combination with cetuximab, in vitro and in vivo using xenograft models. ML and cNK cells were engineered to express anti-EphA2 CAR-CD8A-41BB-CD3z, and functional responses were assessed in vitro against HNSCC cell lines and primary HNSCC tumor cells. Human ML NK cells displayed enhanced IFNγ and TNF production and both short- and long-term killing of HNSCC cell lines and primary targets, compared with cNK cells. These enhanced responses were further improved by cetuximab. Compared with controls, ML NK cells expressing anti-EphA2 CAR had increased IFNγ and cytotoxicity in response to EphA2+ cell lines and primary HNSCC targets. These preclinical findings demonstrate that ML differentiation alone or coupled with either cetuximab-directed targeting or EphA2 CAR engineering were effective against HNSCCs and provide the rationale for investigating these combination approaches in early phase clinical trials for patients with HNSCC.

Genetic distinction between functional tissue-resident and conventional natural killer cells

Tissue-residential natural killer (trNK) cells act as pioneering responders during infectious challenges. However, their discrimination with conventional NK (cNK) cells is still an issue. Through an integrative transcriptome comparison of the two NK subgroups from different tissues, we have defined two genesets capable of efficiently distinguishing them. Based on the two genesets, a fundamental difference between the activation of trNK and cNK is identified and further confirmed. Mechanistically, we have discovered a particular role of chromatin landscape in regulating the trNK activation. In addition, IL-21R and IL-18R are respectively highly expressed by trNK and cNK, indicating a role of cytokine milieu in determining their differential activation. Indeed, IL-21 is particularly critical in accessorily promoting trNK activation using a bunch of bifunctional transcription factors. Together, this study sheds light on the bona fide difference between trNK and cNK, which will further expand our knowledge about their distinct functionalities during immune responses.

Brief IL-12, IL-15, IL-18 activation drives the acquisition of two distinct memory-like NK cell states

Abstract IL-12/15/18 activation induces natural killer (NK) cell differentiation into cytokine-induced memory-like (ML) NK cells with enhanced IFNγ and killing of tumor targets that is antigen independent, rendering them distinct from conventional (cNK) and CMV-induced adaptive NK cells. In leukemia patients, ML NK cells have an excellent safety profile and 47% CR rate (PMID: 32826231). Despite their clinical promise, little is known about the biology underlying ML NK cells. Based on individual variability of human ML NK cell function, we hypothesized that ML NK cells manifest molecular heterogeneity. To this end, we applied Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) to profile RNA and protein expression on the single-cell level, and evaluated NK cell function. Overnight IL-12/15/18 stimulation induced broad activation with distinct transcriptional programs evident in CD56 brightcompared to CD56 dimNK cells. In contrast, following in vitrodifferentiation for 7 days, IL-12/15/18 activated NK cells acquired a unique transcriptional and protein signature, and manifest one of two ML NK cell states (ML-1, ML-2), or remained similar to cNK cells. ML-1 was marked by increased activating receptor expression (NKp46, NKp30, NKp44) while ML-2 had increased expression of inhibitory receptors, CD25, HLA-DR, MKI67, and IFNG. In functional assays, ML-2 cells produced more IFNγ following cytokine stimulation than ML-1 cells. Further, ML-1 and ML-2 had divergent transcription factor profiles suggesting distinct mechanisms of regulation. Collectively, our findings reveal molecular and functional heterogeneity of ML NK cells, and investigations into ML subsets in patients receiving ML NK cell therapy are ongoing. AAI Intersect Fellowship for Computational Scientists & Immunologists (JAF, TAF, AAP), T32GM139799 (JAF), P50CA171963 (TAF, MMB-E), R01CA205239 (TAF), P30CA91842 (TAF)

Abstract 893: Memory like differentiation enhances in vitro and in vivo NK cell responses against colorectal cancer

Abstract Metastatic (m) colorectal cancer (CRC) is an incurable, frequently lethal disease present in approximately 25% of newly diagnosed CRC patients, and almost 50% of the patients with CRC will develop metastatic disease. For mCRC, while systemic therapies (cytotoxic therapy, targeted therapy, immunotherapy or their combinations) have extended patient’s life expectancy, not all patients are candidates for these treatments. Immunotherapy has achieved significant curative effects in patients with solid tumors; however, a considerable proportion of mCRC patients (~90%) are not responsive to immune checkpoint blockade (ICB) leaving a gap in the CRC immunotherapy options. Natural killer (NK) cells are cytotoxic innate lymphoid cells that display potent effector responses against a wide variety of tumor cells; however, they are frequently dysfunctional in cancer patients. NK cells from CRC patients exhibit decreased expression of activating receptors and reduced cytokine production after stimulation with CRC cells with a more accentuated phenotype in advanced stages of the disease. Memory-like (ML) NK cells differentiated after IL-12, IL-15, and IL-18 activation have been shown to overcome limitations associated with deficient tumor recognition and poor anti-tumor activity. In clinical trials, ML NK cells were safe and active against acute myeloid leukemia (Romee R et al, Sci Transl Med, 2016). Preclinically, ML NK cells exhibited improved responses against melanoma (Marin ND et al, Clin Cancer Res, 2021) and ovarian cancers, compared to conventional NK cells. Here, we hypothesized that memory-like differentiation will enhance multiple aspects of the NK cell response against CRC cells. Allogeneic ML NK cells displayed enhanced IFN-γ production against four CRC cell lines DLD-1 (p=0.015), SW480 (p=0.0005), HT-29 (p=0.0005) and HCT116 (p=0.001), as well as primary patient derived CRC tumoroids (p=0.0156), compared to conventional (c) NK cells. ML NK cells also exhibited superior and sustained killing of CRC cell lines over time compared to cNK cells, as measured by Incucyte assays and using CRC tumoroids. Furthermore, IFN-γ production was significantly reduced after blockade of NKG2D, DNAM-1 and NKp46 (p<0.01) revealing mechanistic insights into how ML NK cells recognize CRC targets. Finally, using a xenograft model of CRC in NSG mice, we demonstrated that ML NK cells exhibited superior control of Luciferase expressing HCT116 cells compared to cNK cells (p=0.01) as measured by bioluminescent imaging (BLI). Collectively, these findings demonstrate that ML NK cells exhibit enhanced responses against CRC cells, and thus warrants further investigation in clinical trials for CRC patients, especially those who are not candidates for standard of care therapy or failed ICB. Citation Format: Nancy D. Marin, Michelle Becker-Hapak, Quazim A. Alayo, Melissa Berrien-Elliot, Lynne Marsala, Naomi Sonnek, Miriam T. Jacobs, Jennifer A. Foltz, Alice Zhou, Jennifer Tran, Pamela Wong, Celia Cubit, Kimberly Hwang, Timothy Schappe, Ryan C. Fields, Matthew A. Ciorba, Todd A. Fehniger. Memory like differentiation enhances in vitro and in vivo NK cell responses against colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 893.

Abstract 2906: Memory-like differentiation, tumor targeting monoclonal antibodies, and chimeric antigen receptors enhance natural killer cell responses to head and neck cancer

Abstract Background: Head and neck cancer (HNC) is an aggressive tumor with low response rates to frontline PD-1 blockade. Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers, but are frequently dysfunctional in patients with HNC. New strategies are needed to enhance NK cell responses against HNC. Activation of NK cells with IL-12, 15 and 18 results in memory-like (ML) differentiation improving NK cell responses in hematological malignancies. We hypothesized that ML NK cell differentiation, tumor targeting with cetuximab, and engineering with α-ephA2 chimeric antigen receptor (CAR) would enhance NK cell responses against HNC. Methods: ML NK and conventional (c) NK cells were generated from healthy donors (HD) and evaluated for their ability to produce IFNγ, TNF, degranulate (CD107a) and kill HNC cell lines and primary HNC cells alone or in combination with cetuximab in vitro and in xenograft models. To identify activating receptors involved in NK cell recognition of HNC, blocking experiments were used. ML and cNK cells were engineered to express α-EphA2 CAR-CD8A-41BB-CD3z and functional responses were assessed in vitro against HNC cell lines and primary HNC targets. Results: ML NK cells generated from HD displayed enhanced IFNγ response compared to cNK cells against HNC cell lines SCC1(11±2% vs. 24±1%, mean ± SEM p<0.001), SCC47 (13±2% vs. 36±2%, p<0.01) and primary HNC targets (17±2% vs. 32±3%, p<0.01) which were further enhanced by cetuximab, SCC1 (cNK + cetuximab vs. ML + cetuximab 16±2% vs. 28±3%, p<0.001), SCC47 (39±3% vs. 56±2%, p<0.001), primary HNC targets (23±2% vs. 40±2%, p<0.0001). The combination of ML NK cells and cetuximab was superior to cNK and cetuximab in 51 Cr release assay and IncuCyte® killing assays for HNC cell lines (p<0.01) and primary cells (p<0.01). Findings were consistent in SCC1 HNC xenograft model, where tumor bearing NSG mice treated with cetuximab and ML NK cells had significantly lower tumor burden at 21 days compared to untreated mice, mice that received cNK cells + cetuximab or cetuximab only (p<0.001). Mechanistically, NK cell IFNγ, CD107a and cytotoxicity against HNC targets is partially dependent on activating receptors NKG2D, CD2 and DNAM-1. ML NK cells expressing α-ephA2 CAR were specific to ephA2+ cell lines and demonstrated increased IFNγ (37±4% vs. 16±3%, p<0.001) and CD107a (36±4% vs. 20±1%, p<0.001) against NK resistant ephA2+ UD-SCC2, compared to CAR negative cells. EphA2 CAR ML NK cells had increased CD107a (26±4% vs. 39±4%, p<0.01) and IFNγ (45±5% vs. 66±3%, p<0.001) against primary ephA2+ HNC targets. Conclusion: These pre-clinical findings show that ML differentiation alone or in concert with cetuximab directed targeting or ephA2 CAR engineering, was effective against HNC and provide the rationale for investigation in early phase clinical trials for HNC patients. Citation Format: Miriam T. Jacobs, Pamela Wong, Alice Zhou, Michelle Becker-Hapak, Nancy Marin, Lynne Marsala, Mark Foster, Jennifer Foltz, Celia Cubitt, Jennifer Tran, Carly Neal, David Russler-Germain, Lily Chang, Timothy Schappe, Ethan McClain, Samantha Kersting-Schadek, Carl DeSelm, Melissa Berrien-Elliott, Sidarth V. Puram, Todd A. Fehniger. Memory-like differentiation, tumor targeting monoclonal antibodies, and chimeric antigen receptors enhance natural killer cell responses to head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2906.

WU-NK-101, an enhanced memory natural killer (NK) cell therapy, with cetuximab (Ctx) for the treatment of advanced colorectal cancer (CRC).

170 Background: CRC is the 4th leading cause of global cancer-related deaths, and novel therapeutic strategies for advanced CRC are urgently needed. Adoptive cell therapy (ACT) is effective in treating hematological malignancies; however, ACT in solid tumors is hindered by target antigen identification, restricted migration into tumors, and survival in the tumor microenvironment (TME) due to immunosuppressive signals and scarcity of nutrients. NK cells are central to anti-tumor immunity and can directly eliminate tumor cells without prior sensitization. Through cytokine reprogramming, NK cells can also gain memory-like features that augment their anti-tumor potential. WU-NK-101 is a cytokine-reprogrammed, expanded, cryopreserved, off-the-shelf NK cell product derived from peripheral blood mononuclear cells, with no additional engineering. Methods: WU-NK-101 ± Ctx was evaluated in vitro in 2D cytotoxicity assays in complete (N) and TME-aligned medias. WU-NK-101 cytotoxicity was further assessed against primary CRC surgical samples in native-TME-aligned 3D assays. Proteomic analysis was performed using tandem-mass spectrometry. In vivo efficacy of WU-NK-101 ± Ctx was evaluated in NSG mice bearing LoVo xenograft CRC tumors. Cell trafficking/penetration to TME was measured by tracking labeled WU-NK-101 ± trastuzumab in NSG mice bearing subcutaneous SKOV-3 xenografts. Results: Compared to conventional NK cells (cNK), WU-NK-101 had a unique phenotype consistent with rapid activation and proliferation (higher expression of activating receptors, Ki67, and GZMB), and showed enhanced in vitro cytotoxicity. WU-NK-101 also exhibited potent cytotoxicity against LoVo CRC tumors in vivo, which was further enhanced in combination with Ctx. Antibody combination improved WU-NK-101 penetration, and persistence in TME. WU-NK-101’s metabolic profile was consistent with aerobic (Warburg) glycolysis, potentially facilitating effector functions in the TME. WU-NK-101 also showed enhanced metabolic fitness and flexibility, as proteins in several metabolic pathways were upregulated in TME vs N media. Consistent with this, WU-NK-101 had increased cell-surface expression of nutrient transporters. While cNK and T cells cytotoxicity was significantly suppressed in TME-aligned media, WU-NK-101’s function was not impacted. Conclusions: We show that WU-NK-101 exerted potent activity against CRC, and in combination with Ctx showed improved intra-tumor infiltration/persistence and anti-tumor activity. Also, WU-NK-101 cells had enhanced metabolic fitness/flexibility and decreased susceptibility to immunosuppression, overcoming limitations encountered by ACT for solid tumors. A Phase 1b clinical trial is in development, which may reshape ACT in CRC and other EGFR-expressing tumors. [Table: see text]

The Frequency and Function of NKG2C+CD57+ Adaptive NK Cells in Cytomagalovirus Co-Infected People Living with HIV Decline with Duration of Antiretroviral Therapy.

Human cytomegalovirus (CMV) infection drives the expansion and differentiation of natural killer (NK) cells with adaptive-like features. We investigated whether age and time on antiretroviral therapy (ART) influenced adaptive NK cell frequency and functionality. Flow cytometry was used to evaluate the frequency of adaptive and conventional NK cells in 229 CMV+ individuals of whom 170 were people living with HIV (PLWH). The frequency of these NK cell populations producing CD107a, CCL4, IFN-γ or TNF-α was determined following a 6-h antibody dependent (AD) stimulation. Though ART duration and age were correlated, longer time on ART was associated with a reduced frequency of adaptive NK cells. In general, the frequency and functionality of NK cells following AD stimulation did not differ significantly between treated CMV+PLWH and CMV+HIV- persons, suggesting that HIV infection, per se, did not compromise AD NK cell function. AD activation of adaptive NK cells from CMV+PLWH induced lower frequencies of IFN-γ or TNF-α secreting cells in older persons, when compared with younger persons.

Single-cell RNA sequencing identifies a population of human liver-type ILC1s

Group 1 innate lymphoid cells (ILCs) comprise a heterogeneous family of cytotoxic natural killer (NK) cells and ILC1s. We identify a population of "liver-type" ILC1s with transcriptional, phenotypic, and functional features distinct from those of conventional and liver-resident NK cells as well as from other previously described human ILC1 subsets. LT-ILC1s are CD49a+CD94+CD200R1+, express the transcription factor T-BET, and do not express the activating receptor NKp80 or the transcription factor EOMES. Similar to NK cells, liver-type ILC1s produce IFN-γ, TNF-α, and GM-CSF; however, liver-type ILC1s also produce IL-2 and lack perforin and granzyme-B. Liver-type ILC1s are expanded in cirrhotic liver tissues, and they can be produced from blood-derived ILC precursors invitro in the presence of TGF-β1 and liver sinusoidal endothelial cells. Cells with similar signature and function can also be found in tonsil and intestinal tissues. Collectively, our study identifies and classifies a population of human cross-tissue ILC1s.

ILC1: Development, maturation, and transcriptional regulation.

Type 1 Innate Lymphoid cells (ILC1s) are tissue-resident cells that partake in the regulation of inflammation and homeostasis. A major feature of ILC1s is their ability to rapidly respond after infections. The effector repertoire of ILC1s includes the pro-inflammatory cytokines IFN-γ and TNF-α and cytotoxic mediators such as granzymes, which enable ILC1s to establish immune responses and to directly kill target cells. Recent advances in the characterization of ILC1s have considerably furthered our understanding of ILC1 development and maintenance in tissues. In particular, it has become clear how ILC1s operate independently from conventional natural killer cells, with which they share many characteristics. In this review, we discuss recent developments with regards to the differentiation, polarization, and effector maturation of ILC1s. These processes may underlie the observed heterogeneity in ILC1 populations within and between different tissues. Next, we highlight transcriptional programs that control each of the separate steps in the differentiation of ILC1s. These transcriptional programs are shared with other tissue-resident type-1 lymphocytes, such as tissue-resident memory T cells (TRM ) and invariant natural killer T cells (iNKT), highlighting that ILC1s utilize networks of transcriptional regulation that are conserved between lymphocyte lineages to respond effectively to tissue-invading pathogens.

Putative Predictors of Response to WU-NK-101, an Allogeneic, Enhanced Memory (ML) Natural Killer (NK) Cell Therapy Product, for Relapsed/Refractory (R/R) Acute Myeloid Leukemia (AML)

Strategies to enhance NK-cell activity prior to adoptive transfer into patients with AML have yielded inconsistent clinical benefit. Immune cells in the AML tumor microenvironment (TME) are dysfunctional, in part due to enhanced interferon-γ signaling and acquisition of a senescent-like T/NK phenotype (Vadakekolathu, et al. Sci. Transl. Med. 2020). Cytokine-induced memory-like (CIML) NK cells are more potent for anti-tumor responses than conventional NK (cNK) cells, are well tolerated, and active in patients with R/R AML (NCT#01898793) with complete response (CR)/CR with partial/incomplete hematologic recovery rate of 47% and a median DOR of 9.4 months after a single infusion (Berrien-Elliott, et al. Cancer Discov. 2020). WU-NK-101 is a cell therapy derived from healthy donor NK cells, which are activated to induce a ML-NK cell program, expanded, and frozen to create an off-the-shelf cell therapy. This abstract 1) describes the approach for treatment of R/R AML with WU-NK-101, built upon prior clinical experience with CIML-NKs; 2) characterizes TME gene expression (GE) signatures associated with outcome after the infusion of CIML-NK in R/R AML and 3) functionally characterizes WU-NK-101. Bone marrow (BM) aspirates and trephine biopsies for immune GE profiling and spatially resolved proteomics (IO360® panel, n = 740 genes, and GeoMx® DSP; NanoString Technologies) were collected at baseline (BL) and on treatment (OT) from 15 patients receiving CIML-NK cells on study NCT #01898793. ssGSEA-based scores were computed using the IOBR package in R. Machine learning was used to select features that correlated with response. Cell phenotypes were evaluated by mass cytometry. NK cell function was assayed in either conventional (N-; 20% IMDM) or TME-aligned media. Multi-omics studies were performed by mass spectrometry. Cell trafficking to TME was measured in NSG mouse tumors by tracking fluorescent-labeled WU-NK-101. Correlative studies showed that BL GE signatures reflecting heightened neutrophil infiltration, chemokine receptor expression, and cancer antigen presentation predicted response to CIML-NKs (AUROC = 0.875, 0.875 and 0.850, respectively). We computed an 8-gene signature score (RPTOR, IRF9, TLR1, NLRP3, CD7, IL6R, FUT4, IL1RN) that was significantly higher at BL in responders and that correlated with response duration (Fig. 1A). Immune deconvolution of GE data with CIBERSORT highlighted critical differences in TME cellular composition at BL in responders to CIML-NKs, including a higher inferred abundance of macrophages, gamma/delta T cells and activated dendritic cells (Fig. 1B). In OT samples from responders, glycolytic GE programs as well as purine/pyrimidine and amino acid (AA) metabolism were upregulated compared with BL; no TME modulation was observed in non-responders (Fig. 1C). Analysis of WU-NK-101 cells revealed a unique phenotype compared with cNK cells, including higher expression of activating receptors, CD25, CD69, and GZMB, which positions WU-NK-101 for rapid activation and improved cytotoxicity. The latter hypothesis was confirmed by in vitro assays, with higher half maximal effective concentration (mEC50) for cNK vs WU-NK-101 (mEC50 = 5.2 vs 1.7; P = 0.032; Fig. 1D). The adverse impact of immunosuppressive TME-aligned media on cytotoxicity was evident on cNK but negligible to WU-NK-101 (mEC50 = 16.3 vs 2.0; P = 0.018; Fig. 1D). In N-media, WU-NK-101 used glucose as its main nutritional source; in hypoglycemic TME-aligned media, AA metabolic pathways were upregulated, indicating metabolic adaptability. This was supported by increased cell surface expression of AA transporters. WU-NK-101's homing to the BM averaged 16.5 ± 2.4%, which is 5.5-fold higher than previously reported for feeder-cell+IL-2-expanded NK cells (~3%; Sato et al. Clin. Cancer Res. 2020). In summary, an 8-gene TME signature showed excellent predictive ability for response to ML-NKs. In responders, ML-NK infusion was associated with TME modulation and with metabolic re-programming. Compared to cNK cells, WU-NK-101 had enhanced trafficking to the BM, anti-tumor activity, and a metabolic profile consistent with aerobic glycolysis, potentially contributing to mitigating TME adversity. Due to their resilience to an immunosuppressive TME, WU-NK-101 cells may represent an effective treatment modality for R/R AML; a phase 1 study of WU-NK-101 in R/R AML is in development (NCT #05470140). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal