NK Cell Biology Research Articles

Rhesus Macaque Killer Cell Ig-like Receptor Domain 0 Glycans Impact Surface Expression and Ligand Specificity.

Defining the MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as a model for infectious diseases and comparative immunogenetics. Several rhesus macaque KIRs belong to a phylogenetically distinct group with a three-amino acid deletion in domain 0 (D0). This deletion results in polymorphic differences in potential N-linked glycosylation (PNG) sites adjacent to a predicted KIR-MHC class I contact site. Whereas most KIRs have two tandem PNG sites in D0 (N36FTN39FT), the KIRs containing the deletion only have a single site in this region (N36FT). To discern the contribution of glycosylation to KIR expression and ligand recognition, we constructed PNG mutants for six lineage II KIR genes that eliminate or create sites for N-glycan addition at these locations. The impact of these mutations on total and surface expression was determined by immunoblotting and flow cytometry. Ligand engagement was assessed by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion proteins. We found that N36FT is glycosylated in KIR with a single site, and at least one site is glycosylated in KIRs with two tandem sites. In general, for rhesus KIRs with a single D0 glycosylation site, that site contributes to surface expression. For KIRs with two tandem sites, the first site can contribute to ligand specificity. This study establishes that D0 glycosylation of rhesus macaque KIRs modulates surface expression and contributes to ligand specificity.

Five decades of natural killer cell discovery.

The first descriptions of "non-specific" killing of tumor cells by lymphocytes were reported in 1973, and subsequently, the mediators of the activity were named "natural killer" (NK) cells by Rolf Kiessling and colleagues at the Karolinska Institute in 1975. The activity was detected in mice, rats, and humans that had no prior exposure to the tumors, major histocompatibility complex (MHC) antigen matching of the effectors and tumor cells was not required, and the cells responsible were distinct from MHC-restricted, antigen-specific T cells. In the ensuing five decades, research by many labs has extended knowledge of NK cells beyond an in vitro curiosity to demonstrate their in vivo relevance in host defense against tumors and microbial pathogens and their role in regulation of the immune system. This brief Perspective highlights a timeline of a few selected advancements in NK cell biology from a personal perspective of being involved in this quest.

Absence of ATM leads to altered NK cell function in mice

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm−/−) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm−/− littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm−/− mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells.These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

The lack of either IRF9, or STAT2, has surprisingly little effect on human natural killer cell development and function.

Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.

NK cells and the profile of inflammatory cytokines in the peripheral blood of patients with advanced carcinomas

BackgroundNatural killer (NK) cells are one of the most crucial immune cells that mediate the antitumoral response due to their ability to immediately recognize and eliminate transformed cells. Because of their great cytotoxic activity, the function of NK cells must be robustly regulated to avoid tissue damage. Such regulation is mediated by a coordinated engagement of activating (NKp46) and inhibitory (CD158b) receptors, which tumor cells may use to escape from immunosurveillance. Also, NK cells are generally divided based on surface molecules, such as CD16 and CD56, and can be classified as CD56brightCD16- (regulatory) and CD56dimCD16+ (cytotoxic) NK cells. Here, we aimed to evaluate the frequency and phenotype of circulating NK cells in patients with advanced carcinomas, as well as their systemic cytokine/chemokine and growth factors production. MethodsPeripheral blood was collected from 24 patients with advanced solid cancer during or after treatment and from 10 healthy donors. The frequency and the expression of activating (NKp46) and inhibitory (CD158b) molecules of CD56brightCD16- and CD56dimCD16+ NK cells were assessed by flow cytometry and the multiplex Luminex platform was used to quantify the secreted factors in peripheral blood serum. ResultsCancer patients had a lower frequency of the cytotoxic CD56dim CD16+ NK cells subset in comparison with healthy controls. Also, the regulatory CD56bright CD16- NKs isolated from cancer patients exhibited a significantly lower expression of NKp46. Among 29 immunological and growth factors analyzed in the peripheral blood of oncologic patients, MCP-1, IP-10, and eotaxin, and VEGF they have presented a higher proportion. The Pearson correlation test showed that IL-12p40 positively correlates with CD56brightCD16- NK cells. We also observed a positive correlation between MCP-1 and the activating marker NKp46, as well as a negative correlation between IP-10 and TNF-α and NKp46. CD158b expression in CD56dimCD16+ was positively correlated with EGF and negatively correlated with MIP-1β. ConclusionsTaken together, these results suggest that cancer patients present a shift towards a poorly cytotoxic and less activated NK profile which may contribute to tumor development and progression. The understanding of NK cell biology and soluble factors during tumor development could aid in the design of possible targeting therapeutic approaches.

DAP10 Co-Stimulation Imparts Memory-like Features to CD5 Targeting Cord Blood Derived CAR-NK Cells

Relapsed/refractory T-cell malignancies have a particularly poor prognosis and novel therapies are direly needed. CD5 is a great candidate for adoptive cellular therapy to target T-cell malignancies since it is ubiquitously expressed on T cells with restricted expression on other hematopoietic cells. NK cells are an attractive platform for CAR engineering to target CD5 since, unlike T cells, they do not express CD5 on their surface, which eliminates the risk of fratricide. Another advantage of NK cells for CAR engineering is their safety profile; in contrast to T cells, they do not cause cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS) and they are not associated with graft-versus-host disease (GVHD) in the allogeneic setting, opening the potential for a completely off-the-shelf cellular product to be used at point of care. Therefore, we sought to develop CD5 targeting CAR-NK cells for the treatment of T-cell malignancies. Multiple studies have demonstrated that the choice of co-stimulatory domain in CAR-T cells influences their function, persistence and metabolic profile. In the field of CAR-NK cells, the first constructs tested in the clinic have incorporated CD28, a T cell specific co-stimulatory domain, borrowing from the design of CAR-T cells. There is a sparsity of data regarding the impact of co-stimulatory domains on the proliferation, transcriptomic and proteomic profile, polyfunctionality, metabolism and fitness of CAR-NK cells. In this study, we aimed to do so, by comprehensively studying the impact of co-stimulatory domains including some that are more relevant for NK cell biology, namely DNAX-activating Protein 10 (DAP10), a major adaptor protein and the exclusive signaling intermediate of NKG2D in human NK cells, DNAX-activating Protein 12 (DAP12), an important adaptor molecule that associates with multiple activating receptors (e.g. NKG2C, NKp44, activating KIRs) and NKG2D, one of the most potent NK cytotoxicity receptors which is essential for anti-tumor immunity. Our results show that CD5 CAR-NK cells with DAP10 co-stimulatory domain show enhanced cytotoxicity against CD5+ T-cell leukemia targets even after multiple tumor rechallenges in an Incucyte live-cell imaging assay. They also show augmented polyfunctionality compared to CD5 CAR-NK cells with other co-stimulatory domains in an Isoplexis single-cell secretome assay. Moreover, DAP10 co-stimulation endowed CD5 CAR-NK cells with enhanced metabolic fitness as evidenced by increased oxidative phosphorylation compared to other co-stimulatory molecules. At the epigenetic level, CD5 CAR-NK cells with DAP10 co-stimulation interrogated by sc-ATACseq show enrichment in AP-1 complex and BATF transcription factors related to memory formation and exhaustion resistance. This translates to better in vivo performance as CD5 CAR-NK cells with DAP10 co-stimulatory domain significantly improve tumor control and survival in an NSG mouse model of CD5+ T-cell leukemia (CCRF-CEM) and show evidence of recall response following tumor rechallenge. In conclusion, our data show that DAP10 co-stimulation induces epigenetic reprogramming of CD5 CAR-NK cells leading to enhanced cellular fitness and memory formation ensuing better anti-tumor potential. Based on these preclinical data, a Phase I/II clinical trial evaluating the safety and efficacy of CD5 CAR-NK cells for the treatment of CD5+ malignancies is in preparation.

Perinatal murine cytomegalovirus infection reshapes the transcriptional profile and functionality of NK cells

Infections in early life can elicit substantially different immune responses and pathogenesis than infections in adulthood. Here, we investigate the consequences of murine cytomegalovirus infection in newborn mice on NK cells. We show that infection severely compromised NK cell maturation and functionality in newborns. This effect was not due to compromised virus control. Inflammatory responses to infection dysregulated the expression of major transcription factors governing NK cell fate, such as Eomes, resulting in impaired NK cell function. Most prominently, NK cells from perinatally infected mice have a diminished ability to produce IFN-γ due to the downregulation of long non-coding RNA Ifng-as1 expression. Moreover, the bone marrow’s capacity to efficiently generate new NK cells is reduced, explaining the prolonged negative effects of perinatal infection on NK cells. This study demonstrates that viral infections in early life can profoundly impact NK cell biology, including long-lasting impairment in NK cell functionality.

CDC45 haploinsufficiency as a novel cause of natural killer cell deficiency

Natural killer cell deficiency (NKD) is a primary immunodeficiency where natural killer cells are the primary lineage affected. Individuals with NKD present with malignancies and susceptibility to viral infection, especially from the Herpesviridae family. Though NKD is rare, four genes that are involved in the CDC45-MCM-GINS (CMG) helicase have been described to result in NKD, including MCM4, GINS1, MCM10, and GINS4. The CMG helicase is critical for eukaryotic replication and plays a critical role in maintaining genomic stability. To our knowledge, CDC45 has never been described as a monogenic cause of NKD. However, here we describe a single damaging heterozygous variant in CDC45 in an individual with consistent defects in the NK cell compartment. This individual has a disruption in the ratio of CD56bright to CD56dim cells, severely decreased frequency of adaptive NK cells, and decreased NK cell cytotoxic function. As previously reported for other helicase variants that cause NKD, we found cell cycle defects, increased DNA damage, and impaired DNA damage repair signaling in patient lymphocytes. The description of CDC45 as a cause of NKD contributes to our understanding of the role of the CMG helicase in NK cell maturation, function, and biology.

The DEAD box RNA helicase DDX3X is required in natural killer cells

Abstract The X-chromosome linked RNA helicase DDX3X plays multiple roles in RNA metabolism and antiviral interferon responses. Some viruses, including SARS-CoV-2, usurp the function of DDX3X to support viral genome replication and evasion of antiviral interferon. These findings have led to development of DDX3X inhibitors as potential antiviral agents. However, somatic loss of function mutations in this helicase are linked to neurological disease and malignancies, including a form of non-Hodgkin’s lymphoma initiated in the natural killer (NK) cell lineage. Given the critical role of NK cells in antiviral and antitumor immunity, we sought to determine how inhibition of DDX3X could affect NK-cell biology. Constitutive (Ncr1-iCre) or inducible (Ncr1-iCreER T2) deletion of Ddx3x in Ncr1-expressing cells (i.e. NK cells) in mice resulted in a complete loss of peripheral NK cells. Addition of the DDX3X inhibitor RK-33 to in vitro cultures of NK cells resulted in a similar loss of cellularity, while deletion of Ddx3x blocked development of NK cells from progenitor cells in an in vitro culture on stromal cells. Preliminary analyses suggest that loss of DDX3X results in rapid cell death of committed NK cells. Partial preservation of early stages of NK-cell development in the bone marrow of male but not female Ncr1 DDdx3xsuggests that the Y-chromosome DDX3Y may compensate for loss of DDX3X in supporting NK-cell survival at the earliest stages of differentiation. We identify a crucial role for DDX3 helicases in the development and survival of NK cells that may have implications for the clinical use of DDX3 inhibitors as antiviral agents. R01 AI148080 R01 AR073228

MHC Class I Ligands of Rhesus Macaque Killer Cell Ig-like Receptors.

Definition of MHC class I ligands of rhesus macaque killer cell Ig-like receptors (KIRs) is fundamental to NK cell biology in this species as an animal model for infectious diseases, reproductive biology, and transplantation. To provide a more complete foundation for studying NK cell responses, rhesus macaque KIRs representing common allotypes of lineage II KIR genes were tested for interactions with MHC class I molecules representing diverse Macaca mulatta (Mamu)-A, -B, -E, -F, -I, and -AG alleles. KIR-MHC class I interactions were identified by coincubating reporter cell lines bearing chimeric KIR-CD3ζ receptors with target cells expressing individual MHC class I molecules and were corroborated by staining with KIR IgG-Fc fusion proteins. Ligands for 12 KIRs of previously unknown specificity were identified that fell into three general categories: interactions with multiple Mamu-Bw4 molecules, interactions with Mamu-A-related molecules, including allotypes of Mamu-AG and the hybrid Mamu-B*045:03 molecule, or interactions with Mamu-A1*012:01. Whereas most KIRs found to interact with Mamu-Bw4 are inhibitory, most of the KIRs that interact with Mamu-AG are activating. The KIRs that recognize Mamu-A1*012:01 belong to a phylogenetically distinct group of macaque KIRs with a 3-aa deletion in the D0 domain that is also present in human KIR3DL1/S1 and KIR3DL2. This study more than doubles the number of rhesus macaque KIRs with defined MHC class I ligands and identifies interactions with Mamu-AG, -B*045, and -A1*012. These findings support overlapping, but nonredundant, patterns of ligand recognition that reflect extensive functional diversification of these receptors.

Abstract 2940: Use of NK humanized mouse models for the in vivo evaluation of anti-tumor NK-cell therapies

Abstract Novel immunotherapeutic strategies targeting human Natural Killer (huNK) cells using monoclonal or multi-specific antibodies and immune modulator molecules are under development and there is a pressing need to have suitable mouse models for the evaluation of these therapeutics. Existing humanized mouse models have limitations and do not show an optimal development of innate immune cells such as NK, dendritic or other myeloid cells. We previously described the development of robust mouse models that allow the engraftment and maintenance of fully functional huNK cells using several strains of immunodeficient transgenic mice for human cytokines (IL-15). The optimal parameters (source of immune cells, pre-conditioning regimen, route of administration) were identified, and the models were validated by flow cytometry and in vivo efficacy studies using an anti-CD20 Ab. Here, we report further development and characterization of these models and highlight their value to evaluate a variety of NK based therapeutics. We show that huNK cells can infiltrate a subcutaneous B-cell lymphoma model after treatment with an anti-CD20 Ab. We show that SAR444245 (non-alpha IL2) can activate huNK cells in vivo in all developed huIL-15 transgenic models and confirmed the minimal effective dose by flow-cytometry PD studies. We show the efficacy of both reference and internal ADCC-enhanced antibodies in disseminated tumor models in our humanized huIL-15 transgenic mice. In addition, we performed a deep characterization of huNK cells in huIL-15 transgenic models, using a 28-color flow cytometry panel centered on NK cell biology and profiled them transcriptionally by single cell RNA Seq in a disseminated B-cell lymphoma model. Overall, we have developed robust models sustaining fully functional huNK cells that can be proficiently recruited in vivo by various NK-based therapeutics and can be used to evaluate the efficacy of combination therapies. Citation Format: Pauline Rettman, Laure-Marie Meyer, Ravi Rangara, Anna Ponchet-Lac, Nicolas Moindrot, Angélique Biancotto, Céline Lefranc, Fabien Delahaye, Emilia Rabia, Matteo Cesaroni, Sukhvinder Sidhu, Céline Nicolazzi. Use of NK humanized mouse models for the in vivo evaluation of anti-tumor NK-cell therapies [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 2940.

Abstract 6418: xWU-NK-101 as salvage therapy post immune checkpoint blockade (ICB)

Abstract ICB therapy has transformed cancer treatment; durable responses in difficult-to-treat cancers have been observed. Despite this most patients (pts) don’t respond to ICB (primary resistance), and many pts who initially respond eventually relapse (acquired resistance). Primary resistance is associated with tumor cell extrinsic factors, e.g., the immunosuppressive nature of the tumor microenvironment (TME), while acquired resistance is associated with tumor intrinsic factors, e.g., downregulation of MHC1, preventing T cell recognition. Treatments to overcome ICB resistance are necessary. WU-NK-101 is a PBMC-derived, cytokine-reprogrammed, expanded, and cryopreserved off-the-shelf memory NK cell product. WU-NK-101 cells capture the memory-like NK cell biology of cytokine-induced memory-like (CIML) NK cells, exhibiting enhanced cytotoxicity, metabolic fitness/flexibility, and resistance to TME immunosuppression (Muth et al. EHA 2022; Rutella et al. ESMO 2022). Bone marrow biopsies, collected from R/R AML pts who received CIML-NK cells (NCT01898793), were interrogated using immune gene expression (GE) profiling and spatially resolved proteomics (IO360® panel, n = 740 genes, and GeoMx® DSP; NanoString Technologies). Higher T-cell infiltration was noted post CIML-NK. CIBERSORT deconvoluted GE data inferred higher abundance of macrophages, γδT cells and activated dendritic cells on day 28 post-treatment. GE signatures showed downregulation of NFIL3 and FAM30A (log2 fold-change <1.0; p<0.05) post CIML-NK. TIDE algorithm modelling indicated that lower expression of NFIL3 and FAM30A correlated with high CTL infiltration and improved outcomes in many TCGA tumors (Jiang et al. Nat. Med. 2018). Hence, CIML-NK treatment led to modifications in the TME towards a more T-cell amenable environment. Deleting MHC1 on NALM6 cells significantly improved WU-NK-101 killing compared to WT (p<0.0001), highlighting one mechanism whereby WU-NK-101 overcomes acquired ICB resistance. In transwell assays, co-incubation of tumor cells with WU-NK-101 in the lower chamber led to dose dependent killing; in the upper chamber (tumor cells alone), viability was preserved but a dose-dependent increase of MHC1/PDL1 expression was noted. Similar results were observed using cell-free conditioned media from WU-NK-101 cytotoxicity assays, suggesting that soluble factors released from WU-NK-101 augment MHC1/PDL1 expression. Treatment of tumor cells with IFNγ alone led to similar increases in MHC1/PDL1 expression. Importantly, in vivo experiments revealed that residual tumor cells post WU-NK-101 treatment exhibited higher levels of MHC1/PDL1. Overall, these data highlight a further mechanism through which WU-NK-101 overcomes acquired ICB resistance. In summary, WU-NK-101 has the potential to reverse primary and acquired mechanisms of ICB resistance. A Phase 1b clinical trial of WU-NK-101 as salvage therapy post-ICB is in development. Citation Format: Tom A. Leedom, Barbara Muz, Jaykumar Vadakekolathu, John J. Muth, Xiao-Hua Li, Gregory Watson, Kristaan Magee, Ryan P. Sullivan, Melissa Berrien-Elliott, Todd Fenigher, Sergio Rutella, Matthew L. Cooper, Ayman Kabakibi, Jan K. Davidson-Moncada. xWU-NK-101 as salvage therapy post immune checkpoint blockade (ICB) [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 6418.

Abstract 4688: Comprehensive analysis of natural killer cell-associated markers using MultiOmyxTM immunofluorescence assay

Abstract As a promising alternative platform for cellular immunotherapy, natural killer cells (NK) have recently gained attention as an important type of innate immune regulatory cell. NK cell immunotherapy approaches have been translated into clinical applications, and clinical trials of NK cell infusion in patients with hematological malignancies (HM) and solid tumors have thus far yielded many encouraging clinical results. Understanding the pattern of NK expression and the relationship to different states of NK cells may have direct relevance for immune responses in cancer. Approaches capable of simultaneously detecting NK cells with detailed information on NK differentiation state, however, remain limited. In this study, we used MultiOmyx hyperplexed immunofluorescence (IF) assay to classify and characterize the spatial arrangement of NK cell markers in a pan-cancer cohort including 9 tissue microarrays (TMAs) from breast, head and neck, prostrate, colon, bladder, lung, kidney, ovarian and melanoma indications. The panel includes CD3, CD4, CD8, CD16, CD45, CD56, CD57, CD137, FoxP3, Granzyme B, HLA-E, NKG2A, NKP46 and tumor segmentation marker. The panel enables the detection of NK cells expressing CD56 and/or NKP46 in the 9 different tumor indications. We also studied the expression of activating and inhibiting receptors, such as CD16 and NKG2A, in NK cell population. The active cytotoxic subsets of mature NK cells were evaluated using co-expression of NK cell surface markers with CD57 and the cytotoxic molecule expression in NK cells was assessed using co-expression with Granzyme B. Using proprietary deep-learning-based image analysis, we were able to quantify the densities of these different NK cell population and study the prevalence of these NK cells in different cancer indications included in this study. Many strategies have been developed for exploiting NK-mediated anti-tumor activities. CAR-NK cell therapy and antibodies that directly target NK cell inhibitory receptors such as NKG2A and TIGIT, are currently being evaluated in the clinical trials. The MultiOmyx NK cell panel reported in this study enables the comprehensive profiling of the NK population and can provide greater understanding of NK cell biology during cancer progression. The panel can be further used to explore the efficacy of the NK cell-based immune therapy. Citation Format: Erinn A. Parnell, Jiong Fei, Harry Nunns, Eric Leones, Elaine Yeung, Blair Russell, Flora Sahafi, Qingyan Au. Comprehensive analysis of natural killer cell-associated markers using MultiOmyxTM immunofluorescence assay. [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 4688.

Abstract 1351: Uterine natural killer cells display a unique functional dichotomy in human endometrial cancer

Abstract Endometrial Cancer (EC) is the most common gynecological malignancy in the United States, with striking heterogeneity of risk factors, molecular identities, and outcomes. Two common subtypes of EC are Type 1, which display endometrioid histology and include those with MSI status, and Type 2, which arise from an atrophic background and are more severe in their morbidity. Despite significant research on checkpoint blockade therapies, such as PD-1/PD-L1 blockade to treat Type 1 ECs, variable response rates underscore the need for further immunomodulation. NK cells are cytotoxic innate lymphocytes that cooperate with other cells of the immune system. Despite years of investigation into basic NK cell biology, our understanding of the role(s) of NK cells in human solid tumors is still in its infancy. For example, in head and neck squamous cell carcinoma (HNSCC) indicate that a subset of NK cells has strong anti-tumor function, while a separate subset of NK cells lacks direct cellular cytotoxicity. Considering these findings, we used flow cytometry to evaluate NK cell phenotypes and frequencies in fresh EC patient tumor samples. Our data suggest a functional dichotomy of NK cell subsets with distinct phenotypes, present in both Type 1 and Type 2 EC tumors (Type 1: 35.5 +/- 10.65 % CD16+ vs 73.5 +/- 10.41 % CD16-, n=6, p=0.10; Type 2: 16.23+/- 3.61 CD16+ vs 83.77 +/- 3.65 CD16-, n=3, p<0.05). The first subset expresses CD16, which mediates antibody dependent cellular cytotoxicity, along with T-BET and EOMES, which are required for direct killing through Perforin and Granzymes and thus reminiscent of conventional NK (cNK) cells found in blood. The second subset is CD16- with decreased expression of T-BET and EOMES, and upregulated expression of tissue resident NK cell (trNK)-associated markers including CD200R1, CD103 and CD49a. To understand frequencies of these NK cell subsets in EC, we used immunohistochemistry for CD3, CD56, CD103, and Granzyme B on formalin-fixed, paraffin-embedded tissues from 10 patients: 4 Type 1, 4 Type 2, and 2 benign controls. We utilized CD56 to denote NK cells and found that NK cells infiltrated the Type 1 EC cases more compared to Type 2 (22.1 +/- 7.3% NK cells in Type 1 infiltrate vs 14.8 +/- 2% NK cells in Type 2 infiltrate, n=5, p=0.17). Further, NK cells expressing CD103, denoting trNK cells, could primarily be found in the glands of EC tumors, while NK cells expressing Granzyme B, denoting the cNK cells, were primarily in the stroma and largely absent in the glands (Granzyme B+: 78.6 +/- 3.7% Stroma vs 21.4 +/- 3.7% Glands, n=3, p<0.05; CD103+: 15.6 +/- 4.5% Stroma vs 84.3 +/- .64% Glands, n=3, p=0.076). This work suggests that an NK cell dichotomy exists within EC, with distinct phenotypes and localizations. Ongoing work will include functional studies to assess killing potential and cytokine production of the two subsets, along with single cell RNA sequencing to unravel which NK cells may be friend or foe in EC. Citation Format: Caprice D. Eisele, Courtney J. Riedinger, Jesse J. Kowalski, Bethany L. Mundy-Bosse, Paige M. Porrett, Patrick L. Collins, Casey M. Cosgrove, Aharon G. Freud. Uterine natural killer cells display a unique functional dichotomy in human endometrial 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 1351.

A humanized mouse model to study NK cell biology during HIV infection.

NK cells are an important subset of innate immune effectors with antiviral activity. However, NK cell development and immune responses in different tissues during acute and chronic HIV infection in vivo have been difficult to study due to the impaired development and function of NK cells in conventional humanized mouse models. In this issue of the JCI, Sangur et al. report on a transgenic MISTRG-6-15 mouse model with human IL-6 and IL-15 knocked into the previously constructed MISTRG mice. The predecessor model was deficient in Rag2 and γ chain (γc) with knock-in expression of human M-CSF, IL-3, GM-CSF, and TPO, and transgenic expression of human SIRPα. The researchers studied tissue-specific NK cell immune responses during HIV infection and clearly show that the endogenous human NK cells in the humanized mouse model suppressed HIV-1 replication in vivo. These findings provide insight into harnessing the innate immune response for clinical antiviral therapies.

Decidual natural killer cells and trophoblast cells: cellular, humoral and molecular mechanisms of interaction

Natural killer cells (NK cells) represent a group of lymphocytes of innate immunity. In addition to NK cells of peripheral blood, tissue-resident populations are described. NK cells of the decidual envelope (decidual NK cells) represent one of the local NK cell populations. Decidual NK cells differ in phenotype and function from peripheral blood NK cells. These cells have, mainly, regulatory functions. At the same time they retain the ability to perform cytotoxic effects. In the uterus, NK cells are located closely to the cells of fetal origin, i.e., trophoblast cells, which differentiate from the outer layer of the invading blastocyst. The purpose of the review article was to analyze the literature data on the studies of the molecular interactions between NK cells and trophoblast cells, as well as potential means of regulating these interactions. The review presents currently available data on receptor-mediated effects (due to adhesion molecules and cytotoxic receptors) and distant interactions (involving cytokines, chemokines and growth factors secreted by the both cell types) between NK population and trophoblast cells. The receptors regulating contacts of NK cells and trophoblast cells with extracellular matrix are also considered. The review provides information on activation of signaling pathways in NK cells and trophoblast cells resulting from their interaction with each other and components of the extracellular matrix. Currently, the molecular mechanisms regulating the NK cell functions and their interaction with trophoblast cells have not been studied sufficiently. The authors attempted to consider molecular regulation of the functional activity of NK cells mediated by the molecular complex of RNA polymerase II. We also describe participation of cyclin-dependent CDK8/19 kinases which comprise a part of the mediator complex which provides functioning of immune cells. The data on the participation of CDK8/19 in regulation of intracellular signaling pathways, as well as influence of CDK8/19 on the NK cell functions, are considered. Summarizing the data presented in the literature, one may emphasize that there is an extensive mutual influence of NK cells and trophoblast cells in decidual lining of uterus during pregnancy, thus leading to a changes in phenotype and functions of these cells. Experimental studies are required on the contribution of molecular mechanisms involved in transcription and translation processes to the biology of NK cells, and their role in maintaining interactions between NK cells and trophoblast cells, including the pathways involving CDK8/19.

Comprehensive Exploration of NK Cell Education in ADCC Triggered By a Monoclonal Antibody or a Bispecific Killer Engager Molecule (BiKE)

The responsiveness of NK cells to malignant cells that lack self-HLA class I (HLA-I) expression is controlled by NK cell education governed by the interaction between selected inhibitory NK cell receptors and their cognate HLA-I ligands. However, it remains unclear to what extent education impacts the ability of NK cells to perform antibody-dependent cellular cytotoxicity (ADCC) as previous studies show inconsistent results and are limited in resolution. Moreover, no study has yet addressed if NK cell education impacts ADCC triggered by novel CD16-binding therapeutics such as bispecific killer engagers (BiKEs). In this study we systematically characterized NK cell degranulation against a panel of tumor cell lines with defined HLA-I expression in the presence of either the monoclonal antibody rituximab or a recently developed CD34xCD16 BiKE. Using high-resolution flow cytometry, our data revealed that NK cells educated by one or two KIRs did not have significantly enhanced ADCC capacity over uneducated KIR+ or KIR- NK cells, especially when increasing the antibody concentration. Contrasting co-education by KIRs, NKG2A was consistently associated with improved responsiveness and superior ADCC. Surprisingly, these results were despite that KIR-education correlated with higher CD16 expression that was markedly lower on NKG2A+ NK cells compared to both KIR-educated and uneducated subsets. An extended analysis uncovered that the inhibitory KIR2DL1-C245 receptor, reported to mediate weak NK cell education compared to the KIR2DL1-R245 variant, further repressed the ADCC capacity of NKG2A+ NK cells from donors expressing its ligand HLA C2. This highlights that also inhibitory KIRs can dampen the ADCC potential of NK cells similar to that of activating KIRs which also were discovered to restrict ADCC even when stimulated by its ligand. Finally, our study unveiled that BiKE-triggered ADCC has a similar relationship to NK cell education and its hierarchies as that triggered by a monoclonal antibody. In conclusion, the ADCC capacity of an NK cell is more regulated by restrictive mechanisms of education conferred by activating or selected inhibitory KIRs in individuals carrying the cognate KIR ligand than that of inhibitory KIRs conferring higher education potential. Hence, this study establishes that NK cell education does not enhance the ADCC capacity of an NK cell per se while providing new insights that also inhibitory KIRs can dampen the ADCC potential even when the target cell lacks its ligand. Our results add to the fundamental understanding of NK cell biology as well as to avenues of therapeutic applications for NK cells.

FT555: Off-the-Shelf CAR-NK Cell Therapy Co-Targeting GPRC5D and CD38 for the Treatment of Multiple Myeloma

Recent improvement in patient outcomes with the use of novel cellular immunotherapies for multiple myeloma (MM) has raised the prospect for the emergence of a curative treatment. While BCMA-targeted chimeric antigen receptor (CAR)-T cell therapies have been successful in treating MM, CAR-T cell manufacturing challenges preventing broad patient access and treatment relapse drive the need for additional targeted therapies with emphasis on multi-antigen targeting and off-the-shelf availability. GPRC5D, a tumor-associated orphan G-protein-coupled receptor found to be highly expressed in MM, is a potentially attractive target that has demonstrated promising clinical benefit when targeted via immunotherapy modalities. Here, we describe the development of FT555, an induced pluripotent stem cell (iPSC)-derived CAR-NK (CAR-iNK) cell product with the unique and effective ability to simultaneously co-target GPRC5D and CD38 (an additional tumor-associated antigen of MM) via combination with daratumumab, and which can be mass produced and is available off-the-shelf to support broad patient access. FT555 is a CAR-NK cell derived from an iPSC master cell line that has been multiplexed-engineered at the clonal level to contain four unique modalities; a novel GPRC5D-specific CAR fine-tuned for NK cell biology; a high-affinity, non-cleavable CD16 (hnCD16) to maximize antibody-dependent cellular cytotoxicity (ADCC) when combined with a monoclonal antibody (mAb); a unique IL-15/IL-15 receptor fusion protein (IL15RF) to promote cytokine-independent function; and CD38 knockout to promote NK cell fitness and uniquely prevent anti-CD38 mAb-mediated fratricide. Sourced from a renewable engineered iPSC master cell line, FT555 is a pure population of engineered NK cells (>95% CD56+) exhibiting uniform expression of CAR-GPRC5D (>90%), hnCD16 (>90%) and IL15RF (>90%), with complete elimination of CD38 expression (not detected). In cytotoxicity assays, FT555 shows antigen specificity, dose-dependent potency and importantly, when combined with the anti-CD38 mAb daratumumab, exhibits resistance to fratricide. In a serial restimulation killing assay, FT555 demonstrates persistent tumor-specific activity against GPRC5D-positive MM.1S WT target cells when compared to isogenic GPRC5D knock-out (KO) targets (70.9% control of tumor by AUC against WT targets vs. 3.5% control against GPRC5D KO targets in the third round of stimulation). When used in combination with daratumumab, FT555 targets CD38 and eliminates CAR-resistant GPRC5D KO target cells (57.1% target killing when combined with daratumumab vs. 3.5% target killing in the absence of mAb in the third round of stimulation), demonstrating the capacity for FT555 to target GPRC5D+ and CD38+ cells through CAR and hnCD16 engagement, respectively. In a disseminated xenograft in vivo model of MM where the MM.1S cell line shows comprehensive tumor engraftment, FT555 demonstrates robust killing kinetics and tumor clearance as a single dose, resulting in control of MM progression for up to 42 days and increased survival to 80 days vs. 37 days for the untreated control arm (Figure 1A, 1B, FT555; 99.9% tumor growth inhibition (TGI) at D37, 151% increased lifespan (ILS)). Durability of FT555 is further enhanced with the addition of daratumumab, where tumor growth inhibition deepened and survival improved, with 2 of 5 mice showing complete clearance of tumor cells at Day 80, demonstrating the synergy of anti-tumor activity between CAR and hnCD16 (Figure 1A, 1B, FT555+Dara; 100% TGI at D37, >207% ILS). Furthermore, in a distinct xenograft model of MM, treatment with FT555 resulted in significantly improved TGI against OPM2 tumor targets (FT555; 100% TGI at D51, p < 0.05). Together these studies demonstrate that FT555 is a multiplexed-engineered CAR-NK cell derived from a clonal master iPSC line, which utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy with daratumumab to simultaneously target both GPRC5D and CD38 in a single, standardized, and scalable off-the-shelf platform. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Partial loss-of-function mutations in GINS4 lead to NK cell deficiency with neutropenia.

Human NK cell deficiency (NKD) is a primary immunodeficiency in which the main clinically relevant immunological defect involves missing or dysfunctional NK cells. Here, we describe a familial NKD case in which 2 siblings had a substantive NKD and neutropenia in the absence of other immune system abnormalities. Exome sequencing identified compound heterozygous variants in Go-Ichi-Ni-San (GINS) complex subunit 4 (GINS4, also known as SLD5), an essential component of the human replicative helicase, which we demonstrate to have a damaging impact upon the expression and assembly of the GINS complex. Cells derived from affected individuals and a GINS4-knockdown cell line demonstrate delayed cell cycle progression, without signs of improper DNA synthesis or increased replication stress. By modeling partial GINS4 depletion in differentiating NK cells in vitro, we demonstrate the causal relationship between the genotype and the NK cell phenotype, as well as a cell-intrinsic defect in NK cell development. Thus, biallelic partial loss-of-function mutations in GINS4 define a potentially novel disease-causing gene underlying NKD with neutropenia. Together with the previously described mutations in other helicase genes causing NKD, and with the mild defects observed in other human cells, these variants underscore the importance of this pathway in NK cell biology.

Mucosal-homing natural killer cells are associated with aging in persons living with HIV

SummaryNatural killer (NK) cells are critical modulators of HIV transmission and disease. Recent evidence suggests a loss of NK cell cytotoxicity during aging, yet analysis of NK cell biology and aging in people with HIV (PWH) is lacking. Herein, we perform comprehensive analyses of people aging with and without HIV to determine age-related NK phenotypic changes. Utilizing high-dimensional flow cytometry, we analyze 30 immune-related proteins on peripheral NK cells from healthy donors, PWH with viral suppression, and viremic PWH. NK cell phenotypes are dynamic across aging but change significantly in HIV and on antiretroviral drug therapy (ART). NK cells in healthy aging show increasing ⍺4β7 and decreasing CCR7 expression and a reverse phenomenon in PWH. These HIV-associated trafficking patterns could be due to NK cell recruitment to HIV reservoir formation in lymphoid tissue or failed mucosal signaling in the HIV-infected gut but appear to be tight delineators of age-related NK cell changes.