Adoptive Transfer Of Natural Killer Cells Research Articles

CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS. Murine NK cells were activated and expanded ex vivo with soluble IL-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, GVHD and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray. AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Treatment with combination CD155 and anti-DNAM-1 pretreated alloNK ameliorated survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS. These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT. Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against OS and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

NK cell transfer overcomes resistance to PD-(L)1 therapy in aged mice

BackgroundCancer is the leading cause of death among older adults. Although the integration of immunotherapy has revolutionized the therapeutic landscape of cancer, the complex interactions between age and immunotherapy efficacy remain incompletely defined. Here, we aimed to elucidate the relationship between aging and immunotherapy resistance.MethodsFlow cytometry was performed to evaluate the infiltration of immune cells in the tumor microenvironment (TME). In vivo T cell proliferation, cytotoxicity and migration assays were performed to evaluate the antitumor capacity of tumor antigen-specific CD8+ T cells in mice. Real-time quantitative PCR (qPCR) was used to investigate the expression of IFN-γ-associated gene and natural killer (NK)-associated chemokine. Adoptive NK cell transfer was adopted to evaluate the effects of NK cells from young mice in overcoming the immunotherapy resistance of aged mice.ResultsWe found that elderly patients with advanced non-small cell lung cancer (aNSCLC) aged ≥ 75 years exhibited poorer progression-free survival (PFS), overall survival (OS) and a lower clinical response rate after immunotherapy. Mechanistically, we showed that the infiltration of NK cells was significantly reduced in aged mice compared to younger mice. Furthermore, the aged NK cells could also suppress the activation of tumor antigen-specific CD8+ T cells by inhibiting the recruitment and activation of CD103+ dendritic cells (DCs). Adoptive transfer of NK cells from young mice to aged mice promoted TME remodeling, and reversed immunotherapy resistance.ConclusionOur findings revealed the decreased sensitivity of elderly patients to immunotherapy, as well as in aged mice. This may be attributed to the reduction of NK cells in aged mice, which inhibits CD103+ DCs recruitment and its CD86 expression and ultimately leads to immunotherapy resistance.

Therapeutic restoration of miR-126-3p as a multi-targeted strategy to modulate the liver tumor microenvironment.

Impaired natural killer (NK) cell-mediated antitumor responses contribute to the growth of liver tumors. Expression of a disintegrin and metalloprotease 9 (ADAM9) increases shedding of membrane-bound major histocompatibility complex class I chain-related protein A and results in evasion from NK cell-mediated cytolysis. ADAM9 is also involved in angiogenesis and tumor progression and is a target of miR-126-3p, a tumor suppressor that is downregulated and alters tumor cell behavior in the liver and other cancers. We evaluated the restoration of miR-126-3p and modulation of the miR-126-3p/ADAM9 axis as a therapeutic approach to simultaneously enhance NK cell-mediated cytolysis while targeting both tumor cells and their microenvironment. Precursor miRNAs were loaded into milk-derived nanovesicles to generate therapeutic vesicles (therapeutic milk-derived nanovesicles) for the restoration of functional miR-126-3p in recipient cancer cells. Administration of therapeutic milk-derived nanovesicles increased miR-126-3p expression and reduced ADAM9 expression in target cells and was associated with an increase in membrane-bound major histocompatibility complex class I chain-related protein A. This enhanced NK cell cytolysis in adherent tumor cells and in multicellular tumor spheroids while also impairing angiogenesis and modulating macrophage chemotaxis. Moreover, IV administration of therapeutic milk-derived nanovesicles with adoptive transfer of NK cells reduced tumor burden in orthotopic hepatocellular cancer xenografts in mice. A directed RNA therapeutic approach can mitigate NK cell immune evasion, reduce angiogenesis, and alter the tumor cell phenotype through the restoration of miR-126-3p in liver tumor cells. The pleiotropic effects elicited by this multi-targeted approach to modulate the local tumor microenvironment support its use for the treatment of liver cancer.

Age-dependent natural killer cell and interferon γ deficits contribute to severe pertussis in infant mice.

Many respiratory infections are selectively injurious to infants, yet the etiology of age-associated susceptibility is unknown. One such bacterial pathogen is Bordetella pertussis. In adult mice, innate interferon γ (IFN-γ) is produced by natural killer (NK) cells and restricts infection to the respiratory tract. In contrast, infant pertussis resembles disease in NK cell- and IFN-γ-deficient adult mice that experience disseminated lethal infection. We hypothesized that infants exhibit age-associated deficits in NK cell frequency, maturation, and responsiveness to B. pertussis, associated with low IFN-γ levels. To delineate mechanisms behind age-dependent susceptibility, we compared infant and adult mouse models of infection. Infection in infant mice resulted in impaired upregulation of IFN-γ and substantial bacterial dissemination. B. pertussis-infected infant mice displayed fewer pulmonary NK cells than adult mice. Furthermore, the NK cells in the infant mouse lungs had an immature phenotype, and the infant lung showed no upregulation of the IFN-γ-inducing cytokine IL-12p70. Adoptive transfer of adult NK cells into infants, or treatment with exogenous IFN-γ, significantly reduced bacterial dissemination. These data indicate that the lack of NK cell-produced IFN-γ significantly contributes to infant fulminant pertussis and could be the basis for other pathogen-induced, age-dependent respiratory diseases.

Antitumor activity of genetically engineered NK-cells in non-hematological solid tumor: a comprehensive review.

Recent advancements in genetic engineering have made it possible to modify Natural Killer (NK) cells to enhance their ability to fight against various cancers, including solid tumors. This comprehensive overview discusses the current status of genetically engineered chimeric antigen receptor NK-cell therapies and their potential for treating solid tumors. We explore the inherent characteristics of NK cells and their role in immune regulation and tumor surveillance. Moreover, we examine the strategies used to genetically engineer NK cells in terms of efficacy, safety profile, and potential clinical applications. Our investigation suggests CAR-NK cells can effectively target and regress non-hematological malignancies, demonstrating enhanced antitumor efficacy. This implies excellent promise for treating tumors using genetically modified NK cells. Notably, NK cells exhibit low graft versus host disease (GvHD) potential and rarely induce significant toxicities, making them an ideal platform for CAR engineering. The adoptive transfer of allogeneic NK cells into patients further emphasizes the versatility of NK cells for various applications. We also address challenges and limitations associated with the clinical translation of genetically engineered NK-cell therapies, such as off-target effects, immune escape mechanisms, and manufacturing scalability. We provide strategies to overcome these obstacles through combination therapies and delivery optimization. Overall, we believe this review contributes to advancing NK-cell-based immunotherapy as a promising approach for cancer treatment by elucidating the underlying mechanisms, evaluating preclinical and clinical evidence, and addressing remaining challenges.

KLRC1 knockout overcomes HLA-E-mediated inhibition and improves NK cell antitumor activity against solid tumors.

Natural Killer (NK) cells hold the potential to shift cell therapy from a complex autologous option to a universal off-the-shelf one. Although NK cells have demonstrated efficacy and safety in the treatment of leukemia, the limited efficacy of NK cell-based immunotherapies against solid tumors still represents a major hurdle. In the immunosuppressive tumor microenvironment (TME), inhibitory interactions between cancer and immune cells impair antitumoral immunity. KLRC1 gene encodes the NK cell inhibitory receptor NKG2A, which is a potent NK cell immune checkpoint. NKG2A specifically binds HLA-E, a non-classical HLA class I molecule frequently overexpressed in tumors, leading to the transmission of inhibitory signals that strongly impair NK cell function. To restore NK cell cytotoxicity against HLA-E+ tumors, we have targeted the NKG2A/HLA-E immune checkpoint by using a CRISPR-mediated KLRC1 gene editing. KLRC1 knockout resulted in a reduction of 81% of NKG2A+ cell frequency in ex vivo expanded human NK cells post-cell sorting. In vitro, the overexpression of HLA-E by tumor cells significantly inhibited wild-type (WT) NK cell cytotoxicity with p-values ranging from 0.0071 to 0.0473 depending on tumor cell lines. In contrast, KLRC1 KO NK cells exhibited significantly higher cytotoxicity when compared to WT NK cells against four different HLA-E+ solid tumor cell lines, with p-values ranging from<0.0001 to 0.0154. Interestingly, a proportion of 43.5% to 60.2% of NKG2A- NK cells within the edited NK cell population was sufficient to reverse at its maximum the HLA-E-mediated inhibition of NK cell cytotoxicity. The expression of the activating receptor NKG2C was increased in KLRC1 KO NK cells and contributed to the improved NK cell cytotoxicity against HLA-E+ tumors. In vivo, the adoptive transfer of human KLRC1 KO NK cells significantly delayed tumor progression and increased survival in a xenogeneic mouse model of HLA-E+ metastatic breast cancer, as compared to WT NK cells (p = 0.0015). Our results demonstrate that KLRC1 knockout is an effective strategy to improve NK cell antitumor activity against HLA-E+ tumors and could be applied in the development of NK cell therapy for solid tumors.

IL-12/15/18-induced cell death and mitochondrial dynamics of human NK cells.

Natural killer (NK) cells are lymphocytes with potent antitumor functions and, consequently, several NK cell-based strategies have been developed for cancer immunotherapy. A remarkable therapeutic approach is the adoptive transfer of NK cells stimulated with IL-12, IL-15 and IL-18. This cytokine stimulation endows NK cells with properties that resemble immunological memory and, for this reason, they are known as cytokine-induced memory-like (CIML) NK cells. Very promising results have been reported in clinical trials and yet, there are still unknown aspects of CIML NK cells. Here, we have conducted a preliminary study of their mitochondrial dynamics. Our results show that upon IL-12/15/18 stimulation the viability of NK cells decreased and an increment in mitochondrial superoxide levels was observed. In addition, we found that mitochondria appeared slightly elongated and their cristae density decreased following IL-12/15/18 stimulation, possibly in a process mediated by the low levels of optic atrophy type 1 (OPA1) protein. Interestingly, although mitophagy was slightly impaired, an increase in autophagic flux was observed, which might explain the reduced viability and the accumulation of unfit mitochondria. Our findings could be of relevance in order to design new strategies intended to improve the mitochondrial fitness of IL-12/15/18-stimulated NK cells with the aim of improving their therapeutic efficacy.

Nicotinamide enhances natural killer cell function and yields remissions in patients with non-Hodgkin lymphoma.

Allogeneic natural killer (NK) cell adoptive transfer has shown the potential to induce remissions in relapsed or refractory leukemias and lymphomas, but strategies to enhance NK cell survival and function are needed to improve clinical efficacy. Here, we demonstrated that NK cells cultured ex vivo with interleukin-15 (IL-15) and nicotinamide (NAM) exhibited stable induction of l-selectin (CD62L), a lymphocyte adhesion molecule important for lymph node homing. High frequencies of CD62L were associated with elevated transcription factor forkhead box O1 (FOXO1), and NAM promoted the stability of FOXO1 by preventing proteasomal degradation. NK cells cultured with NAM exhibited metabolic changes associated with elevated glucose flux and protection against oxidative stress. NK cells incubated with NAM also displayed enhanced cytotoxicity and inflammatory cytokine production and preferentially persisted in xenogeneic adoptive transfer experiments. We also conducted a first-in-human phase 1 clinical trial testing adoptive transfer of NK cells expanded ex vivo with IL-15 and NAM (GDA-201) combined with monoclonal antibodies in patients with relapsed or refractory non-Hodgkin lymphoma (NHL) and multiple myeloma (MM) (NCT03019666). Cellular therapy with GDA-201 and rituximab was well tolerated and yielded an overall response rate of 74% in 19 patients with advanced NHL. Thirteen patients had a complete response, and 1 patient had a partial response. GDA-201 cells were detected for up to 14 days in blood, bone marrow, and tumor tissues and maintained a favorable metabolic profile. The safety and efficacy of GDA-201 in this study support further development as a cancer therapy.

Understanding NK cell biology for harnessing NK cell therapies: targeting cancer and beyond.

Gene-engineered immune cell therapies have partially transformed cancer treatment, as exemplified by the use of chimeric antigen receptor (CAR)-T cells in certain hematologic malignancies. However, there are several limitations that need to be addressed to target more cancer types. Natural killer (NK) cells are a type of innate immune cells that represent a unique biology in cancer immune surveillance. In particular, NK cells obtained from heathy donors can serve as a source for genetically engineered immune cell therapies. Therefore, NK-based therapies, including NK cells, CAR-NK cells, and antibodies that induce antibody-dependent cellular cytotoxicity of NK cells, have emerged. With recent advances in genetic engineering and cell biology techniques, NK cell-based therapies have become promising approaches for a wide range of cancers, viral infections, and senescence. This review provides a brief overview of NK cell characteristics and summarizes diseases that could benefit from NK-based therapies. In addition, we discuss recent preclinical and clinical investigations on the use of adoptive NK cell transfer and agents that can modulate NK cell activity.

Sirtuin 6 is a negative regulator of the anti-tumor function of natural killer cells in murine inflammatory colorectal cancer

The immune system plays a crucial role in controlling colorectal cancer (CRC) development. Natural killer (NK) cells are tumoricidal but undergo exhaustion in CRC patients. The current research aims to understand the role of sirtuin 6 (SIRT6) in CRC-associated NK cell exhaustion in a murine inflammatory colorectal cancer model. To this end, inflammatory CRC was induced by treating mice with azoxymethane plus dextran sulfate sodium. The expression of SIRT6 in NK cells in murine mesenteric lymph nodes (mLNs) and the CRC tissue was characterized by Immunoblotting. SIRT6 knockdown was achieved by lentiviral transduction of murine splenic NK cells, followed by evaluation of NK cell proliferation and the expression of cytotoxic mediators using flow cytometry. NK cell cytotoxicity was measured by cytotoxicity assays. Adoptive transfer of murine NK cells was applied to analyze the effect of SIRT6 knockdown in vivo. We found that SIRT6 was up-regulated in infiltrating NK cells in the murine CRC tissue, especially NK cells with an exhausted phenotype and impaired cytotoxicity. SIRT6 knockdown significantly boosted murine splenic NK cell functionality, as evidenced by accelerated proliferation, increased production of cytotoxic mediators, and higher tumoricidal activity both in vitro and in vivo. Furthermore, the adoptive transfer of SIRT6-knockdown NK cells into CRC-bearing mice effectively suppressed CRC progression. Therefore, SIRT6 up-regulation is essential for murine NK cell exhaustion in CRC because it impedes the tumoricidal activity of murine NK cells. Artificial SIRT6 down-regulation could boost the function of infiltrating NK cells to oppress CRC progression in mice.

NK Cell Phenotype Is Associated With Response and Resistance to Daratumumab in Relapsed/Refractory Multiple Myeloma.

The CD38-targeting antibody daratumumab has marked activity in multiple myeloma (MM). Natural killer (NK) cells play an important role during daratumumab therapy by mediating antibody-dependent cellular cytotoxicity via their FcγRIII receptor (CD16), but they are also rapidly decreased following initiation of daratumumab treatment. We characterized the NK cell phenotype at baseline and during daratumumab monotherapy by flow cytometry and cytometry by time of flight to assess its impact on response and development of resistance (DARA-ATRA study; NCT02751255). At baseline, nonresponding patients had a significantly lower proportion of CD16+ and granzyme B+ NK cells, and higher frequency of TIM-3+ and HLA-DR+ NK cells, consistent with a more activated/exhausted phenotype. These NK cell characteristics were also predictive of inferior progression-free survival and overall survival. Upon initiation of daratumumab treatment, NK cells were rapidly depleted. Persisting NK cells exhibited an activated and exhausted phenotype with reduced expression of CD16 and granzyme B, and increased expression of TIM-3 and HLA-DR. We observed that addition of healthy donor-derived purified NK cells to BM samples from patients with either primary or acquired daratumumab-resistance improved daratumumab-mediated MM cell killing. In conclusion, NK cell dysfunction plays a role in primary and acquired daratumumab resistance. This study supports the clinical evaluation of daratumumab combined with adoptive transfer of NK cells.

Intrinsic suppression of type I interferon production underlies the therapeutic efficacy of IL-15-producing natural killer cells in B-cell acute lymphoblastic leukemia

BackgroundType I interferons (IFN-Is), secreted by hematopoietic cells, drive immune surveillance of solid tumors. However, the mechanisms of suppression of IFN-I-driven immune responses in hematopoietic malignancies including B-cell acute lymphoblastic...

Abstract 6671: Simplifying high-parameter phenotypic and functional characterization of cancer immune cells

Abstract Interrogating immune cell composition and function in patients with cancer is critical for making disease prognoses, monitoring clinical efficacy of tumor immunotherapies, identifying novel therapeutic targets, and discovering predictive biomarkers of disease. Both the adaptive and innate arms of the immune system play important roles in generating pro- or anti-tumor milieus. In particular, natural killer (NK) cells are gaining increasing attention as potential cell-based therapies in immuno-oncology, including in adoptive transfer of chimeric antigen receptor (CAR) and iPSC-derived NK cells or off-the-shelf NK cell lines to target multiple myeloma. Since NK cells can also indirectly impact CAR T cell or antibody-based immunotherapies, characterizing these cells using optimized and reproducible assays is critical. CyTOF® is a high-plex flow cytometry technology that exploits metal-isotope-tagged antibodies to probe cellular phenotypes and functions. In contrast to fluorescence-based conventional and spectral flow cytometry, CyTOF experimental workflows are streamlined as autofluorescence is not an issue and signal spillover is minimal, allowing rapid design and application of 40-plus-marker panels. To expand on the increasing clinical and preclinical utility of the 30-marker Maxpar® Direct™ Immune Profiling Assay™ (Maxpar Direct Assay), we developed nine add-on Expansion Panels for deeper phenotyping of specific cell types and activation states, including panels designed to characterize ex vivo and activated myeloid cells, T cells, and NK cells. Peripheral blood mononuclear cells from healthy donors and donors with multiple myeloma were stimulated in vitro, then stained in the 30-antibody Maxpar Direct Assay tube with the NK Cell Expansion Panel (CD181, NKp30, NKp46, PD-1, NKG2A, ICOS, and TIGIT) or the T Cell Panel 3 (OX40, TIGIT, CD69, PD-1, Tim-3, ICOS, and 4-1BB) as drop-in antibodies. Surface staining was followed by intracellular staining with the Basic Activation Expansion Panel antibodies (IL-2, TNFα, IFNγ, perforin, granzyme B) after cell fixation and permeabilization. Anti-CD107a was added during stimulation to measure degranulation. Samples were acquired on a CyTOF XT™ instrument in automated batch acquisition mode. Automated analysis was performed with Maxpar Pathsetter™ software to enumerate immune cell types and quantify marker expression. In addition to the 37 populations identified by Pathsetter with the base Maxpar Direct Assay, the new Expansion Panels allowed deeper profiling of NK and T cells, while the Basic Activation Panel revealed their cytokine responsiveness and cytotoxic potential. Thus, the optimized single-tube Maxpar Direct Assay is a powerful tool that can be further expanded and customized with predefined panels or antibodies to comprehensively study immune cells in health and disease. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Deeqa Mahamed, Michael Cohen, Stephen Li, Lauren Tracey, Huihui Yao, Christina Loh, Leslie Fung. Simplifying high-parameter phenotypic and functional characterization of cancer immune cells. [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 6671.

EBV-Upregulated B7-H3 Inhibits NK cell-Mediated Antitumor Function and Contributes to Nasopharyngeal Carcinoma Progression.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated epithelial malignancy characterized by the presence of prominent infiltration of lymphocytes, including natural killer (NK) cells. Although NK cells can directly target EBV-infected tumor cells without restriction by the MHC, EBV-positive (EBV+) NPC cells often develop resistance mechanisms that allow them to evade immune surveillance by NK cells. Elucidating the mechanisms involved in EBV-induced NK-cell dysfunction will contribute to the design of novel NK cell-based immunotherapies to treat NPC. Herein, we confirmed that the cytotoxic function of NK cells was impaired in EBV+ NPC tissues and found that EBV infection-induced expression of B7-H3 in NPC negatively correlated with NK-cell function. The inhibitory effect of EBV+ tumor expression of B7-H3 on NK-cell function was clarified in vitro and in vivo. Mechanistically, activation of the PI3K/AKT/mTOR signaling pathway via EBV latent membrane protein 1 (LMP1) was responsible for EBV infection-induced upregulation of B7-H3 expression. In an NPC xenograft mouse model with adoptive transfer of primary NK cells, deletion of B7-H3 on tumor cells in combination with anti-PD-L1 treatment restored NK cell-mediated antitumor activity and significantly improved the antitumor efficacy of NK cells. On the basis of our findings, we conclude that EBV infection can inhibit NK cell-mediated antitumor function by inducing upregulation of B7-H3 expression and provide a rationale for NK cell-based immunotherapies in combination of PD-L1 blockade and overcoming the immunosuppression of B7-H3 to treat EBV-associated NPC.

A2AR limits IL-15-induced generation of CD39+ NK cells with high cytotoxicity.

CD39-mediated inhibition of natural killer (NK) cell activity has been demonstrated, but the characteristics of CD39+ NK cells in humans are not known. We investigated the characteristics of human circulating CD39+ NK cells. In healthy donors, the proportion of circulating CD39+ NK cells in total NK cells was relatively low compared with that of CD39- NK cells. Nonetheless, a higher proportion of CD39+ NK cells expressed CD107a. Similarly, a higher proportion of CD39+ NK cells expressed CD107a in patients with hepatitis B virus or patients with hepatocellular carcinoma. Stimulation with NK-sensitive K562 cells or interleukin (IL)-12/IL-18 activated CD39+ NK cells to express higher levels of CD107a, IFN-γ and TNF-α, relative to CD39- NK cells. Importantly, IL-15 induced the generation of CD39+ NK cells. In contrast, A2A adenosine receptor (A2AR) ligation suppressed the generation of CD39+ NK cells by inhibiting IL-15 signaling. These data for the first time demonstrated that A2AR counteracts IL-15-induced generation of human CD39+ NK cells, which have a stronger cytotoxicity than CD39- NK cells. IL-15-induced human CD39+ NK cells might be better choice for immunotherapy based on adoptive transfer of NK cells.

The natural killer cell immunotherapy platform: An overview of the landscape of clinical trials in liquid and solid tumors

The translation of natural killer (NK) cells to the treatment of malignant disease has made significant progress in the last few decades. With a variety of available sources and improvements in both in vitro and in vivo NK cell expansion, the NK cell immunotherapy platform has come into its own. The enormous effort continues to further optimize this platform, including ways to enhance NK cell persistence, trafficking to the tumor microenvironment, and cytotoxicity. As this effort bears fruit, it is translated into a plethora of clinical trials in patients with advanced malignancies. The adoptive transfer of NK cells, either as a standalone therapy or in combination with other immunotherapies, has been applied for the treatment of both liquid and solid tumors, with numerous early-phase trials showing promising results. This review aims to summarize the key advantages of NK cell immunotherapy, highlight several of the current approaches being taken for its optimization, and give an overview of the landscape of clinical trials translating this platform into clinic.

Inhibition of MICA and MICB Shedding Enhances Cytokine-Induced Memory-like NK Cell-Mediated Activity Against Multiple Myeloma

Introduction Natural Killer (NK) cells are important mediators of cytotoxic immune responses against MM, and the efficacy of existing MM therapies relies substantially on NK cell function. However, MM cells develop mechanisms to escape NK cell recognition by loss of ligands for activating receptors and by inducing functional defects. To overcome reduced NK cell functionality, adoptive transfer of cytokine-induced memory-like (CIML) NK cells are of particular interest, as these cells acquire memory-like features resulting in enhanced activity upon restimulation, greater IFN-γ response, and greater anti-tumor cytotoxicity. The success of adoptive transfer of CIML NK cells in MM is dependent on the ability of NK cells to recognize malignant plasma cells (PCs). NKG2D, one of the major activating receptors on NK cells, is upregulated on CIML NK cells. NKG2D plays an important role in the immune recognition of malignant PCs through interaction with its ligands MICA and MICB. However, MM cells evade this recognition by proteolytic shedding of MICA/B from the cell surface, leading to NKG2D internalization. Rationally designed antibodies like the 7C6 monoclonal antibody (mAb) targeting the proteolytic MICA α3 domain stabilize cell surface MICA/B expression and can enhance NK cell recognition and susceptibility to NK cell-mediated cytotoxicity. Furthermore, it has been shown that Bortezomib, an FDA-approved proteasome inhibitor with potent anti-MM activity, augments MICA expression in MM. We hypothesized that use of the 7C6 mAb would allow simultaneous blockade of MICA/B shedding and ADCC by CIML NK cells resulting in enhanced targeting of MM cells, which would further synergize with Bortezomib treatment. Results Flowcytometric analysis of surface MICA/B levels on 10 human myeloma cell lines (HMCLs) demonstrated heterogeneous protein expression. MICA/B transcript levels for corresponding HMCLs were queried from the CCLE database. There was no correlation between transcript and protein expression, suggesting substantial post-translational modifications, such as proteolytic cleavage, determining surface expression levels of MICA/B in MM. To determine if proteolytic cleavage occurs in MM, we treated HMCL with the 7C6 mAb, targeting the proteolytic cleavage site. Flowcytometric analysis of MM.1S revealed that 7C6 was able to upregulate basal expression of MICA/B in a dose-dependent manner as well as inhibit MICA/B shedding. These results suggest proteolytic cleavage of MICA/B occurs in MM and that this process can be inhibited by a MICA/B stabilizing antibody. Previously, it has been shown that MICA/B levels can be upregulated in MM by Bortezomib. As heterogeneity in MICA/B expression levels was seen among HMCLs, we hypothesized that the use of 7C6 would synergize with Bortezomib. Indeed, the addition of Bortezomib to 7C6 substantially increased MICA/B surface expression. To assess the functional consequences of MICA/B stabilization, we examined the effects on NK cell activation in co-culture assays of NK cells with MM.1S pretreated with 7C6 or IC mAb. We observed enhanced NK cell function as assessed by increased CD107a degranulation, IFN-γ and TNF-α production in co-culture assays of conventional NK cells with MM.1S pre-treated with 7C6 vs IC. Next, we measured the activity of CIML NK cells to myeloma targets. CIML NK cells exhibited enhanced CD107a in response to MM targets when compared to control NK cells from the same donors (Figure 1A). Furthermore, CIML NK cells demonstrated significantly higher cytotoxicity against 7C6 pretreated MM.1S, which was dependent on increased MICA/B and NKG2D levels. CIML NK cells in NSG mice treated with 7C6-hIgG1 mAb were significantly more effective in controlling early MM tumor cell outgrowth compared to CIML NK cells in mice treated with isotype control antibodies (Figure 1B). Conclusion Here we demonstrate that inhibition of MICA/B shedding with a novel antibody 7C6 enhances CIML NK cell-mediated function against MM in vitro and in a xenograft mouse model. Preliminary data demonstrate that this treatment strategy could synergize with current treatment regimens including Bortezomib. Our data support the hypothesis that combination strategies to enhance NK cell activation can be effective to enhance NK cell-mediated immune surveillance in MM. This combined approach highlights their potential use as novel immunotherapy in MM. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Down-regulation of MLLT1 super elongation complex subunit impairs the anti-tumor activity of natural killer cells in esophageal cancer

Natural killer (NK) cells actively participate in anti-tumor immunity and are thus regarded as a promising tool in immunotherapy against esophageal cancer (EC). However, the mechanisms regulating NK cell activation and exhaustion have not been completely elucidated. In this study, we characterized the expression and function of MLLT1 super elongation complex subunit (MLLT1) in esophageal NK cells in a mouse EC model. MLLT1 was down-regulated in esophageal NK cells, especially NK cells expressing both T cell immunoglobulin and mucin-domain containing-3 (TIM-3) and lymphocyte activation gene3(LAG-3). In vitro knockdown of MLLT1 in NK cells resulted in significant decreases in the expression of IFN-γ and perforin, as well as impaired NK cell cytotoxicity on tumor cells. Adoptive transfer of MLLT-deficient NK cells into EC-bearing mice showed consistent impairment of NK cell anti-tumor activity, as evidenced by decreases in IFN-γ and perforin but not granzyme B. Furthermore, EC tissue cells, which were enriched from the esophagus of EC-bearing mice, induced down-regulation of MLLT1 in splenic NK cells. This down-regulation was partially restored by a TIM-3 blocking antibody. Therefore, this study indicated that TIM-3 signaling down-regulated MLLT1 in esophageal NK cells, and MLLT1 down-regulation undermined the tumoricidal function of NK cells in EC. Our study unveils a novel mechanism underlying NK cell exhaustion/dysfunction in the EC microenvironment. MLLT1 could be a potential target in future NK cell-mediated immunotherapy against EC.

Abstract LB116: CXCL8/Interleukin-8 orchestrates radiation-induced immune responses in pancreatic adenocarcinoma

Abstract The role of radiotherapy in the treatment of pancreatic adenocarcinoma (PDAC) is controversial. In many other tumor types, radiotherapy is a mainstay therapy whose anti-tumor effects have partially been ascribed to T cell responses. The role of Natural Killer (NK) cells in radiotherapy tumor responses remains unclear. Here, we focused on the role of NK cells in radiotherapy of pancreatic cancer. In a randomized controlled pancreatic cancer trial, we found that CXCL8 serum levels increased under radiotherapy and NK cells positively correlated with prolonged overall patient survival. Accordingly, NK cells preferentially infiltrated irradiated tumors and exhibited CD56dim like cytotoxic transcriptomic states. In preclinical experimental systems in mice and tumor cells, NF-κB and mTOR signaling prompted radiation-induced CXCL8 secretion from senescent tumor cells causing directional migration of CD56dim NK cells into tumors. Thus, we link senescence-associated CXCL8 release to innate immune surveillance of human tumors. Moreover, combined radiotherapy and adoptive NK cell transfer improved tumor control over monotherapies in xenografted mice suggesting NK cells combined with radiotherapy as a rational cancer treatment strategy which may be integrated into PDAC treatment algorithms. Citation Format: Thomas Walle, Joscha A. Kraske, Boyu Liao, Carmen Timke, Benedicte Lenoir, Niels Halama, Peter E. Huber. CXCL8/Interleukin-8 orchestrates radiation-induced immune responses in pancreatic adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB116.

Harnessing allogeneic NK cells: improving outcomes with tailored donor lymphocyte infusion.

Since researchers first began to uncover the mechanisms underlying allogeneic transplantation, the focus has been on T cells. T cells are a major instigator of graft-versus-host disease (GVHD). The clear association between GVHD occurrence and subsequent reduction in relapse supported concentrating on T cells as the masterminds behind graft-versus-tumor (GVT) effects. Recently, an alternative mediator of GVT has taken center stage: natural killer (NK) cells. Part of the appeal of NK cells is their potential to provide antitumor immunity without GVHD. Donor lymphocyte infusion has been the predominant treatment of relapse after allogeneic transplant, but the mix of lymphocytes includes CD8+ T cells and, consequently, a substantial risk for GVHD. In this issue of the JCI, Shapiro and colleagues developed an adoptive NK cell transfer platform to treat relapse after haploidentical allogeneic transplant. The study demonstrated safety, sought to determine resistance mechanisms, and provided avenues for future research.