Cytotoxic Activity Of Natural Killer Cells Research Articles

Immunological nanomaterials to combat cancer metastasis.

Metastasis causes greater than 90% of cancer-associated deaths, presenting huge challenges for detection and efficient treatment of cancer due to its high heterogeneity and widespread dissemination to various organs. Therefore, it is imperative to combat cancer metastasis, which is the key to achieving complete cancer eradication. Immunotherapy as a systemic approach has shown promising potential to combat metastasis. However, current clinical immunotherapies are not effective for all patients or all types of cancer metastases owing to insufficient immune responses. In recent years, immunological nanomaterials with intrinsic immunogenicity or immunomodulatory agents with efficient loading have been shown to enhance immune responses to eliminate metastasis. In this review, we would like to summarize various types of immunological nanomaterials against metastasis. Moreover, this review will summarize a series of immunological nanomaterial-mediated immunotherapy strategies to combat metastasis, including immunogenic cell death, regulation of chemokines and cytokines, improving the immunosuppressive tumour microenvironment, activation of the STING pathway, enhancing cytotoxic natural killer cell activity, enhancing antigen presentation of dendritic cells, and enhancing chimeric antigen receptor T cell therapy. Furthermore, the synergistic anti-metastasis strategies based on the combinational use of immunotherapy and other therapeutic modalities will also be introduced. In addition, the nanomaterial-mediated imaging techniques (e.g., optical imaging, magnetic resonance imaging, computed tomography, photoacoustic imaging, surface-enhanced Raman scattering, radionuclide imaging, etc.) for detecting metastasis and monitoring anti-metastasis efficacy are also summarized. Finally, the current challenges and future prospects of immunological nanomaterial-based anti-metastasis are also elucidated with the intention to accelerate its clinical translation.

A randomized, double-blind, placebo-controlled study on immune improvement effects of ethanolic extract of Echinacea purpurea (L.) Moench in Korean adults.

Echinacea purpurea (L.) Moench (EP), a medicinal plant native to North America, is now cultivated in various regions including Europe. With increasing popularity of Echinacea in Korea recently, a human clinical trial was conducted to evaluate immune-enhancing efficacy and safety of EP 60% ethanolic extract (EPE) in Koreans. Eighty volunteers were recruited for this randomized, double-blind, placebo-controlled clinical trial. They were randomly divided into two groups and given either a daily dose of 200 mg of EPE or a placebo. All participants underwent testing for Natural Killer (NK) cell cytotoxic activity, serum cytokine levels (IL-2, IL-6, IL-10, IL-12, IFN-γ, TNF-α), Wisconsin Upper Respiratory Symptom Survey-21 (WURSS-21), and Multidimensional Fatigue Scale (MFS) during this study to assess changes in outcomes. After 8 weeks of EPE consumption, a significant increase in NK cell cytotoxic activity compared to the placebo was observed. Additionally, serum cytokine levels of IL-2, IFN-γ, and TNF-α also significantly increased following EPE consumption. However, no significant changes were observed in WURSS-21 and MFS before and after EPE consumption. Throughout the 8-week study period, no adverse reactions were reported in relation to EPE consumption, and there were no clinically significant changes in diagnostic laboratory tests or vital signs in the EPE group. These results indicate that consumption of EPE could lead to immune improvement without any adverse effects. This clinical trial was the first to demonstrate beneficial effects of EPE consumption on immunity in Korean adults.

Lacticaseibacillus casei K11 exerts immunomodulatory effects by enhancing natural killer cell cytotoxicity via the extracellular regulated-protein kinase pathway.

Probiotics can serve as immunomodulators that regulate the activation of immune cells. This study aimed to screen potential probiotic strains that can enhance NK cell toxicity to improve host immunity. In this investigation, we examined three potential probiotic strains, namely Lactiplantibacillus plantarum YZX21 (YZX21), Bifidobacterium bifidum FL-276.1 (FL-276.1) and Lacticaseibacillus casei K11 (K11), to assess their capacity in modulating NK cytotoxicity both in vitro and in vivo, while elucidating the underlying mechanisms involved. The findings demonstrated that K11 exhibited superior efficacy in enhancing NK cytotoxicity. Subsequent analysis revealed that K11 significantly augmented the secretion of perforin and granzyme B by NK cells through activation of receptors NKp30 and NKp46 via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, heat-inactivated K11 also enhanced NK cell activity to an extent comparable to live bacteria, with lipoteichoic acid from K11 identified as a crucial factor mediating the activation of NK cell cytotoxicity. Our study suggests that K11 may have potential applications as probiotics or postbiotics for regulating NK cell cytotoxicity to enhance immunity.

Abstract LB363: A novel humanized NKP46 mouse model to support xenograft studies evaluating NK cell-based cancer immunotherapies

Abstract The NKP46 receptor is expressed by all NK cells, and plays a key role in the recognition and activation of NK cells against tumor cells. Cross-linking of NKp46 by monoclonal antibodies triggers both NK cell cytotoxic activity and cytokine release. As the only NCR present in mice, NKP46 is well conserved, but differences between the murine and human NKP46 protein necessitate use of humanized mice for evaluating preclinical efficacy of novel NKP46-targeting antibodies. Previously, we established humanized NKP46 mice on immunocompetent backgrounds for evaluating antibody efficacy in preventing growth of syngeneic tumor models. However, xenograft models in immunodeficient mice have also been widely employed as important in vivo models. Here, we describe a novel humanized NKP46 mouse on the CB-17-SCID background in order to inoculate human tumor cells for therapeutic efficacy testing. In this model, using gene editing technology, the portion of the mouse NKp46 gene encoding the extracellular domain was replaced by the corresponding human NKP46 coding sequences. Human NKP46 protein expression was confirmed on NK cells derived from splenocytes and bone marrow of B-hNKP46 mice(CB17-SCID) by flow cytometry. Subsequently, assessment of leukocyte populations in the humanized model indicated no significant alterations in the percentages of NK cells, dendritic cells, granulocytes, monocytes and macrophages in the spleen, bone marrow or blood of the humanized mice compared with wild-type CB17-SCID mice. Finally, tumor efficacy studies demonstrate that treatment with an anti-human NKP46 antibody significantly reduced BxPC-3 tumor growth at a range of doses in homozygous hNKP46 mice compared to PBS-injected controls. In summary, humanized B-hBKP46 mice(CB17-SCID) provide a suitable model for evaluating the efficacy of anti-human NKP46 antibody candidates. Citation Format: Zhenlan Niu, Yongshun Li, Xiaofei Zhou, Jiawei Yao, Qingcong Lin, Meiqi Zhang, Qingqing Xu. A novel humanized NKP46 mouse model to support xenograft studies evaluating NK cell-based cancer immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB363.

Immunohistochemical Evaluation of NKP46 Receptor Expression and the Number of NK Cells in the Endometrium of Patients with Endometriosis.

Endometriosis is a medical condition that can cause infertility in women. Women with endometriosis experience a decrease in NK cell cytotoxic activity against endometrial cells, ultimately contributing to the spread of these cells. To assess the frequency of NK cells and the expression of the NKP46 receptor in endometrial tissue from patients with endometriosis using immunohistochemistry. 30 endometrial tissue specimens were collected from three groups of cases with mild (n=11), moderate (n=10), and severe endometriosis (n=9), respectively. Additionally, 20 normal endometrial tissue specimens were collected as the control group. Immunohistochemical staining was carried out using specific human monoclonal antibodies against CD56 and NKP46 molecules. Cases with severe endometriosis had a significantly higher number of CD56+ uterine NK cells (26.19±2.50) compared to fertile women (15.02±0.622) and women with mild to moderate endometriosis (p<0.001). However, there was no significant difference between the mild to moderate patients compared with the healthy women (p>0.05). Endometrial NKp46 expression was lower in women with severe endometriosis (0.447±0.0829) compared to fertile women (0.987±0.115, p=0.03). The NKp46+/CD56+ cell ratio was also lower in women with severe endometriosis (0.019±0.003) compared to fertile women (0.072±0.011, p=0.01). Women with severe endometriosis demonstrated an increased rate of infiltrated uterine NK cells and a significant decrease in NKP46 expression compared to fertile women. Therefore, NK cells and the NKp46 receptor may be involved in the development of endometriosis.

CTLA-4 Blockade of Natural Killer Cells Increases Cytotoxicity against Acute Lymphoid Leukaemia Cells Neda.

There is interest in using cytotoxic T lymphocyte antigen-4 (CTLA-4) immunotherapy to treat blood cancers. Unfortunately, patients with acute lymphoblastic leukaemia (ALL) frequently exhibit resistance to treatment and natural killer (NK) cell exhaustion. This study aims to increase the cytotoxic potency of natural killer cells by using CTLA-4 to block the Nalm-6 leukaemia cell line. In this experimental study, NK cells were purified from the peripheral blood mononuclear cells (PBMCs) of 10 healthy people and assessed by flow cytometry for purity and viability. The purified cells were activated overnight at 37°C and 5% CO2 with interleukin-15 (IL-15, 10 ng/ml) followed by evaluation of expressions of CTLA-4, activating and inhibitory receptors, and the release of interferon gamma (IFN-γ) and granzyme B (GZM B). CTLA-4 expression on NK cells from recurrent ALL patients was also evaluated. Finally, the cytotoxic activity of NK cells was assessed after the CTLA-4 blockade. The purity of the isolated cells was 96.58 ± 2.57%. Isolated NK cells activated with IL-15 resulted in significantly higher CTLA-4 expression (8.75%, P<0.05). Similarly, CTLA-4 expression on the surface of NK cells from patients with ALL was higher (7.46%) compared to healthy individuals (1.46%, P<0.05). IL-15 reduced NKG2A expression (P<0.01), and increased expressions of NKP30 (P<0.05) and NKP46 (P<0.01). The activated NK cells released more IFN-γ (P<0.5) and GZM B (P<0.01) compared to unactivated NK cells. Blockade of CTLA-4 enhanced the NK cell killing potential against Nalm-6 cells (56.3%, P<0.05); however, IFN-γ and GZM B levels were not statistically different between the blocked and non-blocked groups. Our findings suggest that CTLA-4 blockage of Nalm-6 cells causes an increase in antitumour activity of NK cells against these cells. Our study also provides evidence for the potential of cancer immunotherapy treatment using blocking anti-CTLA-4 mAbs.

Osteoclasts and Probiotics Mediate Significant Expansion, Functional Activation and Supercharging in NK, γδ T, and CD3+ T Cells: Use in Cancer Immunotherapy.

Our previous studies have introduced osteoclasts (OCs) as major activators of NK cells. It was found that OCs exhibit the capabilities of inducing cell expansion as well as increasing the cytotoxic activity of NK cells by granule release and increasing the secretion of TNF-α and TRAIL, leading to increased lysis of tumors in short-term as well as long-term periods, respectively. OC- induced expanded NK cells were named supercharged NK cells (sNK) due to their significantly high functional activity as well as their significantly higher cell expansion rate. It is, however, unclear whether the OC-mediated effect in NK cells is specific or whether other cytotoxic immune cells can also be expanded and activated by OCs. We chose to focus on γδ T cells and pan T cells, which also include CD8+ T cells. In this paper, we report that OCs are capable of expanding and functionally activating both γδ T cells and pan T cells. Expanded γδ T and pan T cells were capable of secreting high levels of INF-γ, albeit with different dynamics to those of NK cells, and, moreover, they are unable to kill NK-specific targets. Since we used humanized-BLT (hu-BLT) mice as a model of human disease, we next determined whether NK and T cell activation through OCs is also evident in cells obtained from hu-BLT mice. Similar to humans, OCs were capable of increasing the cell expansion and secretion of IFN-γ in the culture of either NK or T cells from hu-BLT mice, providing yet further evidence that these mice are appropriate models to study human disease. Therefore, these studies indicated that CD3+ T or γδ T cells can proliferate and be supercharged by OCs similar to the NK cells; thus, they can be used individually or in combination in the cell therapy of cancers.

Combined use of NK cells and radiotherapy in the treatment of solid tumors.

Natural killer (NK) cells are innate lymphocytes possessing potent tumor surveillance and elimination activity. Increasing attention is being focused on the role of NK cells in integral antitumor strategies (especially immunotherapy). Of note, therapeutic efficacy is considerable dependent on two parameters: the infiltration and cytotoxicity of NK cells in tumor microenvironment (TME), both of which are impaired by several obstacles (e.g., chemokines, hypoxia). Strategies to overcome such barriers are needed. Radiotherapy is a conventional modality employed to cure solid tumors. Recent studies suggest that radiotherapy not only damages tumor cells directly, but also enhances tumor recognition by immune cells through altering molecular expression of tumor or immune cells via the in situ or abscopal effect. Thus, radiotherapy may rebuild a NK cells-favored TME, and thus provide a cost-effective approach to improve the infiltration of NK cells into solid tumors, as well as elevate immune-activity. Moreover, the radioresistance of tumor always hampers the response to radiotherapy. Noteworthy, the puissant cytotoxic activity of NK cells not only kills tumor cells directly, but also increases the response of tumors to radiation via activating several radiosensitization pathways. Herein, we review the mechanisms by which NK cells and radiotherapy mutually promote their killing function against solid malignancies. We also discuss potential strategies harnessing such features in combined anticancer care.

A novel Fc-engineered cathepsin D-targeting antibody enhances ADCC, triggers tumor-infiltrating NK cell recruitment, and improves treatment with paclitaxel and enzalutamide in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) prognosis is poor. Immunotherapies to enhance the antibody-induced natural killer (NK) cell antitumor activity are emerging for TNBC that is frequently immunogenic. The aspartic protease cathepsin D (cath-D), a tumor cell-associated extracellular protein with protumor activity and a poor prognosis marker in TNBC, is a prime target for antibody-based therapy to induce NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC). This study investigated whether Fc-engineered anti-cath-D antibodies trigger ADCC, their impact on antitumor efficacy and tumor-infiltrating NK cells, and their relevance for combinatory therapy in TNBC. Cath-D expression and localization in TNBC samples were evaluated by western blotting, immunofluorescence, and immunohistochemistry. The binding of human anti-cath-D F1M1 and Fc-engineered antibody variants, which enhance (F1M1-Fc+) or prevent (F1M1-Fc-) affinity for CD16a, to secreted human and murine cath-D was analyzed by ELISA, and to CD16a by surface plasmon resonance and flow cytometry. NK cell activation was investigated by flow cytometry, and ADCC by lactate dehydrogenase release. The antitumor efficacy of F1M1 Fc-variants was investigated using TNBC cell xenografts in nude mice. NK cell recruitment, activation, and cytotoxic activity were analyzed in MDA-MB-231 cell xenografts by immunophenotyping and RT-qPCR. NK cells were depleted using an anti-asialo GM1 antibody. F1M1-Fc+ antitumor effect was assessed in TNBC patient-derived xenografts (PDXs) and TNBC SUM159 cell xenografts, and in combination with paclitaxel or enzalutamide. Cath-D expression on the TNBC cell surface could be exploited to induce ADCC. F1M1 Fc-variants recognized human and mouse cath-D. F1M1-Fc+ activated NK cells in vitro and induced ADCC against TNBC cells and cancer-associated fibroblasts more efficiently than F1M1. F1M1-Fc- was ineffective. In the MDA-MB-231 cell xenograft model, F1M1-Fc+ displayed higher antitumor activity than F1M1, whereas F1M1-Fc- was less effective, reflecting the importance of Fc-dependent mechanisms in vivo. F1M1-Fc+ triggered tumor-infiltrating NK cell recruitment, activation and cytotoxic activity in MDA-MB-231 cell xenografts. NK cell depletion impaired F1M1-Fc+ antitumor activity, demonstrating their key role. F1M1-Fc+ inhibited growth of SUM159 cell xenografts and two TNBC PDXs. In combination therapy, F1M1-Fc+ improved paclitaxel and enzalutamide therapeutic efficacy without toxicity. F1M1-Fc+ is a promising immunotherapy for TNBC that could be combined with conventional regimens, including chemotherapy or antiandrogens.

Effects of homo‐ and heterozygous TREM2 mutations on natural killer cell activity

AbstractBackgroundNasu‐Hakola disease (NHD) is a rare genetic neurodegenerative disorder characterised by progressive presenile dementia and bone cysts. Dysfunctions in myeloid‐derived innate immune cells such as microglia due to loss‐of‐function mutations in the TREM2 (trigger receptor 2 expressed on myeloid cells) gene cause the clinical manifestations. Heterozygous carriers have strongly increased risk of developing Alzheimer’s Disease but do not present with NHD symptoms. TREM2 is associated with DAP12 protein, which contains an immunoreceptor tyrosine‐based activation motif that is structurally similar to natural killer (NK) cell receptors. In this study, we aimed to analyse the effects of TREM2 mutation and its effect on the peripheral immune system represented by NK cell functions.MethodSix patients with NHD with a defined homozygous TREM2 mutation, 7 carriers with heterozygous TREM2 mutations and 10 age‐ and gender‐matched controls were included. NK cell subsets, KIR expressions (NKG2A, NKG2D, NKp46), cytotoxic activity (CD107a, Perforin, Granzyme A) and cytokine secretions (IL‐4, IFN‐γ, TNF‐α, IL‐10, IL‐17, TGF‐β) were analysed by flow cytometry.ResultCD3−CD56dimCD16− (p = 0.003) subsets were found to be increased in the NHD group compared to carriers. Activating C‐type lectin receptor NKG2D expression was increased in CD8+ (p = 0.003) NK cell subsets in the NHD group. Inhibiting C‐type lectin receptor NKG2A expression was increased in total (p = 0.011) as well as CD8+ (p = 0.007) NK cell subsets in carriers compared to controls. In total NK cells, intracellular TNF‐α (p = 0.005) and TGF‐β (p = 0.013) levels were decreased, IL‐17 (p = 0.05) and IL‐4 (p = 0.005) levels were increased in the NHD group. When NK cells were grouped due to their CD8 expression, TNF‐α levels were decreased, and IL‐17 levels were increased in both subsets in the NHD group. Cytotoxic activity showed no differentiation in both unstimulated and K562 stimulated conditions between groups.ConclusionUnlike previous case series, NK cell ratios did not differ between groups. Over‐expression of inhibiting receptors in the carriers group, unlike high levels of activating receptors in the NHD, might explain the lack of symptoms in the carriers. The increased ratio of CD3−CD56dimCD16− NK cell subsets that overexpress inhibitory checkpoints might explain the low levels of pro‐inflammatory cytokines in the NHD group.

A CD38/CD28xCD3 Trispecific T-Cell Engager (TCE) As a Potentially Active Agent in Multiple Myeloma Patients Relapsed and/or Refractory (RRMM) to Anti-CD38 Monoclonal Antibodies (mAbs)

Background: Despite great advances in the treatment of RRMM, including anti-CD38 mAbs, patients become resistant and therapeutic options are limited. Some factors involved in resistance to anti-CD38 mAbs have been described. On the one hand, their efficacy is partially dependent on baseline CD38 expression in MM plasma cells (MMPC), but the treatment induces a rapid and sustained CD38 downregulation. On the other hand, their tumor-killing activity relies on NK cells, but its frequency rapidly decreases following initiation of treatment, and persistent NK cells exhibit a dysfunctional phenotype. Given the reduction of CD38 levels in MMPC and the impaired NK-cell activity in resistance to anti-CD38 mAbs, we hypothesized that RRMM patients previously treated with these agents could benefit from T-cell-based immunotherapeutic strategies that depend less on CD38 antigen density and NK cell function. Aim: Evaluate a CD38/CD28xCD3 trispecific TCE (SAR442257) as a potential therapeutic agent in the RRMM setting. Results: CD38 expression in MMPC and NK-cell frequency in bone marrow were significantly reduced in 29 RRMM patients previously exposed to anti-CD38 mAbs vs 45 newly-diagnosed MM (NDMM) patients ( P &amp;lt;.001 and P =.013, respectively). Moreover, the percentage of CD57+ NK cells was significantly lower in RRMM than NDMM patients ( P =.006), suggesting that both CD38 downregulation and a contraction of cytotoxic NK cells may be associated with resistance to anti-CD38 mAbs. By contrast, T cells did not change significantly between stages, which ignited further interest in the preclinical efficacy of the CD38/CD28xCD3 TCE. First, we observed that, contrary to isatuximab and daratumumab, CD38/CD28xCD3 TCE did not reduce surface CD38 levels in the MOLP 8, RPMI 8226 and KMS 12 BM cell lines. Fluorescence microscopy imaging revealed that CD38 was present in cell membrane up to one week of treatment with CD38/CD28xCD3 TCE, while isatuximab induced an internalization of CD38 at 24h. Next, in a co-culture of the OPM-2 cell line with purified T or NK cells from healthy individuals (E:T ratio 2:1), we confirmed that the activity of CD38/CD28xCD3 TCE relied on T but not NK cells. Namely, no direct NK-cell stimulation was found whereas a significant T-cell activation was observed upon CD38/CD28xCD3 TCE treatment. Surprisingly, when OPM-2 were co-cultured with freshly-isolated peripheral blood mononuclear cells (PBMCs, E:T ratio 10:1), CD38/CD28xCD3 TCE induced activation of T- and NK-cells, suggesting a bystander NK-cell activation. This dual stimulation resulted in a significant reduction of OPM-2 cell viability at 48h. Interestingly, we found a comparable tumor-killing activity and T-cell activation in the presence of the CD38 null trispecific isotype (CD3xCD28 ΔCD38) compared with CD38/CD28xCD3 TCE, suggesting that the latter targets CD28 not only in T-cells, but also in OPM-2 cells, which express high CD28 basal levels. Altogether, these data demonstrate the potential dual CD38/CD28 targeting in RRMM patients previously exposed to anti-CD38 mAbs, particularly in those with double positive MMPC. Additionally, CD38 regulation was analyzed in the co-culture system (measured with a non-competing anti-CD38 antibody), and an unaltered and increased CD38 expression was observed in OPM-2 and T-cells, respectively, indicating again T-cell stimulation. We tested the ex vivo efficacy of CD38/CD28xCD3 TCE (48h, 700 pM) in primary bone marrow samples from eight RRMM patients, which were collected after a median of 10 months since the last anti-CD38 treatment (range, 0.5 - 37). T-cell activation and MMPC lysis were found in six and four samples, respectively, which was accompanied by a reduction in the frequency of CD4 T cells (from 100% to 76%; P =.014). A positive and borderline-significant correlation (r = 0.764, P =.057) was found between the response to CD38/CD28xCD3 TCE and the time since last anti-CD38 treatment, and no differences in CD38 expression were observed between CD38/CD28xCD3 TCE responders and non-responders. Conclusions: We observed a reduction in CD38 levels in MMPC and an impaired cytotoxic activity of NK cells in RRMM patients previously exposed to anti-CD38 mAbs. We propose a second targeting of CD38 using T-cell-based immunotherapeutic agents whose efficacy depends less on CD38 antigen density, especially after longer washout periods, as a potential therapeutic strategy in the RRMM setting.

Mitochondrial Fitness and Immune Function of Natural Killer Cells Are Impaired By the Multiple Myeloma (MM) Tumor Cells: The Role of MM Microenvironment Interactions

The immune system including Natural Killer (NK) activity was shown to be impaired in active MM. We and others have previously demonstrated the important role of the bone marrow (BM) microenvironment in MM pathophysiology, however, data on its effects on NK activity in MM is limited. We first assessed the phenotype of the BM-derived NK cells. BM-NK from MM pts (n=10) showed an exhausted phenotypic signature with significantly reduced expression of CD16 (p&amp;lt;0.01), and increased expression of exhaustion markers CD39 (p&amp;lt;0.001) and CD57 (p&amp;lt;0.001) compared to BM-NK cells from healthy controls (n=8). Furthermore, MM-BM NK cells demonstrated reduced mitochondrial membrane potential, suggesting impaired mitochondrial fitness. The dysfunction of the mitochondria led to deficiency of ATP synthesis; mitochondrial ATP levels were reduced in NK cells from MM BM samples in comparison to the normal BMs (p&amp;lt;0.001). Next, the functional response of BM NK cells to the stimulation with IL-2 (2000 IU/ml for 7 and 14 days) was evaluated in vitro. NK cells from MM BM demonstrated reduced proliferation capacity, increased CD39, reduced expression of activating receptors NKp44 and NKp30, and reduced mitochondrial membrane potential upon the activation with IL-2. The impaired mitochondrial fitness correlated with reduced levels of NKp44, indicating a potential link between the decreased mitochondrial activity and the reduced functionality of NK cells. Indeed, in vitro, the cytotoxic activity of IL-2-stimulated MM-BM NK cells against MM cell lines was significantly diminished compared to healthy IL-2-activated BM-NK cells. Glucose and fatty acids (FA) are key substrates of the mitochondrial bioenergetic pathways and essential for NK cell activation. Of note, glucose uptake was increased comparably upon IL-2 stimulation in NK cells from both MM patients and healthy donors. In contrast, MM-BM NK cells demonstrated reduced FA uptake in response to IL-2 stimulation compared to BM NK cells from healthy donors. These data indicate impaired mitochondrial and NK cell properties in the BM MM niche. Next, we established an in vitro model to interrogate the relationship between NK cell mitochondrial and immune dysfunction within the MM BM microenvironment. Specifically, we cultured MM cell lines (n=5) with the NK92 cell line or primary IL-2-activated or not activated blood-derived NK cells, and assessed NK cell mitochondrial fitness, metabolic activity, and immune functional properties. Co-culture with MM cells significantly impaired mitochondrial mass and mitochondrial membrane potential in NK cells, decreased mitochondrial ATP levels, while upregulating MM mitochondrial ROS in NK cells, indicating that MM cells induce mitochondrial damage and impair ATP synthesis in NK cells. Mitochondrial fitness and energy production are pivotal for NK cell persistence and cytotoxicity. In accordance, direct interaction with MM cells suppressed NK cell proliferation in vitro, inducing G0/G1 cell cycle arrest. Moreover, MM-induced mitochondrial impairment in NK cells correlated with reduced responsiveness of MM cells to NK-induced killing, while short-term exposure to MM cells significantly diminished the anti-myeloma cytotoxic activity of NK cells. Accordingly, CD39 expression was upregulated, while NKp44 levels significantly decreased in NK cells upon interaction with MM cells. To further characterize the molecular mechanisms associated with MM-mediated NK cell mitochondrial impairment, we assessed the gene expression profile of MM-exposed sorted NK92 cells versus unexposed cells. A distinct metabolic signature characterized by reduced expression of genes involved in the mitochondrial activity was detected in MM-exposed NK cells. Thus, genes involved in respiratory activity and mitochondrial dynamics were downregulated post-MM exposure. Furthermore, a profound increase in fructose-1,6-biphosphatase 1 (FBP1) expression was detected in MM-exposed NK cells. FBP1 is the rate-limiting enzyme for gluconeogenesis that simultaneously inhibits glycolysis, possibly interfering with NK cell metabolic activity and immune function. Altogether, our data suggest that MM BM microenvironment and direct interactions with malignant plasma cells impair NK mitochondrial fitness, diminishing energy production, increasing oxidative stress, and driving exhaustion with reduced anti-tumor capacity in NK cells.

A CRISPR activation screen identifies MUC-21 as critical for resistance to NK and T cell-mediated cytotoxicity

BackgroundImmunotherapy has significantly advanced cancer treatments, but many patients do not respond to it, partly due to immunosuppressive mechanisms used by tumor cells. These cells employ immunosuppressive ligands to evade detection and elimination by the immune system. Therefore, the discovery and characterization of novel immunosuppressive ligands that facilitate immune evasion are crucial for developing more potent anti-cancer therapies.MethodsWe conducted gain-of-function screens using a CRISPRa (CRISPR activation) library that covered the entire human transmembrane sub-genome to identify surface molecules capable of hindering NK-mediated cytotoxicity. The immunosuppressive role and mechanism of MUC21 were validated using NK and T cell mediated cytotoxicity assays. Bioinformatics tools were employed to assess the clinical implications of mucin-21 (MUC21) in cancer cell immunity.ResultsOur genetic screens revealed that MUC21 expression on cancer cell surfaces inhibits both the cytotoxic activity of NK cells and antibody-dependent cellular cytotoxicity, but not affecting complement-dependent cytotoxicity. Additionally, MUC21 expression hinders T cell activation by impeding antigen recognition, thereby diminishing the effectiveness of the immune checkpoint inhibitor, anti-PD-L1. Moreover, MUC21 expression suppress the antitumor function of both CAR-T cells and CAR-NK cells. Mechanistically, MUC21 facilitates immune evasion by creating steric hindrance, preventing interactions between cancer and immune cells. Bioinformatics analysis revealed elevated MUC21 expression in lung cancer, which correlated with reduced infiltration and activation of cytotoxic immune cells. Intriguingly, MUC21 expression was higher in non-small cell lung cancer (NSCLC) tumors that were non-responsive to anti-PD-(L)1 treatment compared to responsive tumors.ConclusionsThese findings indicate that surface MUC21 serves as a potent immunosuppressive ligand, shielding cancer cells from NK and CD8+T cell attacks. This suggests that inhibiting MUC21 could be a promising strategy to improve cancer immunotherapy.

The aryl hydrocarbon receptor maintains antitumor activity of liver resident natural killer cells after partial hepatectomy in C57BL/6J mice.

Liver-resident natural killer (lr-NK) cells are distinct from conventional NK cells and exhibit higher cytotoxicity against hepatoma via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the mechanism by which partial hepatectomy (PH) significantly suppresses TRAIL expression in lr-NK cells remains unclear. This study aimed to investigate the PH influence on the function and characteristics of liver-resident NK (lr-NK) cells using a PH mouse model. Here, we report that PH alters the differentiation pattern of NK cells in the liver, and an aryl hydrocarbon receptor (AhR) molecule is involved in these changes. Treatment with the AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) inhibited the maturation of NK cells. FICZ increased the immature subtype proportion of NK cells with high TRAIL activity and decreased the mature subtype of NK cells with low TRAIL activity. Consequently, FICZ increased the expression of TRAIL and cytotoxic activity of NK cells in the liver, and this effect was confirmed even after hepatectomy. The participation of AhR promoted FoxO1 expression in the mTOR signaling pathway involved in the maturation of NK cells, resulting in TRAIL expression. Our findings provide direct in-vivo evidence that partial hepatectomy affects lrNK cell activity through NK cell differentiation in the liver. Perioperative therapies using an AhR agonist to improve NK cell function may reduce the recurrence of hepatocellular carcinoma after hepatectomy.

Engineering NK-CAR.19 cells with the IL-15/IL-15Rα complex improved proliferation and anti-tumor effect in vivo.

Natural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary. In this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence. We demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model. Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.

Reduction of Ca2+ Entry by a Specific Block of KCa3.1 Channels Optimizes Cytotoxic Activity of NK Cells against T-ALL Jurkat Cells.

Degranulation mediated killing mechanism by NK cells is dependent on store-operated Ca2+ entry (SOCE) and has optimum at moderate intracellular Ca2+ elevations so that partial block of SOCE optimizes the killing process. In this study, we tested the effect of the selective blocker of KCa3.1 channel NS6180 on SOCE and the killing efficiency of NK cells from healthy donors and NK-92 cells against T-ALL cell line Jurkat. Patch-clamp analysis showed that only one-quarter of resting NK cells functionally express KCa3.1 current, which increases 3-fold after activation by interleukins 15 and 2. Nevertheless, blockage of KCa3.1 significantly reduced SOCE and intracellular Ca2+ rise induced by IL-15 or target cell recognition. NS6180 (1 μM) decreased NK degranulation at zero time of coculture with Jurkat cells but already after 1 h, the degranulation reached the same level as in the control. Monitoring of target cell death by flow cytometry and confocal microscopy demonstrated that NS6180 significantly improved the killing ability of NK cells after 1 h in coculture with Jurkat cells and increased the Jurkat cell fraction with apoptotic and necrotic markers. Our data evidence a strong dependence of SOCE on KCa3.1 activity in NK cells and that KCa3.1 specific block can improve NK cytotoxicity.

P-339 Activities and changes in the number of natural killer cells in endometriosis: systematic review and meta-analysis

Abstract Study question What are the differences in peripheral, uterine, and peritoneal NK (uNK, pNK, pfNK) cell counts/percentages, and activities in women with endometriosis compared controls Summary answer The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference in women with endometriosis compared controls What is known already NK cells play an important role in the pathogenesis of endometriosis Study design, size, duration This systematic review and meta-analysis included 36 experimental studies (case control and cross sectional). Narrative review is also conducted on the NK cell activity, cytokine expression, regulations and receptors. Participants/materials, setting, methods Women suffering with endometriosis confirmed with laparoscopy and/or pathology was the cases whereas, women having different pelvic pathology such as myoma, ovarian cyst, healthy woman and verified not having endometriosis with laparoscopy and/or pathology was considered as controls for this study. Databases (PubMed, Web of Science, Scopus, Google scholar, and EMBASE through OVID) were used to search for the available studies. RevMan 5.4 and STATA 14 were used to analyze the data. Main results and the role of chance The mean percentage level of uNK, pNK, and peritoneal NK (pfNK) cells has no significant difference (standard mean difference SMD 0.13, 95%CI -0.35, 0.62; P = 0.59, I2 74%; total 393 women, 6 studies), (SMD 0.27, 95%CI -0.07, 0.61; P = 0.12, I2 80%; total 825 women, 11 studies), and (SMD 0.31, 95%CI -0.68, 01.29; P = 0.59, I2 94%; total 404 women,7 studies) in endometriosis patients compared with controls respectively. The pooled NK (uNK, pNK, pfNK) cell cytotoxicity/activity level is significantly higher in controls compared with women with endometriosis (MD 5.43, 95%CI 2.29, 8.57; P &amp;lt; 0.007, I2 50%; total 323 women, 7 studies). The NK cytotoxicity/activity level in early stages of endometriosis has no significant difference compared with advanced stages of endometriosis (MD 2.29, 95%CI –1.98-6.57; P = 0.29, I2 87%; total 263 women, 7 studies). There are variations in studies conducted in NK cell activities in endometriosis: broadly categorized as NK cell cytotoxicity, cytokine expression and NK cell regulation and receptors (Inhibition and activation). The cytotoxic activity of NK cells decreased in women with endometriosis and correlated with the severity of the disease, however the cytokine expression and inhibitor receptors are equivocal across studies. Limitations, reasons for caution We unable to conduct sub-group analysis to investigate the cytotoxic activity of NK cell in women with endometriosis and its stages in different samples such as pNK, uNK, and pfNK due to the lack of studies for the analysis. Wider implications of the findings This study revealed that the activity of NK cells in women with endometriosis is significantly lower than controls.Though the mean difference of NK cell count and percentage has no significant difference, the cytotoxicity activity of NK cell is higher in controls compared with women with endometriosis Trial registration number CRD42022384060

Early TRAIL-engagement elicits potent multimodal targeting of melanoma by CD34+ progenitor cell-derived NK cells

Umbilical cord blood (UCB) CD34+ progenitor cell-derived natural killer (NK) cells exert efficient cytotoxicity against various melanoma cell lines. Of interest, the relative cytotoxic performance of individual UCB donors was consistent throughout the melanoma panel and correlated with IFNγ, TNF, perforin and granzyme B levels. Importantly, intrinsic perforin and Granzyme B load predicts NK cell cytotoxic capacity. Exploring the mode of action revealed involvement of the activating receptors NKG2D, DNAM-1, NKp30, NKp44, NKp46 and most importantly of TRAIL. Strikingly, combinatorial receptor blocking led to more pronounced inhibition of cytotoxicity (up to 95%) than individual receptor blocking, especially in combination with TRAIL-blocking, suggesting synergistic cytotoxic NK cell activity via engagement of multiple receptors which was also confirmed in a spheroid model. Importantly, lack of NK cell-related gene signature in metastatic melanomas correlates with poor survival highlighting the clinical significance of NK cell therapies as a promising treatment for high-risk melanoma patients.

Human NK cells internalize recombinant major stress protein HSP70

Heat shock proteins 70 kDa (HSP70) protect intracellular proteins from the damaging effects of stress factors of various natures. Moreover, HSP70 play an important role in the vital activity of cells under normal physiological conditions, performing chaperone functions. These functions are realized in the intracellular space; however, in some cases, these proteins are also found on the cell surface and in the extracellular environment. The causes and mechanisms of HSP70 translocation to the cell surface and secretion into the extracellular space have not yet been well understood, but such an unusual localization of HSP70 activates the immune system. The surface HSP70 and their extracellular pool stimulate the cytotoxic activity of NK cells. However, direct experimental evidence for the internalization of HSP70 molecules by NK cells has not yet been demonstrated. This paper presents the results of the interaction of the extracellular HSP70 pool with NK cells from the peripheral blood. The results demonstrated the confirmation of the internalization of exogenous HSP70 molecules by NK cells. To this end, fluorescently labeled recombinant stress-inducible human HSP70 were obtained. The electrophoretic data indicated the absence of protein degradation during the labeling process, the purity and stability of the modified protein. To assess the interaction of HSP70 with NK cells, the fluorescently labeled HSP70 was added to an in vitro culture of NK cells isolated by magnetic separation from the peripheral blood mononuclear fraction and analyzed by confocal microscopy. This analysis indicated that living NK cells internalize extracellular HSP70 with localization both in lysosomes and in phagosomes. Our experiments illustrated for the first time the process of penetration of the extracellular form of HSP70 into these cells. The results suggest that the activation of NK cells under the action of exogenous HSP70 could be associated with the internalization of these protein molecules.

Development of anti-GD2 Antibody-producing Mesenchymal Stem Cells as Cellular Immunotherapy.

Using the tyrosine hydroxylase (TH)-MYCN mouse neuroblastoma (NB) model, we have previously reported the accumulation of mouse mesenchymal stem cells (mMSCs) on tumors in vivo and the antitumor effect of mMSCs transfected with a small molecule (IFN-β) expression gene. In this study, we have developed novel MSCs secreting anti-disialoganglioside GD2 antibody (anti-GD2-MSCs) and evaluated their antitumor effects in vitro. We generated an anti-GD2 antibody construct (14.G2a-Fcx2-GFP) incorporating FLAG-tagged single-chain fragment variable against GD2 fused to a linker sequence, a fragment of the constant portion of human IgG1, and GFP protein. The construct was lentivirally transduced into mMSCs and the transduction efficiency was assessed by GFP expression. The secretion of FLAG-tagged anti-GD2 antibody was detected by Western blotting using anti-FLAG antibody. Antibody binding capacity was confirmed by flow cytometry. Antibody-dependent cellular cytotoxicity (ADCC) was evaluated using human NB cells and human natural killer (NK) cells to assess whether the antitumor activity was enhanced in the presence of the produced antibodies. The transduction efficiency of anti-GD2-MSCs was more than 90%. anti-GD2-MSCs secreted antibodies extracellularly and these antibodies had high affinity to GD2-expressing human NB cells. ADCC assays showed that the addition of antibodies secreted from anti-GD2-MSCs significantly increased the cytotoxic activity of NK cells against NB cells. Newly developed anti-GD2-MSCs produced functional antibodies that have affinity to the GD2 antigen on NB cells and can induce ADCC-mediated cytotoxicity. Anti-GD2-MSCs based cellular immunotherapy has the potential to be a novel therapeutic option for intractable NB.