HLA class I NK-epitopes and KIR diversities in patients with multiple myeloma.

Multiple myeloma (MM) is a hematological malignancy caused by the clonal expansion of malignant plasma cells in the bone marrow. Myeloma cells are susceptible to killing by natural killer (NK) cells, but NK cells fail to control disease progression, suggesting immunosuppression. The activation threshold of NK-effector function is regulated by interaction between KIRs and self-HLA class I, during a process called "education" to ensure self-tolerance. NK cells can respond to diseased cells based on the absence of HLA class I expression ("Missing-self" hypothesis). The HLA and KIR repertoire is extremely diverse; thus, the present study aimed to characterize potential variances in genotypic composition of HLA Class I NK-epitopes and KIRs between MM patients and healthy controls. Genotypic expression of KIR and HLA (HLA-C group-C1/C2 and Bw4 motifs (including HLA-A*23, A*24, A*32) were analyzed in 172 MM patients and 195 healthy controls. Compared to healthy controls, we did not observe specific KIR genes or genotypes, or HLA NK-epitopes with higher prevalence among MM patients. The presence of all three HLA NK-epitopes (C1+C2+Bw4+) was not associated with MM occurrence. However, MM patients were more likely to be C1-/C2+/Bw4+ (p = 0.049, OR 1.996). In line with this, there was a trend of increased genetic co-occurrence of Bw4 and KIR3DL1 in MM patients (p = 0.05, OR 1.557). Furthermore, MM patients were more likely to genetically express both C2/KIR2DL1 and Bw4/KIR3DL1 (p = 0.019, OR 2.453). Our results reveal an HLA NK-epitope combination that is associated with the occurrence of MM. No specific KIR genotypes were associated with MM.

Antibody-dependent cellular cytotoxicity-inducing antibodies enhance the natural killer cell anti-cancer response against patient-derived pancreatic cancer organoids.

Introduction: Pancreatic cancer is associated with poor prognosis, and limited treatment options are available for the majority of patients. Natural killer (NK) cells in combination with antibodies inducing antibody-dependent cell-mediated cytotoxicity (ADCC) could be a highly effective new therapeutic option in pancreatic cancer. Accurate predictive preclinical models are needed to develop successful NK cell immunotherapy. Tumor organoids, in vitro 3D organ-like structures that retain important pathophysiological characteristics of the in vivo tumor, may provide such a model. In the current study, we assessed the cytotoxic potential of adoptive NK cells against human pancreatic cancer organoids. We hypothesized that NK cell anti-tumor responses could be enhanced by including ADCC-triggering antibodies. Methods: We performed cytotoxicity assays with healthy donor-derived IL-2-activated NK cells and pancreatic cancer organoids from four patients. A 3D cytotoxicity assay using live-cell-imaging was developed and enabled real-time assessment of the response. Results: We show that NK cells migrate to and target pancreatic cancer organoids, resulting in an increased organoid death, compared to the no NK cell controls (reaching an average fold change from baseline of 2.1±0.8 vs 1.4±0.6). After 24-hours of co-culture, organoid 2D growth increased. Organoids from 2 out of 4 patients were sensitive to NK cells, while organoids from the other two patients were relatively resistant, indicating patient-specific heterogeneity among organoid cultures. The ADCC-inducing antibodies avelumab (anti-PD-L1) and trastuzumab (anti-HER2) increased NK cell-induced organoid cell death (reaching an average fold change from baseline of 3.5±1.0 and 4.5±1.8, respectively). Moreover, combination therapy with avelumab or trastuzumab resulted in complete disintegration of organoids. Finally, inclusion of ADCC-inducing antibodies was able to overcome resistance in NK-organoid combinations with low or no kill. Discussion: These results support the use of organoids as a relevant and personalized model to study the anti-tumor response of NK cells in vitro and the potential of ADCC-inducing antibodies to enhance NK cell effector function.

An in vitro model to monitor natural killer cell effector functions against breast cancer cells derived from human tumor tissue.

Adoptive natural killer (NK) cell-based immunotherapy poses a promising treatment approach in cancer. Despite minimal toxicities associated with NK cell infusion, the potential of NK cell therapy is inhibited by the immunosuppressive tumor microenvironment (TME). Multiple approaches to improve anti-cancer NK cell effector functions are being investigated. While much of this preclinical research is currently performed with commercially available tumor cell lines, this approach lacks the influence of the TME and heterogeneity of the primary tumor in patients. Here, we describe a comprehensive protocol for NK cell cytotoxicity- and degranulation assays against tumor cells derived from primary breast cancer tissue. Treatments to boost NK cell anti-tumor effector functions can be implemented in this model. Moreover, by using culture supernatants in follow up assays or by including additional cell types in the co-culture system, other NK cell effector mechanisms that further orchestrate innate and adaptive immunity could be studied.

Inhibitory receptors for HLA class I as immune checkpoints for natural killer cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy.

Natural killer (NK) cells mediate potent anti-tumor responses, which makes them attractive targets for immunotherapy. The anti-tumor response of endogenous- or allogeneic NK cells can be enhanced through clinically available monoclonal antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is regulated by interaction of inhibitory receptors with classical- and non-classical human leukocyte antigens (HLA) class I molecules. Inhibitory receptors of the killer immunoglobulin-like receptor (KIR) family interact with HLA-A, -B or -C epitopes, while NKG2A interacts with the non-classical HLA-E molecule. Both types of inhibitory interactions may influence the strength of the ADCC response. In the present review, we provide an overview of the effect of inhibitory KIRs and NKG2A on NK cell-mediated ADCC, which highlights the rationale for combination strategies with ADCC triggering antibodies and interference with the NK cell relevant inhibitory immune checkpoints, such as KIR and NKG2A.

The Immunomodulatory Role of Hypoxic Tumor-Derived Extracellular Vesicles.

Tumor-associated immune cells frequently display tumor-supportive phenotypes. These phenotypes, induced by the tumor microenvironment (TME), are described for both the adaptive and the innate arms of the immune system. Furthermore, they occur at all stages of immune cell development, up to effector function. One major factor that contributes to the immunosuppressive nature of the TME is hypoxia. In addition to directly inhibiting immune cell function, hypoxia affects intercellular crosstalk between tumor cells and immune cells. Extracellular vesicles (EVs) play an important role in this intercellular crosstalk, and changes in both the number and content of hypoxic cancer-cell-derived EVs are linked to the transfer of hypoxia tolerance. Here, we review the current knowledge about the role of these hypoxic cancer-cell-derived EVs in immunosuppression. In addition, we provide an overview of hypoxia-induced factors (i.e., miRNA and proteins) in tumor-derived EVs, and their role in immunomodulation.

ADCC-Inducing Antibody Trastuzumab and Selection of KIR-HLA Ligand Mismatched Donors Enhance the NK Cell Anti-Breast Cancer Response.

Natural killer (NK)-cell-based immunotherapies are an attractive treatment option for cancer. We previously showed that alloreactive mouse NK cells cured mice of 4T1 breast cancer. However, the tumor microenvironment can inhibit immune responses, and these suppressive factors must be overcome to unfold the NK cells' full anti-tumor potential. Here, we investigated the combination of antibody-dependent cellular cytotoxicity (ADDC) and the selection of KIR-HLA-ligand mismatched NK cells to enhance NK cell anti-breast cancer responses in clinically relevant settings. Donor-derived and IL-2-activated NK cells were co-cultured with patient-derived breast cancer cells or cell lines MCF7 or SKBR3 together with the anti-HER2 antibody trastuzumab. NK cells mediated anti-breast cancer cytotoxicity under normoxic and hypoxic conditions. Under both conditions, trastuzumab vigorously enhanced NK cell degranulation (CD107a) against HER2-overexpressing SKBR3 cells, but we observed a discrepancy between highly degranulating NK cells and a rather modest increase in cytotoxicity of SKBR3. Against patient-derived breast cancer cells, the anti-tumor efficacy was rather limited, and HLA class I expression seemed to contribute to inhibited NK cell functionality. KIR-ligand-mismatched NK cells degranulated stronger compared to the matched NK cells, further highlighting the role of HLA. In summary, trastuzumab and KIR-ligand-mismatched NK cells could be two strategies to potently enhance NK cell responses to breast cancer.

Genome-scale screens identify factors regulating tumor cell responses to natural killer cells.

To systematically define molecular features in human tumor cells that determine their degree of sensitivity to human allogeneic natural killer (NK) cells, we quantified the NK cell responsiveness of hundreds of molecularly annotated 'DNA-barcoded' solid tumor cell lines in multiplexed format and applied genome-scale CRISPR-based gene-editing screens in several solid tumor cell lines, to functionally interrogate which genes in tumor cells regulate the response to NK cells. In these orthogonal studies, NK cell-sensitive tumor cells tend to exhibit 'mesenchymal-like' transcriptional programs; high transcriptional signature for chromatin remodeling complexes; high levels of B7-H6 (NCR3LG1); and low levels of HLA-E/antigen presentation genes. Importantly, transcriptional signatures of NK cell-sensitive tumor cells correlate with immune checkpoint inhibitor (ICI) resistance in clinical samples. This study provides a comprehensive map of mechanisms regulating tumor cell responses to NK cells, with implications for future biomarker-driven applications of NK cell immunotherapies.

Consumption of Nitrate-Rich Beetroot Juice with or without Vitamin C Supplementation Increases the Excretion of Urinary Nitrate, Nitrite, and N-nitroso Compounds in Humans.

Consumption of nitrate-rich beetroot juice (BRJ) by athletes induces a number of beneficial physiological health effects, which are linked to the formation of nitric oxide (NO) from nitrate. However, following a secondary pathway, NO may also lead to the formation of N-nitroso compounds (NOCs), which are known to be carcinogenic in 39 animal species. The extent of the formation of NOCs is modulated by various other dietary factors, such as vitamin C. The present study investigates the endogenous formation of NOCs after BRJ intake and the impact of vitamin C on urinary NOC excretion. In a randomized, controlled trial, 29 healthy recreationally active volunteers ingested BRJ with or without additional vitamin C supplements for one week. A significant increase of urinary apparent total N-nitroso Compounds (ATNC) was found after one dose (5 to 47 nmol/mmol: p < 0.0001) and a further increase was found after seven consecutive doses of BRJ (104 nmol/mmol: p < 0.0001). Vitamin C supplementation inhibited ATNC increase after one dose (16 compared to 72 nmol/mmol, p < 0.01), but not after seven daily doses. This is the first study that shows that BRJ supplementation leads to an increase in formation of potentially carcinogenic NOCs. In order to protect athlete's health, it is therefore important to be cautious with chronic use of BRJ to enhance sports performances.

RAGE deficiency does not affect non-alcoholic steatohepatitis and atherosclerosis in Western type diet-fed Ldlr-/- mice.

Non-alcoholic fatty liver disease is a spectrum of liver diseases ranging from steatosis only to non-alcoholic steatohepatitis (NASH). The latter is characterized by hepatic inflammation, which increases the risk of cardiovascular disease. It is poorly understood which factors contribute to the onset of hepatic inflammation characterizing the progression from steatosis to NASH. Previously, we demonstrated increased advanced glycation endproducts (AGEs) in the livers of NASH patients. We hypothesise that AGEs play a key role in NASH development by activating their proinflammatory receptor, RAGE. RAGE-deficient mice and wildtype littermates, both on Ldlr-/- background, were fed a Western type diet (WTD) for 3 or 12 weeks. Flow cytometry, histology, gene expression and AGE measurements were performed to evaluate the effects of RAGE deficiency. RAGE-deficient mice displayed reduced weight gain and visceral fat expansion compared to control mice. No difference in adipose tissue inflammation was observed between groups. RAGE deficiency did not affect WTD-induced monocytosis, circulating lipids or hepatic steatosis. WTD-induced hepatic neutrophil and macrophage accumulation and atherosclerotic plaque development was comparable between control and RAGE-deficient mice. No difference in AGE levels was observed. RAGE does not seem to play a major role in the development of NASH or atherosclerosis in a hyperlipidemic mouse model.