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Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1–CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Beata Kaleta,1 Mieszko Lachota,1,2 Jacek Łukaszkiewicz,3 Anna Woźniacka,4 Jarosław Bogaczewicz5 1Department of Clinical Immunology, Medical University of Warsaw, Warsaw, Poland; 2Doctoral School, Medical University of Warsaw, Warsaw, Poland; 3Department of Biochemistry and Clinical Chemistry, Medical University of Warsaw, Warsaw, Poland; 4Department of Dermatology and Venereology, Medical University of Lodz, Lodz, Poland; 5University of Economics and Human Sciences in Warsaw, Warsaw, PolandCorrespondence: Beata KaletaDepartment of Clinical Immunology, Medical University of Warsaw, Nowogrodzka 59 Street, Warsaw, 02-006, PolandTel +48 600 301 690Fax +48 502 21 59Email beata.kaleta@wum.edu.plPurpose: Atopic dermatitis (AD) is a chronic, relapsing inflammatory disease, caused by environmental and genetic factors, which lead to immunological abnormalities. Osteopontin (OPN), also named secreted phosphoprotein 1 (SPP1), is a protein involved in the pathogenesis of numerous autoimmune and inflammatory conditions. However, its role in AD has not been fully elucidated. Therefore, we aim to gain an insight into the role of OPN in AD pathogenesis through investigating its gene single nucleotide polymorphisms (SNPs) and their possible associations with disease clinical features.Patients and Methods: A total of 182 Caucasian participants (45 AD patients and 137 gender- and age-matched controls) were studied. Genomic DNA was isolated from peripheral blood samples. Genotyping for the rs1126616 C>T, rs1126772 A>G, rs9138 A>C, and rs3841116 T>G SNPs was performed by real time polymerase chain reaction (RT-PCR).Results: The frequency of the minor TT genotype and the T allele of rs1126616 C>T was higher in AD patients compared to controls (P = 0.019, OD = 4.86, 95% CI = 1.46– 16.20, and P = 0.047, OR = 1.77, 95% CI = 1.04– 3.00, respectively) and was associated with the higher prevalence of asthma (P = 0.017, OR = 3.73, 95% CI = 0.71– 19.67, and P = 0.004, OR = 3.96, 95% CI = 1.53– 10.25, respectively). Likewise, the minor GG genotype and the G allele of rs1126772 A>G were more frequent in AD patients (P = 0.026, OR = 3.27, 95% CI = 1.29– 8.33, and P = 0.013, OR = 1.94, 95% CI = 1.18– 3.21, respectively) and were associated with the increased incidence of asthma (P = 0.016, OR = 5.06, 95% CI = 1.14– 22.49, and P = 0.002, OR = 4.40, 95% CI = 1.71– 11.35, respectively). Furthermore, haplotype frequency estimation determined the four-loci haplotype TGCT, as a significant risk factor for AD compared to controls (P = 0.031, OR = 9.48, 95% CI = 1.23– 71.91).Conclusion: Our results suggest that the variation in the OPN gene might be associated with AD and increased incidence of asthma in Caucasians. Further studies should be conducted to look into the possible role of OPN as a biomarker for AD.Keywords: asthma, atopic dermatitis, gene, osteopontin, polymorphism, AD, OPN

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy caused by accumulation of immature B cells. Advances in the field, especially in genomic studies and new treatment development, significantly improved the prognosis of B-ALL over the last decades. However, the B-ALL subtype harboring Philadelphia chromosome t(9;22)(q34;q11) (Ph+ B-ALL) still remains a therapeutic challenge despite available targeted treatment with tyrosine kinase inhibitors (TKIs). The novel therapeutic regimens for adult B-ALL often include rituximab (RTX) - a monoclonal anti-CD20 antibody - for patients with >20% CD20+ blasts. Moreover, drug combinations including approved TKIs and antibodies are being tested in clinical trials. Since some TKIs display immune effector cell inhibition, investigating their immunomodulatory effects in the context of combined chemo-immunotherapies is clinically relevant for the rational combination design. We found that blasts isolated from Ph+ B-ALL patients showed the highest CD20 expression on mRNA level among defined B-ALL genetic subtypes. As Ph+ CD20+ patients may be qualified for treatment with TKIs along with RTX, this result prompted the need of investigating the effects of TKIs on immune effectors. We therefore compared how first-, second- and third-generation TKIs (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) and allosteric inhibitor asciminib recently approved for the treatment of chronic myeloid leukemia (CML) affect innate and antibody-mediated cytotoxicity. B-ALL cell lines, patient-derived lymphoblasts propagated in NSG mice (primografts) and ex vivo whole blood assays were used for testing the efficacy of effector cell-mediated mechanisms in the presence of TKIs. The TKIs were compared in NK-cell based natural cytotoxicity tests, antibody-dependent cytotoxicity (ADCC) tests and phagocytosis assays with monocyte-derived macrophages. Dasatinib was also tested using whole blood ex vivo ADCC assays before and after administration of the TKI. Out of six tested TKIs, asciminib presented the most favorable profile, causing slight or no effector cell inhibition in both innate and antibody-mediated responses. In contrast, dasatinib significantly decreased the immune cells activity in all tests, reducing natural and RTX-mediated cytotoxicity of NK cells against B-ALL blasts. Dasatinib and ponatinib significantly inhibited in vitro phagocytosis of primograft B-ALL cells. To a lesser extent, cytotoxicity and phagocytosis were suppressed by imatinib, bosutinib and nilotinib. Importantly, we confirmed that the orally administered dasatinib impairs the NK cell activity in patients’ blood. Our findings indicate that the TKIs differentially impact RTX-mediated effector mechanisms. The novel, allosteric TKI asciminib could be preferentially used in combination with RTX-based immunotherapy. This work was supported by the Polish National Science Centre grant 2019/35/B/NZ5/01428 and by the Ministry of Education and Science within “Regional Initiative of Excellence” program 013/RID/2018/19." Citation Format: Krzysztof Domka, Martyna Poprzeczko, Zuzanna Urbanska, Lukasz Komorowski, Agata Pastorczak, Klaudyna Fidyt, Agnieszka Dabkowska, Mieszko Lachota, Magdalena Winiarska, Karolina Siudakowska, Elzbieta Patkowska, Łukasz Sędek, Bartosz Perkowski, Beata Krzymieniewska, Malgorzata Firczuk. Optimizing the therapeutic potential of tyrosine kinase inhibitors in chemo-immunotherapy of B-cell acute lymphoblastic leukemia involving rituximab [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 3324.

Tumor-infiltrating immune cells are capable of effective cancer surveillance, and their abundance is linked to better prognosis in numerous tumor types. However, in uveal melanoma (UM), extensive immune infiltrate is associated with poor survival. This study aims to decipher the role of different tumor-infiltrating cell subsets in UM in order to identify potential targets for future immunotherapeutic treatment. We have chosen the TCGA-UVM cohort as a training dataset and GSE22138 as a testing dataset by mining publicly available databases. The abundance of 22 immune cell types was estimated using CIBERSORTx. Then, to determine the significance of tumor-infiltrating cell subsets in UM, we built a multicell type prognostic signature, which was validated in the testing cohort. The created signature was built upon the negative prognostic role of CD8+ T cells and M0 macrophages and the positive role of neutrophils. Based on the created signature score, we divided the patients into low- and high-risk groups. Kaplan-Meier, Cox, and ROC analyses demonstrated superior performance of our risk score compared to either clinical or pathologic characteristics of both cohorts. Further, we found the molecular pathways associated with cancer immunoevasion and metastasis to be enriched in the high-risk group, explaining both the lack of adequate immune surveillance despite increased infiltration of CD8+ T cells as well as the higher metastatic potential. Genes associated with tryptophan metabolism (IDO1 and KYNU) and metalloproteinases were among the most differentially expressed between the high- and low-risk groups. Our correlation analyses interpreted in context of published in vitro data strongly suggest the central role of CD8+ T cells in shifting the UM tumor microenvironment towards suppressive and metastasis-promoting. Therefore, we propose further investigations of IDO1 and metalloproteinases as novel targets for immunotherapy in lymphocyte-rich metastatic UM patients.

Chemokines play an integral role in cancer immunoevasion by modulating leukocyte infiltration. They selectively increase the migration of immunosuppressive cells while impairing the infiltration of cytotoxic lymphocytes with potent antitumor activity. Some cancer-associated chemokines may also enhance tumor progression by acting as growth factors or promoting epithelial-mesenchymal transition. Here, we acquired fresh human glioblastoma (GBM) tissues to define chemokine expression patterns in GBM tumors by integrating bulk and single-cell RNA sequencing of both tumor and healthy brain tissues along with Luminex secretome profiling of GBM primary cell cultures. Extensive mapping of GBM-associated chemokines has enabled us to gain insight into intratumoral chemokine-mediated cell-to-cell communication. Among others, we identified CCL7 and midkine (MDK) to play a crucial role in the formation of the hostile tumor microenvironment (TME) in GBM. We hypothesize that these chemoattractants can facilitate the GBM tumor growth by recruiting M2 macrophages, promoting epithelial-mesenchymal transition, and activating the prosurvival PI3K-Akt pathway. Indeed, high expression of both chemokines was linked to poor overall survival. Additionally, by integrating the data, we further identified the cellular source of the most abundantly secreted chemokines in GBM tumors. Since immune cells require TME infiltration to mediate an appropriate antitumor response, impaired tumor infiltration is one of the critical limiting factors for cancer cell therapy. Our chemokine atlas provides novel targets to enhance adoptive cell therapies by improving the potential of effector cells for tumor homing and anticancer activities in GBM. This research was funded by the National Science Centre, Poland (2020/37/B/NZ6/02191), the Polish Ministry of Science and Higher Education (grant number DI2018 020548), and the National Centre for Research and Development (STRATEGMED3/307326/6/NCBR/2017).

Purpose To evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV). Methods Nine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 μm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 μg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation. Results IMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1wt/gfp mice. In vitro IMD-0354 (10–25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity. Conclusion The present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD.

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-kappa B signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-kappa B activation through selective blockade of the IKK complex I kappa B kinase beta (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNF alpha-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-alpha expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-kappa B by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-kappa B signaling in the development of pathologic corneal neovascularization. Funding Agencies|Swedish Research Council [2012-2472]; Swedish Foundation Stiftelsen Synframjandets Forskningsfond/Ogonfonden; Svenska Sallskapet for Medicinsk Forskning; Linkoping Universitet; Jeanssons Stiftelser

Natural Killer (NK) cells play an essential role in cancer surveillance and have a unique capability of spontaneous cytotoxicity against cancer cells. The human NK cell repertoire is functionally diversified through a tightly regulated differentiation process characterized by an early transition from CD56bright to CD56dim NK cells, followed by coordinated changes in expression of inhibitory receptors, including NKG2A and killer cell immunoglobulin-like receptors (KIR). The acquisition of self HLA class I binding KIRs during NK cell differentiation tunes the cytotoxic potential of NK cells in a process termed education, characterized by increased loading of granzyme B in dense core granules. Although NK cell differentiation and education are critical determinants of the functional potential of the cell, little is known about how these events shape the migratory behavior of NK cells. To mediate appropriate and directed immune response against cancer, NK cells must be capable of migration to the tumor site. This process is mediated by chemokines, which guide cell migration by binding to their specific receptors. For example, in multiple myeloma, CXCR3 and CCR5 ligands (MIG, IP-10, and MIP-1a) are significantly upregulated in the bone marrow compared to healthy controls, affecting the composition of immune cells in the tumor microenvironment. In order to delineate the homing patterns of distinct NK cell subsets, we used high-dimensional flow cytometry combined with functional assays to map the NK cell chemokine receptor expression and migratory behavior. We screened resting and cytokine/feeder cell stimulated peripheral blood NK cells for the expression of a panel of 20 chemokine receptors (A). Based on CD56, CD57, NKG2A, and KIR expression, NK cells were divided into 6 phenotypically and functionally distinct subsets that were ordered according to their differentiation status (B). We found that the expression of CX3CR1, CXCR1, CXCR2, and CMKLR1 gradually increased during differentiation, whereas the expression of CXCR3, CCR7, and CCR5 was lower in more differentiated NK cells. CXCR4, CCR4, and CCR2 expression was relatively uniform across all subsets. Interestingly, CCR1 and CXCR6 were expressed mainly on less differentiated NKG2A+ CD56dim NK cells (B). Next, we stratified the chemokine receptor expression on mature KIR+ NK cells based on the expression of self (educated) or non-self KIR (uneducated). Educated NK cells expressed CXCR1, CX3CR1, CCR5, and CMKLR1 at higher levels than the uneducated NK cells. Conversely, CXCR3 was expressed at lower levels on educated NK cells (C). No difference was observed for CXCR2 expression. To determine whether the observed differences in chemokine receptor expression translate into altered chemokine responsiveness between the subsets, we combined the transwell system with multicolor flow cytometry. We found that the chemokine-induced migration capability of NK cells correlated closely with the expression level of corresponding chemokine receptor, leading to subset specific responses to various chemokine gradients (D). The present results show that peripheral blood NK cell chemokine receptor profile changes in a coordinated fashion during NK cell differentiation and is further influenced by the expression of self-specific KIR. Interestingly, receptors which expression declines during NK cell differentiation (CCR5, CCR7, and CXCR3) are commonly associated with adaptive T cell responses to viruses, whereas receptors that are upregulated along the differentiation axis (CXCR1, CXCR2, CX3CR1, CMKLR1) are typical for neutrophils and macrophages as a part of the innate immune response. Thus, our results suggest that NK cell differentiation and education processes together shape the NK cell migratory capabilities to promote homing of the most functional NK cell subsets to the site of inflammation and serve as the first line of defense in the immune response to pathogens and tumors. Figure Disclosures Malmberg: Fate Therapeutics: Consultancy, Patents & Royalties; Vycellix: Membership on an entity's Board of Directors or advisory committees.

Simple Summary Sarcomas are a group of aggressive tumors originating from mesenchymal tissues. Patients with advanced disease have poor prognosis due to the ineffectiveness of current treatment protocols. A subset of lymphocytes called natural killer (NK) cells is capable of effective surveillance and clearance of sarcomas, constituting a promising tool for immunotherapeutic treatment. However, sarcomas can cause impairment in NK cell function, associated with enhanced tumor growth and dissemination. In this review, we discuss the molecular mechanisms of sarcoma-mediated suppression of NK cells and their implications for the design of novel NK cell-based immunotherapies against sarcoma. Abstract Natural killer (NK) cells are innate lymphoid cells with potent antitumor activity. One of the most NK cell cytotoxicity-sensitive tumor types is sarcoma, an aggressive mesenchyme-derived neoplasm. While a combination of radical surgery and radio- and chemotherapy can successfully control local disease, patients with advanced sarcomas remain refractory to current treatment regimens, calling for novel therapeutic strategies. There is accumulating evidence for NK cell-mediated immunosurveillance of sarcoma cells during all stages of the disease, highlighting the potential of using NK cells as a therapeutic tool. However, sarcomas display multiple immunoevasion mechanisms that can suppress NK cell function leading to an uncontrolled tumor outgrowth. Here, we review the current evidence for NK cells’ role in immune surveillance of sarcoma during disease initiation, promotion, progression, and metastasis, as well as the molecular mechanisms behind sarcoma-mediated NK cell suppression. Further, we apply this basic understanding of NK–sarcoma crosstalk in order to identify and summarize the most promising candidates for NK cell-based sarcoma immunotherapy.

Simple Summary Natural killer cells are able to effectively eliminate tumor cells without previous sensitization, therefore the interest in their application in tumor immunotherapy has recently increased. However, tumor cells synergize with tumor-associated cells to create a specific immunosuppressive niche, which limits the activity of NK cells. In this review, we provide a detailed description of molecular mechanisms responsible for NK cells’ cytotoxic machinery. Moreover, we shortly characterize the tumor microenvironment and summarize how various metabolic factors within the tumor niche modulate antitumor capacity of NK cells. Moreover, we discuss the potential strategies and implications for the novel antitumor therapies augmenting NK cells functions. Abstract NK cells have unique capabilities of recognition and destruction of tumor cells, without the requirement for prior immunization of the host. Maintaining tolerance to healthy cells makes them an attractive therapeutic tool for almost all types of cancer. Unfortunately, metabolic changes associated with malignant transformation and tumor progression lead to immunosuppression within the tumor microenvironment, which in turn limits the efficacy of various immunotherapies. In this review, we provide a brief description of the metabolic changes characteristic for the tumor microenvironment. Both tumor and tumor-associated cells produce and secrete factors that directly or indirectly prevent NK cell cytotoxicity. Here, we depict the molecular mechanisms responsible for the inhibition of immune effector cells by metabolic factors. Finally, we summarize the strategies to enhance NK cell function for the treatment of tumors.

Inflammation in the normally immune-privileged cornea can initiate a pathologic angiogenic response causing vision-threatening corneal neovascularization. Inflammatory pathways, however, are numerous, complex and are activated in a time-dependent manner. Effective resolution of inflammation and associated angiogenesis in the cornea requires knowledge of these pathways and their time dependence, which has, to date, remained largely unexplored. Here, using a model of endogenous resolution of inflammation-induced corneal angiogenesis, we investigate the time dependence of inflammatory genes in effecting capillary regression and the return of corneal transparency. Endogenous capillary regression was characterized by a progressive thinning and remodeling of angiogenic capillaries and inflammatory cell retreat in vivo in the rat cornea. By whole-genome longitudinal microarray analysis, early suppression of VEGF ligand-receptor signaling and inflammatory pathways preceded an unexpected later-phase preferential activation of LXR/RXR, PPARα/RXRα and STAT3 canonical pathways, with a concurrent attenuation of LPS/IL-1 inhibition of RXR function and Wnt/β-catenin signaling pathways. Potent downstream inflammatory cytokines such as Cxcl5, IL-1β, IL-6 and Ccl2 were concomitantly downregulated during the remodeling phase. Upstream regulators of the inflammatory pathways included Socs3, Sparc and ApoE. A complex and coordinated time-dependent interplay between pro- and anti-inflammatory signaling pathways highlights a potential anti-inflammatory role of LXR/RXR, PPARα/RXRα and STAT3 signaling pathways in resolving inflammatory corneal angiogenesis. Electronic supplementary material The online version of this article (10.1007/s10456-018-9604-y) contains supplementary material, which is available to authorized users.

Introduction Dasatinib is a potent small molecule kinase inhibitor targeting BCR-ABL kinase - oncogenic driver in Philadelphia chromosome-positive (Ph+) cases of chronic myelogenous leukemia (CML). In addition to BCR-ABL kinase, it also targets a broad array of other kinases, affecting not only leukemia cells but also immune cells. Just one hour after dasatinib oral administration a rapid increase of NK, NKT, T and B cells is observed in peripheral blood. Dasatinib has been also shown to influence NK cell cytotoxicity, however, the results are discordant. Some groups observe potentiation of NK cell cytotoxic activity while others strong inhibitory effects. These inconsistencies may be explained by differences in in vitro protocols used to study this phenomenon. Aim Our study aims to investigate dasatinib influence on immune cells in whole blood assays resembling physiological conditions observed in patients. In particular, we want to investigate dasatinib effect on NK cell mobilization, degranulation, and anti-tumor immunity. In light of clinical and pre-clinical studies involving dasatinib and the importance of NK cells in cancer, it is crucial to establish the mechanisms and kinetics of dasatinib immunomodulatory activity. Methods Before and one hour after first dasatinib administration peripheral blood was collected from CML patients. Collected whole blood was directly added to the target K562 cell line. After co-incubation, erythrocytes were lysed, cells were stained with a panel of monoclonal antibodies and analyzed with flow cytometry. A relative increase in lymphocyte count was determined by Trucount Tubes (BD). Dasatinib effect on NK cells in vitro was studied with degranulation and cytotoxicity assays using NK cells isolated from healthy volunteers PBMCs. Dasatinib at clinically relevant concentrations (20-200mM) was used to assess its effect on NK cell degranulation, cytotoxicity, cytokine, and chemokine production with flow cytometry upon staining with anti-CD107a, TNF-α, IFN-γ and CCL-4 monoclonal antibodies. For in vivo experiments C57BL/6 mice were inoculated with EL4 tumor cell line stably expressing luciferase and human CD20. 3 days after tumor inoculation mice were treated with dasatinib or vehicle intraperitoneally (i.p.) in a dose of 30 mg/kg. To monitor tumor growth, mice were injected with luciferin and imaged using the IVIS system. Results In agreement with previous reports, we confirm NK, NKT, T and B cell count increase in peripheral blood after dasatinib administration. To evaluate how dasatinib influences NK cell cytokine and chemokine production we stimulated NK cells with K562 cell line. Production of major proinflammatory cytokines secreted by NK cells, TNF-α and IFN-γ, was inhibited by dasatinib treatment. Production of MIP-1β (CCL4), a chemokine secreted by NK cells attracting a broad spectrum of immune cells to inflammation sites, was also profoundly decreased. According to our findings, dasatinib presence during the cytotoxicity assay, in a dose-dependent manner, inhibits NK cell cytotoxicity. However, 24-hour dasatinib pretreatment increases their cytotoxic potential. To better mimic the physiological conditions we used whole blood degranulation assay which closely resembles patient settings, including dasatinib concentration. One hour after dasatinib intake we observed a potent inhibitory effect of dasatinib on NK cell degranulation. Additionally, we observed a shift in NK cell subpopulations - dasatinib present during degranulation assay decreases CD16⁻ NK cell number. Finally, we evaluated the influence of high-dose dasatinib treatment on tumor rejection in mice. Mice treated with dasatinib exhibit significantly increased tumor growth compared with vehicle-treated mice. Conclusions Using whole blood degranulation assay and in vitro degranulation and cytotoxicity assays we report that dasatinib effect on NK cell cytotoxicity is dose- and time-dependent. Our results indicate that dasatinib has a dual effect on NK cell degranulation and affects other NK cell functions including cytokine production and migration. Further studies are needed to evaluate the significance of these findings. Disclosures No relevant conflicts of interest to declare.