Inhibition of NK cell cytotoxicity by tubular epithelial cell expression of Clr-b and Clr-f

Dasatinib Effect on NK Cells and Anti-Tumor Immunity

Murine cytomegalovirus (MCMV) infection of mice induces early cytokines. Although certain of these can directly inhibit viral replication, they also can promote defense by activating NK cells. MCMV induces IFN-alphabeta-dependent NK cell cytotoxicity and IL-12-dependent NK cell IFN-gamma production. Studies were initiated to define cytokine-mediated NK and T cell-independent antiviral defense and specific cytokine-elicited NK cell responses during MCMV infections. IFN-alphabeta, TNF, IL-12, and IFN-gamma were all shown to be induced 2 days after infection of immunocompetent mice. Infections of NK and T cell-deficient mice demonstrated that virus-induced IFN-alphabeta, TNF, and IL-12, but not IFN-gamma, were produced independently of these populations, and that IL-12 production occurred in the absence of detectable IFN-gamma. In vivo neutralization studies of IFN-alphabeta, TNF, and IL-12 showed that each of these factors had NK and T cell-independent antiviral functions, as well as specific effects on NK cell responses. Examination of NK cell cytotoxicity, blastogenesis, and IFN-gamma production demonstrated that: IL-12 was required for NK cell IFN-gamma production but not blastogenesis and cytotoxicity; IFN-alphabeta was necessary for NK cell blastogenesis and cytotoxicity but not IFN-gamma production; and TNF facilitated IFN-gamma production but inhibited NK cell cytotoxicity. This work defines the biologic consequences of early cytokine expression during viral infection.

To the editor: Murine gammaherpesvirus 68 (MHV-68) escapes from NK-cell-mediated immune surveillance by a CEACAM1-mediated immune evasion mechanism.

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 < 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

NK cells are important innate immune cells with potent cytotoxicity that can be activated by type I IFN from the host once infected. How NK cell cytotoxicity is activated by type I IFN and then tightly regulated remain to be fully elucidated. MicroRNAs (miRNAs, or miRs) are important regulators of innate immune response, but the full scale of miRNome in human NK cells remains to be determined. In this study, we reported an in-depth analysis of miRNomes in resting and IFN-α-activated human NK cells, found two abundant miRNAs, miR-378 and miR-30e, markedly decreased in activated NK cells by IFN-α, and further proved that miR-378 and miR-30e directly targeted granzyme B and perforin, respectively. Thus, IFN-α activation suppresses miR-378 and miR-30e expression to release cytolytic molecule mRNAs for their protein translation and then augments NK cell cytotoxicity. Importantly, the phenomena are also confirmed in human NK cells activated by other cytokines and even in the sorted CD16(+)CD56(dim)CD69(+) human NK cell subset. Finally, miR-378 and miR-30e were proved to be suppressors of human NK cell cytotoxicity. Taken together, our results reveal that downregulated miR-378 and miR-30e during NK cell activation are negative regulators of human NK cell cytotoxicity, providing a mechanistic explanation for regulation of NK cell function by miRNAs.

Bone marrow‐derived mesenchymal stem cells inhibit NK cell function via Tim‐3/galectin‐9 in multiple myeloma patients

Chemotherapy is currently regarded as a potential ally for cancer immunotherapy. Several anticancer agents, including classical chemotherapeutic compounds, are able to enforce tumor specific immune responses either by inducing the immunogenic death of tumor cells or by modulating key cells for immune suppression or activation. To determine whether Temozolomide (TMZ), the standard chemotherapeutic agent for glioblastoma and malignant gliomas, could induce tumor immunogenicity, we treated murine GL261 cells with 50 and 150 microM TMZ in vitro for 2, 8 and 20 hours. Flow cytometry showed that the NKG2D ligand, involved in NKG2D-mediated NK cell recognition of tumor cells, was highly expressed by TMZ-treated GL261, whereas GL261 treated with vehicle showed only a weak expression (P < 0.01). B7-H3, an inhibitory molecule for NK cells, significantly decreased in TMZ-treated GL261 cells (P < 0.01). TGF-beta1 and TGF-beta2 concentrations in the supernatant from GL261 treated with TMZ significantly decreased, as shown by ELISA (from 23.2 ± 2.5 and 478.4 ± 19.3 in vehicle to 13.2 ± 2.2 and 283.8 ± 25.9 in TMZ-treated cells, respectively, P < 0.0001). We therefore studied the effects of TMZ on anti-tumor NK cell response by treating mice 7 days after intracranial implantation of GL261 gliomas with intraperitoneal injections of TMZ (5mg/kg) or vehicle (control mice) for 5 days. Five mice were sacrificed 20 hours after treatment on days 1-5: brain, spleen and blood were harvested and analyzed by flow cytometry. Trafficking of NKp46+ NK1.1+ CD3- NK cells in blood but not in spleen and their homing ability into the brain significantly increased in TMZ-treated compared to control mice after the second administration of TMZ (7.5 ± 1.4 vs 2.3 ± 1.2, P = 0.001; 10.9 ± 0.4 vs. 3.9 ± 0.6, P = 0.005, respectively). Notably TMZ led to an enrichment of CD11bhigh CD27high NK cells, the most potent effector cells. To verify NK anti-glioma activity, NK1.1 positive cells were isolated from blood and brain of treated and control mice using magnetic separation and incubated with GL261 cells. NK cell cytotoxicity from TMZ-treated mice was significantly higher than that NK cells from control mice. These results support the contention that chemotherapy may induce enhancement of NK cell response and open new opportunities to design novel combined therapies reversing the immune suppressive role of glioblastoma and unmasking the therapeutic potential of NK responses. Citation Format: Serena Pellegatta, Gabriele Cantini, Sara Pessina, Gaetano Finocchiaro. NK cell activation and cytotoxicity can be enhanced with chemotherapy in a murine model of glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr A60.

NK Cell Anti-Tumor Reactivity Is Impaired by Platelet-Derived TGF-B through NKG2D Downregulation

Osteosarcoma and Ewing's sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-β. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.

Membrane Associated TGF-β1 on Leukemia Blast-Derived Microvesicles In Sera of Acute Myeloid Leukemia Patients Suppresses NK Cell Function

BackgroundHuman major histocompatibility complex class I-related chain A (MICA) plays a dual role in adaptive and innate immune responses. Increasing evidence demonstrates that MICA is closely correlated with acute and chronic kidney allograft rejection. Therefore, understanding the activation mechanisms of MICA is important in kidney transplantation. We previously demonstrated that ischemia/reperfusion injury (IRI) could up-regulate MICA expression on mouse kidney allografts. Since hypoxia-inducible factor-1 (HIF-1) is the master regulator of cellular adaptive responses to hypoxia during IRI, here we investigate whether HIF-1 could up-regulate MICA expression and its influence on NK cell cytotoxicity.ResultsWe find that HIF-1alpha plays an important role in up-regulating MICA expression, inducing IFNgamma secretion and NK cell cytotoxicity during hypoxia/reoxygenation. First, we generated a HIF-1alphaDELTAODD-expressing adenovirus to stably and functionally express HIF-1alpha in human renal proximal tubular epithelial (HK-2) cells under normoxia conditions. HIF-1alpha over-expression in HK-2 cells induces MICA expression and enhances NK cell cytotoxic activity towards cells that express HIF-1alpha. Second, we used a hypoxia/reoxygenation cell model to simulate IRI in vitro and found that the suppression of HIF-1alpha by RNAi induces down-regulation of MICA expression and inhibits NK cytotoxicity. In antibody blocking experiments, an anti-MICA mAb was able to down-regulate NK cell cytotoxic activity towards HK-2 cells that over-expressed HIF-1alpha. Moreover, when NK cells were co-cultured with the HK-2 cells expressing MICA, which was up-regulated by over-expression of HIF-1alpha, there was a significant increase in the secretion of IFNgamma. In the presence of the blocking MICA mAb, IFNgamma secretion was significantly decreased.ConclusionsThese results demonstrate that hypoxia/reoxygenation-promoted MICA expression on HK-2 cells is through a HIF-1 pathway. The increased IFNgamma secretion and enhanced NK cell cytotoxicity was mainly due to the surface expression of MICA induced by over-expression of HIF-1alpha. This study enhances our understanding of MICA activation mechanisms during kidney transplantation and provides insights into how IRI can influence transplant outcome. Moreover, these findings might be also important for developing strategies to reduce the effect of MICA in kidney transplant outcomes in the future.

Tumor infiltrating NK cell (TINK) subsets and functional molecules in patients with breast cancer

ABSTRACTNatural killer (NK) cells are essential for killing transformed and virally infected cells. To prevent auto-reactivity, NK cell activation is inhibited by inhibitory receptors that activate the tyrosine phosphatase SHP-1, which dephosphorylates signaling molecules crucial for NK cell activation. Initially, only a single SHP-1 substrate was identified in NK cells, the GEF VAV1. We recently demonstrated that under inhibitory conditions, LAT, PLCγ1 and PLCγ2 serve as novel SHP-1 substrates in NK cells. Furthermore, we showed that during NK cell inhibition, LAT is ubiquitylated by c-Cbl and Cbl-b, leading to its proteasomal degradation, abolishing NK cell cytotoxicity. Here, we address the mechanism through which the Cbl proteins are activated following inhibitory receptor engagement. We demonstrate that during NK cell inhibition, the expression level of the Cbl proteins significantly increases. These data suggest that inhibitory KIR receptors regulate the stability of the Cbl proteins, thereby enabling Cbl-mediated inhibition of NK cell cytotoxicity.

Natural killer (NK) cells provide an attractive platform for development of effective cancer immunotherapies. NK cells are known for their ability to kill tumor cells and do not elicit graft-versus-host disease, making them a potential source of ‘off-the-shelf’ allogeneic cell therapy. NK cells are also amenable to CRISPR genomic engineering to enhance the antitumor activity of NK cells by increasing their cytotoxicity, overcoming suppression within the tumor microenvironment, or promoting their persistence and homing to tumor sites. Cytokine inducible SH2-containing protein (CISH) is a NK cell checkpoint for IL-15 mediated NK survival, proliferation, cytotoxicity, and anti-tumor immunity. The E3 ubiquitin ligase CBLB is also a negative regulator of NK cell function and has been shown to mediate TGF-β sensitivity by downregulating inhibitory SMAD7 in primary T cells. We hypothesized that knockout of both CISH and CBLB would not only improve NK cell effector function and but also render NK cells resistant to TGF-β mediated suppression. In this study, we utilized CRISPR-Cas9 ribonucleoproteins (RNPs) to disrupt CISH and CBLB genes in isolated peripheral blood NK cells from healthy donors. Given that CD70 expression is present on activated NK cells, to target CD70 on renal cell carcinoma (RCC) with CAR NK cells, CD70 was knocked out in NK cells to avoid fratricide. Western blotting, FACS and TIDE/Amplicon NGS Sequencing data confirmed all three genes were successfully disrupted. Then we expanded these edited NK cells by using IL-2 and stimulation using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. Furthermore, we were able to transduce CRISPR/Cas9 edited NK cells to express a CD70-CAR construct and membrane bound IL-15. CAR expression was assessed by flow cytometry. In vitro cytotoxicity was measured using the IncuCyte S3 live cell analysis system. CD70/CISH/CBLB triple knockout CD70-CAR NK cells could be produced efficiently and exhibited similar persistence as CD70/CISH or CD70/CBLB double knockout CD70-CAR NK cells in culture. Cytotoxicity assays demonstrated that CD70/CISH/CBLB triple knockout CD70-CAR NK cells had greater tumor growth control after multiple rechallenges. In the presence of exogenous TGF-β, CD70/CISH/CBLB triple knockout CD70-CAR NK cells showed greater resistance to TGF-β inhibition of cytotoxicity. In summary, we show CD70/CISH/CBLB triple knockout CD70-CAR NK cells demonstrate enhanced anti-tumor activity against relevant solid tumor cell lines and provide greater resistance to tumor microenvironment inhibition. These data support the further exploration of CD70/CISH/CBLB triple gene knockout CD70 CAR NK cells for clinical application. Citation Format: Chao Guo, Yanying Fan, Alexander Aronov, Qi Zhang, Sombeet Sahu, Mary-Lee Dequéant, Changan Guo, Sushant Karnik, Glenn D. Leary, Chandirasegaran Massilamany, Ivan H. Chan, James B. Trager. CBLB, CISH and CD70 multiplexed gene knockout with CRISPR/Cas9 enhances cytotoxicity of CD70-CAR NK cells and provides greater resistance to TGF-β for cancer immunotherapy [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 5512.

Combination Therapy with Daratumumab and CAR-NK Targeting CS1 for Multiple Myeloma