Memory-like differentiation enhances NK cell responses against colorectal cancer.

Metastatic (m) colorectal cancer (CRC) is an incurable disease with a poor prognosis and thus remains an unmet clinical need. Immune checkpoint blockade (ICB)-based immunotherapy is effective for mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H) mCRC patients, but it does not benefit the majority of mCRC patients. NK cells are innate lymphoid cells with potent effector responses against a variety of tumor cells but are frequently dysfunctional in cancer patients. Memory-like (ML) NK cells differentiated after IL-12/IL-15/IL-18 activation overcome many challenges to effective NK cell anti-tumor responses, exhibiting enhanced recognition, function, and in vivo persistence. We hypothesized that ML differentiation enhances the NK cell responses to CRC. Compared to conventional (c) NK cells, ML NK cells displayed increased IFN-γ production against both CRC cell lines and primary patient-derived CRC spheroids. ML NK cells also exhibited improved killing of CRC target cells in vitro in short-term and sustained cytotoxicity assays, as well as in vivo in NSG mice. Mechanistically, enhanced ML NK cell responses were dependent on the activating receptor NKG2D as its blockade significantly decreased ML NK cell functions. Compared to cNK cells, ML NK cells exhibited greater antibody-dependent cytotoxicity when targeted against CRC by cetuximab. ML NK cells from healthy donors and mCRC patients exhibited increased anti-CRC responses. Collectively, our findings demonstrate that ML NK cells exhibit enhanced responses against CRC targets, warranting further investigation in clinical trials for mCRC patients, including those who have failed ICB.

Absence of ATM leads to altered NK cell function in mice.

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

Natural Killer Cells Reprogram Myeloid-Derived Suppressor Cells to Induce TNF-α Release via NKG2D-Ligand Interaction after Cryo-Thermal Therapy.

In our previous studies, a novel cryothermal therapy (CTT) was developed to induce systemic long-term anti-tumor immunity. Natural killer (NK) cells were found to play an important role in CTT-induced long-term immune-mediated tumor control at the late stage after CTT, but the underlying mechanism is unclear. Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that have potent immunosuppressive effects on T cells and weaken the long-term benefits of immunotherapy. Consequently, overcoming MDSC immunosuppression is essential for maintaining the long-term efficacy of immunotherapy. In this study, we revealed that NK cells considerably diminish MDSC accumulation at the late stage after CTT, boost T cell production, increase T cell activation, and promote MDSC maturation, culminating in Th1-dominant CD4+ T cell differentiation and enhancing NK and CD8+ T cell cytotoxicity. Additionally, NK cells activate ERK signaling in MDSCs through NKG2D-ligand interaction to increase the activity of tumor necrosis factor (TNF)-α converting enzyme (TACE)-cleaved membrane TNF-α. Furthermore, Increased TACE activity releases more soluble TNF-α from MDSCs to promote MDSC maturation. In our studies, we propose a novel mechanism by which NK cells can overcome MDSC-induced immunosuppression and maintain CTT-induced persistent anti-tumor immunity, providing a prospective therapeutic option to improve the performance of cancer immunotherapy.

Targeting NKG2D ligands in glioblastoma with a bispecific T-cell engager is augmented with conventional therapy and enhances oncolytic virotherapy of glioma stem-like cells.

BACKGROUND: Glioblastoma (GBM) almost invariably becomes resistant towards conventional treatment of radiotherapy and temozolomide (TMZ) chemotherapy, partly due to subpopulations of intrinsically resistant glioma stem-like cells (GSC). The oncolytic herpes simplex virus-1 G207 is a promising approach for GBM virotherapy although its efficacy in patients with GBM is often limited. Natural killer group 2 member D ligands (NKG2DLs) are minimally expressed by healthy cells but are upregulated by the DNA damage response (DDR) and in malignant cells with chronic DDR signaling, resulting in innate immune activation. METHODS: We have designed a bispecific T-cell engager (BiTE) capable of cross-linking CD3 on T cells with NKG2DL-expressing GBM cells. We then engineered the G207 virus to express the NKG2D BiTE and secrete it from infected cells. The efficacy of the free BiTE and BiTE delivered by G207 was evaluated in combination with conventional therapies in GBM cells and against patient-derived GSCs in the context of T-cell activation and target cell viability. RESULTS: NKG2D BiTE-mediated cross-linking of GBM cells and T cells causes antigen-independent T-cell activation, pro-inflammatory cytokine release, and tumor cell death, thereby combining direct viral oncolysis with BiTE-mediated cytotoxicity. Surface NKG2DL expression was further elevated on GBM cells following pretreatment with sublethal doses of TMZ and radiation to induce the DDR, increasing sensitivity towards G207-NKG2D BiTE and achieving synergistic cytotoxicity. We also demonstrate a novel strategy for targeting GSCs that are non-permissive to G207 infection but remain sensitive to NKG2D BiTE. CONCLUSIONS: We propose a potential model for targeting GSCs in heterogeneous tumors, whereby differentiated GBM cells infected with G207-NKG2D BiTE produce NKG2D BiTE locally, directing T-cell cytotoxicity towards the GSC subpopulations in the tumor microenvironment.

Tinostamustine (EDO-S101), an Alkylating Deacetylase Inhibitor, Enhances the Efficacy of Daratumumab in Multiple Myeloma by Upregulation of CD38 and NKG2D Ligands.

Multiple myeloma is a malignancy characterized by the accumulation of malignant plasma cells in bone marrow and the production of monoclonal immunoglobulin. A hallmark of cancer is the evasion of immune surveillance. Histone deacetylase inhibitors have been shown to promote the expression of silenced molecules and hold potential to increase the anti-MM efficacy of immunotherapy. The aim of the present work was to assess the potential effect of tinostamustine (EDO-S101), a first-in-class alkylating deacetylase inhibitor, in combination with daratumumab, an anti-CD38 monoclonal antibody (mAb), through different preclinical studies. Tinostamustine increases CD38 expression in myeloma cell lines, an effect that occurs in parallel with an increment in CD38 histone H3 acetylation levels. Also, the expression of MICA and MICB, ligands for the NK cell activating receptor NKG2D, augments after tinostamustine treatment in myeloma cell lines and primary myeloma cells. Pretreatment of myeloma cell lines with tinostamustine increased the sensitivity of these cells to daratumumab through its different cytotoxic mechanisms, and the combination of these two drugs showed a higher anti-myeloma effect than individual treatments in ex vivo cultures of myeloma patients' samples. In vivo data confirmed that tinostamustine pretreatment followed by daratumumab administration significantly delayed tumor growth and improved the survival of mice compared to individual treatments. In summary, our results suggest that tinostamustine could be a potential candidate to improve the efficacy of anti-CD38 mAbs.

THEMIS2 Impairs Antitumor Activity of NK Cells by Suppressing Activating NK Receptor Signaling.

NK cells are cytotoxic innate lymphocytes that play a critical role in antitumor immunity. NK cells recognize target cells by using a repertoire of activating NK receptors and exert the effector functions. Although the magnitude of activation signals through activating NK receptors controls NK cell function, it has not been fully understood how these activating signals are modulated in NK cells. In this study, we found that a scaffold protein, THEMIS2, inhibits activating NK receptor signaling. Overexpression of THEMIS2 attenuated the effector function of human NK cells, whereas knockdown of THEMIS2 enhanced it. Mechanistically, THEMIS2 binds to GRB2 and phosphorylated SHP-1 and SHP-2 at the proximity of activating NK receptors DNAM-1 and NKG2D. Knockdown of THEMIS2 in primary human NK cells promoted the effector functions. Furthermore, Themis2-deficient mice showed low metastatic burden in an NK cell-dependent manner. These findings demonstrate that THEMIS2 has an inhibitory role in the antitumor activity of NK cells, suggesting that THEMIS2 might be a potential therapeutic target for NK cell-mediated cancer immunotherapy.

[Effect of Spatholobi Caulis extract from ethyl acetate on immune killing function of NK cells].

This study aims to investigate the regulatory effect of the Spatholobi Caulis extract from ethyl acetate(SEA) on natural killer(NK) cells under physiological conditions and elucidate the underlying mechanism. The C57BL/6 mice were randomized into NC and SEA groups, and NK-92 cells were respectively treated with 0, 25, 50, and 100 µg·mL~(-1) SEA. The body weight and immune organ index of the mice were compared between groups. The lactate dehydrogenase(LDH) assay was employed to examine the cytotoxicity of NK-92 cells treated with SEA and the killing activity of mouse NK cells against YAC-1 cells. The cell-counting kit-8(CCK-8) was used to examine the impact of SEA on the proliferation of NK-92 cells. Flow cytometry was employed to measure the number of NK cells in the peripheral blood as well as the expression levels of natural killer group 2 member A(NKG2A) and natural killer group 2 member D(NKG2D). The enzyme-linked immunosorbent assay(ELISA) was performed to determine the interferon(IFN)-γ secretion in the serum. Semi-quantitative PCR was conducted to determine the mRNA levels of NKG2A, NKG2D, and IFN-γ in spleen cells. Western blot was employed to investigate the involvement of phosphoinositide 3-kinase(PI3K)/extracellular regulated protein kinase 1(ERK1) signaling pathway. The results showed that SEA exhibited no adverse effects on the body, while significantly enhance the number of NK cells and augment the cytotoxicity of NK-92 cells against YAC-1 cells. Moreover, it suppressed the expression of NKG2A, enhanced the expression of NKG2D, promoted IFN-γ secretion, and upregulated the protein levels of PI3K and ERK. The findings suggest that SEA has the potential to enhance the immune recognition and effector function of NK cells by increasing the cell number, modulating the expression of functional receptors, and promoting IFN-γ secretion via the PI3K/ERK signaling pathway.

Double-negative T cells ameliorate psoriasis by selectively inhibiting IL-17A-producing γδlow T cells.

BACKGROUND: Psoriasis is a chronic immune-mediated skin condition. Although biologic treatments are effective in controlling psoriasis, some patients do not respond or lose response to these therapies. Thus, new strategies for psoriasis treatment are still urgently needed. Double-negative T cells (DNT) play a significant immunoregulatory role in autoimmune diseases. In this study, we aimed to evaluate the protective effect of DNT in psoriasis and explore the underlying mechanism. METHODS: We conducted a single adoptive transfer of DNT into an imiquimod (IMQ)-induced psoriasis mouse model through tail vein injection. The skin inflammation and IL-17A producing γδ T cells were evaluated. RESULTS: DNT administration significantly reduced the inflammatory response in mouse skin, characterized by decreased skin folds, scales, and red patches. After DNT treatment, the secretion of IL-17A by RORc+ γδlow T cells in the skin was selectively suppressed, resulting in an amelioration of skin inflammation. Transcriptomic data suggested heightened expression of NKG2D ligands in γδlow T cells within the mouse model of psoriasis induced by IMQ. When blocking the NKG2D ligand and NKG2D (expressed by DNT) interaction, the cytotoxic efficacy of DNT against RORc+IL17A+ γδlow T cells was attenuated. Using Ccr5-/- DNT for treatment yielded evidence that DNT migrates into inflamed skin tissue and fails to protect IMQ-induced skin lesions. CONCLUSIONS: DNT could migrate to inflamed skin tissue through CCR5, selectively inhibit IL-17-producing γδlow T cells and finally ameliorate mouse psoriasis. Our study provides feasibility for using immune cell therapy for the prevention and treatment of psoriasis in the clinic.

RAC1high NK cell-based immunotherapy in hepatocellular carcinoma via STAT3-NKG2D axis.

Natural killer (NK) cells exert an indispensable role in innate immune responses against cancer progression, however NK cell dysfunction has been rarely reported in hepatocellular carcinoma (HCC). This study sought to uncover the immunoregulatory mechanisms of tumor-infiltrating NK cells in HCC. A consensus NK cell-based signature (NKS) was constructed using integrative machine learning algorithms based on multi-omics data of HCC patients. HCC tumors had lower numbers of infiltrating NK cells than para-tumor normal liver tissues. Based on the NK cell-associated genes, the NKS was built for HCC prognostic prediction and clinical utilities. Drug targets and novel compounds were then identified for high-NKS groups. RAC1 was confirmed as the hub gene in the NKS genes. RAC1 was upregulated in HCC tumors and positively correlated with shorter survival time. RAC1 overexpression in NK-92 cells facilitated the cancer-killing capacity by the anticancer cytotoxic effectors and the upregulated NKG2D. The survival time of PDX-bearing mice was also prolonged upon NK-92RAC1 cells. Mechanistically, RAC1 interacted with STAT3 and facilitated its activation, thereby enabling its binding to the promoter region of NKG2D and functioning as a transcriptional regulator in NK-92 via molecular docking, Co-IP assay, CHIP and luciferase experiments. Collectively, our study describes a novel function of RAC1 in potentiating NK cell-mediated cytotoxicity against HCC, highlighting the clinical utilities of NKS score and RAC1high NK cell subset in HCC immunotherapy.

NK Cell-Monocyte Cross-talk Underlies NK Cell Activation in Severe COVID-19.

NK cells in the peripheral blood of severe COVID-19 patients exhibit a unique profile characterized by activation and dysfunction. Previous studies have identified soluble factors, including type I IFN and TGF-ß, that underlie this dysregulation. However, the role of cell-cell interactions in modulating NK cell function during COVID-19 remains unclear. To address this question, we combined cell-cell communication analysis on existing single-cell RNA sequencing data with in vitro primary cell coculture experiments to dissect the mechanisms underlying NK cell dysfunction in COVID-19. We found that NK cells are predicted to interact most strongly with monocytes and that this occurs via both soluble factors and direct interactions. To validate these findings, we performed in vitro cocultures in which NK cells from healthy human donors were incubated with monocytes from COVID-19+ or healthy donors. Coculture of healthy NK cells with monocytes from COVID-19 patients recapitulated aspects of the NK cell phenotype observed in severe COVID-19, including decreased expression of NKG2D, increased expression of activation markers, and increased proliferation. When these experiments were performed in a Transwell setting, we found that only CD56bright CD16- NK cells were activated in the presence of severe COVID-19 patient monocytes. O-link analysis of supernatants from Transwell cocultures revealed that cultures containing severe COVID-19 patient monocytes had significantly elevated levels of proinflammatory cytokines and chemokines, as well as TGF-ß. Collectively, these results demonstrate that interactions between NK cells and monocytes in the peripheral blood of COVID-19 patients contribute to NK cell activation and dysfunction in severe COVID-19.

A novel MICA/B-targeted chimeric antigen receptor augments the cytotoxicity of NK cells against tumor cells.

Chimeric antigen receptor (CAR)-modified immune cells have emerged as a promising approach for cancer treatment, but single-target CAR therapy in solid tumors is limited by immune escape caused by tumor antigen heterogeneity and shedding. Natural killer group 2D (NKG2D) is an activating receptor expressed in human NK cells, and its ligands, such as MICA and MICB (MICA/B), are widely expressed in malignant cells and typically absent from healthy tissue. NKG2D plays an important role in anti-tumor immunity, recognizing tumor cells and initiating an anti-tumor response. Therefore, NKG2D-based CAR is a promising CAR candidate. Nevertheless, the shedding of MICA/B hinders the therapeutic efficacy of NKG2D-CARs. Here, we designed a novel CAR by engineering an anti-MICA/B shedding antibody 1D5 into the CAR construct. The engineered NK cells exhibited significantly enhanced cytotoxicity against various MICA/B-expressing tumor cells and were not inhibited by NKG2D antibody or NKG2D-Fc fusion protein, indicating no interference with NKG2D-MICA/B binding. Therefore, the developed 1D5-CAR could be combined with NKG2D-CAR to further improve the obstacles caused by MICA/B shedding.

Evasion of NKG2D-mediated cytotoxic immunity by sarbecoviruses.

SARS-CoV-2 and other sarbecoviruses continue to threaten humanity, highlighting the need to characterize common mechanisms of viral immune evasion for pandemic preparedness. Cytotoxic lymphocytes are vital for antiviral immunity and express NKG2D, an activating receptor conserved among mammals that recognizes infection-induced stress ligands (e.g., MIC-A/B). We found that SARS-CoV-2 evades NKG2D recognition by surface downregulation of MIC-A/B via shedding, observed in human lung tissue and COVID-19 patient serum. Systematic testing of SARS-CoV-2 proteins revealed that ORF6, an accessory protein uniquely conserved among sarbecoviruses, was responsible for MIC-A/B downregulation via shedding. Further investigation demonstrated that natural killer (NK) cells efficiently killed SARS-CoV-2-infected cells and limited viral spread. However, inhibition of MIC-A/B shedding with a monoclonal antibody, 7C6, further enhanced NK-cell activity toward SARS-CoV-2-infected cells. Our findings unveil a strategy employed by SARS-CoV-2 to evade cytotoxic immunity, identify the culprit immunevasin shared among sarbecoviruses, and suggest a potential novel antiviral immunotherapy.

Aberrant phenotypes of circulating γδ-T cells may be involved in the onset of systemic lupus erythematosus.

OBJECTIVE: Human gamma-delta T cells (γδ-T cells) play crucial roles in both innate and adaptive immune responses. However, much less is known about the immune status of γδT cells in systemic lupus erythematosus (SLE) patients. The objective of this study was to explore potential relationships between the frequency of γδ-T-cell subpopulations and disease activity, autoantibody titres and renal involvement in patients with SLE. METHODS: Circulating γδ-T cells and their subsets (Vδ1+ T cells, Vδ2+ T cells and γδ-T-cell subpopulations defined by expression of surface receptors, including NKG2D, NKp30, NKp46 and PD-1), were identified via flow cytometry. Sixty active SLE patients were selected, including 41 new-onset and 19 relapsing cases. One hundred healthy controls (HCs) were enrolled as the control group. Percentages of these cell subsets in SLE patients and HCs and their relationships with disease activity were analysed. Twenty-two of the 41 new-onset SLE patients were assessed before and after treatment. Changes in the frequencies of these cell subsets and their relationships with renal involvement were also analysed. RESULTS: Compared with that in HCs, the percentage of total γδ-T cells among CD3+ T cells in SLE patients was significantly lower. An imbalance in the proportions of Vδ1+ and Vδ2+ T cells among γδ-T cells was observed. The proportion of Vδ1+ T cells among γδ-T cells was significantly greater in SLE patients than in HCs, while the proportion of Vδ2+ T cells was significantly lower. Expression levels of PD-1, NKG2D, NKp30 and NKp46 in Vδ1+ T cells and Vδ2+ T cells from SLE patients were generally significantly increased, except for expression of NKG2D in Vδ2+ T cells. Moreover, Vδ2+ T cells, Vδ1+ T cells and Vδ1+PD-1+ T cells were associated with disease activity, and an increase in Vδ2+ T-cell frequency and a decrease in PD-1 expression by γδ-T cells might be associated with effective treatment. Interestingly, our results indicated that Vδ2+ T cells and their Vδ2+NKp30+ T-cell subpopulation might be associated with renal involvement in SLE. CONCLUSION: A broad range of anomalies in the proportions of γδ-T-cell subsets and γδ-T cells in SLE patients may be involved in the pathogenesis of SLE. There is a strong association between Vδ2+ T cells and their Vδ2+NKp30+ T-cell subpopulation and LN occurrence. Our results indicate that γδ-T cells and their subpopulations might be key players in disease immunopathology and renal involvement in SLE.

Lower NKG2D expression in hepatic natural killer cells predicts poorer prognosis for chronic hepatitis B patients with cirrhosis.

OBJECTIVE: Natural killer cells (NK) acts a central player of the immune system in liver cirrhosis. The aim of this study was to examine the expression of activating intra-hepatic NK cell group 2D (NKG2D) in patients with chronic hepatitis B (CHB) and analyzed the correlation between NKG2D expression and prognosis of liver cirrhosis in these patients. METHODS: This was a cross-section study. Subjects with liver biopsy or sponge hemangioma surgery were included. The primary outcome was the NKG2D expression on intra-hepatic NK cells and their subtype cells in patients with CHB-related liver cirrhosis. Subsequently, the correlation of expression of NKG2D and clinical characteristic indicators were assayed RESULTS: Among 38 subjects, 11 (28.95%) normal liver sections adjacent the sponge hemangioma (healthy group) were collected during surgery, and 27 (71.05%) CHB-cirrhosis tissues (Cirrhosis group) were preserved after liver biopsy. Compared with healthy group, sections from cirrhosis group revealed more severe inflammation and collagen deposition and lower NKG2D expression in hepatic NK cells. The proportion of hepatic NK cells and the mean fluorescence intensity (MFI) of NKG2D on hepatic NK cells showed a positive correlation with serum albumin (Alb) level, platelet (Plt) count. Moreover, they had a significantly negative correlation with patient prothrombin time (PT), international standardized ratio (INR), the sirius red positive stained area and fibrosis stages. CONCLUSIONS: Lower NKG2D expression in intra-hepatic NK cells may be predictive of poorer prognosis of CHB patients with cirrhosis.

Secondary Sites of the C-type Lectin-Like Fold.

C-type lectins are a large superfamily of proteins involved in a multitude of biological processes. In particular, their involvement in immunity and homeostasis has rendered them attractive targets for diverse therapeutic interventions. They share a characteristic C-type lectin-like domain whose adaptability enables them to bind a broad spectrum of ligands beyond the originally defined canonical Ca2+-dependent carbohydrate binding. Together with variable domain architecture and high-level conformational plasticity, this enables C-type lectins to meet diverse functional demands. Secondary sites provide another layer of regulation and are often intricately linked to functional diversity. Located remote from the canonical primary binding site, secondary sites can accommodate ligands with other physicochemical properties and alter protein dynamics, thus enhancing selectivity and enabling fine-tuning of the biological response. In this review, we outline the structural determinants allowing C-type lectins to perform a large variety of tasks and to accommodate the ligands associated with it. Using the six well-characterized Ca2+-dependent and Ca2+-independent C-type lectin receptors DC-SIGN, langerin, MGL, dectin-1, CLEC-2 and NKG2D as examples, we focus on the characteristics of non-canonical interactions and secondary sites and their potential use in drug discovery endeavors.

NK-92MI Cells Engineered with Anti-claudin-6 Chimeric Antigen Receptors in Immunotherapy for Ovarian Cancer.

Background: The application of chimeric antigen receptor (CAR) NK cells in solid tumors is hindered by lack of tumor-specific targets and inefficient CAR-NK cell efficacy. Claudin-6 (CLDN6) has been reported to be overexpressed in ovarian cancer and may be an attractive target for CAR-NK cells immunotherapy. However, the feasibility of using anti-CLDN6 CAR-NK cells to treat ovarian cancer remains to be explored. Methods: CLDN6 expression in primary human ovarian cancer, normal tissues and cell lines were detected by immunohistochemistry and western blot. Two types of third-generation CAR NK-92MI cells targeting CLDN6, CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) and CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB) were constructed by lentivirus transfection, sorted by flow cytometry and verified by western blot and qPCR. OVCAR-3, SK-OV-3, A2780, Hey and PC-3 cells expressing the GFP and luciferase genes were transduced. Subcutaneous and intraperitoneal tumor models were established via NSG mice. The ability of CLDN6-CAR NK cells to kill CLDN6-positive ovarian cancer cells were evaluated in vitro and in vivo by live cell imaging and bioluminescence imaging. Results: Both CLDN6-CAR1 and CLDN6-CAR2 NK-92MI cells could specifically killed CLDN6-positive ovarian cancer cells (OVCAR-3, SK-OV-3, A2780 and Hey), rather than CLDN6 negative cell (PC-3), in vitro. CLDN6-CAR1 NK-92MI cells with domains containing self-activated elements (NKG2D, 2B4) exhibited stronger cytotoxicity than CLDN6-CAR2 NK-92MI cells with classical domains (CD28, 4-1BB). Furthermore, CLDN6-CAR1 NK cells could effectively eliminate ovarian cancer cells in subcutaneous and intraperitoneal tumor models. More importantly, CAR-NK cells combined with immune checkpoint inhibitors, anti-PD-L1, could synergistically enhance the antitumor efficacy of CLDN6-targeted CAR-NK cells. Conclusions: These results indicate that CLDN6-CAR NK cells possess strong antitumor activity and represent a promising immunotherapeutic modality for ovarian cancer.

Soluble MICA in endometriosis pathophysiology: Impairs NK cell degranulation and effector functions.

PROBLEM: Endometriosis exhibits several immune dysfunctions, including deficient natural killer (NK) cell cytotoxicity. MICA (MHC class I chain-related molecule A) is induced by biological stress and soluble MICA (sMICA) negatively modulates the expression of the activating receptor, NKG2D, reducing NK cells activities. We investigated the involvement of soluble MICA in NK cell-deficient activity in endometriosis. METHODS OF STUDY: sMICA levels (serum and peritoneal fluid-PF) were evaluated by ELISA. Circulating NK cell subsets quantification and its NKG2D receptor expression, NK cell cytotoxicity and CD107a, IFN-γ and IL-10 expressions by NK cells stimulated with K562 cells were determined by flow cytometry. RESULTS: We found higher sMICA levels (serum and PF) in endometriosis, especially in advanced and deep endometriosis. Endometriosis presented lower percentages of CD56dim CD16+ cytotoxic cells and impaired NK cell responses upon stimulation, resulting in lower CD107a and IFN-γ expressions, and deficient NK cell cytotoxicity. NK cell stimulation in the MICA-blocked condition (mimicking the effect of sMICA) showed decreased cytotoxicity in initial endometriosis stages and the emergence of a negative correlation between CD107a expression and sMICA levels. CONCLUSIONS: We suggest that soluble MICA is a potential player in endometriosis pathophysiology with involvement in disease progression and severity, contributing to NK cell impaired IFN-γ response and degranulation. NK cell compartment exhibits multiple perturbations, including quantitative deficiency and impaired cytotoxicity, contributing to inadequate elimination of ectopic endometrial tissue.

Dual Targeting of Glioblastoma Cells with Bispecific Killer Cell Engagers Directed to EGFR and ErbB2 (HER2) Facilitates Effective Elimination by NKG2D-CAR-Engineered NK Cells.

NKG2D is an activating receptor of natural killer cells that recognizes stress-induced ligands (NKG2DL) expressed by many tumor cells. Nevertheless, NKG2DL downregulation or shedding can still allow cancer cells to evade immune surveillance. Here, we used lentiviral gene transfer to engineer clinically usable NK-92 cells with a chimeric antigen receptor (NKAR) which contains the extracellular domain of NKG2D for target recognition, or an NKAR, together with the IL-15 superagonist RD-IL15, and combined these effector cells with recombinant NKG2D-interacting bispecific engagers that simultaneously recognize the tumor-associated antigens epidermal growth factor receptor (EGFR) or ErbB2 (HER2). Applied individually, in in vitro cell-killing assays, these NKAB-EGFR and NKAB-ErbB2 antibodies specifically redirected NKAR-NK-92 and NKAR_RD-IL15-NK-92 cells to glioblastoma and other cancer cells with elevated EGFR or ErbB2 levels. However, in mixed glioblastoma cell cultures, used as a model for heterogeneous target antigen expression, NKAR-NK cells only lysed the EGFR- or ErbB2-expressing subpopulations in the presence of one of the NKAB molecules. This was circumvented by applying NKAB-EGFR and NKAB-ErbB2 together, resulting in effective antitumor activity similar to that against glioblastoma cells expressing both target antigens. Our results demonstrate that combining NK cells carrying an activating NKAR receptor with bispecific NKAB antibodies allows for flexible targeting, which can enhance tumor-antigen-specific cytotoxicity and prevent immune escape.

Elevated levels of cell-free NKG2D-ligands modulate NKG2D surface expression and compromise NK cell function in severe COVID-19 disease.

Introduction: It is now clear that coronavirus disease 19 (COVID-19) severity is associated with a dysregulated immune response, but the relative contributions of different immune cells is still not fully understood. SARS CoV-2 infection triggers marked changes in NK cell populations, but there are contradictory reports as to whether these effector lymphocytes play a protective or pathogenic role in immunity to SARS-CoV-2. Methods: To address this question we have analysed differences in the phenotype and function of NK cells in SARS-CoV-2 infected individuals who developed either very mild, or life-threatening COVID-19 disease. Results: Although NK cells from patients with severe disease appeared more activated and the frequency of adaptive NK cells was increased, they were less potent mediators of ADCC than NK cells from patients with mild disease. Further analysis of peripheral blood NK cells in these patients revealed that a population of NK cells that had lost expression of the activating receptor NKG2D were a feature of patients with severe disease and this correlated with elevated levels of cell free NKG2D ligands, especially ULBP2 and ULBP3 in the plasma of critically ill patients. In vitro, culture in NKG2DL containing patient sera reduced the ADCC function of healthy donor NK cells and this could be blocked by NKG2DL-specific antibodies. Discussion: These observations of reduced NK function in severe disease are consistent with the hypothesis that defects in immune surveillance by NK cells permit higher levels of viral replication, rather than that aberrant NK cell function contributes to immune system dysregulation and immunopathogenicity.

Immune evasion by proteolytic shedding of natural killer group 2, member D ligands in Helicobacter pylori infection.

Background: Helicobacter pylori (H. pylori) uses various strategies that attenuate mucosal immunity to ensure its persistence in the stomach. We recently found evidence that H. pylori might modulate the natural killer group 2, member 2 (NKG2D) system. The NKG2D receptor and its ligands are a major activation system of natural killer and cytotoxic T cells, which are important for mucosal immunity and tumor immunosurveillance. The NKG2D system allows recognition and elimination of infected and transformed cells, however viruses and cancers often subvert its activation. Here we aimed to identify a potential evasion of the NKG2D system in H. pylori infection. Methods: We analyzed expression of NKG2D system genes in gastric tissues of H. pylori gastritis and gastric cancer patients, and performed cell-culture based infection experiments using H. pylori isogenic mutants and epithelial and NK cell lines. Results: In biopsies of H. pylori gastritis patients, NKG2D receptor expression was reduced while NKG2D ligands accumulated in the lamina propria, suggesting NKG2D evasion. In vitro, H. pylori induced the transcription and proteolytic shedding of NKG2D ligands in stomach epithelial cells, and these effects were associated with specific H. pylori virulence factors. The H. pylori-driven release of soluble NKG2D ligands reduced the immunogenic visibility of infected cells and attenuated the cytotoxic activity of effector immune cells, specifically the anti-tumor activity of NK cells. Conclusion: H. pylori manipulates the NKG2D system. This so far unrecognized strategy of immune evasion by H. pylori could potentially facilitate chronic bacterial persistence and might also promote stomach cancer development by allowing transformed cells to escape immune recognition and grow unimpeded to overt malignancy.