Peripheral Blood Natural Killer Cells Research Articles

Single-cell transcriptome sequencing to explore the infiltration and disappearance of NK cells in liver cancer.

e14520 Background: The emergence of tumor immunotherapy such as immune checkpoint blocking has greatly promoted the development of Hepatocellular carcinoma (HCC) treatment. However, the response rate of HCC patients to immunotherapy is not high, and the probability of adverse reactions is high. Natural killer (NK) cells play a crucial role in inhibiting the proliferation and metastasis of tumor cells and are expected to become a new hope for tumor therapy. Methods: The HCC single-cell transcriptome atlas was constructed by using the tumor tissues, adjacent tissues, peripheral blood of HCC patients. To emulate the hypoxic state within the tumor, NK cells were treated with rotenone, and the post-rotenone treatment changes of NK cells in transcriptome levels were evaluated using qPCR. The proliferation, apoptosis, and cytotoxicity of NK cells were assessed via flow cytometry. The study also investigated the role of c-Jun N-terminal protein kinase (JNK) in NK cell function by applying the JNK inhibitor tanzisertib (CC-930) in vitro. The impact of JNK inhibition on the tumor-killing potential of NK cells was examined in vivo using tumor-bearing mice NCG (NOD/ShiLtJ-Prkdcem26Cd52Il2rgem26Cd22/Gpt). Results: Analysis of the source of NK cell subsets found that the specific NK cells in tumor tissues were tissue-resident NK cells (trNK). Both RNA velocity analysis and pseudo-time sequence analysis suggested that trNK cells differentiated from PBMC-1 NK cells. Upon treatment of PBMC-1 NK cells with Tanzisertib, we observed inhibition of NK cell apoptosis. Through the subcutaneous tumor formation experiment of NCG mice, Tanzisertib-treated NK cells were transferred into tumor-bearing mice. Notably, we observed a significant reduction in tumor weight (Before treatment: 0.19g, 0.19g, 0.22g, 0.24g, 0.26g, 0.27g; After treatment: 0.16g, 0.17g, 0.17g, 0.19g, 0.21g, 0.23g; P value = 0.049). TUNEL assay confirmed a significant increase number of apoptotic cells within the tumor and an increased number of infiltrated NK cells. Conclusions: The NK cells in HCC tumors are likely derived from the infiltration of NK cells in peripheral blood. Importantly, the inhibition of JNK function can significantly inhibit the apoptosis of NK cells and enhance the killing ability of NK cells against tumor cells in vivo.

Cytotoxic function of human natural killer cells is transiently inhibited by transforming growth factor beta (TGFβ).

e14557 Background: Natural killer (NK) cells can eliminate early neoplastic cells, however, their efficacy as an adoptive therapy for solid tumors is limited. We have shown that NK cells recovered from human kidney tumors are CD56pos/CD16dim/neg, poorly cytotoxic, and produce vascular endothelial growth factor (VEGF). These features are analogous to human decidual NK (dNK) cells that support the development and growth of the placenta during implantation. Transforming growth factor beta (TGFβ) is found at high levels in the decidua and kidney tumor environment, and is known to convert peripheral blood NK (pNK) cells to dNK-like cells in vitro. We hypothesized that TGFβ-mediated inhibition of NK cell proliferation and function is transient and therefore reversible. Methods: pNK cells were isolated from healthy donors (HD) by negative selection or purchased from ATCC (NK-92; CRL-2407). RT-qPCR and flow cytometry were used to assess NK markers: CD56, CD16 NKp46, CD49a, CD69; TGFβ receptors R1, R2, R3; VEGF; and cytokines IFNγ, TNFα. NK cells were cultured for 4-5 days with proliferative cytokines IL2 or IL15 alone and in combination, and without or with TGFβ (2-5 ng/mL). Cytotoxic function of NK cells (effectors) was quantified by lysis of human K562 cells engineered to express firefly luciferase (targets). Statistics (ANOVA/t-test) were performed using GraphPad Prism with p≤0.05 considered significant. Results: pNK cells from HD were CD56pos/CD16pos and NK-92 cells were CD56pos/CD16neg as reported. Both expressed all three TGFβ receptors, required IL2 or IL15 to survive in culture and were potently cytotoxic against K562 targets. TGFβ treatment of NK cells cultured with IL2 or IL2 plus IL15 suppressed proliferation by 20% and potently inhibited cytotoxic ability (85% reduction; p≤0.05). TGFβ caused significant changes in CD49a (35% increase), CD69 (45% increase) and IFNγ (50% decrease) but had no effect on NKp46, VEGF, or TNFα. TGFβ-mediated changes in NK cell phenotype and function reverted to normal when NK cells were returned to standard culture conditions for 4-5 days. Conclusions: Despite differences in CD16 expression, pNK cells and NK-92 cells are potently cytotoxic and represent an effective anti-cancer therapy. TGFβ limits this potential by blocking the proliferative effects of IL2 and IL15 and quelling cytotoxic function. TGFβ caused increased expression of integrin CD49a, a marker of tissue resident immune cells, and the early lymphocyte activation marker CD69. Augmentation of these surface markers was concomitant with diminution of IFN-γ, a cytokine critical for cell-mediated responses to tumors. These collective changes are akin to dNK cells that are not cytotoxic and instead support the implant. Importantly, all alterations were restored if NK cells were no longer exposed to TGFβ. Thus, inhibition of TGFβ signaling could preserve NK function in the tumor environment.

Efficiently targeting neuroblastoma with the combination of anti-ROR1 CAR NK cells and N-803 in vitro and in vivo in NB xenografts

The prognosis for children with recurrent and/or refractory neuroblastoma (NB) is a dismal. The receptor tyrosine kinase like orphan receptor 1 (ROR1), which is highly expressed on the surface of NB cells, provides a potential target for novel immunotherapeutics. Anti-ROR1 CAR engineered ex vivo expanded peripheral blood natural killer (Anti-ROR1 CAR exPBNK) cells represent this approach. N-803 is an IL15 superagonist with enhanced biological activity. In this study, we investigated the in-vitro and in-vivo anti-tumor effects of anti-ROR1 CAR exPBNK cells with or without N-803 against ROR1+ NB models. Compared to mock exPBNK cells, anti-ROR1 CAR exPBNK cells had significantly enhanced cytotoxicity against ROR1+ NB cells, and N-803 further increased cytotoxicity. High dimensional analysis revealed that N-803 enhanced Stat5 phosphorylation and Ki67 levels in both exPBNK and anti-ROR1 CAR exPBNK cells with or without NB cells. In-vivo, anti-ROR1 CAR exPBNK plus N-803 significantly (p < 0.05) enhanced survival in human ROR1+ NB xenografted NSG mice compared to anti-ROR1 CAR exPBNK alone. Our results provide the rationale for further development of anti-ROR1 CAR exPBNK cells plus N-803 as a novel combination immunotherapeutic for patients with recurrent and/or refractory ROR1+ NB.

Investigating into microbiota in the uterine cavity of the unexplained recurrent pregnancy loss patients in early pregnancy

IntroductionThe majority of unexplained recurrent pregnancy loss (URPL) cases have been attributed to immune abnormalities. Inappropriate changes in microbiota could lead to immune disorders. However, the specific role of uterine cavity microbiota in URPL remains unclear, and only a limited number of related studies are available for reference. MethodsWe utilized double-lumen embryo transfer tubes to collect uterine cavity fluid samples from pregnant women in their first trimester. Subsequently, we conducted 16S rRNA sequencing to analyze the composition and abundance of the microbiota in these samples. ResultsFor this study, we enlisted 10 cases of URPL and 28 cases of induced miscarriages during early pregnancy. Microbial communities were detected in all samples of the URPL group (100 %, n = 10), whereas none were found in the control group (0 %, n = 28). Among the identified microbes, Lactobacillus and Curvibacter were the two most dominant species. The abundance of Curvibacter is correlated with the number of NK cells in peripheral blood (r = −0.759, P = 0.018). DiscussionThis study revealed that during early pregnancy, Lactobacillus and Curvibacter were the predominant colonizers in the uterine cavity of URPL patients and were associated with URPL. Consequently, alterations in the dominant microbiota may lead to adverse pregnancy outcomes.

Unraveling the early transcriptional signature of peripheral blood NK cell transition to uterine residency

Abstract Tissue-resident uterine NK cells are essential for successful pregnancy, yet the molecular mechanisms guiding their tissue residency programming early after infiltration of blood-based precursors remain unknown. We examined the transition of CD56+ peripheral blood NK cells to a uterine resident state by performing scRNA-seq on enriched CD56+ cells from a matched peripheral blood (pb) and endometrial (e) biopsy taken during the secretory phase. Our integrated analysis comparing eNK vs pbNK cells identified a tissue adaptation signature. Key components of this signature included NR4A2 (94% vs 0%, p=9.46e-22), FOS (90% vs 9%, p=9.05e-14), and JUNB (77% vs 16%, p=3.15e-07) respectively. This signature also mirrored genes previously identified in activated murine intestinal CD8+ TRM cells early after LCMV infection. This signature was found in all endometrial CD56+ cells, including CD56bright CD16- eNKs, NKTs, and conventional eNKs (CD56dimCD16+). Pathway analysis implicated IL-17 and IL-18 cytokines, along with NFAT and VEGF signals in tissue residency programming. In conclusion, these data expand our understanding of early transcriptional programs in lymphocyte tissue adaptation, revealing the involvement of cytokines beyond TGF-β. Our data further indicate that signals associated with early tissue adaptation are conserved across species and cell types independent of an antigen receptor.

Prognostic significance of natural killer cell depletion in predicting progressive fibrosing interstitial lung disease in idiopathic inflammatory myopathies.

Interstitial lung disease (ILD) is one of the common extramuscular involvement in idiopathic inflammatory myopathies (IIMs) (1). Several patients develop a progressive fibrosing ILD (PF-ILD) despite conventional treatment, resulting in a progressive deterioration in their quality of life (2). Here, we investigated the clinical and immune characteristics of IIM-ILD and risk factors for PF-ILD in IIM, mainly in anti-melanoma differentiation-associated protein 5 (anti-MDA5+) dermatomyositis (DM) and anti-synthetase syndrome (ASS). Here, a prospective cohort of 156 patients with IIM-ILD were included in the longitudinal analysis and divided into the PF-ILD (n=65) and non-PF-ILD (n=91) groups, and their baseline clinical characteristics were compared. Univariate and multivariate Cox analyses were performed to identify the variables significantly associated with pulmonary fibrosis progression in the total cohort, then anti-MDA5+ DM and ASS groups separately. Peripheral blood lymphocyte counts, including T, B, and NK cell counts, were significantly lower in the PF-ILD group than in the non-PF-ILD group. This characteristic is also present in the comparison between patients with anti-MDA5+ DM and ASS. The multivariate Cox regression analysis revealed that age > 43.5 years [HR: 7.653 (95% CI: 2.005-29.204), p = 0.003], absolute NK cell count < 148 cells/μL [HR: 6.277 (95% CI: 1.572-25.067), p = 0.009] and absolute Th cell count < 533.2 cells/μL [HR: 4.703 (95% CI: 1.014-21.821), p = 0.048] were independent predictors of progressive fibrosing during 1-year follow-up for patients with anti-MDA5+ DM, while absolute count of NK cells < 303.3 cells/µL [HR: 19.962 (95% CI: 3.108-128.223), p = 0.002], absolute count of lymphocytes < 1.545×109/L [HR: 9.684 (95% CI: 1.063-88.186), p = 0.044], and ferritin > 259.45 ng/mL [HR: 6 (95% CI: 1.116-32.256), p = 0.037] were independent predictors of PF-ILD for patients with ASS. Patients with anti-MDA5+ DM and ASS have independent risk factors for PF-ILD. Lymphocyte depletion (particularly NK cells) was significantly associated with PF-ILD within 1-year of follow-up for IIM-ILD.

Generation of an Inhibitory NK Cell Subset by TGF-β1/IL-15 Polarization.

NK cells have been shown to exhibit inflammatory and immunoregulatory functions in a variety of healthy and diseased settings. In the context of chronic viral infection and cancer, distinct NK cell populations that inhibit adaptive immune responses have been observed. To understand how these cells arise and further characterize their immunosuppressive role, we examined invitro conditions that could polarize human NK cells into an inhibitory subset. TGF-β1 has been shown to induce regulatory T cells invitro and invivo; we therefore investigated if TGF-β1 could also induce immunosuppressive NK-like cells. First, we found that TGF-β1/IL-15, but not IL-15 alone, induced CD103+CD49a+ NK-like cells from peripheral blood NK cells, which expressed markers previously associated with inhibitory CD56+ innate lymphoid cells, including high expression of GITR and CD101. Moreover, supernatant from ascites collected from patients with ovarian carcinoma also induced CD103+CD49a+ NK-like cells invitro in a TGF-β-dependent manner. Interestingly, TGF-β1/IL-15-induced CD103+CD56+ NK-like cells suppressed autologous CD4+ T cells invitro by reducing absolute number, proliferation, and expression of activation marker CD25. Collectively, these findings provide new insight into how NK cells may acquire an inhibitory phenotype in TGF-β1-rich environments.

MUC16 can Predict the Pregnancy Outcomes in Human and Intraperitoneal Administration of MUC16 can Rescue Pregnancy Losses in Mouse Models.

Mucin 16 (MUC16) participates in the process of embryo implantation, but few studies have examined the association between MUC16 and pregnancy loss. To investigate this association, the expression of MUC16 in serum and decidua was compared between women with pregnancy loss and ongoing pregnancies. In vitro experiments and animal models were used to explore the role and underlying mechanisms of MUC16 in pregnancy loss. In human study, the expression of MUC16 in serum and decidua was both consistently lower in the women with pregnancy loss compared with those in women with ongoing pregnancies. In vitro experiments revealed the interaction of MUC16 with peripheral blood natural killer (pNK) cells. MUC16 changed the phenotype and reduced the pro-inflammation ability of pNK cells. MUC16 also inhibited the cytotoxicity of pNK cells through the Src homology region 2 domain-containing phosphatase-1/extracellular signal-regulated kinase (SHP-ERK) pathway. Furthermore, MUC16 promoted the migration, invasion and tube formation of trophoblast cells by co-culturing together with pNK cells. In vivo experiments, the mouse model of abortion was used to further confirm that intraperitoneal administration of MUC16 could rescue the pregnancy loss. This study reveals the still-unknown connection between MUC16 and pNK cells and indicates that MUC16 provides a novel method for future prediction and treatment of unfavorable pregnancy outcomes.

Short-chain fatty acids induced lung tumor cell death and increased peripheral blood CD4+ T cells in NSCLC and control patients ex vivo.

Despite therapy advances, one of the leading causes of cancer deaths still remains lung cancer. To improve current treatments or prevent non-small cell lung cancer (NSCLC), the role of the nutrition in cancer onset and progression needs to be understood in more detail. While in colorectal cancer, the influence of local microbiota derived SCFAs have been well investigated, the influence of SCFA on lung cancer cells via peripheral blood immune system should be investigated more deeply. In this respect, nutrients absorbed via the gut might affect the tumor microenvironment (TME) and thus play an important role in tumor cell growth. This study focuses on the impact of the short-chain fatty acid (SCFA) Sodium Butyrate (SB), on lung cancer cell survival. We previously described a pro-tumoral role of glucose on A549 lung adenocarcinoma cell line. In this study, we wanted to know if SB would counteract the effect of glucose and thus cultured A549 and H520 in vitro with and without SB in the presence or absence of glucose and investigated how the treatment with SB affects the survival of lung cancer cells and its influence on immune cells fighting against lung cancer. In this study, we performed cell culture experiments with A549, H520 and NSCLC-patient-derived epithelial cells under different SB levels. To investigate the influence on the immune system, we performed in vitro culture of peripheral mononuclear blood cells (PBMC) from control, smoker and lung cancer patients with increasing SB concentrations. To investigate the effect of SB on lung tumor cells, we first analyzed the effect of 6 different concentrations of SB on A549 cells at 48 and 72 hours cell culture. Here we found that, SB treatment reduced lung cancer cell survival in a concentration dependent manner. We next focused our deeper analysis on the two concentrations, which caused the maximal reduction in cell survival. Here, we observed that SB led to cell cycle arrest and induced early apoptosis in A549 lung cancer cells. The expression of cell cycle regulatory proteins and A549 lung cancer stem cell markers (CD90) was induced. Additionally, this study explored the role of interferon-gamma (IFN-γ) and its receptor (IFN-γ-R1) in combination with SB treatment, revealing that, although IFN-γ-R1 expression was increased, IFN-γ did not affect the efficacy of SB in reducing tumor cell viability. Furthermore, we examined the effects of SB on immune cells, specifically CD8+ T cells and natural killer (NK) cells from healthy individuals, smokers, and NSCLC patients. SB treatment resulted in a decreased production of IFN-γ and granzyme B in CD8+ T cells and NK cells. Moreover, SB induced IFN-γ-R1 in NK cells and CD4+ T cells in the absence of glucose both in PBMCs from controls and NSCLC subjects. Overall, this study highlights the potential of SB in inhibiting lung cancer cell growth, triggering apoptosis, inducing cell cycle arrest, and modulating immune responses by activating peripheral blood CD4+ T cells while selectively inducing IFN-γ-R1 in NK cells in peripheral blood and inhibiting peripheral blood CD8+ T cells and NK cells. Thus, understanding the mechanisms of action of SB in the TME and its influence on the immune system provide valuable insights of potentially considering SB as a candidate for adjunctive therapies in NSCLC.

The proportion of CD161 on CD56+ NK cells in peripheral circulation associates with clinical features and disease activity of primary Sjögren's syndrome.

The purpose of this study was to examine the proportion of CD161 on CD56+ natural killer (NK) cells in peripheral blood of primary Sjögren's syndrome (pSS) and investigate its clinical relevance of pSS. The proportion of CD56+ NK cells and CD161 on CD56+ NK cells was detected by flow cytometry in 31 pSS patients and 29 healthy controls (HCs). The correlations between the proportion of CD161+CD56+ NK cells and clinical features and disease activity of pSS were further analyzed. Meanwhile, we drew the receiver operating characteristic curve to evaluate the diagnostic value of CD161+CD56+ NK cells in pSS. In addition, we evaluated the differences in the effects of CD161+ cells and CD161- cells in peripheral blood on the function of CD56+ NK cells in 5 pSS patients. The proportion of CD56+ NK cells and CD161+CD56+ NK cells decreased markedly in pSS patients compared to HCs. The correlation analysis showed that the proportion of CD161+CD56+ NK cells negatively correlated with white blood cells, Immunoglobulin A (IgA), IgM, IgG, European League Against Rheumatism Sjogren's Syndrome Patient Reported Index andEuropean League Against Rheumatism Sjogren's Syndrome Disease Activity Index, and positively correlated with complement C4. The proportion of CD161+CD56+ NK cells in pSS patients with decayed tooth, fatigue, arthralgia, skin involvement, primary biliary cirrhosis, interstitial lung disease, anti-SSA/Ro60 positive, anti-SSB positive and high IgG was lower than that in negative patients. Furthermore, compared with inactive patients, the proportion of CD161+CD56+ NK cells decreased obviously in active patients. The area under the curve was 0.7375 (p = .0016), the results indicated that CD161+CD56+ NK cells had certain diagnostic values for pSS. In addition, the proportion of CD86, HLA-DR, Ki67, FasL, TNF-α, and IFN-γ on CD161+CD56+ NK cells was lower than that on CD161-CD56+ NK cells in the peripheral blood of pSS patients. This study suggested that the proportion of CD56+ NK cells and CD161+CD56+ NK cells decreased significantly in pSS patients, and the proportion of CD161+CD56+ NK cells negatively associated with the clinical features and disease activity of pSS patients. CD161 expression inhibited the function of CD56+ NK cells in peripheral blood of pSS patients. The CD161+CD56+ NK cells may present as a potential target for therapy and a biomarker of disease activity in pSS.

Abstract 1326: Pre-clinical development of ARB011: A CDH17 targeting allogeneic nonviral RNA-based “Flash” CAR-NK therapy for gastrointestinal cancer

Abstract Background: Treatment for advanced gastrointestinal (GI) cancers has remained challenging, therefore, novel immunotherapies using chimeric antigen receptor (CAR)-T cells are being developed as a new hope for the patients. However, despite the clinical successes in hematologic malignancies, the overall safety and efficacy against solid tumors of CAR-T cells have yet to be established, and the manufacturing of autologous CAR-T cells requires lengthy cell expansion and viral transduction processes. In view of these technical challenges, we have developed ARB011, an RNA-based allogeneic CAR-NK therapy targeting GI cancers expressing cadherin-17 (CDH17), a promising therapeutic target with its overexpression associated with poor prognosis in patients with GI cancers. The use of allogeneic NK cells would mitigate the risk of graft-versus-host disease. Moreover, engineering NK cells with nonviral CAR-encoding mRNA would provide a rapid large-scale manufacturing platform with minimal risk of abnormal genetic alteration mediated by viral integration. Methods: To generate the ARB011 CAR-NK cells, ex vivo expanded human peripheral blood NK (Magicell-NK, Medigen, Taiwan) cells were electroporated with mRNA encoding a humanized anti-CDH17 scFv fused with transactivation domains. Flow cytometry, in vitro cytotoxicity assay, and cytokine release assay were performed to evaluate the expression and functionality of ARB011. To examine the developability of ARB011 as an off-the-shelf cell product, cryopreservation and large-scale electroporation were also studied. Results: Flow cytometry analysis revealed efficient electroporation with a high level of CDH17 CAR expression (&amp;gt;80%) and optimal CAR-NK cell viability (60%-80%). Following electroporation, cytotoxicity of ARB011 was tested against gastric (AGS and NCI-N87), colorectal (DLD-1 and T84), and liver (MHCC-97H) cancer cells by co-culturing at 1:1 and 2:1 effector/target ratio for 12-24h. ARB011 showed significant cytotoxicity against CDH17+ but not CDH17- cancer cells. The CDH17-sepcific cytotoxicity was accompanied with elevated levels of CD107a or LAMP-1 expression on CAR-NK cells and IFN-γ and TNF-α releases. Interestingly, cryopreserved ARB011 maintained optimal cell viability, CAR expression, and potent CDH17 target-specific cytolytic activity. Lastly, large-scale electroporation showed the establishment of up-scaling process of ARB011 production for clinical application. Conclusions: Overall, the findings suggest that ARB011 could be a novel off-the-shelf, CAR-NK therapy that warrants further studies in animal models and in clinical trials. Citation Format: Jaydeep Roy, Nga Tin Lin, Kronos Chow, Chih Ya Yang, James C.L. Lin, John M. Luk, Kwong F. Wong. Pre-clinical development of ARB011: A CDH17 targeting allogeneic nonviral RNA-based “Flash” CAR-NK therapy for gastrointestinal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1326.

Abstract 3615: Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells

Abstract Background: Neuroblastoma (NB) is a common malignant extracranial solid tumor in children, responsible for 11% of cancer deaths in pediatric patients under 15 (Smith et al, Cancer, 2014). High-risk neuroblastoma is a metastatic disease in children aged 18 months or older with L2, M, or MS disease with amplification of the MYCN oncogene (Chuang Pediatr Blood Cancer 2021). Recent analyses by COG show that HR Neuroblastoma still has a 5-year event free survival of 51% despite an extensive treatment regimen (Steven et al, ASCO, 2022). To address improved tumor targeting and overcome antigen escape, chimeric antigen receptor (CAR) technology to Natural Killer (NK) cell therapy has been adapted (Chu/Cairo, et al, Frontiers in Oncology, 2019). TGFβ is a potent mechanism of the NK cell anti-tumor resistance and is present at elevated levels in the NB tumor microenvironment (TME) (Foltz, Cancers, 2018). Studies demonstrated that blockade of TGF-β signaling in NK cells caused the accumulation of NK cells that produce IFN-γ and neutralization of TGF-β prevented NKG2D downregulation and also restored NK cell anti-tumor reactivity (Nayyar/Chu/Cairo, et al Frontierls in Oncology, 2018). ROR1, a transmembrane protein is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al Cancer Immunol Res, 2015). Objective: To determine in vitro cytotoxicity against neuroblastoma cells by ex vivo expanded TGFβ imprinting (TGFβi) NK cells. Methods: TGFβi NK cells are generated by culturing expanded NK cells with irradiated K562-41BBL-mbIL21 in 50 IU/mL IL-2 plus 10 ng/mL TGFβ every 2-3 days for two weeks. NK and TGFβi NK cells specific in-vitro cytotoxicity were compared via luciferase reporter assay against ROR1+ NB cell lines as we previously described (Chu/Cairo, et al, JITC, 2022). Cytokine/chemokine levels secreted by NK and TGFβi NK cells were compared using ELISA assays (Chu/Cairo, et al, JITC, 2022). Results: We have successfully expanded TGFβi NK cells. These TGFβi NK cells showed significantly enhanced in vitro cytotoxicity against NB cells compared to NK cells under TGFβ environment (20+2.4% vs 0%; p&amp;lt;0.001) at E:T=3:1 and consistently, TGFβ-imprinting NK secreted significantly high level of TNF-alpha (393.7pg/ml+16.77pg/ml) against NB cells (p&amp;lt;0.001) compared to NK cells (79.7pg/ml+14.7pg/ml) under TGFβ environment. Conclusion: Our data indicate TGFβi NK cells may be a promising novel therapy to overcome the immunosuppression of TGFβ in the tumor microenvironment for pediatric NB tumors. We will further engineer these TGFβi NK cells to express anti-ROR1 CAR to enhance the targeting specificity against NB tumors and investigate the anti-tumor effects of anti-ROR1 CAR TGFβi NK cells against NB tumors in-vitro and in-vivo (Funded by U54 CA232561). Citation Format: Jessica A. Ayala-Cuesta, Yaya Chu, Keira Foley, Meijuan Tian, Kayleigh Klose, Alyssa S. Mendelowitz, Timothy P. Cripe, Dean A. Lee, Mitchell S. Cairo. Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3615.

Abstract 1240: Enhancing NK cell function in the ‘cold’ tumor microenvironment of prostate cancer with a novel tri-specific killer engager

Abstract Introduction: Natural killer (NK) cells are associated with good prognosis in patients with metastatic castration-resistant prostate cancer (mCRPC). However, NK cell function is hindered in the immunosuppressive and hypoxic tumor microenvironment (TME) of mCRPC. To improve NK cell-mediated responses against mCRPC, we designed a novel Tri-specific Killer Engager (TriKE®) that engages with NK cell activating receptor CD16, binds to prostate-specific membrane antigen (PSMA) that is highly and specifically expressed on mCRPC, and has an interleukin (IL)-15 moiety that is essential for NK cell survival, proliferation, and priming. Methods: PSMA TriKE was produced using the mammalian expression system and tested using healthy donor and prostate cancer patient-derived NK cells. Flow cytometry-based functional and dye dilution proliferation assays were used to compare activation and proliferation of NK cells treated with PSMA TriKE or IL-15. NK cell cytolytic capacity against C4-2 cells, a PSMA-expressing prostate cancer line, was measured using IncuCyte live cell imaging. In various assays, hypoxic (1% oxygen) culture condition and cytokine-induced myeloid-derived suppressor cells (MDSC) were incorporated to better examine PSMA TriKE function in the physiological setting of mCRPC. In vivo testing of the PSMA TriKE was performed using C4-2 xenograft model and expanded NK cells in NSG mice. Results: PSMA TriKE significantly enhanced expansion of peripheral blood NK cells derived from healthy donors up to 8-fold. Additionally, PSMA TriKE induced significantly higher NK cell degranulation and intracellular IFNγ and TNFα buildup, when compared to IL-15 treatment, after incubation with C4-2 cells. Responses of NK cells derived from prostate cancer patients were equivalent to healthy controls. Although NK cell activation was not observed against PSMA knockout (KO) C4-2, indicating specificity of PSMA TriKE treatment, bystander killing of PSMA-KO C4-2 was achieved with PSMA TriKE treatment when wildtype C4-2 cells were cocultured. This suggests potential PSMA TriKE benefit in controlling tumor antigen escape, through natural cytotoxicity once primed. In settings that mimic the TME of mCRPC, NK cells treated with PSMA TriKE in prolonged exposure to hypoxia showed retention of cytotoxicity against C4-2 while IL-15 treated NK cells showed greatly impaired cytotoxicity. Similarly, MDSC suppressed NK cell cytotoxicity in the presence of IL-15, but PSMA TriKE treatment abrogated MDSC-induced suppression. Finally, PSMA TriKE enhanced tumor control and improved survival of mice as compared to IL-15 and no treatment groups in vivo. Conclusion: PSMA TriKE demonstrates potential in overcoming suppression of NK cells in the TME of mCRPC and is a promising candidate for advanced prostate cancer therapy. Citation Format: Shee Kwan Phung, Yvette Soignier, Nicholas Zorko, Rhett L. Waller, Josh Walker, Trygve Nelson, Carly Selleck, Laura Bendzick, Philippa Kennedy, Jeffrey S. Miller, Martin Felices. Enhancing NK cell function in the ‘cold’ tumor microenvironment of prostate cancer with a novel tri-specific killer engager [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1240.

The Ratio of Peripheral Blood Natural Killer Cells is not a Solid Surrogate Immune Index in Unexplained Recurrent Miscarriage.

Immunotherapies targeting peripheral natural killer (pbNK) cells in unexplained recurrent miscarriage (uRM) remain controversial. We hypothesized that the change in pbNK cell count might be a result of innate immune responses rather than a cause. To explore whether the pbNK count is significantly different in women testing positive than those testing negative for commonly studied autoimmune markers. Peripheral blood samples were collected from 302 eligible patients with uRM for the antinuclear antibody (ANA) testing determined by the enzyme-linked immunosorbent assay (ELISA), anti-thyroid peroxidase antibody (TPO-Ab) testing and anti-thyroglobulin antibody (Tg-Ab) testing determined by the chemiluminescent immunoassay, and pbNK cell testing determined by flow cytometry. The patients were divided into two groups according to the pbNK normal range, and the comparative analysis entailed an examination of the prevalence rates of autoantibodies within the high pbNK group and the normal pbNK group, followed by a comprehensive investigation into the potential correlations between autoantibodies and pbNK cells. There was a positive association between TPO-Ab positivity and high pbNK cells (p=0.016, OR=5.097, 95% CI 1.356-19.159), while there was a negative association between ANA positivity and high pbNK cells (p=0.013, OR=0.293, 95% CI 0.111-0.773). TPO-Ab-positive patients had a higher pbNK cell count compared with TPO-Ab-negative patients, while ANA-positive patients had a lower pbNK cell count compared with ANA-negative patients. The change in pbNK cell count may be a consequence of immune responses, and there should be careful consideration in applying it as an immunotherapeutic index.

The changes and its significance of peripheral blood NK cells in patients with tuberculous meningitis.

Tuberculous meningitis (TBM) is the most severe form of tuberculosis (TB). The purpose of this study was to explore the relationship between the number of natural killer (NK) cells and adaptive immune status, and disease severity in TBM patients. We conducted a retrospective study on 244 TB patients and 146 healthy control subjects in the 8th Medical Center of the PLA General Hospital from March 2018 and August 2023. The absolute count of NK cells in the peripheral blood of TBM patients was significantly lower than that in normal controls (NC), latent tuberculosis infection (LTBI), and non-severe TB (NSTB) patients (p < 0.05). The proportion of TBM patients (48.7%) with a lower absolute count of NK cells than the normal reference value was significantly higher than that in NC (5.2%) and LTBI groups (4.0%) (p < 0.05), and slightly higher than that in NSTB group (36.0%) (p > 0.05). The absolute counts of lymphocyte subsets in TBM combined with other active TB group, etiology (+) group, IGRA (-) group, and antibody (+) group were lower than that in simple TBM group, etiology (-) group, IGRA (+) group, and antibody (-) group, respectively. The CD3+ T, NK, and B cells in BMRC-stage III TBM patients were significantly lower than those in stage I and stage II patients (p < 0.05). The counts of CD3+ T, CD4+ T, and B cells in the etiology (+) group were significantly lower than those in the etiology (-) group (p < 0.05). The absolute counts of lymphocyte subsets in the peripheral blood of TBM patients were significantly decreased, especially in NK cells. The reduction of these immune cells was closely related to the disease severity and had a certain correlation with cellular and humoral immune responses. This study helps to better understand the immune mechanism of TBM and provides reliable indicators for evaluating the immune status of TBM patients in clinical practice.

NK92 cells and peripheral blood NK cells respond oppositely upon dasatinib treatment.

Natural killer (NK) cell is a valuable tool for immunotherapy in cancer treatment, both the cultured cell line NK92 and primary NK cells are widely studied and used in research and clinical trials. Clinical observations witnessed the improvement of patients' NK cells in terms of cell counts and cytotoxic activity upon dasatinib treatment, an approved drug for chronic myeloid leukaemia and Ph+ acute lymphocytic leukaemia. Several studies supported the clinical observations, yet others argued a detrimental effect of dasatinib on NK cells. Due to the complex conditions in different studies, the definite influence of dasatinib on NK92 and primary NK cells remains to be settled. Here, we used a well-defined in vitro system to evaluate the effects of dasatinib on NK92 cells and peripheral blood (PB)-NK cells. By co-culturing NK cells with dasatinib to test the cell counts and target cell-killing activities, we surprisingly found that the chemical influenced oppositely on these two types of NK cells. While dasatinib suppressed NK92 cell proliferation and cytotoxic activity, it improved PB-NK-killing tumour cells. RNA sequencing analysis further supported this finding, uncovering several proliferating and cytotoxic pathways responding invertedly between them. Our results highlighted an intrinsic difference between NK92 and PB-NK cells and may build clues to understand how dasatinib interacts with NK cells in vivo.

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

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. 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. 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. 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.

Integrated Analysis of Single-Cell and Bulk RNA Sequencing Data Reveals Memory-like NK Cell Subset Associated with Mycobacterium tuberculosis Latency.

Natural killer (NK) cells are innate-like lymphocytes that belong to the family of type-1 innate lymphoid cells and rapidly respond to virus-infected and tumor cells. In this study, we have combined scRNA-seq data and bulk RNA-seq data to define the phenotypic and molecular characteristics of peripheral blood NK cells. While the role of NK cells in immune surveillance against virus infections and tumors has been well established, their contribution to protective responses to other intracellular microorganisms, such as Mycobacterium tuberculosis (Mtb), is still poorly understood. In this study, we have combined scRNA-seq data and bulk RNA-seq data to illuminate the molecular characteristics of circulating NK cells in patients with active tuberculosis (TB) disease and subjects with latent Mtb infection (LTBI) and compared these characteristics with those of healthy donors (HDs) and patients with non-TB other pulmonary infectious diseases (ODs). We show here that the NK cell cluster was significantly increased in LTBI subjects, as compared to patients with active TB or other non-TB pulmonary diseases and HD, and this was mostly attributable to the expansion of an NK cell population expressing KLRC2, CD52, CCL5 and HLA-DRB1, which most likely corresponds to memory-like NK2.1 cells. These data were validated by flow cytometry analysis in a small cohort of samples, showing that LTBI subjects have a significant expansion of NK cells characterized by the prevalence of memory-like CD52+ NKG2C+ NK cells. Altogether, our results provide some new information on the role of NK cells in protective immune responses to Mtb.

Aberrant NK cell profile in gestational diabetes mellitus with fetal growth restriction.

Gestational diabetes mellitus (GDM) is a gestational disorder characterized by hyperglycemia, that can lead to dysfunction of diverse cells in the body, especially the immune cells. It has been reported that immune cells, specifically natural killer (NK) cells, play a crucial role in normal pregnancy. However, it remains unknown how hyperglycemia affects NK cell dysfunction thus participates in the development of GDM. In this experiment, GDM mice were induced by an intraperitoneal injection of streptozotocin (STZ) after pregnancy and it has been found that the intrauterine growth restriction occurred in mice with STZ-induced GDM, accompanied by the changed proportion and function of NK cells. The percentage of cytotoxic CD27-CD11b+ NK cells was significantly increased, while the proportion of nourished CD27-CD11b- NK cells was significantly reduced in the decidua of GDM mice. Likewise, the same trend appeared in the peripheral blood NK cell subsets of GDM patients. What's more, after intrauterine reinfusion of NK cells to GDM mice, the fetal growth restriction was alleviated and the proportion of NK cells was restored. Our findings provide a theoretical and experimental basis for further exploring the pathogenesis of GDM.

Androgen receptor blockade to activate NK cells and to upregulate the surface expression of HLA-E in prostate cancer cell lines.

190 Background: The blockade of the androgen receptor (AR) pathway is an effective treatment for prostate cancer (PCa), but most patients progress to castration-resistant prostate cancer (mCRPC). AR signaling modulates CD8+ T cytotoxic function, revealing the immune-modulatory function of the AR pathway. We have described the activation of NK cells by AR inhibitors and the potential synergistic effect with anti-NKG2a combination.1 Strategies to activate NK cells can be limited by the expression of HLA-E, a ligand of the inhibitory checkpoints on NK cells NKG2A. We investigated the mechanisms of AR-dependent modulation of HLA-E on tumor cells and the ADT-enhancing effect on patient-derived NK cells. Methods: PC cell lines were treated with second-generation androgen pathway inhibitors in vitro (enzalutamide 10uM, darolutamide 15 uM), and the expression of HLA-E was evaluated by flow cytometry. To evaluate the modulation of HLA-E by AR, the AR-negative cell lines (PC3 and DU145) were stably transduced with an inducible AR system. The pan HDAC inhibitors vorinostat (0.4 uM) and panobinostat (2.5 uM) were used to evaluate the regulation of HLA-E by epigenetics. We analyzed the activation status of paired peripheral blood NK cells isolated from patients with PCa prior to and post-androgen deprivation therapy (ADT). The patients (n=6) had a median time between collection of 26.3±2.3 days. Results: The AR-responsive LNCaP cell line displayed an increase in surface expression of HLA-E upon treatment with enzalutamide (Enza) or darolutamide (Daro) ([C]: 12.1±1.3%, Enza: 22.3±3.3%, Daro: 19.1±1.1%, p=0.004). AR blockade did not change the surface expression of HLA-E of AR negative PC3 ([C]: 10.5±2.7%, Daro: 13.1±2.3%, p=0.17) or DU145 cell lines ([C]: 3.5±1.7%, Daro: 2.5±0.6%, p=0.12). Transduction of AR in PC3 and DU145 lead to upregulation of HLA-E expression with AR blockade (PC3-AR+ [C]: 13.1±3.7%, Daro: 43.1±5.3%, DU145-AR+ [C]: 4.5±1.4%, Daro: 27.2±3.6%, p=0.001). The upregulation of HLA-E upon AR blockade was suppressed if cell lines were co-treated with vorinostat or panobinostat. Patient-derived peripheral blood NK cells displayed enhanced cytotoxic activity after ADT (expression of Granzyme B pre-ADT: 12.3±2.3%, post-ADT: 36.5±5.7% p=0.0017, Perforin pre-ADT: 3±0.6%, post-ADT: 26.5±3.7% p=0.001), and upregulation of the inhibitory checkpoint NKG2a (pre-ADT: 5.2±1.2%, post-ADT: 12.3±1.4%, p=0.04). Conclusions: Androgen inhibitors upregulate the expression of HLA-E in PCa cell lines by an AR-dependent mechanism regulated by epigenetics. ADT promotes peripheral blood patient-derived NK cell activation and upregulation of inhibitory NKG2A receptor. These findings support further investigative approaches targeting the HLA-E and NKG2A in mCRPC. 1. Schwermann, AACR 2023.