Activating Receptors Research Articles

Natural Killer-Based Therapy: A Prospective Thought for Cancer Treatment Related to Diversified Drug Delivery Pathways.

Immunotherapy has been a research hotspot due to its low side effects, long-lasting efficacy, and wide anti-tumor spectrum. Recently, NK cell-based immunotherapy has gained broad attention for its unique immunological character of tumor identification and eradication and low risk of graft-versus-host disease and cytokine storm. With the cooperation of a drug delivery system (DDS), NK cells activate tumoricidal activity by adjusting the balance of the activating and inhibitory signals on their surface after drug-loaded DDS administration. Moreover, NK cells or NK-derived exosomes can also be applied as drug carriers for distinct modification to promote NK activation and exert anti-tumor effects. In this review, we first introduce the source and classification of NK cells and describe the common activating and inhibitory receptors on their surface. Then, we summarize the strategies for activating NK cells in vivo through various DDSs. Finally, the application prospects of NK cells in tumor immunotherapy are also discussed.

Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy.

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca 2+ influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylate SYK genes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination of SYK resulted in more robust Ca ++ influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereas ZAP70 deletion diminished these responses. By contrast, ZAP70 depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca 2+ flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion of SYK in NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablate SYK or ZAP70 . Deletion of SYK resulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.

CAR memory-like NK cells targeting the membrane proximal domain of mesothelin demonstrate promising activity in ovarian cancer.

Epithelial ovarian cancer (EOC) remains one of the most lethal gynecological cancers. Cytokine-induced memory-like (CIML) natural killer (NK) cells have shown promising results in preclinical and early-phase clinical trials. In the current study, CIML NK cells demonstrated superior antitumor responses against a panel of EOC cell lines, increased expression of activation receptors, and up-regulation of genes involved in cell cycle/proliferation and down-regulation of inhibitory/suppressive genes. CIML NK cells transduced with a chimeric antigen receptor (CAR) targeting the membrane-proximal domain of mesothelin (MSLN) further improved the antitumor responses against MSLN-expressing EOC cells and patient-derived xenograft tumor cells. CAR arming of the CIML NK cells subtanstially reduced their dysfunction in patient-derived ascites fluid with transcriptomic changes related to altered metabolism and tonic signaling as potential mechanisms. Lastly, the adoptive transfer of MSLN-CAR CIML NK cells demonstrated remarkable inhibition of tumor growth and prevented metastatic spread in xenograft mice, supporting their potential as an effective therapeutic strategy in EOC.

An Extended Flow Cytometry Evaluation of ex Vivo Expanded NK Cells Using K562.Clone1, a Feeder Cell Line Manufactured in Brazil

Natural killer (NK) cells play a crucial role in the immune system's response against cancer. However, the challenge of obtaining the required quantity of NK cells for effective therapeutic response necessitates the development of strategies for their ex vivo expansion. This study aimed to develop a novel feeder cell line, K562.Clone1, capable of promoting the ex vivo expansion of NK cells while preserving their cytotoxic potential. he K562 leukemic cell line was transduced with mbIL-21 and 4-1BBL proteins to generate K562.Clone1 cells. NK cells were then co-cultured with these feeder cells, and their expansion rate was monitored over 14 days. The cytotoxic potential of the expanded NK cells was evaluated against acute myeloid leukemia blasts and tumor cell lines of leukemia and glial origin. Statistical analysis was performed to determine the significance of the results. The K562.Clone1 co-cultured with peripheral NK showed a significant increase in cell count, with an approximate 94-fold expansion over 14 days. Expanded NK cells demonstrated cytotoxicity against the tested tumor cell lines, indicating preservation of their cytotoxic characteristics. Additionally, the CD56, CD16, inhibitory KIRs, and activation receptors were conserved and present in a well-balanced manner. The study successfully developed a feeder cell line, K562.Clone1, that effectively promotes the expansion of NK cells ex vivo while maintaining their cytotoxic potential. This development could significantly contribute to the advancement of NK cell therapy, especially in Brazil.

Cataloging circulating CD3+CD56+ NKT-like cells through a series of stimulating (NKG2D and DNAM-1) and inhibitory (PD-1, TIGIT, and Tim-3) immune checkpoint receptors in women diagnosed with precancerous cervical lesions or invasive cervical carcinoma

Persistent human papillomavirus infection is associated with the development of premalignant lesions that can eventually lead to cervical cancer. In this study, we evaluated the expression of activating (NKG2D, DNAM-1) and inhibitory immune checkpoints receptors (PD-1, TIGIT, and Tim-3) in peripheral blood NKT-like (CD3+CD56+) lymphocytes from patients with cervical carcinoma (CC, n = 19), high-grade lesions (HG, n = 8), low-grade lesions (LG, n = 19) and healthy donors (HD, n = 17) using multiparametric flow cytometry. Dimensional data analysis showed four clusters within the CD3+CD56+ cells with different patterns of receptor expression. We observed upregulation of CD16 in CC and HG patients in one of the clusters. In another, TIGIT was upregulated, while DNAM-1 was downregulated. Throughout manual gating, we observed that NKT-like cells expressing activating receptors also co-express inhibitory receptors (PD-1 and TIGIT), which can affect the activation of these cells. A deeper characterization of the functional state of the cells may help to clarify their role in cervical cancer, as will the characterization of the NKT-like cells as cytotoxic CD8+ T cells or members of type I or type II NKT cells.

The HHV-6B U20 glycoprotein binds ULBP1, masking it from recognition by NKG2D and interfering with natural killer cell activation.

Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1.Structural modeling of the U20 - ULBP1 complex indicates some similarities to the m152-RAE1γ complex.

CD155 blockade enhances allogeneic natural killer cell-mediated antitumor response against osteosarcoma.

Allogeneic bone marrow transplant (alloBMT) is curative for hematologic malignancies through the graft-versus-tumor (GVT) effect but has been ineffective for solid tumors like osteosarcoma (OS). OS expresses CD155 which interacts strongly with inhibitory receptors TIGIT and CD96 but also binds to activating receptor DNAM-1 on natural killer (NK) cells. CD155 has never been targeted after alloBMT. Combining adoptively transferred allogeneic NK (alloNK) cells with CD155 blockade after alloBMT may enhance a GVT effect against OS. Murine NK cells were activated and expanded ex vivo with soluble IL-15/IL-15Rα. AlloNK and syngeneic NK (synNK) cell phenotype, cytotoxicity, cytokine production, and degranulation against CD155-expressing murine OS cell line K7M2 were assessed in vitro. Mice bearing pulmonary OS metastases underwent alloBMT and alloNK cell infusion with anti-CD155 either before or after tumor induction, with select groups receiving anti-DNAM-1 pretreated alloNK cells. Tumor growth, GVHD and survival were monitored, and differential gene expression of lung tissue was assessed by RNA microarray. AlloNK cells exhibited superior cytotoxicity against CD155-expressing OS compared to synNK cells, and this activity was enhanced by CD155 blockade. CD155 blockade increased alloNK cell degranulation and interferon gamma production through DNAM-1. In vivo, CD155 blockade with alloNK infusion increased survival when treating OS that relapsed after alloBMT. No benefit was seen for treating established OS before alloBMT. Treatment with combination CD155 and anti-DNAM-1 pretreated alloNK ameliorated survival and tumor control benefits seen with CD155 blockade alone. RNA microarray showed mice treated with alloNK and CD155 blockade had increased expression of cytotoxicity genes and the NKG2D ligand H60a, whereas mice treated with anti-DNAM-1 pretreated alloNK cells resulted in upregulation of NK cell inhibitory receptor genes. Whereas blocking DNAM-1 on alloNK abrogated cytotoxicity, blocking NKG2D had no effect, implying DNAM-1:CD155 engagement drives alloNK activation against OS. These results demonstrate the safety and efficacy of infusing alloNK cells with CD155 blockade to mount a GVT effect against OS and show benefits are in part through DNAM-1. Defining the hierarchy of receptors that govern alloNK responses is critical to translating alloNK cell infusions and immune checkpoint inhibition for solid tumors treated with alloBMT. Allogeneic bone marrow transplant (alloBMT) has yet to show efficacy in treating solid tumors, such as osteosarcoma (OS). CD155 is expressed on OS and interacts with natural killer (NK) cell receptors, such as activating receptor DNAM-1 and inhibitory receptors TIGIT and CD96 and has a dominant inhibitory effect on NK cell activity. Targeting CD155 interactions on allogeneic NK cells could enhance anti-OS responses, but this has not been tested after alloBMT. CD155 blockade enhances allogeneic natural killer cell-mediated cytotoxicity against OS and improved event-free survival after alloBMT in an in vivo mouse model of metastatic pulmonary OS. Addition of DNAM-1 blockade abrogated CD155 blockade-enhanced allogeneic NK cell antitumor responses. These results demonstrate efficacy of allogeneic NK cells combined with CD155 blockade to mount an antitumor response against CD155-expressing OS. Translation of combination adoptive NK cell and CD155 axis modulation offers a platform for alloBMT treatment approaches for pediatric patients with relapsed and refractory solid tumors.

CD56bright NK cell expansion correlated with EBV reactivation control post allogeneic hematopoietic stem cell transplantation.

Natural killer (NK) cells are equipped with anti-Epstein-Barr virus (EBV) function, however, whether EBV infection will affect NK cells reconstitution after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains unclear. To identify the characteristics of NK cells, we prospectively enrolled 11 patients who occurred EBV reactivation post allo-HSCT and 11 patients without EBV infection as control. We found that that EBV infection induced the expansion of CD56bright and NKG2A+KIR- NK subsets,and decreased the cytotoxicity function of NK cells. The frequency of NKG2A+KIR- NK cells were higher in patients who progressed into post-transplant lymphoproliferative disorder (PTLD) than EBV viremia patients, which also correlated with decreased proliferation and cytotoxic function. By screening the activation receptors of NK cells, we found the DNAM-1+CD56bright NK cells is significantly increased after EBV stimulation, further we demonstrated that DNAM-1 is essential for EBV induced NK cells activation as the cytokine release against EBV-transformed lymphoblastoid cell lines(EBV-LCLs) of CD56bright NK cells were significantly decreased after DNAM-1 blockade. NK cells infusion suppressed the progression of EBV-related tumor mice model. A prospective cohort indicated that old donor age was an independent risk factor for EBV infection. Rapid CD56bri expansion and high expression of DNAM-1 on CD56bri NK cells in response to EBV reactivation correlated with rapid EBV clearance post allo-HSCT in patients with younger donors. In summary, our data showed that high expression of DNAM-1 receptors on NK cell may participate protective CD56bri NK cells response to EBV infection after allo-HSCT.

Expression of a single inhibitory Ly49 receptor is sufficient to license NK cells for effector functions.

Natural killer (NK) cells recognize target cells through germline-encoded activation and inhibitory receptors enabling effective immunity against viruses and cancer. The Ly49 receptor family in the mouse and killer immunoglobin-like receptor family in humans play a central role in NK cell immunity through recognition of MHC class I and related molecules. Functionally, these receptor families are involved in licensing and rejection of MHC-I-deficient cells through missing-self. The Ly49 family is highly polymorphic, making it challenging to detail the contributions of individual Ly49 receptors to NK cell function. Herein, we showed mice lacking expression of all Ly49s were unable to reject missing-self target cells in vivo, were defective in NK cell licensing, and displayed lower KLRG1 on the surface of NK cells. Expression of Ly49A alone on a H-2Dd background restored missing-self target cell rejection, NK cell licensing, and NK cell KLRG1 expression. Thus, a single inhibitory Ly49 receptor is sufficient to license NK cells and mediate missing-self in vivo.

Proteasome inhibitors FHND6091 enhance the ability of NK cells to kill tumor cells through multiple mechanisms

The immune system has a strong connection to tumors. When a tumor cell is recognized as an abnormal cell by the immune system, the immune system may initiate an immune response to kill the tumor cell. In this study, RNA sequencing was performed on multiple myeloma (MM) cells treated with the proteasome inhibitor FHND6091. The transcriptional changes induced by FHND6091 in RPMI8226 cells aligned notably with immune response activation and results indicated upregulation of cGAS-STING pathway-related genes in the FHND6091-treated group. In vivo and in vitro experiments had demonstrated that FHND6091 stimulated the immunoreaction of MM cells via activation of the cyclic guanosine monophosphate-adenosine synthase/stimulator of interferon genes (cGAS-STING) pathway. This activation resulted in the generation of type-I interferons and the mobilization of natural killer (NK) cells. Notably, FHND6091 upregulated the levels of calreticulin and the protein ligands UL16-binding protein 2/5/6, MHC class I chain-related A (MICA), and MICB on the surface of MM cells. Subsequently, upon engaging with the surface activation receptors of NK cells, these ligands triggered NK cell activation, leading to the subsequent elimination of tumor cells. Thus, our findings elucidated the mechanism whereby FHND6091 exerted its immunotherapeutic activity as a STING agonist, enhancing the killing ability of NK cells against tumor cells.

EVEREST-2: A seamless phase 1/2 study of A2B694, a mesothelin (MSLN) logic-gated Tmod CAR T-cell therapy, in patients with solid tumors that show MSLN expression and human leukocyte antigen (HLA)-A*02 loss of heterozygosity (LOH).

TPS2699 Background: Despite the success in hematologic cancers, chimeric antigen receptor (CAR) T-cell therapies are challenging to implement in solid tumors owing to a lack of tumor-specific targets that discriminate cancer from normal cells. MSLN expression normally is limited to the mesothelium of major body cavities but can be upregulated in diverse solid tumor types (TCGA 2022), making it a potential target for cancer therapy. MSLN-targeted cell approaches, including CAR T-cell and T-cell receptor fusion therapies, have shown promising clinical activity; however, on-target, off-tumor toxicity including fatal events have occurred (1-3). A2B694 is an autologous logic-gated, MSLN-targeted Tmod CAR T-cell therapy that addresses the challenges of on-target, off-tumor toxicity by combining 2 CARs: an activating and blocking receptor. The activator recognizes MSLN present on the surface of both tumor and normal cells; the blocker binds HLA-A*02 and prevents CAR T-cell activity. Thus, in patients with both germline HLA-A*02 and tumor-associated HLA-A*02 LOH, the blocker prevents on-target, off-tumor toxicity on normal cells owing to retained HLA-A*02 expression (4). Through this unique discriminatory mechanism, A2B694 may provide a therapeutic window to treat patients with MSLN-expressing solid tumors exhibiting HLA-A*02 LOH. Methods: EVEREST-2 (NCT06051695) is a first-in-human, phase 1/2, open-label, nonrandomized study to evaluate the safety and efficacy of A2B694 in adults with recurrent unresectable, locally advanced, or metastatic cancers with MSLN expression, including non-small cell lung cancer, colorectal cancer, pancreatic cancer, ovarian cancer, mesothelioma, or other solid tumors with MSLN expression. Eligible patients should have received ≥1 line of prior therapy such as checkpoint inhibitor, molecular targeted, or chemotherapy. Enrollment to EVEREST-2 occurs through the prescreening study BASECAMP-1 (NCT04981119), which identifies patients with tumor-associated HLA-A*02 LOH via next-generation sequencing (Tempus AI, Inc.). Eligible patients enroll in the BASECAMP-1 study and undergo leukapheresis. A2B694 is manufactured from cryopreserved T cells when clinically appropriate for patients. The primary objective of phase 1 is to evaluate the safety and tolerability of A2B694 and identify the recommended phase 2 dose (RP2D). The dose-expansion phase will confirm RP2D and collect biomarker data to further characterize A2B694. Phase 2 will assess overall response rate per RECIST v1.1. 1. Beatty, et al. Gastroenterology. 2018. 2. Haas, et al. Mol Ther. 2023. 3. Hong, et al. ESMO 2021. Abstract 9590. 4. Hamburger, et al. Mol Immunol. 2020. Clinical trial information: NCT06051695 .

Association of overexpression of TNF receptor superfamily members (CD40, BAFFR, LTβR and RANK) with favorable prognosis in patients with colorectal adenocarcinoma.

e15516 Background: Numerous studies by our and other groups have highlighted the implication of NF-κB in colorectal cancer (CRC) through the deterioration of classical and alternative pathway signaling. The alternative pathway of NF-κB can be activated by members of TNF superfamily, such as lymphotoxin β (LTβ), tumor necrosis factor ligand superfamily member 5 (CD40L), B-cell activation factor (BAFF) and receptor activator of NF-κB ligand (RANKL). We have previously shown that alternative pathway of NF-κΒ is deteriorated in CRC. In the present study, we investigated the clinical significance of the aforementioned receptors in patients with CRC as well as their relation with effector transcription factors NF-κB2 and RELB. Methods: Gene expression of CD40, BAFFR, LT β R, RANK, NF- κ B2 and RELB, quantified by using real-time qRT-PCT and specific primers and probes, was assessed in 97 cancerous and 61 paired non-neoplastic tumor-adjacent formalin-fixed paraffin-embedded (FFPE) tissue specimens from CRC patients who were surgically managed in the University Hospital of Patras, Greece. Concurrently, we assessed protein expression of the same molecules by immunohistochemistry in the same cohort of patients. Both, mRNA and protein levels of the receptors were analyzed in association with clinicopathological parameters and clinical outcome of the patients in terms of time to disease progression. Results: Cytoplasmic expression of all molecules was higher in cancer compared to tumor-adjacent epithelial cells ( p= 0.047 for CD40, p= 0.007 for BAFFR, p< 0.001 for LTβR and p= 0.003 for RANK) and in stromal cells ( p< 0.001 for all molecules), while the reverse pattern (lower in neoplastic epithelial cells) was observed for nuclear expression ( p< 0.001 for all molecules). Similarly, mRNA levels of LTβR and RANK were higher in cancer compared to adjacent non-neoplastic tissues ( p= 0.019 and p= 0.037, respectively). Notably, patients with high mRNA levels of all studied receptors had significantly longer time to disease progression ( p= 0.026 for CD40, p= 0.023 for BAFFR, p= 0.027 for LTβR and p= 0.038 for RANK). In addition, mRNA levels of BAFFR, LTβR and RANK levels were higher in ulcerative tumors compared to polypoid tumors ( p= 0.002, p= 0.005 and p= 0.011, respectively). RELB mRNA levels were positively correlated with mRNA levels of CD40, BAFFR and LTβR in cancerous ( p< 0.001 for all molecules) and non-cancerous tissues (p < 0.001 for all molecules). Surprisingly, NF-κB2 mRNA levels were positively correlated with BAFFR and LTβR mRNA levels in malignant tissues, while in non-malignant tissues were positively correlated with all receptors’ mRNA levels ( p< 0.001 for all molecules). Conclusions: This study suggests a prognostic value for CD40, BAFFR, LTβR and RANK expression in patients with CRC and further supports the role of the alternative pathway of NF-κB in CRC.

Autologous, oncolytic virus-activated killer (VAK) cell therapy to treat malignant pleural and peritoneal effusions.

e20549 Background: OH2 is an attenuated oncolytic Herpes Simplex-2 virus expressing GM-CSF. OH2 replicates only in cancer cells, causing their lysis, which reverses the immunosuppressive tumor microenvironment, leading to tumor regression. OH2 monotherapy is safe and efficacious in phase I-III trials of 300+ patients across several cancer indications. We previously showed that OH2 treated briefly with UV light (UV-OH2) is unable to form plaques yet still able to infect cancer cells and to potently activate human PBMCs, leading to increased antitumor activity in vitro and in animal models. Methods: Translating these findings in humans, 12 stage IV lung cancer patients with malignant pleural or peritoneal effusions were enrolled in an Investigator-Initiated Trial (NCT05565014). Lymphocytes from each patient’s effusions were isolated, incubated in vitro with UV-OH2 for 36-48h, and then re-infused into the patient's pleural or peritoneal cavity. Each patient received 2 cycles of 3 or up to 6 infusions of this Virus Activated Killer (VAK) Autologous Cell Therapy. Results: Effusion volume (EV) measurement and local and whole-body tumor assessments were performed at the beginning and 28 days post VAK treatment. On D28, EV remained stable in 3 patients (SD), whereas 4 of 12 patients recorded a reduction of EV (PR). Notably, subject S004’s PR was maintained for 210 days post-treatment. Evaluation of target lesions per RECIST revealed 4 subjects with PR and 7 with an SD in target lesions. There was no toxicity or treatment-related adverse events. The median patient OS was 384 days (12.8 months), which is up to 4 times the average published OS for stage IV lung cancer with malignant pleural effusions. RNA-Seq analysis indicated increased expression of many T and NK cell effectors and NK cell activating receptors. Importantly, average autologous CD8+ T lymphocytes rose from 53% to 74% in the effusions post VAK-treatment, thereby confirming activation of the patient’s immune system by UV-OH2 (preliminary clinical PoC). Conclusions: In sum, our VAK therapy offers a cost-effective, extremely safe, and patient-friendly autologous treatment that dramatically increases overall survival in patients with essentially no options. Larger scale studies are in the works. Clinical trial information: NCT05565014 .

Induced CD8α identifies human NK cells with enhanced proliferative fitness and modulates NK cell activation.

The surface receptor CD8α is present on 20%-80% of human (but not mouse) NK cells, yet its function on NK cells remains poorly understood. CD8α expression on donor NK cells was associated with a lack of therapeutic responses in patients with leukemia in prior studies, thus, we hypothesized that CD8α may affect critical NK cell functions. Here, we discovered that CD8α- NK cells had improved control of leukemia in xenograft models compared with CD8α+ NK cells, likely due to an enhanced capacity for proliferation. Unexpectedly, we found that CD8α expression was induced on approximately 30% of previously CD8α- NK cells following IL-15 stimulation. These induced CD8α+ (iCD8α+) NK cells had the greatest proliferation, responses to IL-15 signaling, and metabolic activity compared with those that sustained existing CD8α expression (sustained CD8α+) or those that remained CD8α- (persistent CD8α-). These iCD8α+ cells originated from an IL-15Rβhi NK cell population, with CD8α expression dependent on the transcription factor RUNX3. Moreover, CD8A CRISPR/Cas9 deletion resulted in enhanced responses through the activating receptor NKp30, possibly by modulating KIR inhibitory function. Thus, CD8α status identified human NK cell capacity for IL-15-induced proliferation and metabolism in a time-dependent fashion, and its presence had a suppressive effect on NK cell-activating receptors.

#2191 Platelet function in chronic kidney disease; can flow cytometry guide in the difficult balance between risk for bleeding versus thrombotic events?

Abstract Background and Aims Increased risk of bleeding is a well-known complication of chronic kidney disease (CKD). However, these patients also have an increased risk of thrombotic events. Several studies have implicated both impaired and enhanced platelet function in patients with chronic kidney disease, but results vary between different studies and methods. Flow cytometry has emerged as a new method to study platelet function. Our aim was to investigate platelet function using flow cytometry in patients with chronic kidney disease. Method 20 patients were included between February and May 2023. Pre-dialytic patients with an eGFR between 8-20 mL/min/1,73 m2 were included, mean eGFR 14.7 mL/min/1,73 m2. Patients with platelet levels >100 × 109/L were included. Medication in the form of anticoagulants, NSAIDs, platelet inhibitors or SSRIs were used as exclusion criteria. Platelet activation assay was performed with flow cytometry. The expression of activation markers PAC-1 (activated GPIIb/IIIa), CD62P (P-selectin) and CD63 (dense granules) were analysed before and after activation with ADP, CRP-XL (collagen stimulating peptide, activates GPVI) or TRAP-6 (stimulates platelet activating receptor 1, PAR1). Platelets from 20 healthy control subjects were analysed for comparison. Results The level and size of platelets (MPV) were comparable in both groups. There were significantly higher levels of D-dimer in the CKD group vs healthy controls (1,17 mg/L FEU vs 0,27 mg/L FEU). Except for the decreased expression of activated GPIIb-IIIa receptor after activation with ADP and CRP-XL in one patient, the platelet activation did not significantly differ between CKD patients and healthy controls. Conclusion No significant differences in the activation of platelets were observed between the CKD population and the healthy controls when analysed by flow cytometry. Elevated levels of D-dimer in CKD patients have previously been described and are believed to be due to increased fibrin formation, lysis and decreased renal clearance. Further studies in a larger patient population, including thrombocytopenic patients are planned, with greater focus to be placed on dense granula quantities which may correlate with decreased levels in the CKD population.

Essential growth factor receptors for fibroblast homeostasis and activation: Fibroblast Growth Factor Receptor (FGFR), Platelet Derived Growth Factor Receptor (PDGFR), and Transforming Growth Factor β Receptor (TGFβR).

Fibroblasts are cells of mesenchymal origin that are found throughout the body. While these cells have several functions, their integral roles include maintaining tissue architecture through the production of key extracellular matrix components, and participation in wound healing after injury. Fibroblasts are also key mediators in disease progression during fibrosis, cancer, and other inflammatory diseases. Under these perturbed states, fibroblasts can activate into inflammatory fibroblasts or contractile myofibroblasts. Fibroblasts require various growth factors and mitogenic molecules for survival, proliferation, and differentiation. While the activity of mitogenic growth factors on fibroblasts in vitro was characterized as early as the 1970s, the proliferation and differentiation effects of growth factors on these cells in vivo are unclear. Recent work exploring the heterogeneity of fibroblasts raises questions as to whether all fibroblast cell states exhibit the same growth factor requirements. Here, we will examine and review existing studies on the influence of fibroblast growth factor receptors (FGFRs), platelet-derived growth factor receptors (PDGFRs), and transforming growth factor β receptor (TGFβR) on fibroblast cell states.

In vitro study on device-induced damage to blood cellular components and degradation of von Willebrand factor in a CentriMag pump-assisted circulation.

High mechanical shear stress (HMSS) generated by blood pumps during mechanical circulatory support induces blood damage (or function alteration) not only of blood cell components but also of plasma proteins. In the present study, fresh, healthy human blood was used to prime a blood circuit assisted by a CentriMag centrifugal pump at a flow rate of 4.5 L/min under three pump pressure heads (75, 150, and 350 mm Hg) for 4 h. Blood samples were collected for analyses of plasma-free hemoglobin (PFH), von Willebrand factor (VWF) degradation and platelet glycoprotein (GP) IIb/IIIa receptor shedding. The extent of all investigated aspects of blood damage increased with increasing cross-pump pressure and duration. Loss of high-molecular-weight multimers (HMWM)-VWF in Loop 2 and Loop 3 significantly increased after 2 h. PFH, loss of HMWM-VWF, and platelet GPIIb/IIIa receptor shedding showed a good linear correlation with mean shear stress corresponding to the three pump pressure heads. HMSS could damage red blood cells, cause pathological VWF degradation, and induce platelet activation and platelet receptor shedding. Different blood components can be damaged to different degrees by HMSS; VWF and VWF-enhanced platelet activation may be more susceptible to HMSS.

Natural killer cells display unique, non-cytotoxic profiles in human endometrial cancer

Abstract Endometrial cancer (EC) responds variably to immune checkpoint blockade, warranting study of the tumor microenvironment (TME). Natural killer (NK) cells are cytotoxic innate lymphocytes that can directly kill tumor cells, yet their diversity, locations, and functions in the EC TME are unknown. CITE-seq analysis of fresh EC tumor samples revealed multiple NK cell populations in the TME, with distinct patterns of surface activating and inhibitory receptor expression and less cytolytic granule expression by tumor-derived conventional NK (cNK) and tissue-resident NK (trNK) cells compared to autologous blood cNK. Ex vivo flow cytometry revealed significantly lower expression of cytolytic granules by TME-derived NK cell subsets (granzyme B MFI: blood cNK 1.5e5 +/- 5.4e4, tumor cNK 7.9e4 +/- 2.9e4 p<0.05, tumor trNK 3.7e4 +/- 1.4e4 p<0.05, n=10; perforin MFI: blood cNK 1.3e4 +/- 3.1e3, tumor cNK 1.0e4 +/- 3.2e3 p=0.19, tumor trNK 3.5e3 +/-1.3e3 p<0.01, n=8). To identify NK cell subsets within the TME, we performed immunohistochemistry using CD3, CD56, granzyme B, and CD103 to demarcate cNK and trNK, respectively. We observed granzyme B+ cells primarily in the stroma (78.6 +/- 3.7% Stroma, 21.4 +/- 3.7% Glands, n=10, p<0.05) and CD103+ cells primarily among tumor cells (15.6 +/- 4.5% Stroma, 84.3 +/- 0.64% Glands, n=10, p=0.07). These data suggest that both cNK and trNK cell cytotoxicity is suppressed in the EC TME and ongoing work seeks to elucidate the mechanisms.

Regulation of NK cell effector function by Acly

Abstract Natural Killer (NK) cells, crucial for viral immunity and tumor clearance, are influenced by metabolism. A prior study revealed that cytokine stimulation boosts the citrate-malate shuttle and cytosolic acetyl-CoA through ATP citrate lyase (ACLY) in NK cells. Acetyl-CoA is vital for fatty acid synthesis and protein acetylation, including histones. To explore the role of ACLY in NK cell function, we generated an inducible NK-specific Acly knockout mouse model. ACLY loss in NKp46+ NK cells did not alter maturation or IFN-γ production in naïve NK cells. However, ACLY-deficient NK cells exhibited notable proliferation defects in homeostatic and IL-15-stimulated conditions, associated with impaired glycolysis. The stimulation and priming of NK cells through IL-15 is an important mechanism for enhancing effector and anti-tumor functions. In addition to proliferation defect, IL-15-primed ACLY-deficient NK cells showed reduced IFN-γ responses to DAP12-associated activating receptors, specifically NKG2D, Ly49H, and Ly49D. RNA-seq revealed ACLY-deficient NK cells failed to upregulate DAP12 expression with IL-15 stimulation, confirmed at the protein level. These findings suggest the importance of ACLY in NK cell proliferation and potentially DAP12-driven effector functions. Ongoing experiments are focused on investigating how ACLY and cytosolic acetyl-CoA are linked to DAP12 expression, and the effects of this during in vivo effector functions.

Age-related dynamics of CD8+ regulatory T cells: Implications of NKG2D in Immune Aging

Abstract Aging is a complex biological process that impacts various physiological functions, including the immune system. Our study investigated the impact of aging on CD8+ regulatory T cells(CD8+Treg), which showed a pattern of initial increase up to 12 months, then a decline by 24 months in a C57BL/6 mouse model, unlike memory T cells, which consistently increase with age. Functionally, CD8+Treg, irrespective of age, did not produce cytokines in response to TCR stimulation, highlighting a stark functional contrast with memory cells. However, upon IL-15 stimulation, CD8+Tregs, unlike their memory cell counterparts, demonstrated enhanced cytokine production. Transcriptomic analysis revealed an abundance of Klrk1 (killer cell lectin-like receptor subfamily K member 1)gene transcripts in aging CD8+Tregs. Klrk1 gene encodes Natural Killer Group2 Member D (NKG2D), an activating receptor NK cells express. This upregulation was specific to CD8+CD122hiLy49+ and absent in CD8+CD122hiLy49-. To explore this correlation further, we utilized a Klrk1-/- mouse and observed an increased CD8+Treg population, suggesting a potential negative regulatory role of NKG2D in CD8+Treg homeostasis. Transcriptome analysis of Klrk1-/- CD8+Tregs showed enrichment of aging-related gene signatures, further corroborating the link between NKG2D and the aging process. Our findings suggest NKG2D's involvement in aging and immune dysfunction, providing avenues for intervention in the elderly.