Natural Killer Cell Cytotoxicity Research Articles

Abstract PO4-13-09: Paclitaxel affects the efficacy of PD-1 blockade by interfering with lipid metabolism reprogramming in patients with breast cancer

Abstract Background: The question of how chemotherapeutic drugs affect the efficacy of PD-1/PD-L1 antibodies in patients with breast cancer is still pending. In a previously single-cell sequencing study, we have demonstrated that paclitaxel reduces the level of CXCL13+ T cells and other immune cells critical for atezolizumab to work, leading to inferior efficacy of atezolizumab in triple negative breast cancer (TNBC). How paclitaxel affects these important immune cells remains obscure. Recently, plasma metabolomic and proteomics analysis have shown that patients’ systemic metabolic signatures is correlated with the response of PD-1/PD-L1 antibody. As such, understanding the differential metabolic influences of the diverse chemotherapeutic drugs that comprise an immune response to TNBC offers an opportunity to appropriately select chemotherapeutic backbones for ICIs. Methods: We collected pre- and post-treatment plasma samples from breast cancer patients for metabolomic and proteomic analysis, including four patient cohorts: two independent cohorts (discovery cohort and validation cohort) in which patients were treated with ICIs, one cohort in which patients were treated with paclitaxel, and one cohort of patients treated with nab-paclitaxel. Patients were divided into responder group (R, tumor shrinkage) or non-responder group (NR, tumor increase) according to the change in tumor size after treatment relative to baseline. This was followed by integrating metabolomics and proteomics with single-cell transcriptome, along with in vitro experiments, to shed light on the mechanisms of action leading to ICIs resistance in breast cancer patients. Results: In the discovery cohort, the metabolites that mostly differed between the R and NR groups at baseline were lysoglycerophospholipids. These results were further validated in the validation cohort of patients. The dynamic changes of lysoglycerophospholipids were still significantly different between the R and the NR groups after ICIs treatment in the discovery cohort. It’s worth noting that the level of lysophosphatidylcholines (LPCs), the most prominent lysoglycerophospholipids, increased significantly in the R group, while no significant changes among LPCs were found in the NR group after ICIs treatment. In the validation cohort, the median PFS of LPClow patients was 1.8 months versus 6.8 months in the LPChigh group (P = 0.0068). After one cycle of paclitaxel treatment, the metabolic status of patients changed dramatically, especially the levels of lipids. It is notable that most LPCs were significantly downregulated after paclitaxel treatment. However, in patients who received nab-paclitaxel, few significantly changed lipids were detected after one cycle nab-paclitaxel treatment compared with their baseline conditions. Interestingly, the levels of LPCs were upregulated after one cycle nab-paclitaxel treatment, but decreased after paclitaxel treatment. Single-cell omics results showed that in patients' PBMCs, CXCR4+ NK cells were the most closely related to plasma LPC levels, while FGFBP2+ NK cells were in the tumor microenvironment. We isolated and expanded NK cells from PBMCs for in vitro experiments, and found that LPC stimulation can significantly upregulate the expression of CXCR4+ in NK cells, and further increase the chemotaxis and cytotoxicity of NK cells. Conclusions: In breast cancer patients receiving ICIs, the systemic lipid metabolism status, especially lysoglycerophospholipids, is closely related to the efficacy of immunotherapy. The key molecule LPC, the most prominent lysoglycerophospholipids, may promote the efficacy of immunotherapy by activating NK cells in breast cancer patients. Paclitaxel may impair the efficacy of immunotherapy by affecting the systemic lipid metabolism status, especially by reducing plasma LPC levels, so it is not a suitable partner for ICIs. Citation Format: Hongnan Mo, Jiashu Han, Jingtong Zhai, Xiaoying Sun, Xiuwen Guan, Haili Qian, Fei Ma. Paclitaxel affects the efficacy of PD-1 blockade by interfering with lipid metabolism reprogramming in patients with breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-09.

Intratumoral CD38+CD19+B cells associate with poor clinical outcomes and immunosuppression in patients with pancreatic ductal adenocarcinoma

The widespread involvement of tumor-infiltrating B cells highlights their potential role in tumor behavior. However, B cell heterogeneity in PDAC remains unexplored. Studying TIL-Bs in PDAC aims to identify new treatment strategies. We performed single-cell RNA sequencing to study the heterogeneity of B cells in PDAC. The prognostic and immunologic value of the identified CD38+ B cells was explored in FUSCC (n=147) and TCGA (n=176) cohorts. Flow cytometry was conducted to characterize the relationship between CD38+ B cells and other immune cells, as well as their phenotypic features. Invitro and in vivo experiments were performed to assess the putative effect of CD38+ B cells on antitumor immunity. The presence of CD38+ B cells in PDAC was associated with unfavorable clinicopathological features and poorer overall survival (p<0.001). Increased infiltration of CD38+ B cells was accompanied by reduced natural killer (NK) cells (p=0.021) and increased regulatory T cells (p=0.016). Molecular profiling revealed high expression of IL-10, IL-35, TGF-β, GZMB, TIM-1, CD5 and CD21, confirming their putative regulatory B cell-like features. Co-culture experiments demonstrated suppression of NK cell cytotoxicity by CD38+ B cell-derived IL-10 (p<0.001). Finally, invivo experiments suggested adoptive transfer of CD38+ B cells reduced antitumor immunity and administration of a CD38 inhibitor hampered tumor growth (p<0.001). We discovered regulatory B cell-like CD38+ B cell infiltration as an independent prognostic factor in PDAC. The use of CD38 inhibitor may provide new possibilities for PDAC immunotherapy. This study was supported by the National Natural Science Foundation of China (U21A20374), Shanghai Municipal Science and Technology Major Project (21JC1401500), Scientific Innovation Project of Shanghai Education Committee (2019-01-07-00-07-E00057), Special Project for Clinical Research in the Health Industry of the Shanghai Health Commission (No. 20204Y0265) and Natural Science Foundation of Shanghai (23ZR1479300).

Granzyme serine proteases in inflammation and rheumatic diseases.

Granzymes (granule-secreted enzymes) are a family of serine proteases that have been viewed as redundant cytotoxic enzymes since their discovery more than 30 years ago. Predominantly produced by cytotoxic lymphocytes and natural killer cells, granzymes are delivered into the cytoplasm of target cells through immunological synapses in cooperation with the pore-forming protein perforin. After internalization, granzymes can initiate cell death through the cleavage of intracellular substrates. However, evidence now also demonstrates the existence of non-cytotoxic, pro-inflammatory, intracellular and extracellular functions that are granzyme specific. Under pathological conditions, granzymes can be produced and secreted extracellularly by immune cells as well as by non-immune cells. Depending on the granzyme, accumulation in the extracellular milieu might contribute to inflammation, tissue injury, impaired wound healing, barrier dysfunction, osteoclastogenesis and/or autoantigen generation.

Effect of Nigella sativa L. extract and thymoquinone on the genes responsible for cell proliferation, migration and NK cell cytotoxicity in breast cancer

Effect of Nigella sativa L. extract and thymoquinone on the genes responsible for cell proliferation, migration and NK cell cytotoxicity in breast cancer

Defining the Effects of PKC Modulator HIV Latency-Reversing Agents on Natural Killer Cells

Background: Latency reversing agents (LRAs) such as protein kinase C (PKC) modulators can reduce rebound-competent HIV reservoirs in small animal models. Furthermore, administration of natural killer (NK) cells following LRA treatment improves this reservoir reduction. It is currently unknown why the combination of a PKC modulator and NK cells is so potent and whether exposure to PKC modulators may augment NK cell function in some way. Methods: Primary human NK cells were treated with PKC modulators (bryostatin-1, prostratin, or the designed, synthetic bryostatin-1 analog SUW133), and evaluated by examining expression of activation markers by flow cytometry, analyzing transcriptomic profiles by RNA sequencing, measuring cytotoxicity by co-culturing with K562 cells, assessing cytokine production by Luminex assay, and examining the ability of cytokines and secreted factors to independently reverse HIV latency by co-culturing with Jurkat-Latency (J-Lat) cells. Results: PKC modulators increased expression of proteins involved in NK cell activation. Transcriptomic profiles from PKC-treated NK cells displayed signatures of cellular activation and enrichment of genes associated with the NFκB pathway. NK cell cytotoxicity was unaffected by prostratin but significantly decreased by bryostatin-1 and SUW133. Cytokines from PKC-stimulated NK cells did not induce latency reversal in J-Lat cell lines.  Conclusions: Although PKC modulators have some significant effects on NK cells, their contribution in “kick and kill” strategies is likely due to upregulating HIV expression in CD4+ T cells, not directly enhancing the effector functions of NK cells. This suggests that PKC modulators are primarily augmenting the “kick” rather than the “kill” arm of this HIV cure approach.

Identification of a novel form of caspase-independent cell death triggered by BH3-mimetics in diffuse large B-cell lymphoma cell lines

BH3-mimetics represent promising anti-cancer agents in tumors that rely on the anti-apoptotic function of B-Cell Lymphoma 2 (BCL2) proteins, particularly in leukemia and lymphoma cells primed for apoptosis. Mechanistically, BH3-mimetics may displace pro-apoptotic binding partners thus inducing BAX/BAK-mediated mitochondrial permeabilization followed by cytochrome c release, activation of the caspase cascade and apoptosis. Here, we describe a novel mode of caspase-independent cell death (CICD) induced by BH3-mimetics in a subset of diffuse large B-cell lymphoma (DLBCL) cells. Of note, rather than occurring via necroptosis, CICD induced immediately after mitochondrial permeabilization was associated with transcriptional reprogramming mediated by activation of c-Jun N-terminal Kinase (JNK) signaling and Activator Protein 1 (AP1). Thereby, CICD resulted in the JNK/AP1-mediated upregulation of inflammatory chemokines and increased migration of cytotoxic Natural Killer (NK) cells. Taken together, our study describes a novel mode of CICD triggered by BH3-mimetics that may alter the immune response towards dying cells.

Lacticaseibacillus casei K11 exerts immunomodulatory effects by enhancing natural killer cell cytotoxicity via the extracellular regulated-protein kinase pathway.

Probiotics can serve as immunomodulators that regulate the activation of immune cells. This study aimed to screen potential probiotic strains that can enhance NK cell toxicity to improve host immunity. In this investigation, we examined three potential probiotic strains, namely Lactiplantibacillus plantarum YZX21 (YZX21), Bifidobacterium bifidum FL-276.1 (FL-276.1) and Lacticaseibacillus casei K11 (K11), to assess their capacity in modulating NK cytotoxicity both in vitro and in vivo, while elucidating the underlying mechanisms involved. The findings demonstrated that K11 exhibited superior efficacy in enhancing NK cytotoxicity. Subsequent analysis revealed that K11 significantly augmented the secretion of perforin and granzyme B by NK cells through activation of receptors NKp30 and NKp46 via the extracellular signal-regulated kinase (ERK) pathway. Furthermore, heat-inactivated K11 also enhanced NK cell activity to an extent comparable to live bacteria, with lipoteichoic acid from K11 identified as a crucial factor mediating the activation of NK cell cytotoxicity. Our study suggests that K11 may have potential applications as probiotics or postbiotics for regulating NK cell cytotoxicity to enhance immunity.

Optimization of PD-1 / PD-L1 Blockade to Increase NK Cells Cytotoxicity in Killing Cancer Cells: Article Review”

Anti-PD-1 and anti-PD-L1 are therapies that have shown success in cancer treatment. However, while treating cancer with PD-1/PD-L1 blockade is similar, the clinical response rate is still low in certain cancers at around 40%. Therefore, therapeutic strategies are needed to increase PD-L1 expression and optimize PD-1/PD-L1 blockade therapy, so as to improve satisfactory therapeutic results. In tumors, IFNγ induces the expression of PD-L1 which is a cytokine secreted by Natural Killer cells. A study showed that PM21-NK cells induced large amounts of IFNγ and transfected PM21-NK cells adaptively induced PD-L1 expression. In in vitro experiments, anti-PD-L1 treatment had no direct effect on cytotoxicity or cytokine secretion by PD-1 negative PM21-NK cells in response to PD-L1+ targets. However, in vivo a significant increase in the antitumor effect of Natural Killer cells was found when combined with anti-PD-L1. PD-L1 blockade also resulted in increased persistence of Natural Killer cells in vivo and retention of their cytotoxic phenotype. These results support the use of anti-PD-L1 in combination with Natural Killer cell therapy regardless of baseline tumor PD-L1 status and suggest that Natural Killer cell therapy will likely increase the applicability of anti-PD-L1 treatment. In this review article, the author will discuss the method of optimizing PD-L1 blockade combined with Natural Killer cell therapy to increase the efficacy of treatment on cancer stem cells in the dormant phase.

Immunoglobulin-like transcript 2 as an impaired anti-tumor cytotoxicity marker of natural killer cells in patients with hepatocellular carcinoma.

Natural killer (NK) cells play a pivotal role in immune surveillance in the liver. We aimed to identify potential targets for NK cell-mediated immune intervention by revealing the functional molecules on NK cells in HCC patients. To evaluate the impact of aging on NK cell phenotypes, we examined NK cells from healthy volunteers (HVs) of various ages. Because ILT2 expression on CD56dim NK cells increased with increasing age, we enrolled age-matched HCC patients and HVs. We determined the NK cell phenotypes in blood mononuclear cells (PBMCs) and intrahepatic lymphocytes (IHLs) from cancerous and non-cancerous tissues. We evaluated cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) of NK cells in vitro. ILT2-positive CD56dim NK cells in PBMCs were increased in HCC patients compared with HVs. In HCC patients, ILT2-positive CD56dim NK cells were increased in cancerous IHLs compared with non-cancerous IHLs and PBMCs. We examined the impact of macrophage migration inhibitory factor (MIF) on ILT2 expression in co-cultures of HCC cells and NK cells. The enhanced expression of ILT2 on CD56dim NK cells from HCC patients was inhibited by masking antibodies against MIF and CXCR4. ILT2-positive CD56dim NK cells exhibited lower capacities for cytotoxicity and ADCC than ILT2-negative cells, which were partially restored by ILT2 blockade. In HCC patients, ILT2 is a signature molecule for cancerous CD56dim NK cells with impaired cytolytic capacity. The MIF-CXCR4 interaction is associated with ILT2 induction on CD56dim NK cells and ILT2 serves as a target for functional NK cell restoration.

Optimizing glutamine concentration enhances ex vivo expansion of natural killer cells through improved redox status.

Amino acids are vital components of the serum-free medium that influence the expansion and function of NK cells. This study aimed to clarify the relationship between amino acid metabolism and expansion and cytotoxicity of NK cells. Based on analyzing the mino acid metabolism of NK-92 cells and Design of Experiments (DOE), we optimized the combinations and concentrations of amino acids in NK-92 cells culture medium. The results demonstrated that NK-92 cells showed a pronounced demand for glutamine, serine, leucine, and arginine, in which glutamine played a central role. Significantly, at a glutamine concentration of 13 mM, NK-92 cells expansion reached 161.9 folds, which was significantly higher than 55.5 folds at 2.5 mM. Additionally, under higher glutamine concentrations, NK-92 cells expressed elevated levels of cytotoxic molecules, the level of cytotoxic molecules expressed by NK-92 cells was increased and the cytotoxic rate was 68.42%, significantly higher than that of 58.08% under low concentration. In view of the close relationship between glutamine metabolism and intracellular redox state, we investigated the redox status within the cells. This study demonstrated that intracellular ROS levels in higher glutamine concentrations were significantly lower than those under lower concentration cultures with decreased intracellular GSH/GSSG ratio, NADPH/NADP+ ratio, and apoptosis rate. These findings indicate that NK-92 cells exhibit improved redox status when cultured at higher glutamine concentrations. Overall, our research provides valuable insights into the development of serum-free culture medium for ex vivo expansion of NK-92 cells.

Deletion of the TMEM30A gene enables leukemic cell evasion of NK cell cytotoxicity

Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.

477 Evaluating the therapeutic efficacy of combination IL-12 and trabectedin for the treatment of triple negative breast cancer

OBJECTIVES/GOALS: IL-12 has potent immune effects but the presence of myeloid-derived suppressor cells (MDSC) can inhibit IL-12-induced NK cell cytotoxicity. Thus, we hypothesized that combining IL-12 with trabectedin, an immunosuppressive myeloid cell depleting agent, would improve its therapeutic efficacy in triple negative breast cancer (TNBC). METHODS/STUDY POPULATION: Combination IL-12 and trabectedin was tested in the 4T1 mouse model of TNBC. 4T1 cells were injected into the mammary fat pad of female BALB/cj mice. When tumors reached 50 mm3, mice were randomly divided into 4 groups and treated with PBS, IL-12 (0.5 μg/mouse 3x/wk), 0.15 mg/kg trabectedin weekly or the combination. Tumor volumes were measured by calipers. Mass cytometry was performed on spleens and tumors using a 35-antibody panel. Plasma IFN-γ levels were measured by ELISA. The role of NK cells was evaluated via depletion with anti-asialo-GM1. The Luminex Discovery Assay was used to measure plasma cytokines and immunohistochemistry was performed for CD4 and CD8a. Linear/nonlinear mixed effects modeling was used for in vivo data analysis and applicable t- or ANOVA tests were used for in vitrodata analysis. RESULTS/ANTICIPATED RESULTS: Combination IL-12 and trabectedin led to a significant reduction in tumor burden compared to single-agent IL-12, trabectedin and control treatments (all p&lt;0.001), as well as higher levels of IFN-γ (all p&lt;0.04). One combination treated mouse had complete tumor regression. Splenic MDSC were significantly decreased in combination treated mice. NK depletion abrogated the effects of combination therapy. Compared to mice receiving a control antibody, NK depletion of combination treated mice led to lower levels of CCL5 (p&lt;0.01) and CXCL10 (p&lt;0.001) and significantly higher tumor burden (p=0.001). CD8+T cell levels were significantly higher in combination treated mice compared to those receiving IL-12 (p&lt;0.01), and these levels were decreased when mice were depleted of NK cells (p=0.01). DISCUSSION/SIGNIFICANCE: TNBC represents 15% of all breast cancer diagnoses and is associated with a worse prognosis compared to other subtypes. Black women are twice as likely to be diagnosed with TNBC and more likely to die from disease than White women. Thus, there is an increasing need to develop additional therapeutic options for this disease.

Natural killer cell subsets and their functional molecules in peripheral blood of the patients with breast cancer.

Natural killer (NK) cells, CD3- lymphocytes, are critical players in cancer immune surveillance. This study aimed to assess two types of CD3- NK cell classifications (subsets), that is, convectional subsets (based on CD56 and CD16 expression) and new subsets (based on CD56, CD27, and CD11b expression), and their functional molecules in the peripheral blood of patients with breast cancer (BC) in comparison with healthy donors (HDs). Thirty untreated females with BC and 20 age-matched healthy women were enrolled. Peripheral blood samples were collected and directly incubated with fluorochrome-conjugated antibodies against CD3, CD56, CD16, CD27, CD11b, CD96, NKG2C, NKG2D, NKp44, CXCR3, perforin, and granzyme B. Red blood cells were then lysed using lysing solution, and the stained cells were acquired on four-color flow cytometer. Our results indicated 15% of lymphocytes in peripheral blood of patients with BC and HDs had NK cells phenotype. However, the frequency of total NK cells (CD3-CD56+), and NK subsets (based on conventional and new classifications) was not significantly different between patients and HDs. We observed mean fluorescent intensity (MFI) of CXCR3 in total NK cells (p = .02) and the conventional cytotoxic (CD3-CD56dim CD16+) NK cells (p = .03) were significantly elevated in the patients with BC compared to HDs. Despite this, the MFI of granzyme B expression in conventional regulatory (CD3-CD56brightCD16- /+) NK cells and CD3-CD56-CD16+ NK cells (p = .03 and p = .004, respectively) in the patients was lower than healthy subjects. The higher expression of chemokine receptor CXCR3 on total NK cells in patients with BC may be associated with increased chemotaxis-related NK cell infiltration. However, lower expression of granzyme B in conventional regulatory NK cells and CD3-CD56-CD16+ NK cells in the patients compared to HDs suggests reduced cytotoxic activity of the NK cells in BC. These results might demonstrate accumulating NK subsets with a dysfunctional phenotype in the peripheral blood of patients with BC.

Targeting TRIM29 As a Negative Regulator of CAR-NK Cell Effector Function to Improve Antitumor Efficacy of These Cells: A Perspective.

Natural killer (NK) cells are among the most important cells in innate immune defense. In contrast to T cells, the effector function of NK cells does not require prior stimulation and is not MHC restricted. Therefore, chimeric antigen receptor (CAR)-NK cells are superior to CAR-T cells. The complexity of the tumor microenvironment (TME) makes it necessary to explore various pathways involved in NK cell negative regulation. CAR-NK cell effector function can be improved by inhibiting the negative regulatory mechanisms. In this respect, the E3 ubiquitin ligase tripartite motif containing 29 (TRIM29) is known to be involved in reducing NK cell cytotoxicity and cytokine production. Also, targeting TRIM29 may enhance the antitumor efficacy of CAR-NK cells. The present study discusses the negative effects of TRIM29 on NK cell activity and genomic deletion or suppression of the expression of TRIM29 as a novel approach to optimize CAR-NK cell-based immunotherapy.

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.

High-resolution KIR and HLA genotyping in three Chinese ethnic minorities reveals distinct origins.

Polymorphism of killer-cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands impacts the effector activity of cytotoxic NK cell and T cell subsets. Therefore, understanding the extent and implications of KIR and HLA class I genetic polymorphism across various populations is important for immunological and medical research. In this study, we conducted a high-resolution investigation of KIR and HLA class I diversity in three distinct Chinese ethnic minority populations. We studied the She, Yugur, and Tajik, and compared them with the Zhejiang Han population (Zhe), which represents the majority Southern Han ethnicity. Our findings revealed that the Tajik population exhibited the most diverse KIR copy number, allele, and haplotype diversity among the four populations. This diversity aligns with their proposed ancestral origin, closely resembling that of Iranian populations, with a relatively higher presence of KIR-B genes, alleles, and haplotypes compared with the other Chinese populations. The Yugur population displayed KIR distributions similar to those of the Tibetans and Southeast Asians, whereas the She population resembled the Zhe and other East Asians, as confirmed by genetic distance analysis of KIR. Additionally, we identified 12.9% of individuals across the three minority populations as having KIR haplotypes characterized by specific gene block insertions or deletions. Genetic analysis based on HLA alleles yielded consistent results, even though there were extensive variations in HLA alleles. The observed variations in KIR interactions, such as higher numbers of 2DL1-C2 interactions in Tajik and Yugur populations and of 2DL3-C1 interactions in the She population, are likely shaped by demographic and evolutionary mechanisms specific to their local environments. Overall, our findings offer valuable insights into the distribution of KIR and HLA diversity among three distinct Chinese ethnic minority populations, which can inform future clinical and population studies.

Structural Characterization and Immunological Activity of Polysaccharide Degradation Products from Phlebopus portentosus

Phlebopus portentosus is an edible and medicinal mushroom with a delicious taste and high nutritional value. The oligosaccharides derived from P. portentosus may be the material basis for its biological activity. The degradation of polysaccharide and the maintenance of its activity after degradation are key steps in related research. This study applied an acid degradation method to prepare P. portentosus refined polysaccharide (PPRP) with a smaller molecular weight, and the optimal hydrolysis conditions determined were a temperature of 80 °C, an acid concentration of 2 mol/L, and a hydrolysis time of 2 h. The polysaccharide structure and immune activity were then further investigated. The results showed that the PPRP comprised two fractions with approximate weights of 61,600 Da and 5500 Da. The monosaccharide composition of PPRP was mannose, rhamnose, glucose, and galactose, with a molar ratio of 1.00: 22.24: 2.93: 1.03. The major functional groups included O-H, C-H, C-O, and C-O-C. The glycosidic bond types were mainly α- and β-glycosidic bonds. Cell experiments demonstrated that PPRP could significantly increase the proliferation of macrophages and enhance the cytotoxicity of NK cells. Moreover, PPRP also significantly promoted the proliferation of B lymphocytes and T lymphocytes, especially at a concentration of 200 μg/mL. This study furnishes scientific evidence underlining the significant potential of PPRP in immune activity, thereby serving as a material basis and scientific bedrock for further investigations into the mechanism of P. portentosus oligosaccharide activity.

NK-cell-elicited gasdermin-D-dependent hepatocyte pyroptosis induces neutrophil extracellular traps that facilitates HBV-related acute-on-chronic liver failure.

Hepatitis B virus (HBV) infection is a major etiology of acute-on-chronic liver failure (ACLF). At present, the pattern and regulation of hepatocyte death during HBV-ACLF progression are still undefined. Evaluating the mode of cell death and its inducers will provide new insights for developing therapeutic strategies targeting cell death. In this study, we aimed to elucidate whether and how immune landscapes trigger hepatocyte death and lead to the progression of HBV-related ACLF. We identified that pyroptosis represented the main cell death pattern in the liver of patients with HBV-related ACLF. Deficiency of MHC-I in HBV-reactivated hepatocytes activated cytotoxic NK cells, which in turn operated in a perforin/granzyme-dependent manner to trigger GSDMD/caspase-8-dependent pyroptosis of hepatocytes. Neutrophils selectively accumulated in pyroptotic liver, and HMGB1 derived from pyroptotic liver constituted an important factor triggering generation of pathogenic extracellular traps in neutrophils (NETs). Clinically, elevated plasma levels of MPO-DNA complexes were a promising prognostic biomarker for HBV-related ACLF. More importantly, targeting GSDMD pyroptosis-HMGB1 release in the liver abrogates NETs that intercept the development of HBV-related ACLF. Studying the mechanisms that selectively modulate GSDMD-dependent pyroptosis, as well as its immune landscapes, will provide a novel strategy for restoring liver function of HBV-related ACLF patients.

Decreased LDHB expression in breast tumor cells causes NK cell activation and promotes tumor progression.

Abnormal metabolism is the underlying reason for breast cancer progression. Decreased lactate dehydrogenase B (LDHB) has been detected in breast cancer but the function of LDHB remains unknown. Western blot was used to analyze LDHB expression in breast cancer cells. The impact of LDHB on tumor cell migration and invasion was determined using Transwell assays, wound healing assays, and a mouse lung metastasis model. Subcutaneous tumor formation, a natural killer (NK) cell cytotoxicity assay, and flow cytometry evaluated NK cell activation. Immunofluorescence and quantitative real-time PCR detected NK cell activation markers. Kaplan-Meier analysis evaluated the effect of immune cell infiltration on prognosis. Single-sample gene set enrichment analysis determined NK cell activation scores. A support vector machine predicted the role of LDHB in NK cell activation. In this study we showed that LDHB inhibits the breast cancer cell metastasis and orchestrates metabolic reprogramming within tumor cells. Our results revealed that LDHB-mediated lactic acid clearance in breast cancer cells triggers NK cell activation within the tumor microenvironment. Our findings, which were confirmed in a murine model, demonstrated that LDHB in tumor cells promotes NK cell activation and ultimately results in the eradication of malignant cells. Clinically, our study further validated that LDHB affects immune cell infiltration and function. Specifically, its expression has been linked to enhanced NK cell-mediated cytotoxicity and improved patient survival. Furthermore, we identified LDHB expression in tumors as an important predictor of NK cell activation, with strong predictive ability in some cancers. Our results suggest that LDHB is a promising target for activating the tumor immune microenvironment in breast cancer, where LDHB-associated lactic acid clearance leads to increased NK cell activity. This study highlights the critical role of LDHB in regulating immune responses and its potential as a therapeutic target for breast cancer.

Abstract 4699: Evaluating extracellular matrix invasion and cytotoxicity of natural killer cells in a novel co-culture assay

Abstract Background: The complex tumor microenvironment (TME) in solid tumors presents unique challenges for lymphocytes used in cancer immunotherapy. The extracellular matrix (ECM) in TME can limit the recruitment of lymphocytes and restrict access to cancer cells. This study utilized Matrigel to study the kinetics of natural killer cell invasion and cytotoxicity. We hypothesized that the kinetics of tumor cell killing by the NKs would depend on the distance for invasion and rely on matrix-metalloproteinases (MMPs) for remodeling the ECM. Methods: Increasing volumes (50-100 µL/well) of Matrigel (6 mg/mL) representing increasing distance for invasion was layered over MCF7-red target tumor cells expressing nuclear-localized mKate2 (red fluorescent protein). NK92 (Creative Bioarray) or eGFP-NK92 (BPS Bioscience) were seeded over the solidified Matrigel layer at E:T of 3:1. The broad spectrum MMP inhibitor, Ilomostat (GM6001, 2 µM and 10 µM), was added to the wells to investigate to role of MMPs. Label-free impedance and live cell imaging data was collected in real time on xCELLigence RTCA eSight to evaluate NK cell invasion and cytotoxicity. Percent cytolysis was calculated on the RTCA software from normalized cell impedance readings and imaging data using the formula [(1-(treated/untreated)*100]. Results: Impedance reading increases as the MCF7-red cells settle, attach, and proliferate. The readings plateau as the cells reach confluence. The addition of Matrigel modulates the impedance signature of MCF7 cells. Importantly, Matrigel delays cytolysis by NK92 cells added at 24h. The delay increases with increasing volume of Matrigel representing increasing invasion distance. The time to achieve 60% killing with respect to controls (KT60) was 67, 76 and 89 hours for 50, 75 and 100 µL/well, respectively. Consistent with the role of MMPs in invasion, percent cytolysis calculated independently from normalized impedance readings and live cell imaging data confirmed both delayed and reduced target cell killing by GFP-NK92 cells in the presence of Ilomostat. Interestingly, only a few GFP-NK92 were detected by live cell imaging when there was increased target cell death. These included highly active NK cells that made multiple contacts with different MCF7 red targets in clusters suggesting serial killing activity. Conclusions: The observations that only a few NK92 cells are detected during the killing of susceptible target cells and that cytolysis is not abrogated by the inhibition of MMPs suggest a potential role for distal effectors like cytokines, independent of invasion. This is a novel 2.5D assay that demonstrates the potential of RTCA eSight platform for studying the invasion and cytotoxicity function of various immune cells through both real-time impedance recording and live-cell time-lapse imaging simultaneously. (For Research Use Only. Not for use in diagnostic procedures. RA45236.3472453704) Citation Format: Jyothi Thyagabhavan Mony, Xiaoyu Zhang. Evaluating extracellular matrix invasion and cytotoxicity of natural killer cells in a novel co-culture assay [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 4699.