Regulation Of Immune Responses Research Articles

Identification of molecular characteristics of hepatocellular carcinoma with microvascular invasion based on deep targeted sequencing.

As an indicator of tumor invasiveness, microvascular invasion (MVI) is a crucial risk factor for postoperative relapse, metastasis, and unfavorable prognosis in hepatocellular carcinoma (HCC). Nevertheless, the genetic mechanisms underlying MVI, particularly for Chinese patients, remain mostly uncharted. We applied deep targeted sequencing on 66 Chinese HCC samples. Focusing on the telomerase reverse transcriptase (TERT) promoter (TERTp) and TP53 co-mutation (TERTp+/TP53+) group, gene set enrichment analysis (GSEA) was used to explore the potential molecular mechanisms of the TERTp+/TP53+ group on tumor progression and metastasis. Additionally, we evaluated the tumor immune microenvironment of the TERTp+/TP53+ group in HCC using multiplex immunofluorescence (mIF) staining. Among the 66 HCC samples, the mutated genes that mostly appeared were TERT, TP53, and CTNNB1. Of note, we found 10 cases with TERTp+/TP53+, of which nine were MVI-positive and one was MVI-negative, and there was a co-occurrence of TERTp and TP53 (p < 0.05). Survival analysis demonstrated that patients with the TERTp+/TP53+ group had lower the disease-free survival (DFS) (p = 0.028). GSEA results indicated that telomere organization, telomere maintenance, DNA replication, positive regulation of cell cycle, and negative regulation of immune response were significantly enriched in the TERTp+/TP53+ group (all adjusted p-values (p.adj) < 0.05). mIF revealed that the TERTp+/TP53+ group decreased CD8+ T cells infiltration (p = 0.25) and enhanced PDL1 expression (p = 0.55). TERTp+/TP53+ was significantly enriched in MVI-positive patients, leading to poor prognosis for HCC patients by promoting proliferation of HCC cell and inhibiting infiltration of immune cell surrounding HCC. TERTp+/TP53+ can be utilized as a potential indicator for predicting MVI-positive patients and poor prognosis, laying a preliminary foundation for further exploration of co-mutation in HCC with MVI and clinical treatment.

What is slough? Defining the proteomic and microbial composition of slough and its implications for wound healing.

Slough is a well-known feature of non-healing wounds. This pilot study aims to determine the proteomic and microbiologic components of slough as well as interrogate the associations between wound slough components and wound healing. Ten subjects with slow-to-heal wounds and visible slough were enrolled. Aetiologies included venous stasis ulcers, post-surgical site infections and pressure ulcers. Patient co-morbidities and wound healing outcome at 3-months post-sample collection was recorded. Debrided slough was analysed microscopically, through untargeted proteomics, and high-throughput bacterial 16S-ribosomal gene sequencing. Microscopic imaging revealed wound slough to be amorphous in structure and highly variable. 16S-profiling found slough microbial communities to associate with wound aetiology and location on the body. Across all subjects, slough largely consisted of proteins involved in skin structure and formation, blood-clot formation and immune processes. To predict variables associated with wound healing, protein, microbial and clinical datasets were integrated into a supervised discriminant analysis. This analysis revealed that healing wounds were enriched for proteins involved in skin barrier development and negative regulation of immune responses. While wounds that deteriorated over time started off with a higher baseline Bates-Jensen Wound Assessment Score and were enriched for anaerobic bacterial taxa and chronic inflammatory proteins. To our knowledge, this is the first study to integrate clinical, microbiome, and proteomic data to systematically characterise wound slough and integrate it into a single assessment to predict wound healing outcome. Collectively, our findings underscore how slough components can help identify wounds at risk of continued impaired healing and serves as an underutilised biomarker.

PROGNOSTIC SIGNIFICANCE OF SOCS IN REGULATING CANCER PROGRESSION

Suppressor of cytokine signalling (SOCS) proteins have emerged as crucial regulators in cancer progression, exhibiting significant prognostic implications. This review explores the prognostic significance of SOCS in various cancer types and their role in modulating key signalling pathways involved in tumour growth, metastasis, and immune evasion. Understanding the intricate interplay between SOCS proteins and cancer progression provides valuable insights into developing targeted therapies and prognostic markers for improved patient management and outcomes. SOCS proteins are pivotal regulators of immune responses and inflammation, but their dysregulation has been implicated in cancer development and progression. We explore the mechanisms by which SOCS proteins exert their effects on oncogenic signalling pathways, such as JAK/STAT, PI3K/AKT, and MAPK pathways, highlighting their potential as therapeutic targets. Furthermore, understanding the complex interplay between SOCS proteins and cancer holds promise for the development of novel therapeutic strategies and precision medicine approaches tailored to individual patients.

Type I/II Immune Balance Contributes to the Protective Effect of AIF-1 on Hepatic Immunopathology Induced by Schistosoma japonicum in a Transgenic Mouse Model.

Schistosomiasis is the second most debilitating neglected tropical disease in the world. Liver egg granuloma and fibrosis are the main damage of schistosomiasis. In this study, the role of allograft inflammatory factor-1 (AIF-1) in liver pathology and its regulation in immune responses were investigated in a transgenic mouse infected with Schistosoma japonicum. We found that AIF-1 overexpression reduced worm burden and decreased egg granuloma sizes and serum alanine aminotransferase levels, along with inhibited hepatic collagen deposition and serum hydroxyproline levels during S. japonicum infection. Moreover, AIF-1 overexpression resulted in an increased ratio of Th1/Th2, increased levels of IFN-γ and T-bet, and lower levels of GATA-3 in the spleen, accompanied by increased M1 percentages, decreased M2 percentages, and thus a higher ratio of M1/M2 in the peritoneal cavity and liver. AIF-1 induced CD68 and iNOS mRNA expression and protein levels of cytoplasmic p-P38 and nuclear NF-κB, along with enhanced levels of TNF-α and TGF-β in macrophages in vitro. Moreover, the hepatic pathology had a negative correlation with Th1/Th2 and M1/M2 ratios in the infected mice. The findings reveal that the beneficial role of AIF-1 in alleviating hepatic damage is related to restoring type I/II immune balance in S. japonicum infection.

Myeloid-derived suppressor cell mitochondrial fitness governs chemotherapeutic efficacy in hematologic malignancies

Myeloid derived suppressor cells (MDSCs) are key regulators of immune responses and correlate with poor outcomes in hematologic malignancies. Here, we identify that MDSC mitochondrial fitness controls the efficacy of doxorubicin chemotherapy in a preclinical lymphoma model. Mechanistically, we show that triggering STAT3 signaling via β2-adrenergic receptor (β2-AR) activation leads to improved MDSC function through metabolic reprograming, marked by sustained mitochondrial respiration and higher ATP generation which reduces AMPK signaling, altering energy metabolism. Furthermore, induced STAT3 signaling in MDSCs enhances glutamine consumption via the TCA cycle. Metabolized glutamine generates itaconate which downregulates mitochondrial reactive oxygen species via regulation of Nrf2 and the oxidative stress response, enhancing MDSC survival. Using β2-AR blockade, we target the STAT3 pathway and ATP and itaconate metabolism, disrupting ATP generation by the electron transport chain and decreasing itaconate generation causing diminished MDSC mitochondrial fitness. This disruption increases the response to doxorubicin and could be tested clinically.

Exploring the Role of Extracellular Vesicles in the Pathogenesis of Tuberculosis.

Tuberculosis (TB) remains a significant global health concern, necessitating accurate diagnosis and treatment monitoring. Extracellular vesicles (EVs), including exosomes, play crucial roles in disease progression, with their associated genes serving as potential biomarkers and therapeutic targets. Leveraging publicly available RNA-Seq datasets of TB patients and healthy controls (HCs), to identify differentially expressed genes (DEGs) and their associated protein-protein interaction networks and immune cell profiles, the common EV-related DEGs were identified and validated in the GSE42830 and GSE40553 datasets. We have identified nine common EV-related DEGs (SERPINA1, TNFAIP6, MAPK14, STAT1, ITGA2B, VAMP5, CTSL, CEACAM1, and PLAUR) upregulated in TB patients. Immune cell infiltration analysis revealed significant differences between TB patients and HCs, highlighting increased proportions of various immune cells in TB patients. These DEGs are involved in crucial cellular processes and pathways related to exocytosis and immune response regulation. Notably, VAMP5 exhibited excellent diagnostic performance (AUC-0.993, sensitivity-93.8%, specificity-100%), with potential as a novel biomarker for TB. The EV-related genes can serve as novel potential biomarkers that can distinguish between TB and HCs. VAMP5, which functions in exosome biogenesis and showed significant upregulation in TB, can be targeted for therapeutic interventions and treatment outcomes.

Species-specific responses during Seoul orthohantavirus infection in human and rat lung microvascular endothelial cells.

Seoul orthohantavirus (SEOV) is a rat-borne zoonotic virus that is transmitted via inhalation of aerosolized infectious excreta, and can cause hemorrhagic fever with renal syndrome (HFRS) in humans worldwide. In rats, SEOV predominantly exists as a persistent infection in the absence of overt clinical signs. Lack of disease in rats is attributed to downregulation of pro-inflammatory and upregulation of regulatory host responses. As lung microvascular endothelial cells (LMECs) represent a primary target of infection in both human and rats, infections in these cells provide a unique opportunity to study the central role of LMECs in the dichotomy between pathogenicity in both species. In this study, host responses to SEOV infection in primary human and rat LMECs were directly compared on a transcriptional level. As infection of rat LMECs was more efficient than human LMECs, the majority of anti-viral defense responses were observed earlier in rat LMECs. Most prominently, SEOV-induced processes in both species included responses to cytokine stimulus, negative regulation of innate immune responses, responses to type I and II interferons, regulation of pattern recognition receptor signaling and MHC-I signaling. However, over time, in the rat LMECs, responses shifted from an anti-viral state towards a more immunotolerant state displayed by a PD-L1, B2M-, JAK2-focused interaction network aiding in negative regulation of cytotoxic CD8-positive T cell activation. This suggests a novel mechanism by which species-specific orthohantavirus-induced endothelium and T cell crosstalk may play a crucial role in the development of acute disease in humans and persistence in rodents.

Preclinical characterization of Pan-NKG2D ligand-binding NKG2D receptor decoys

NKG2D and its ligands are critical regulators of protective immune responses controlling infections and cancer, defining a crucial immune signaling axis. Current therapeutic efforts targeting this axis almost exclusively aim at enhancing NKG2D-mediated effector functions. However, this axis can drive disease processes when dysregulated, in particular, driving stem-like cancer cell reprogramming and tumorigenesis through receptor/ligand self-stimulation on tumor cells. Despite complexities with its structure and biology, we developed multiple novel engineered proteins that functionally serve as axis-blocking NKG2D “decoys” and report biochemical, structural, in vitro, and in vivo evaluation of their functionality.

MRNA epitranscriptomics.

Epitranscriptomics refers to chemical changes in RNAs and includes numerous chemical types with varying stoichiometry and functions. RNA modifications are highly diverse in chemistry and respond in cell-type- and cell-state-dependent manners that enable and facilitate the execution of a wide array of biological functions. This includes roles in the regulation of transcription, translation, chromatin maintenance, immune response, and many other processes. This special issue presents the past, present, and future of epitranscriptomics research with a focus on mRNA. It includes perspectives from experts in the field, with the goal of encouraging discussions and debates that will further advance this area of research and enable it to realize its full potential in basic research and applications to human health and disease.

Gut microbiota and prospects for probiotics in paediatric celiac disease

Celiac disease is a chronic disease, with predominant damage to the small intestine and provoked by gluten consumption in genetically predisposed individuals. Previous large-scale studies suggest a possible role of the gut microbiota in the pathogenesis of this disease. Changes in the qualitative and quantitative characteristics of microbiota of different genesis can lead to impaired regulation of immune response, which, in turn, may contribute to the development of gluten intolerance. In this context, it is relevant to analyse the data on changes in the intestinal microbiota in celiac disease and the possibility of its correction with probiotics as one of the strategies for the treatment of children with celiac disease, suffering from celiac disease.

Regulatory SVA retrotransposons and classical HLA genotyped-transcripts associated with Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative and polygenic disorder characterised by the progressive loss of neural dopamine and onset of movement disorders. We previously described eight SINE-VNTR-Alu (SVA) retrotransposon-insertion-polymorphisms (RIPs) located and expressed within the Human Leucocyte Antigen (HLA) genomic region of chromosome 6 that modulate the differential co-expression of 71 different genes including the HLA classical class I and class II genes in a Parkinson's Progression Markers Initiative (PPMI) cohort. In the present study, we (1) reanalysed the PPMI genomic and transcriptomic sequencing data obtained from whole blood of 1521 individuals (867 cases and 654 controls) to infer the genotypes of the transcripts expressed by eight classical HLA class I and class II genes as well as DRA and the DRB3/4/5 haplotypes, and (2) examined the statistical differences between three different PD subgroups (cases) and healthy controls (HC) for the HLA and SVA transcribed genotypes and inferred haplotypes. Significant differences for 57 expressed HLA alleles (21 HLA class I and 36 HLA class II alleles) up to the three-field resolution and four of eight expressed SVA were detected at p<0.05 by the Fisher's exact test within one or other of three different PD subgroups (750 individuals with PD, 57 prodromes, 60 individuals who had scans without evidence of dopamine deficits [SWEDD]), when compared against a group of 654 HCs within the PPMI cohort and when not corrected by the Bonferroni test for multiple comparisons. Fourteen of 20 significant alleles were unique to the PD-HC comparison, whereas 31 of the 57 alleles overlapped between two or more different subgroup comparisons. Only the expressed HLA-DRA*01:01:01 and -DQA1*03:01:01 protective alleles (PD v HC), the -DQA1*03:03:01 risk (HC v Prodrome) or protective allele (PD v Prodrome), the -DRA*01:01:02 and -DRB4*01:03:02 risk alleles (SWEDD v HC), and the NR_SVA_381 present genotype (PD v HC) at a 5% homozygous insertion frequency near HLA-DPA1, were significant (Pc<0.1) after Bonferroni corrections. The homologous NR_SVA_381 insertion significantly decreased the transcription levels of HLA-DPA1 and HLA-DPB1 in the PPMI cohort and its presence as a homozygous genotype is a risk factor (Pc=0.012) for PD. The most frequent NR_SVA_381 insertion haplotype in the PPMI cohort was NR_SVA_381/DPA1*02/DPB1*01 (3.7%). Although HLA C*07/B*07/DRB5*01/DRB1*15/DQB1*06 was the most frequent HLA 5-loci phased-haplotype (n, 76) in the PPMI cohort, the NR_SVA_381 insertion was present in only six of them (8%). These data suggest that expressed SVA and HLA gene alleles in circulating white blood cells are coordinated differentially in the regulation of immune responses and the long-term onset and progression of PD, the mechanisms of which have yet to be elucidated.

Foot-and-mouth disease virus VP1 degrades YTHDF2 through autophagy to regulate IRF3 activity for viral replication

ABSTRACT Many viruses, including foot-and-mouth disease virus (FMDV), can promote the degradation of host proteins through macroautophagy/autophagy, thereby promoting viral replication. However, the regulatory mechanism between autophagy and innate immune responses is not fully understood during FMDV infection. Here, we found that the host GTPBP4/NOG1 (GTP binding protein 4) is a negative regulator of innate immune responses. GTPBP4 deficiency promotes the antiviral innate immune response, resulting in the ability of GTPBP4 to promote FMDV replication. Meanwhile, GTPBP4-deficient mice are more resistant to FMDV infection. To antagonize the host’s antiviral immunity, FMDV structural protein VP1 promotes the expression of GTPBP4, and the 209th site of VP1 is responsible for this effect. Mechanically, FMDV VP1 promotes autophagy during virus infection and interacts with and degrades YTHDF2 (YTH N6-methyladenosine RNA binding protein F2) in an AKT-MTOR-dependent autophagy pathway, resulting in an increase in GTPBP4 mRNA and protein levels. Increased GTPBP4 inhibits IRF3 binding to the Ifnb/Ifn-β promoter, suppressing FMDV-induced type I interferon production. In conclusion, our study revealed an underlying mechanism of how VP1 negatively regulates innate immunity through the autophagy pathway, which would contribute to understanding the negative regulation of host innate immune responses and the function of GTPBP4 and YTHDF2 during FMDV infection. Abbreviation: 3-MA:3-methyladenine; ACTB: actin beta; ATG: autophagy related; ChIP:chromatin immunoprecipitation; CQ: chloroquine; DAPI:4’,6-diamidino-2-phenylindole; dpi: days post-infection; EV71:enterovirus 71; FMDV: foot-and-mouth disease virus; GTPBP4/NOG1: GTPbinding protein 4; HIF1A: hypoxia inducible factor 1 subunit alpha;hpt:hours post-transfection; IFNB/IFN-β:interferon beta; IRF3: interferon regulatory factor 3; MAP1LC3/LC3:microtubule associated protein 1 light chain 3; MAVS: mitochondriaantiviral signaling protein; MOI: multiplicity of infection; MTOR:mechanistic target of rapamycin kinase; m6A: N(6)-methyladenosine;qPCR:quantitativePCR; SIRT3:sirtuin 3; SQSTM1/p62: sequestosome 1; STING1: stimulator ofinterferon response cGAMP interactor 1; siRNA: small interfering RNA;TBK1: TANK binding kinase 1; TCID50:50% tissue culture infectious doses; ULK1: unc-51 like autophagyactivating kinase 1; UTR: untranslated region; WT: wild type; YTHDF2:YTH N6-methyladenosine RNA binding protein F2

Abstract 1394: The CXCL10-CXCR3 axis as Jekyll and Hyde: A regulator of metastasis and immune response in osteosarcoma

Abstract The CXCL10-CXCR3 axis is known to promote tumor growth and metastasis via autocrine signaling, while it can also elicit anti-tumor responses by paracrine signaling. However, its roles are still elusive in osteosarcoma (OS), the most common malignant bone tumor in children. In this study, we utilized in vitro assays, mouse models, and gene expression analysis to characterize the roles of the CXCL10-CXCR3 axis in OS. To understand the autocrine signaling, we performed in vitro phenotypic assays on three OS cell lines with and without CXCL10. The results showed that the chemokine increased AKT phosphorylation and tumor cell migration. Using a CRISPR-based CXCR3 deletion mutant, we demonstrated that the lack of the receptor inhibited OS tumor growth and pulmonary metastasis in an orthotopic xenograft mouse model. The results indicate that the CXCL10-CXCR3 axis is sufficient and necessary to promote aggressive phenotypes in OS via autocrine signaling. Next, using gene expression datasets from two cohorts of OS patients, we showed that high expression of CXCL10 or its cognate receptor CXCR3 was associated with a better prognosis. Since CXCL10 is known to recruit CXCR3+ immune cells to fight against cancer, we further found that the expression of the T cell marker CD3D was associated with a better prognosis. These results suggest that the chemokine may also play a protective role in osteosarcoma by recruiting anti-tumor immune cells via paracrine signaling. Lastly, we have previously reported that a high circulating level of CXCL10 correlates with a poor prognosis in osteosarcoma patients. In this study, we demonstrated that increasing the circulating CXCL10 level in immunodeficient orthotopic xenograft mouse models of OS did not significantly promote the development of pulmonary metastases, suggesting circulating CXCL10 may exert its chemotactic effect mainly on immune cells, not on tumor cells. Taken together, we propose a model that tumor expression of CXCL10 promotes osteosarcoma growth and metastasis development via autocrine signaling. The increase of the local level of CXCL10 attracts CXCR3+ immune cells to the tumor site and promotes anti-tumor function via paracrine signaling. Metastatic cells colonize the lungs and cause tissue damage and inflammation, which increases the production of CXCL10 in the circulation. The higher circulating CXCL10 recruits T cells away from the primary tumor site and indicates the occurrence of metastasis and, hence, correlates with a worse outcome in patients. Our research highlights the importance of understanding the opposite effects of CXCL10 on tumor and immune cells as well as in the tumor microenvironment and circulation. Identification of the mechanistic differences in the CXCL10-CXCR3 signaling between tumor and immune cells will provide a novel therapeutic approach against tumor cells while promoting anti-tumor immune response in OS. Citation Format: Benjamin B. Gyau, Xiang Chen, Junyan Wang, Margaret A. Clement, Angela M. Major, Jun Xu, M. John Hicks, Tsz-Kwong Man. The CXCL10-CXCR3 axis as Jekyll and Hyde: A regulator of metastasis and immune response in osteosarcoma [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 1394.

Abstract 6003: The TNF-α-induced protein 3-interacting protein1 (TNIP1) suppresses HGF mediated NF-kB pathway activation and growth in gastric cancer cells

Abstract Background: TNF-α-induced protein 3-interacting protein 1 (TNIP1), an integral component in the regulation of immune responses and cellular pathways, is gaining increased attention in the scientific community. Its ability to interact with A20, a crucial ubiquitin-editing enzyme, forms a significant part of the body's mechanism to control inflammation and prevent unregulated immune responses, which are pivotal in the pathogenesis of autoimmune diseases. TNIP1's role extends beyond autoimmunity; it is also implicated in the regulation of cancer cell proliferation and survival, making it a potential target in cancer therapy. However, TNIP1 has not yet been elucidated on the tumorigenesis, proliferation, and invasion of human gastric cancer, and needs to be researched and discovered. In this study, the HGF-mediated association of TNIP1, nuclear factor kappa-B (NF-κB), and matrix metalloproteinase 9 (MMP9) and cancer cell proliferation and invasion were investigated in two types of human gastric cancer cell lines. Methods: In this study, cell culture, cDNA microarray analysis, western blotting, Real-Time Polymerase chain reaction, Zymography, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, TNIP1 knock-down with short hairpin RNA (shRNA), apoptosis, reagents and antibodies, standard two chamber invasion assay. Results: In our study on gastric cancer cells, we found that hepatocyte growth factor (HGF) stimulation altered TNIP1 expression at both mRNA and protein levels, indicating a dynamic interaction between HGF and TNIP1. The use of LY294002, a PI3K/AKT pathway inhibitor, revealed TNIP1's involvement in the NF-kB pathway, showing changes in NFkB and TNIP1 expression post-HGF stimulation. TNIP1-shRNA expressing cells exhibited altered expression of p53, BCL-2, NFkB, and MMP9, markers crucial for tumor progression and metastasis. Notably, TNIP1 knockdown led to increased cell proliferation and enhanced invasion capabilities in gastric cancer cells. Furthermore, TNIP1 knockdown cells showed a significant decrease in apoptosis rates upon HGF treatment, as confirmed by propidium iodide staining and FACS analysis. Conclusions: In conclusion, our results demonstrate that TNIP1 is closely associated with the NF-kB pathway and plays a critical role in modulating tumor invasion and apoptosis in gastric cancer cells. These findings suggest that TNIP1 could be a potential therapeutic target for the treatment of gastric cancer, providing new insights into the mechanisms of tumor progression and the development of targeted therapies. Citation Format: Jiyoon Jung, Sungae Koh, Kyunghee Lee. The TNF-α-induced protein 3-interacting protein1 (TNIP1) suppresses HGF mediated NF-kB pathway activation and growth in gastric cancer 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 6003.

Abstract 7517: Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging

Abstract Background: Programmed cell death protein 1 (PD1) and its ligand PD-L1 are pivotal components of the immune checkpoint machinery, playing a crucial role in the regulation of immune responses in cancer. The interaction between PD1 and PD-L1 is a significant therapeutic target in oncology, as its inhibitors have shown improved outcomes across a variety of malignancies. However, the treatment response to immunotherapies such as pembrolizumab has shown a modest response (~30%) in previous studies. This could be explained by the lack of molecular insight pertaining to the interaction of PD1 on T-cells and PD-L1 in the tumor cells in malignant tissues at the time of clinical diagnosis. The recent development of in situ Proximity Ligation Assay (isPLA), marks a significant advancement in the field. This method allows for the visualization of direct protein interactions within tissues, for example between PD1 and PD-L1. This adds an important layer of information compared to multiplexed immunofluorescence, which is useful for deep phenotyping of cell types in a tissue but lacks the ability to detect direct interactions between specific cells. Methods: PD-L1-PD1 interactions in human FFPE tonsil and bladder cancer tissue were identified using the NaveniFlex Tissue Atto647N kit, following the guidelines provided by the manufacturer. This method is capable of detecting proteins within a proximity of less than 40 nm, utilizing oligonucleotide-antibody conjugates that generate an enhanced fluorescent signal. After completing the Naveni® detection process, we performed multiplexed immunofluorescence (IF) using the Phenocycler platform and a panel of 14 barcoded antibodies, to generate isPLA and multiplexed IF data on the same tissue section. Results &amp; Discussion: We have successfully established an automated workflow for the isPLA protocol. In this work, we aim to continue this effort and combine the isPLA, with downstream highly multiplexed immunofluorescence using the Phenocycler platform. We have generated proof of concept data for the successful integration of the two methods, using tonsil and bladder cancer tissue to detect protein interactions between PD1 and PD-L1, as well as phenotypic profiling of the tumor microenvironment with a 14 plex panel. This panel allows us to phenotype the relevant cell types (B cells, dendritic cells, T cells, macrophages, tumor cells and their proliferation status) and subsets of T-cells like cytotoxic CD8+ cells. The integration of the isPLA and multiplexed IF is poised to significantly advance our understanding of the TME and hopefully aid in patient stratification for immune checkpoint inhibition therapy. The clinical significance of this method integration will later be explored in a retrospective cohort of muscle invasive bladder cancer, receiving immune checkpoint inhibition therapy with pembrolizumab as a second line treatment. Citation Format: Tony Ullman, David Krantz, Hampus Elofsson, Carolina Oses Sepulveda, Agata Zieba-Wicher, Päivi Östling, Anders Ullén, Subham Basu, Charlotte Stadler. Integrating in situ proximity ligation assay for PD1-PDL1 interaction with multiplexed immunofluorescence imaging [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 7517.

Abstract 1337: Characterization of anti-AXL CAR-NK cell therapy for the treatment of melanoma

Abstract AXL is a receptor tyrosine kinase (RTK) that is commonly expressed in metastatic melanoma and several other forms of cancer. Activation of AXL signaling is linked to tumor cell growth, invasion, metastatic transformation, drug resistance, and immune response regulation. However, treatment of metastatic melanoma continues to present clinical challenges due to its capacity to rapidly develop drug resistance, which manifests under the influence of AXL signaling. As such, AXL may be a viable antigen target for immunotherapies. Despite their successes in hematological malignancies, chimeric antigen receptor (CAR)-T cell therapies remain limited by their susceptibility to causing off-target toxicity. In contrast, natural killer (NK) cells are not associated with such risks, which makes them clinically appealing as an alternative to T cells. However, studies on the application of CAR-NK cells to treat solid tumors remain acutely scarce. To explore the clinical viability of CAR-NK cells for the treatment of metastatic melanoma, AXL-specific CAR-NK cells were generated by transfecting NK cells obtained from NK cell lines. Preliminary data from a cytotoxicity assay indicated that the cytotoxicity of CAR-NK92-MI cells against AXL-positive melanoma cell lines was significantly stronger (up to a 10-fold difference) than the corresponding parental cells. Further in vitro characterization of this CAR-NK cell line and other CAR-NK cell lines, as well as in vivo evaluation of their efficacy in a mouse xenograft model of melanoma metastasis, is underway. Citation Format: Winston Hibler, Glenn Merlino, Yan Lin Yu. Characterization of anti-AXL CAR-NK cell therapy for the treatment of melanoma [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 1337.

Abstract 105: Understanding the role of β2-adrenergic receptor signaling on type II innate lymphoid cells in cancer

Abstract Increased norepinephrine (NE) due to chronic stress can activate adrenergic receptors, specifically β2-adrenergic receptors (β2AR), to produce an immunosuppressive tumor microenvironment (TME). Previous work by our lab demonstrated that tumor burden can be reduced by blocking β2AR signaling. Immune cells express β2AR and we have shown that the increased β2AR signaling leads to increased T cell exhaustion and increased inhibitory activity of myeloid-derived suppressor cells (MDSCs). In general, the resulting immunosuppression leads to a modest but significant reduction in tumor growth. However, the lack of a dramatic reduction in tumor growth reveals the complex nature of adrenergic signaling dependent regulation of the cancer immune response. Recent literature revealed that innate lymphoid cells (ILCs), especially type II ILCs (ILC2s), express high levels of β2AR. Therefore, we evaluated whether β2AR regulates ILC2s within the TME and how that affects tumor prognosis. We generated a genetic conditional knockout murine model where β2AR expression in ILC2s was deleted by crossing IL5cre expressing mice to β2ARflox/flox mice (IL5creβ2ARf/f). AT3 breast cancer cell line was injected into the 4th mammary fat pad and tumor volume was measured every 2-3 days. Tumor infiltrating ILC2s (CD45+Lineage−CD90.2+CD127+ST2+) were analyzed by flow cytometry at D36 post-tumor implantation. Notably, IL5creβ2ARf/f mice had increased AT3 breast cancer tumor growth compared to control mice. Flow cytometric analysis of the tumor revealed an increased frequency of GATA3+ ILC2s within the TME. These GATA3+ ILC2s lacking β2AR expressed higher levels of the IL33 receptor, ST2, and a higher percentage of IL13+ cells compared to WT GATA3+ ILC2 cells. We also observe a trend toward increased MDSC population within the TME of mice lacking β2AR in ILC2s. These results reveal an anti-tumor function of β2AR signaling through the suppression of ILC2 activity within the TME that warrants further investigation. This work has been in part supported by NIH Grants F30 CA284763 (to J.C.), K99 HL155792 (to H.M.), and RO1 CA205246 (to E.R.) Citation Format: Jee Eun Choi, Cameron R. MacDonald, Saeed Daneshmandi, Jianmin Wang, Nathan T. Roberts, Caitlin M. James, Philip L. McCarthy, Hemn Mohammadpour, Elizabeth A. Repasky. Understanding the role of β2-adrenergic receptor signaling on type II innate lymphoid cells in 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 105.

Abstract 177: Targeting murine or human KLRG1 exerts potent anti-tumor efficacy involving both CD8T cells and NK cells

Abstract Clinical response to anti-PD-1 immunotherapy is correlated with patients who have a T cell-inflamed tumor microenvironment. Some patients with immune cell infiltration fail to respond, and some patients experiencing an initial clinical response develop acquired resistance. It is likely that additional immune-regulatory pathways need to be targeted to expand the efficacy of immunotherapies. To identify a broader array of therapeutic relevant immunotherapy molecules in T cell-inflamed tumors, our lab utilized gene expression profiling to characterize tumor antigen-specific dysfunctional CD8+ T cells in the tumor microenvironment. One molecule that was found to be overexpressed on these CD8+ T cells was C-type lectin inhibitory receptor, KLRG1. KLRG1 is an ITIM domain-containing receptor mostly explored on NK cells, and the known ligands are N- and E- cadherins which are broadly expressed in solid cancers. Analysis of single cell RNA-Seq data from tumor biopsies of melanoma patients classified as responders or non-responders to anti-PD-1 therapy revealed significantly higher expression of KLRG1 expression on tumor-infiltrating T cell subsets in non-responders, suggesting a possible salvage immune suppressive role. To determine the potential functional relevance of KLRG1 on tumor control, we generated KLRG1 knockout (KO) mice, then implanted tumors and assessed tumor growth rate in vivo. KLRG1 KO mice showed markedly slowed B16 melanoma tumor growth compared to WT mice, arguing it is a critical negative regulator of anti-tumor immunity. Both antigen-specific CD8+ T cells and NK cells were expanded in these tumors, and depletion of CD8+ T cells or NK cells eliminated this tumor control. Genetic deletion of N-cadherin from B16 cells also resulted in slowed tumor growth, suggesting that tumor cells are providing the functionally relevant ligand. To assess the degree of inhibitory function of human KLRG1, we generated a humanized KLRG1 knock-in mouse model. We confirmed that human KLRG1 can biochemically interact with murine cadherins. Knock-in mice with the humanized KLRG1 protein had significantly increased tumor growth compared to both WT and KLRG1 KO mice, indicating a stronger immunosuppressive effect of human KLRG1 compared to murine KLRG1. An antagonistic antibody blocking interaction of human KLRG1 with its ligands was generated, which potentiated human T cell activation in vitro. Administration of anti-human KLRG1 Ab to tumor-bearing humanized KLRG1 mice led to markedly reduced tumor growth. In summary, our data support KLRG1 as a functionally critical negative regulator of the anti-tumor immune response and that blocking KLRG1-cadherin interactions has potential as a therapeutic candidate. Citation Format: Tyler A. Jones, Stephen Sinicrope, Kei Yasuda, Jan Pinkas, Thomas F. Gajewski. Targeting murine or human KLRG1 exerts potent anti-tumor efficacy involving both CD8T cells and NK 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 177.

Abstract 6819: Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma

Abstract Intrahepatic cholangiocarcinoma (CCA) is an aggressive biliary tract cancer that carries unfavorable prognosis. 10-20% of intrahepatic CCAs harbor a mutation in IDH1 that can be targeted with mutant IDH1-specific inhibitors. However, objective and durable responses to treatment with mutant IDH1 inhibitors are rare in CCA. Mutant IDH1 has neomorphic enzymatic activity that produces the oncometabolite D-2-hydroxyglutarate (D-2-HG). D-2-HG promotes biliary tumor formation through cancer cell-intrinsic mechanisms but is also a paracrine factor in the tumor microenvironment (TME). Our group and others have identified that IDH1-mutant CCAs are not responsive to immunotherapy treatment approaches in part due to decreased CD8+ T cell infiltration and an increase in immunosuppressive macrophage cells in the TME. To better understand the immunosuppressive features of the IDH1-mutant CCA TME, we generated an isogenic cell line panel of IDH1-mutant (MUT) and IDH1-wild type (WT) mouse cholangiocarcinoma cells in the SB1 cell line background through a CRISPR homology directed repair approach. Compared to IDH1-WT cells, IDH1-MUT cells produce high levels of D-2-HG and both cell line panels readily form tumors in immunocompetent C57BL/6 mice. Tumors formed from IDH1-mut cells have reduced CD8+ T cell infiltration compared to IDH1-WT tumors, a finding that correlates with analysis of human CCA samples. Using cytometry by time of flight (CyTOF), we identified that CD8+ T cells in IDH1-MUT tumors have decreased expression of key regulators of the antitumor immune response including EOMES, TBET, and GZMB compared to those isolated from IDH1-WT tumors. Analysis of secreted factors from these IDH1-MUT and IDH1-WT cells identified that IDH1-MUT cells release increased levels of CCL2, a chemokine shown to diminish the antitumor immune response across multiple cancer types, compared to IDH1-WT cells. Further, IDH1-MUT tumors grown in syngeneic, immunocompetent mice have increased CCL2 staining by IHC compared to IDH1-WT tumors. CCL2 expression is also increased in a preliminary analysis of IDH1-mutant human CCA primary tumors. Treatment of mice harboring orthotopic liver tumors formed from IDH1-MUT cells with a CCL2 neutralizing antibody resulted in decreased tumor size compared to isotype control. Together, our data suggest that CCL2 is a mediator of immunosuppression in the IDH1-mutant CCA TME and that modulation of CCL2 activity may improve outcomes in this rare disease subtype. Further work to understand the mechanisms through which IDH1 mutations regulate CCL2 levels in these tumors is underway. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Kiyoko Yoshima, Daniel J. Zabransky, Mark Yarchoan. Exploring the impact of IDH1-mutations on antitumor immunity in intrahepatic cholangiocarcinoma [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 6819.

Abstract 2039: Cellular characterization of small molecule PD-L1 inhibitors reveal their novel mechanisms of action

Abstract Introductions: Immune checkpoint inhibitors, including PD-1/L1, are key regulators of immune response and promising targets in cancer immunotherapy. Like anti-PD-1/L1 antibodies, small molecule PD-L1 inhibitors that have been discovered by us and others could also efficiently block PD-1 and PD-L1 interaction and exhibit anti-tumor efficacy in preclinical and clinically settings. In this study, we explore the cellular mechanism of small molecule PD-L1 inhibitors, unveiling their novel mechanisms of action in the regulation of PD-L1 and its functions. Materials and Methods: The cellular effects of the small molecule PD-L1 inhibitor on PD-L1 protein and RNA levels were assessed through Western blot and real-time qPCR experiments. HTRF assay and cellular luciferase reporter assay were used to evaluate the blockade of PD-1 and PD-L1 interaction in vitro and in cells. Flow cytometry and confocal microscopy were used to investigate the internalization of PD-L1 upon treatment with inhibitors. MSD assay was used to evaluate the alteration of soluble PD-L1 proteins. Results: Small molecule PD-L1 inhibitors discovered by Abbisko Therapeutics potently blocked PD-1/PD-L1 interaction in vitro and in cellular system. It could also rapidly and strongly induce PD-L1 internalization in cells. Further profiling suggested that small molecule PD-L1 inhibitors specifically disrupt PD-L1 N-glycosylation, resulting in aberrant PD-L1 glycosylation. This, in turn, may hinder the proper translocation of newly synthesized PD-L1 to the cell surface, thereby abolishing its interaction with PD-1 and functions in immune regulation. Conclusion: Taken together, these results for the first time revealed the distinctive mechanisms of our small molecule PD-L1 inhibitors. With their multi-layer inhibitory effects stemming from various mechanisms, small molecule PD-L1 inhibitors may offer potentially improved activities and an alternative therapeutic treatment for the cancer patients. Citation Format: Weiling Pan, Yongxian Zhang, Fangfei Qi, Wenqun Xin, Zhigang Feng, Zhui Chen, Haiyan Ying. Cellular characterization of small molecule PD-L1 inhibitors reveal their novel mechanisms of action [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 2039.