Killer Cell Lectin Research Articles

KLRD1 (CD94): A Prognostic Biomarker and Therapeutic Candidate in Head and Neck Squamous Cell Carcinoma.

Background: Killer Cell Lectin Like Receptor D1 (KLRD1) plays a crucial role in antitumor immunity. However, its expression patterns across various cancers, its relationship with patient prognosis, and its potential as an immunotherapy target remain inadequately understood. Methods: We analyzed KLRD1 expression across various cancer types using multi-omics data from The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), and Gene Expression Omnibus (GEO) databases, correlating it with patient prognosis. Single-cell RNA sequencing data were employed to further explore KLRD1 expression in natural killer (NK) cells and exhausted CD8+ T cells (CD8Tex). Functional enrichment analyses using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) identified the biological processes and pathways associated with KLRD1. Immune infiltration analysis, conducted via CIBERSORT, assessed the relationship between KLRD1 expression and immune cell infiltration within the tumor microenvironment. Furthermore, the Tracking Tumor Immunophenotype (TIP) meta-server and Easier tool were employed to assess the role of KLRD1 in the cancer immunity cycle and to predict immunotherapy responses. Drug sensitivity was predicted using tools like CellMiner and the Genomics of Drug Sensitivity in Cancer (GDSC) database to explore the link between KLRD1 expression and responsiveness to various anticancer drugs. Results: KLRD1 exhibits significant differential expression and strong prognostic value across cancers, particularly as an independent prognostic factor in head and neck squamous cell carcinoma (HNSC). Single-cell analysis revealed high expression of KLRD1 in NK and CD8Tex cells, indicating its critical role in antitumor immune responses. Functional enrichment analyses showed that KLRD1 is involved in several immune-related signaling pathways, including NK cell-mediated cytotoxicity and T cell receptor pathways. Immune infiltration analysis further confirmed a positive correlation between KLRD1 expression and the infiltration of various immune cells. Moreover, higher KLRD1 expression in HNSC is associated with enhanced immune pathway activity, increased sensitivity to cell division inhibitors, and the identification of arachidonyltrifluoromethane as a potential compound to counteract its oncogenic effects. Conclusion: In HNSC, KLRD1 is a key prognostic marker and potential target for personalized immunotherapy.

Diagnostic potential of genomic blood biomarkers of pulmonary fibrosis in a prospective cohort.

Fibrotic interstitial lung diseases (ILDs) result from excessive deposition of extracellular matrix (ECM) proteins in the lung, causing irreversible damage to the lung architecture. Clinical management of ILDs differs depending on the diagnosis, but differentiation between subtypes can be difficult and better clinical biomarkers are needed. In this study, we use a 166-gene NanoString assay to investigate whether there are ILD subtype-specific transcripts in whole blood. We identified one transcript, killer cell lectin like receptor 1 (KLRF1), as differentially expressed between idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated ILD (SSc-ILD), and identified two transcripts (VCAN, LTK) associated with IPF expression against other ILD subtypes. These findings were validated by examining their expression in ILD lung, with KLRF1 expression significantly higher in SSc-ILD compared to IPF and hypersensitivity pneumonitis (HP) samples. Taken together, this pilot study provides support for the use of the peripheral transcriptome in identifying diagnostic biomarkers of ILD with biological relevance.

Single Cell Droplet-Based Efficacy and Transcriptomic Analysis of a Novel Anti-KLRG1 Antibody for Elimination of Autoreactive T Cells.

Progress in developing improvements in the treatment of autoimmune disease has been gradual, due to challenges presented by the nature of these conditions. Namely, the need to suppress a patient's immune response while maintaining the essential activity of the immune system in controlling disease. Targeted treatments to eliminate the autoreactive immune cells driving disease symptoms present a promising new option for major improvements in treatment efficacy and side effect management. Monoclonal antibody therapies can be applied to target autoreactive immune cells if the cells possess unique surface marker expression patterns. Killer cell lectin like receptor G1 (KLRG1) expression on autoreactive T cells presents an optimal target for this type of cell depleting antibody therapy. In this study, we apply a variety of in vitro screening methods to determine the efficacy of a novel anti-KLRG1 antibody at mediating specific natural killer (NK) cell mediated antibody-dependent cellular cytotoxicity (ADCC). The methods include single-cell droplet microfluidic techniques, allowing timelapse imaging and sorting based on cellular interactions. Included in this study is the development of a novel method of sorting cells using a droplet-sorting platform and a fluorescent calcium dye to separate cells based on CD16 recognition of cell-bound antibody. We applied this novel sorting method to visualize transcriptomic variation between NK cells that are or are not activated by binding the anti-KLRG1 antibody using RNA sequencing. The data in this study reveals a reliable and target-specific cytotoxicity of the cell depleting anti-KLRG1 antibody, and supports our droplet-sorting calcium assay as a novel method of sorting cells based on receptor activation.

Cathepsin W, T-cell receptor-associated transmembrane adapter 1, lymphotactin and killer cell lectin like receptor K1 are sensitive and specific RNA biomarkers of canine epitheliotropic lymphoma.

Cutaneous T-cell lymphoma (CTCL) is an uncommon type of lymphoma involving malignant skin-resident or skin-homing T cells. Canine epitheliotropic lymphoma (EL) is the most common form of CTCL in dogs, and it also spontaneously arises from T lymphocytes in the mucosa and skin. Clinically, it can be difficult to distinguish early-stage CTCLs apart from other forms of benign interface dermatitis (ID) in both dogs and people. Our objective was to identify novel biomarkers that can distinguish EL from other forms of ID, and perform comparative transcriptomics of human CTCL and canine EL. Here, we present a retrospective gene expression study that employed archival tissue from biorepositories. We analyzed a discovery cohort of 6 canines and a validation cohort of 8 canines with EL which occurred spontaneously in client-owned companion dogs. We performed comparative targeted transcriptomics studies using NanoString to assess 160 genes from lesional skin biopsies from the discovery cohort and 800 genes from the validation cohort to identify any significant differences that may reflect oncogenesis and immunopathogenesis. We further sought to determine if gene expression in EL and CTCL are conserved across humans and canines by comparing our data to previously published human datasets. Similar chemokine profiles were observed in dog EL and human CTCL, and analyses were performed to validate potential biomarkers and drivers of disease. In dogs, we found enrichment of T cell gene signatures, with upregulation of IFNG, TNF, PRF1, IL15, CD244, CXCL10, and CCL5 in EL in dogs compared to healthy controls. Importantly, CTSW, TRAT1 and KLRK1 distinguished EL from all other forms of interface dermatitis we studied, providing much-needed biomarkers for the veterinary field. XCL1/XCL2 were also highly specific of EL in our validation cohort. Future studies exploring the oncogenesis of spontaneous lymphomas in companion animals will expand our understanding of these disorders. Biomarkers may be useful for predicting disease prognosis and treatment responses. We plan to use our data to inform future development of targeted therapies, as well as for repurposing drugs for both veterinary and human medicine.

Development of a novel tumor microenvironment-related radiogenomics model for prognosis prediction in hepatocellular carcinoma.

The tumour microenvironment (TME) has occupied a potent position in the tumorigenesis and tumor progression of hepatocellular carcinoma (HCC). Radiogenomics is an emerging field that integrates imaging and genetic information, thus offering a novel class of non-invasive biomarkers with diagnostic, prognostic, and treatment response. However, optimal evaluation methodologies for radiogenomics in patients with HCC have not been well established. Therefore, this study aims to develop a radiogenomics models, associating contrast-enhanced computed tomography (CECT) based radiomics features and transcriptomics data with TME, to increase predictive precision for overall survival (OS) in patients with HCC. Transcriptome profiles of 365 patients with HCC from The Cancer Genome Atlas (TCGA)-HCC cohort were used to obtain TME-related genes by differential expression analysis. TME-related radiomics features of 53 patients with HCC from The Cancer Imaging Archive (TCIA)-HCC cohort matched with the TCGA-HCC cohort were screened via correlation analysis. Furthermore, a radiogenomics score-based prognostic model was constructed using the least absolute shrinkage and selection operator (LASSO) Cox regression analysis in the TCIA-HCC cohort. Finally, the ability to predict prognosis and the value of the model in identifying the abundance of immune cell infiltration were investigated. A radiogenomics prognostic model was developed, which incorporated 1 radiomics feature [original_gray-level co-occurrence matrix (glcm)_inverse difference normalized (Idn)] and 3 genes [spen paralogue and orthologue C‑terminal domain containing 1 (SPOCD1); killer cell lectin like receptor B1 (KLRB1); G protein-coupled receptor 182 (GPR182)]. The model performed satisfactorily in the training and test sets [1-year, 2-year, 3-year area under the curve (AUC) of 0.81, 0.85 and 0.87 in the training set, respectively; and 0.73, 0.83, and 0.84 in the test set, respectively]. Moreover, the model showed that higher radiogenomics scores were associated with worse OS and lower levels of immune infiltration. The novel CECT-based radiogenomics model may provide valuable insights for prognostic stratification and TME assessment of patients with HCC.

Analysis on tumor immune microenvironment and construction of a prognosis model for immune-related skin cutaneous melanoma.

Malignant melanoma is a highly malignant and heterogeneous skin cancer. Although immunotherapy has improved survival rates, the inhibitory effect of tumor microenvironment has weakened its efficacy. To improve survival and treatment strategies, we need to develop immune-related prognostic models. Based on the analysis of the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Sequence Read Archive (SRA) database, this study aims to establish an immune-related prognosis prediction model, and to evaluate the tumor immune microenvironment by risk score to guide immunotherapy. Skin cutaneous melanoma (SKCM) transcriptome sequencing data and corresponding clinical information were obtained from the TCGA database, differentially expressed genes were analyzed, and prognostic models were developed using univariate Cox regression, the LASSO method, and stepwise regression. Differentially expressed genes in prognostic models confirmed by real-time reverse transcription PCR (real-time RT-PCR) and Western blotting. Survival analysis was performed by using the Kaplan-Meier method, and the effect of the model was evaluated by time-dependent receiver operating characteristic curve as well as multivariate Cox regression, and the prognostic model was validated by 2 GEO melanoma datasets. Furthermore, correlations between risk score and immune cell infiltration, Estimation of STromal and Immune cells in MAlignant Tumor tissues using Expression data (ESTIMATE) score, immune checkpoint mRNA expression levels, tumor immune cycle, or tumor immune micro-environmental pathways were analyzed. Finally, we performed association analysis for risk score and the efficacy of immunotherapy. We identified 4 genes that were differentially expressed in TCGA-SKCM datasets, which were mainly associated with the tumor immune microenvironment. A prognostic model was also established based on 4 genes. Among 4 genes, the mRNA and protein levels of killer cell lectin like receptor D1 (KLRD1), leukemia inhibitory factor (LIF), and cellular retinoic acid binding protein 2 (CRABP2) genes in melanoma tissues differed significantly from those in normal skin (all P<0.01). The prognostic model was a good predictor of prognosis for patients with SKCM. The patients with high-risk scores had significantly shorter overall survival than those with low-risk scores, and consistent results were achieved in the training cohort and multiple validation cohorts (P<0.001). The risk score was strongly associated with immune cell infiltration, ESTIMATE score, immune checkpoint mRNA expression levels, tumor immune cycle, and tumor immune microenvironmental pathways (P<0.001). The correlation analysis showed that patients with the high-risk scores were in an inhibitory immune microenvironment based on the prognostic model (P<0.01). The immune-related SKCM prognostic model constructed in this study can effectively predict the prognosis of SKCM patients. Considering its close correlation to the tumor immune microenvironment, the model has some reference value for clinical immunotherapy of SKCM.

CRISPR/Cas9 Gene Editing of Immune Checkpoint Receptor NKG2A Improves the Efficacy of Primary CD33-CAR-NK Cells Against AML

BACKGROUND: CD33-targeting chimeric antigen receptor (CAR)-T cells already showed utility for the treatment of AML. Yet clinical application of CD33-CAR-T cells remains challenging due to potential side effects and its restriction to autologous cell preparations. In contrast, natural killer (NK) cells can be safely administered to HLA-mismatched recipients without severe side effects. Recently, we reported on the successful generation of primary CD33-CAR-NK cells, which are highly effective against AML in vitro and in vivo in AML-xenograft models (Albinger et al., Blood Cancer J 2022). However, CAR-NK cell function can be impaired by high levels of the inhibitory immune checkpoint receptor NKG2A (natural killer group 2A) expressed on NK cells (Bexte et al., Oncoimmunology 2022). By applying a CRISPR/Cas9 gene editing for knockout (KO) of NKG2A, we significantly improved CD33-CAR-NK cell functionality in vitro and in vivo. METHODS: CD33-targeting CAR-NK cells were generated by lentiviral transduction. KO of the NKG2A-encoding killer cell lectin like receptor C1 (KLRC1) locus was performed using CRISPR-Cas9 technology. The CD33-CAR- and NKG2A-expression as well as cytotoxicity were analysed using flow cytometry after feeder cell-free, IL-15/IL-2-based expansion. The in vivo-efficacy was evaluated in OCI-AML2 (GFP+, Luc+) xenografted NSG-SGM3 mouse models. RESULTS: Lentiviral transduction resulted in up to 60% CD33-CAR-positive NK cells, while KLRC1 gene disruption resulted in 50% reduction of NKG2A expression. Cite-Seq and qPCR analysis revealed a distinct gene regulation pattern in CD33-CAR- and CD33-CAR-NKG2A-KO-NK cells and CAR-KO-NK cells showed significantly higher elimination of CD33+/HLA-E+ OCI-AML2 cells in in vitro cytotoxicity assays compared to NKG2A-KO- or CD33-CAR-NK cells. Furthermore, a reduction of leukemic burden was observed in vivo following a single injection of a low dose (3x106 cells) of CAR-KO-NK cells compared to NKG2A-KO or CD33-CAR-NK cell treatment in an AML-xenografted mouse model. A double injection led to a complete elimination of AML and leukemia-initiating cells in the bone marrow of mice treated with CAR-KO NK cells, which was confirmed by bone marrow re-engraftment analysis. CONCLUSION: Removing an inhibitory receptor in CAR-NK cells showed a highly beneficial effect for the treatment of AML. This double genetic modification has the potential to enable NK cells to bypass the suppressive effect not only in the tumor microenvironment in context of AML, but also in a broad range of malignant diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Myelin oligodendrocyte glycoprotein-associated disease is associated with BANK1, RNASET2 and TNIP1 polymorphisms

AimHere we aimed to compare association of common immune-related genetic variants with three autoimmune central nervous system (CNS) demyelinating diseases, namely myelin oligodendrocyte glycoprotein-associated disease (MOGAD), multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD). MethodsIn this retrospective cross-sectional study, 26 common immune-related single nucleotide polymorphisms were genotyped in 102 patients with MOGAD, 100 patients with MS, 198 patients with NMOSD and 541 healthy control subjects recruited from Guangzhou, China. ResultsAmong all tested genetic variations, one polymorphism, B cell scaffold protein with ankyrin repeats 1 (BANK1) rs4522865 was associated with multiple disorders, namely MOGAD (OR = 1.94, 95% CI:1.19–3.17, P = 0.0059) and NMOSD (OR = 1.69, 95% CI:1.17–2.45). Besides BANK1 rs4522865, two other non-HLA loci, ribonuclease T2 (RNASET2) rs9355610 (OR = 0.47, 95% CI: 0.26–0.85) and TNFAIP3 interacting protein 1 (TNIP1) rs10036748 (OR = 1.76, 95% CI: 1.16–2.71), were associated with MOGAD. In addition, NMOSD was associated with signal transducer and activator of transcription 4 (STAT4) rs7574865 (OR = 1.58, 95% CI: 1.12–2.24) and general transcription factor Iii (GTF2I) rs73366469 (OR = 1.60, 95% CI:1.12–2.29), while MS was associated with a killer cell lectin like receptor G1 (KLRG1) rs1805673 (OR = 0.61, 95% CI: 0.40–0.94) and T-box transcription factor 21 (TBX21) rs17244587 (OR = 2.25, 95% CI: 1.25–4.06). ConclusionThe current study suggests for the first time three non-HLA susceptibility loci for MOGAD. In addition, comparison of association of 26 immune-related polymorphisms with three autoimmune CNS demyelinating diseases demonstrates substantial difference in genetic basis of those disorders.

Expression of KLRG1 on subpopulations of lymphocytes in the peripheral blood of patients with locally advanced nasopharyngeal carcinoma and prognostic analysis

AbstractObjectiveWe aimed to investigate the relationship between the expression of killer cell lectin like receptor G1 (KLRG1) in peripheral blood lymphocytes, and the clinical characteristics and prognosis in patients with locally advanced nasopharyngeal carcinoma (NPC), and to dynamically evaluate the changes of KLRG1 expression after treatment.MethodsA total of 82 patients with locally advanced NPC and 44 healthy individuals were recruited in this study. The expression of KLRG1 on T, natural killer, and natural killer T cells in peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry. Its relationship with clinical characteristics and prognosis of patients, as well as changes in KLRG1 expression in PBMCs after treatment, were investigated.ResultsThe expression of KLRG1 in CD8+ T cells in PBMCs of NPC patients was 46.50 (29.10, 69.80)%, significantly higher than 32.50 (17.38, 61.00) % in healthy controls (Z = −2.290, p = 0.022). The expression of KLRG1 in CD3+ CD56+ natural killer T cells in PBMCs of patients was 29.50 (6.21, 78.00) %, significantly higher than 1.47 (0.62, 8.88)% in healthy controls (Z = −5.371, p &lt; 0.001). The expression levels of KLRG1(+)CD8+ T cells in PBMCs of patients were higher in the age ≥50 years (Z = −3.413, p &lt; 0.001), pathology of keratinizing squamous cell carcinoma (H = 10.541, p = 0.005, and EGFR(+) groups (Z = −2.006, p = 0.043). Patients with higher KLRG1(+) expression in CD8+ T cells had a lower progression‐free survival (p = 0.029) and a lower distant metastasis‐free survival (p = 0.023). KLRG1 expression on T cells increased after radiotherapy.ConclusionKLRG1 can be harnessed as a potential target in designing novel therapy to encompass a broader range of NPC patients.

Metabotropic Glutamate Receptor 5 in Natural Killer Cells Attenuates Liver Fibrosis by Exerting Cytotoxicity to Activated Stellate Cells.

The important roles of glutamate and metabotropic glutamate receptor 5 (mGluR5) in HSCs have recently been reported in various liver diseases; however, the mechanism linking the glutamine/glutamate metabolism and mGluR5 in liver fibrosis remains unclear. Here, we report that mGluR5 activation in natural killer (NK) cells attenuates liver fibrosis through increased cytotoxicity and interferon-γ (IFN-γ) production in both mice and humans. Following 2-week injection of carbon tetrachloride (CCl4 ) or 5-week methionine-deficient and choline-deficient diet, liver fibrosis was more aggravated in mGluR5 knockout mice with significantly decreased frequency of NK cells compared with wild-type mice. Consistently, NK cell-specific mGluR5 knockout mice had aggravated CCl4 -induced liver fibrosis with decreased production of IFN-γ. Conversely, in vitro activation of mGluR5 in NK cells significantly increased the expression of anti-fibrosis-related genes including Ifng, Prf1 (perforin), and Klrk1 (killer cell lectin like receptor K1) and the production of IFN-γ through the mitogen-activated extracellular signal-regulated kinase/extracellular signal-related kinase pathway, contributing to the increased cytotoxicity against activated HSCs. However, we found that the uptake of glutamate was increased in activated HSCs, resulting in shortage of extracellular glutamate and reduced stimulation of mGluR5 in NK cells. Consequently, this could enable HSCs to evade NK cell cytotoxicity in advanced liver fibrosis. In vivo, pharmacologic activation of mGluR5 accelerated CCl4 -induced liver fibrosis regression by restoring NK cell cytotoxicity. In humans, mGluR5 activation enhanced the cytotoxicity of NK cells isolated from healthy donors, but not from patients with cirrhosis with significantly reduced mGluR5 expression in NK cells. mGluR5 plays important roles in attenuating liver fibrosis by augmenting NK cell cytotoxicity, which could be used as a potential therapeutic target for liver fibrosis.

Functions and clinical significance of KLRG1 in the development of lung adenocarcinoma and immunotherapy

BackgroundAs a marker of differentiation, Killer cell lectin like receptor G1 (KLRG1) plays an inhibitory role in human NK cells and T cells. However, its clinical role remains inexplicit. This work intended to investigate the predictive ability of KLRG1 on the efficacy of immune-checkpoint inhibitor in the treatment of lung adenocarcinoma (LUAD), as well as contribute to the possible molecular mechanisms of KLRG1 on LUAD development.MethodsUsing data from the Gene Expression Omnibus, the Cancer Genome Atlas and the Genotype-Tissue Expression, we compared the expression of KLRG1 and its related genes Bruton tyrosine kinase (BTK), C-C motif chemokine receptor 2 (CCR2), Scm polycomb group protein like 4 (SCML4) in LUAD and normal lung tissues. We also established stable LUAD cell lines with KLRG1 gene knockdown and investigated the effect of KLRG1 knockdown on tumor cell proliferation. We further studied the prognostic value of the four factors in terms of overall survival (OS) in LUAD. Using data from the Gene Expression Omnibus, we further investigated the expression of KLRG1 in the patients with different responses after immunotherapy.ResultsThe expression of KLRG1, BTK, CCR2 and SCML4 was significantly downregulated in LUAD tissues compared to normal controls. Knockdown of KLRG1 promoted the proliferation of A549 and H1299 tumor cells. And low expression of these four factors was associated with unfavorable overall survival in patients with LUAD. Furthermore, low expression of KLRG1 also correlated with poor responses to immunotherapy in LUAD patients.ConclusionBased on these findings, we inferred that KLRG1 had significant correlation with immunotherapy response. Meanwhile, KLRG1, BTK, CCR2 and SCML4 might serve as valuable prognostic biomarkers in LUAD.

Natural killer cells in the human lung tumor microenvironment display immune inhibitory functions

BackgroundNatural killer (NK) cells play a crucial role in tumor immunosurveillance through their cytotoxic effector functions and their capacity to interact with other immune cells to build a coordinated antitumor...

Oral cancer cell‑derived exosomes modulate natural killer cell activity by regulating the receptors on these cells.

Oral cancer (OC) is the most common type of head and neck malignant tumor. Tumor-derived exosomes induce a complex extracellular environment that affects tumor immunity. In the present study, exosomes were isolated from OC cell lines (WSU-HN4 and SCC-9) by ultrafiltration and the protein content of these oral cancer-derived exosomes (OCEXs) was analyzed by mass spectrometry, which revealed the enrichment of transforming growth factor (TGF)-β1. Natural killer (NK) cells were examined by flow cytometry following co-culture with OCEXs. The expression of killer cell lectin like receptor K1 (KLRK1; also known as NKG2D, as used herein) and natural cytotoxicity triggering receptor 3 (NCR3; also known as NKp30, as used herein) in NK cells was found to be significantly upregulated following co-culture with the OCEXs for 1 day, whereas this expression decreased at 7 days. Killer cell lectin like receptor C1 (KLRC1; also known as NKG2A; as used herein) expression exhibited an opposite trend at 1 day. In addition, NK cell cytotoxicity against the OC cells was enhanced at 1 day, but was attenuated at 7 days. TGF-β1 inhibited the function of NK cells at 7 days, whereas it had no obvious effects at 1 and 3 days. On the whole, the findings of the present study reveal changes in NK cell function and provide new insight into NK cell dysfunction.

Abstract 5717: LncRNA RP11-291B21.2 is associated with Durvalumab response in NSCLC and BLCA cancers

Abstract Introduction Long noncoding RNAs (lncRNAs) are interacting with oncogenes, tumor suppressors and immune genes by regulating their expression and activity of relevant transcription factors, RNA-binding proteins, and microRNAs. Dysregulated lncRNAs in immune cells are associated with excessive or uncontrolled inflammation, and play vital roles in innate immune responses, carcinogenesis and tumor microenvironment (TME). Understanding potential associations of lncRNAs with bladder cancer (BLCA) and non-small cell lung cancer (NSCLC) patients treated by ICB agents, such as durvalumab, is emerging. Methods RNAseq data from CP1108 (NCT01693562) clinical trial samples (97 NSCLC samples and 66 BLCA samples treated by durvalumab monotherapy) were processed using a comprehensive annotated genome (GRCh38.p12, release 28). The normalized lncRNA expression was reported, significantly differentially expressed lncRNAs between RECIST responders and non-responders in both BLCA and NSCLC patients were then identified using R limma package. Single-cell RNAseq data analysis was done with R Seurat package. Correlation analysis between interested lncRNAs and known oncogenic or immune genes was carried out using Spearman's rho metric in both CP1108 and TCGA RNAseq datasets. Kaplan-Meier survival analysis (OS and PFS) was performed and log-rank p-value was reported. Results Eight lncRNAs (RP4-740C4.7, AL1333245.1, RP11-284F21.10, RP5-1139B12.4, RP11-291B21.2, CTD-3064M3.7, TRG-AS1, and RP11-291B21.2) were identified to be differentially expressed between durvalumab responders and non-responders in both CP1108 BLCA and NSCLC tumors. RP11-291B21.2 is the most interested lncRNAs since 6 of 7 its upstream proximity genes (up to 300kb) are killer cell lectin like receptors (KLRs): KLRC1, KLRC2, KLRC3, KLRC4, KLRK1, GABARAPL1, and KLRD1 and they are all significantly correlated with the expression of RP11-291B21.2. Co-expression analysis has shown that lncRNA RP11-291B21.2 is highly correlated with multiple key immune genes, including NK / T cells markers and IFNg induced genes, suggesting its important role in regulating tumor microenvironment. The analysis of scRNAseq data of NSCLC tumors suggests RP11-291B21.2 is dominantly expressed in CD8+ T cells, especially in exhaustive T cells. RP11-291B21.2 is also confirmed to be highly expressed (FC&amp;gt;1.8, p&amp;lt;0.03) in exhausted CD8+ T cells both 40h and day 6 after stimulation in the in vitro experiments. Survival analysis has revealed high expression of RP11-291B21.2 is associated with better survival for patients treated by durvalumab in NSCLC and BLCA (HR=~0.3), but not for patients treated with chemotherapy in TCGA datasets. In conclusion, high expression of RP11-291B21.2 predicts better survival to NSCLC and BLCA patients treated by durvalumab which may suggest that RP11-291B21.2 is a potential lncRNA biomarker to PD1/PDL1 blockage. Citation Format: Song Wu, Sarah Hsu, Qu Zhang, Lydia Greenlees, Mike Xiao, Ashok Gupta. LncRNA RP11-291B21.2 is associated with Durvalumab response in NSCLC and BLCA cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5717.

MICA and KLRK1 genes and their impact in cervical intraepithelial neoplasia development in the southern Brazilian population

Cervical carcinoma and cervical intraepithelial neoplasia (CIN) are associated with persistent infection by oncogenic subtypes of HPV (Human Papillomavirus). Factors linked to immunity, genetics and others like oral contraceptive use, sexual behavior, coinfections with other microorganisms and smoking seem to influence the mechanisms that determine regression or progression to CIN and cervical cancer. We investigated the effect of the MHC class I chain-related gene A (MICA) and Killer Cell Lectin Like receptor K1 (KLRK1) genes on cervical cancer and CIN lesions susceptibility in a group of 195 patients from southern Brazil. There were found a significantly higher number of ex-smokers in the control group (p = 0.005). There were more oral contraceptives (OC) users in the patient group. MICA*008:01/04 allele showed a significant difference between patient and control groups (p = 0.03; OR = 0.63, 95% CI 0.41–0.96), as well as MICA*018:01(p = 0.004, OR = 0.15, 95% CI 0.03–0.64) and MICA*002:01/020 (p = 0.01; OR = 0.60, 95% CI 0.40–0.88). We also analyzed cases and controls according to the MICA-129 genotypes (Met/Val). There was found a difference (p = 0.02) with the Met/Val genotype in a higher frequency in controls and Val/Val and Val/MICA del at a higher frequency in the patient group. For the KLRK1 gene there was no significant difference between groups.

Construction of a risk score prognosis model based on hepatocellular carcinoma microenvironment.

BACKGROUNDHepatocellular carcinoma (HCC) is a common cancer with a poor prognosis. Previous studies revealed that the tumor microenvironment (TME) plays an important role in HCC progression, recurrence, and metastasis, leading to poor prognosis. However, the effects of genes involved in TME on the prognosis of HCC patients remain unclear. Here, we investigated the HCC microenvironment to identify prognostic genes for HCC.AIMTo identify a robust gene signature associated with the HCC microenvironment to improve prognosis prediction of HCC.METHODSWe computed the immune/stromal scores of HCC patients obtained from The Cancer Genome Atlas based on the ESTIMATE algorithm. Additionally, a risk score model was established based on Differentially Expressed Genes (DEGs) between high‐ and low‐immune/stromal score patients.RESULTSThe risk score model consisting of eight genes was constructed and validated in the HCC patients. The patients were divided into high- or low-risk groups. The genes (Disabled homolog 2, Musculin, C-X-C motif chemokine ligand 8, Galectin 3, B-cell-activating transcription factor, Killer cell lectin like receptor B1, Endoglin and adenomatosis polyposis coli tumor suppressor) involved in our risk score model were considered to be potential immunotherapy targets, and they may provide better performance in combination. Functional enrichment analysis showed that the immune response and T cell receptor signaling pathway represented the major function and pathway, respectively, related to the immune-related genes in the DEGs between high- and low-risk groups. The receiver operating characteristic (ROC) curve analysis confirmed the good potency of the risk score prognostic model. Moreover, we validated the risk score model using the International Cancer Genome Consortium and the Gene Expression Omnibus database. A nomogram was established to predict the overall survival of HCC patients.CONCLUSIONThe risk score model and the nomogram will benefit HCC patients through personalized immunotherapy.

O,O'-diethyl-(S,S)-ethylenediamine-N,N'-di-2-(3-cyclohexyl)propanoate dihydrochloride enhances influx of effective NK and NKT cells in murine breast cancer

Background/Aim. O,O'-diethyl-(S,S)-ethylenediamine- N,N'-di-2-(3-cyclohexyl)propanoate dihydrochloride (DEEDCP) has been found to possess promising cytotoxic activity against various tumor cell lines. Also, DE-EDCP reduces tumor progression by several mechanisms such as triggering tumor cell death and inhibition of cell proliferation. The aim of present study was to further evaluate antitumor activity of DE-EDCP by investigating effects on migratory potential of tumor cells and anti-tumor immune response. Methods. Migratory potential of DE-EDCP was evaluated by scratch wound assay. Female BALB/c mice were inoculated with 4T1 breast cancer cells and treatment with DE-EDCP started five days following orthotopic tumor implantation. The frequency and phenotype of tumorinfiltrating natural killer (NK) and natural killer T (NKT) cells were analyzed by flow cytometry. Results. DE-EDCP inhibited migratory potential of highly metastatic 4T1 cells. DE-EDCP facilitated accumulation of CD3+CD49+ NKT cells and CD3-CD49+ NK cells in tumor microenvironment. DE-EDCP treatment led to significant decrement of tumor infiltrating anergic NKT cells expressing cytotoxic Tlymphocyte- associated protein 4 (CTLA-4), killer cell lectin like receptor G1 (KLRG-1) and programmed cell death protein- 1 (PD-1). Mice given DE-EDCP had significantly increased percentages of tumoricidal fas ligand (FasL) positive NK cells. Conclusion. DE-EDCP inhibits murine breast cancer progression through direct effects on tumor cells and by facilitating anti-tumor immunity. DE-EDCP enhances accumulation, promotes tumoricidal phenotype and maintenances responsiveness of NK and NKT cells in 4T1 murine breast cancer.

ILC2 transfers to apolipoprotein E deficient mice reduce the lipid content of atherosclerotic lesions

BackgroundExpansion of type 2 innate lymphoid cells (ILC2s) in hypercholesterolaemic mice protects against atherosclerosis while different ILC2 subsets have been described (natural, inflammatory) based on their suppression of tumorigenicity 2 (ST2) and killer-cell lectin like receptor G1 (KLRG1) expression. The aim of the current study is to characterize the interleukin 25 (IL25)-induced splenic ILC2 population (Lin−CD45+IL17RB+ICOS+IL7raintermediate) and address its direct role in experimental atherosclerosis by its adoptive transfer to hypercholesterolaemic apolipoprotein E deficient (apoE−/−) mice.ResultsImmunomagnetically enriched, FACS-sorted ILC2s from the spleens of IL-25 treated apoE−/− mice were stained for KLRG1 and ST2 directly upon cell obtainment or in vitro cell expansion for flow cytometric analysis. IL25-induced splenic ILC2s express high levels of both KLRG1 and ST2. However, both markers are downregulated upon in vitro cell expansion. In vitro expanded splenic ILC2s were intraperitoneally transferred to apoE−/− recipients on high fat diet. ApoE−/− mice that received in vitro expanded splenic ILC2s had decreased lipid content in subvalvular heart and brachiocephalic artery (BCA) plaques accompanied by increased peritoneal B1 cells, activated eosinophils and alternatively activated macrophages (AAMs) as well as anti-phosphorylcholine (PC) immunoglobulin (Ig) M in plasma.ConclusionsWith the current data we designate the IL25-induced ILC2 population to decrease the lipid content of atherosclerotic lesions in apoE−/− mice and we directly link the induction of B1 cells and the atheroprotective anti-PC IgM antibodies with ILC2s.

A Comprehensive Analysis of Key Immune Checkpoint Receptors on Tumor-Infiltrating T Cells From Multiple Types of Cancer.

Background: Cancer patients often display dysfunctional antitumor T-cell responses. Because noteworthy benefits of immune checkpoint pathway blockade, such as programmed cell death protein 1 (PD-1) inhibitors, have been achieved in multiple advanced cancers, the next critical question is which mono-blockade or combinatorial blockade regimens may reinvigorate antitumor T-cell immunity in those cancer patients while limiting immune-related adverse effects.Method: This study recruited, in total, 172 primary cancer patients (131 were blood-tumor-matched patients) who were treatment-naïve prior to the surgeries or biopsies covering the eight most prevalent types of cancer. With access to fresh surgical samples, this study simultaneously investigated the ex vivo expression level of eight known immune checkpoint receptors [PD-1, cytotoxic T-lymphocyte antigen-4 [CTLA-4], T-cell immunoglobulin and mucin-domain containing-3 [Tim-3], 2B4, killer cell lectin like receptor G1 [KLRG-1], TIGIT, B- and T-lymphocyte attenuator [BTLA], and CD160] on tumor-infiltrating T cells (TILs) and paired circulating T cells in blood from a 131-patient cohort.Results: We found increased an expression of PD-1 and Tim-3 but a decreased expression of BTLA on TILs when compared with peripheral blood from multiple types of cancer. Moreover, our co-expression analysis of key immune checkpoint receptors delineates “shared” subsets as PD-1+Tim-3+TIGIT+2B4+KLRG-1–CTLA-4– and PD-1+TIGIT+2B4+Tim-3–KLRG-1–CTLA-4– from bulk CD8 TILs. Furthermore, we found that a higher frequency of advanced differentiation stage T cells (CD27–CCR7–CD45RA–) among the “shared” subset (PD-1+Tim-3+TIGIT+2B4+KLRG-1–CTLA-4–) in bulk CD8 TILs was associated with poorly differentiated cancer type in cervical cancer patients.Conclusions: To our knowledge, our study is the first comprehensive analysis of key immune checkpoint receptors on T cells in treatment-naïve, primary cancer patients from the eight most prevalent types of cancer. These findings might provide useful information for future design of mono-blockade/combinatorial blockades and/or genetically modified T-cell immunotherapy.

Identification of potential transcriptomic markers in developing pediatric sepsis: a weighted gene co-expression network analysis and a case\u2013control validation study

BackgroundSepsis represents a complex disease with the dysregulated inflammatory response and high mortality rate. The goal of this study was to identify potential transcriptomic markers in developing pediatric sepsis by a co-expression module analysis of the transcriptomic dataset.MethodsUsing the R software and Bioconductor packages, we performed a weighted gene co-expression network analysis to identify co-expression modules significantly associated with pediatric sepsis. Functional interpretation (gene ontology and pathway analysis) and enrichment analysis with known transcription factors and microRNAs of the identified candidate modules were then performed. In modules significantly associated with sepsis, the intramodular analysis was further performed and “hub genes” were identified and validated by quantitative real-time PCR (qPCR) in this study.Results15 co-expression modules in total were detected, and four modules (“midnight blue”, “cyan”, “brown”, and “tan”) were most significantly associated with pediatric sepsis and suggested as potential sepsis-associated modules. Gene ontology analysis and pathway analysis revealed that these four modules strongly associated with immune response. Three of the four sepsis-associated modules were also enriched with known transcription factors (false discovery rate-adjusted P < 0.05). Hub genes were identified in each of the four modules. Four of the identified hub genes (MYB proto-oncogene like 1, killer cell lectin like receptor G1, stomatin, and membrane spanning 4-domains A4A) were further validated to be differentially expressed between septic children and controls by qPCR.ConclusionsFour pediatric sepsis-associated co-expression modules were identified in this study. qPCR results suggest that hub genes in these modules are potential transcriptomic markers for pediatric sepsis diagnosis. These results provide novel insights into the pathogenesis of pediatric sepsis and promote the generation of diagnostic gene sets.