Higher CXCL10 Research Articles

Selective inhibition of HDAC6 promotes bladder cancer radiosensitization and mitigates the radiation-induced CXCL1 signalling

BackgroundAlthough trimodality therapy resecting tumours followed by chemoradiotherapy is emerged for muscle-invasive bladder cancer (MIBC), chemotherapy produces toxicities. Histone deacetylase inhibitors have been identified as an effective strategy to enhance cancer radiotherapy (RT).MethodsWe examined the role of HDAC6 and specific inhibition of HDAC6 on BC radiosensitivity by performing transcriptomic analysis and mechanism study.ResultsHDAC6 knockdown or HDAC6 inhibitor (HDAC6i) tubacin exerted a radiosensitizing effect, including decreased clonogenic survival, increased H3K9ac and α-tubulin acetylation, and accumulated γH2AX, which are similar to the effect of panobinostat, a pan-HDACi, on irradiated BC cells. Transcriptomics of shHDAC6-transduced T24 under irradiation showed that shHDAC6 counteracted RT-induced mRNA expression of CXCL1, SERPINE1, SDC1 and SDC2, which are linked to cell migration, angiogenesis and metastasis. Moreover, tubacin significantly suppressed RT-induced CXCL1 and radiation-enhanced invasion/migration, whereas panobinostat elevated RT-induced CXCL1 expression and invasion/migration abilities. This phenotype was significantly abrogated by anti-CXCL1 antibody, indicating the key regulator of CXCL1 contributing to BC malignancy. Immunohistochemical evaluation of tumours from urothelial carcinoma patients supported the correlation between high CXCL1 expression and reduced survival.ConclusionUnlike pan-HDACi, the selective HDAC6i can enhance BC radiosensitization and effectively inhibit RT-induced oncogenic CXCL1-Snail-signalling, thus further advancing its therapeutic potential with RT.

Interferon and interferon-induced cytokines as markers of impending clinical progression in ANA+ individuals without a systemic autoimmune rheumatic disease diagnosis

BackgroundElevated levels of interferons (IFNs) are a characteristic feature of systemic autoimmune rheumatic diseases (SARDs) and may be useful in predicting impending symptomatic progression in anti-nuclear antibody-positive (ANA+) individuals lacking a SARD diagnosis. Typically, these are measured by their effect on gene expression in the blood, which has limited their utility in clinical settings. Here, we assessed whether the measurement of serum IFN-α or selected IFN-induced cytokines accurately mirrors IFN-induced gene expression in ANA+ individuals and investigated their utility as biomarkers of clinical progression.MethodsA total of 280 subjects were studied, including 50 ANA− healthy controls, 160 ANA+ individuals without a SARD diagnosis (96 asymptomatic, 64 with undifferentiated connective tissue disease), and 70 SARD patients. IFN-induced gene expression was measured by nanoString and cytokine levels by ELISA or Simoa. ANA+ individuals lacking a SARD diagnosis who had the new onset of SARD criteria over the subsequent 2 years were defined as progressors.ResultsMeasurement of IFN-α levels by high-sensitivity ELISA or Simoa correlated much better with IFN-induced gene expression than measurement of CXCL-10 or Galectin-9 levels. Despite this, high CXCL-10 and Galectin-9 levels were better predictors of subsequent progression in ANA+ individuals than measures of IFN-α or IFN-induced gene expression with the optimal combination of predictive cytokines (CXCL-10 and IFN-α as measured by ELISA), resulting in a specificity and positive predictive value of 100%.ConclusionEasily performed ELISA assays for CXCL-10 and IFN-α can be used to predict ANA+ individuals at high risk of imminent symptomatic progression.

Prognostic Value of CXCL12 in Non-Small Cell Lung Cancer Patients Undergoing Tumor Resection

Adjuvant chemotherapy is commonly indicated in lung cancer patients undergoing surgical therapy because tumor recurrence is frequent. A biomarker that can predict tumor recurrence in the postoperative period is currently unavailable. CXCR4 receptor and its ligand CXCL12 play important roles in metastasis. This study investigated the value of tumor CXCL12 expression to predict prognosis and indicate adjuvant chemotherapy in non-small cell lung cancer patients. This study enrolled 82 non-small cell lung cancer patients. The expression of CXCL12 was evaluated by immunohistochemistry. The degree of CXCL12 expression was assessed using the Allred score system. Among all subjects, the progression-free survival and overall survival were significantly prolonged in cancer patients with low tumor expression of CXCL12 compared to patients with high tumor expression. Multivariate analysis showed that the increased level of CXCL12 is a significant predictor of progression-free survival and overall survival in NSCLC patients. Among subjects with high tumor CXCL12 expression, progression-free survival and overall survival were significantly improved in patients treated with adjuvant chemotherapy compared to untreated patients. These results suggest the potential value of tumor CXCL12 expression as a marker to predict prognosis and to indicate adjuvant chemotherapy after surgical tumor resection in non-small cell lung cancer patients.

Loss of bone morphogenetic protein signaling in fibroblasts results in CXCL12-driven serrated polyp development

Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling–CXCL12 interaction could have a role in the etiology of serrated polyp formation.

PD-L1 Expression in High-Risk Early-Stage Colorectal Cancer-Its Clinical and Biological Significance in Immune Microenvironment.

Programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule that can regulate immune responses in the tumor microenvironment (TME); however, the clinical applications of PD-L1 in early-stage colorectal cancer (CRC) remain unclear. In this study, we aimed to investigate the relationship between PD-L1 expression and survival outcome and explore its relevant immune responses in CRC. PD-L1 expression was evaluated by immunohistochemical staining to determine the tumor proportion score and combined positive score (CPS) in a Taiwanese CRC cohort. The oncomine immune response research assay was conducted for immune gene expression analyses. CRC datasets from the TCGA database were reappraised for PD-L1-associated gene enrichment analyses using GSEA. The high expression of PD-L1 (CPS ≥ 5) was associated with longer recurrence-free survival (p = 0.031) and was an independent prognostic factor as revealed by multivariate analysis. High PD-L1 expression was related to six immune-related gene signatures, and CXCL9 is the most significant overexpressed gene in differential analyses. High CXCL9 expression correlated with increased infiltration levels of immune cells in the TME, including CD8+ T lymphocytes and M1 macrophages. These findings suggest that high PD-L1 expression is a prognostic factor of early-stage CRC, and CXCL9 may play a key role in regulating PD-L1 expression.

CXCR4-CXCL12-CXCR7 and PD-1/PD-L1 in Pancreatic Cancer: CXCL12 Predicts Survival of Radically Resected Patients.

Pancreatic ductal adenocarcinoma (PDAC) is currently the most deadly cancer. Although characterized by 5-20% of neoplastic cells in the highly fibrotic stroma, immunotherapy is not a valid option in PDAC treatment. As CXCR4-CXCL12 regulates tumor invasion and T-cell access and PD-1/PD-L1 controls immune tolerance, 76 PDACs were evaluated for CXCR4-CXCL12-CXCR7 and PD-1/PD-L1 in the epithelial and stromal component. Neoplastic CXCR4 and CXCL12 discriminated PDACs for recurrence-free survival (RFS), while CXCL12 and CXCR7 discriminated patients for cancer-specific survival (CSS). Interestingly, among patients with radical resection (R0), high tumor CXCR4 clustered patients with worse RFS, high CXCL12 identified poor prognostic patients for both RFS and CSS, while stromal lymphocytic-monocytic PD-L1 associated with improved RFS and CSS. PD-1 was only sporadically expressed (<1%) in focal lymphocyte infiltrate and does not impact prognosis. In multivariate analysis, tumoral CXCL12, perineural invasion, and AJCC lymph node status were independent prognostic factors for RFS; tumoral CXCL12, AJCC Stage, and vascular invasion were independent prognostic factors for CSS. CXCL12's poor prognostic meaning was confirmed in an additional perspective-independent 13 fine-needle aspiration cytology advanced stage-PDACs. Thus, CXCR4-CXCL12 evaluation in PDAC identifies prognostic categories and could orient therapeutic approaches.

Could Plasma CXCL12 Predict Ventricular Dysfunction in Patients with Severe Myocardial Infarction?

Plasma level of chemokine CXCL12 can predict adverse cardiovascular outcomes in patients with coronary artery disease, but data on its relationship with severity of coronary stenosis in cases of severe myocardial infarction (MI) are scarce and conflicting. The objective of this study was to investigate link between plasma CXCL12 levels and different grades of left ventricular ejection fraction (LVEF) in statin-treated and -untreated patients with severe MI. A total of 198 consecutive patients with first-time severe MI (ST-elevated myocardial infarction [STEMI], n = 121 and non-ST-elevated myocardial infarction [NSTEMI], n = 77) were recruited from Coronary Care Unit, King Abdulaziz University Hospital. They have one to two coronary arteries blocked ≥50%, or three arteries blocked 30 to 49%. Demographic and clinical criteria were collected and plasma CXCL12 level was measured. No correlations were detected between demographic and clinical criteria and CXCL12 level. While troponin peaks and LVEF significantly differed between STEMI and NSTEMI patients, CXCL12 level showed nonsignificant changes. Plasma CXCL12 levels decreased significantly in statin-treated patients compared with those untreated. From receiver operating characteristic (ROC) analysis, high CXCL12 levels were associated with no statin therapy. For STEMI and NSTEMI patients, area under the receiver operating characteristic curve for CXCL12 test were 0.685 and 0.820, while sensitivity and specificity values were 75.9 and 54.8%, and 73.1 and 84%, respectively. Plasma CXCL12 levels showed nonsignificant changes with different ranges of LVEF and troponin peaks. In patients with severe MI, irrespective of statin therapy, plasma CXCL12 showed no correlation with different ranges of LVEF suggesting that it cannot predict left ventricular dysfunction in these cases. However, cross-sectional design of this study is a limitation.

Knee osteoarthritis phenotypes based on synovial fluid immune cells correlate with clinical outcome trajectories

Knee osteoarthritis (KOA) is a highly heterogeneous disease encompassing a wide range of clinical phenotypes. Phenotypes based on immune cells and protein pattern in synovial fluid (SF) and their relationship to clinical trajectories have not been described. To assess phenotypes based on immune cells and protein pattern of SF in KOA. SF-derived immune cells were investigated in 119 patients with KOA using flow cytometry. Immune-phenotypes (iPhen) were determined by multivariate patient similarity network analysis and related to clinical trajectory (3-6 months post-sampling) along with protein pattern and macrophage chemokine receptors. Four iPhen were detected based on the distribution of T-lymphocytes, monocyte-macrophage lineage cells and activated CD8+ T-lymphocytes. The 'activated' phenotype (n=17) had high T-lymphocytes but low monocyte-macrophage lineage cells and neutrophils, all highly activated, and showed improved symptoms in 70% patients. The 'lymphoid progressive' phenotype (n=31) had high neutrophils, low lymphocytes and monocyte-macrophage lineage cells, low activation and was associated with lower pain levels. The 'myeloid progressive' phenotype (n=35) had high NK and monocyte-macrophage lineage cells but low T-lymphocytes and activation. The 'aggressive' phenotype (n=36) had high lymphocytes, macrophages, NK cells and neutrophils and high activation, and only 39% of patients improved during follow-up. Low CXCR4 and CCR7 expression on macrophages and high CXCL10 in SF were linked to improved clinical trajectory. We identified four immune-phenotypes that were associated with different clinical trajectories in KOA patients. How these phenotypes can be targeted therapeutically deserves further investigation.

1661MO Genomic somatic copy number alterations drive adaptive tumor immune suppression and primary resistance to anti-PD1 + anti-angiogenics in pleural mesothelioma

In patients with advanced pleural malignant mesothelioma (MM), Pembrolizumab [P] in combination with Nintedanib [N] synergize with a response rate of 24% (PEMBIB trial initial results reported at ESMO Congress 2021), superior to [P] alone (8% in MM in the KN158 trial). Here we report new data from the biomarker analysis performed in this trial (NCT02856425). 30 MM patients were treated with [N] (150mg bid) & [P] (200mg IV Q3W). Fresh tumor biopsies were collected in media and immediately processed at baseline and at C2D1 for measuring proteins released in the biopsy supernatant and immune cell infiltrates (by Flow Cytometry). Frozen and fixed tumor biopsies were also analyzed by WES, FISH, RNAseq and IHC. Circulating cytokines & immune cells were measured on plasma and fresh whole blood at baseline and on-treatment. At 6 months, patients remaining in stable disease or objective tumor response per RECIST1.1 were categorized as having Durable Clinical Benefit (DCB). All differences reported here are statistically significant with a p value at least <0,05. Higher PD-L1 on tumor cells, higher tumor CD45+, CD3+ & CD8+ T-cells characterized the DCB group at baseline. Unexpectedly, active recruitment of CD8+ T-cells, with higher gene expression of EOMES, T-BET, IFNg, & GzmB, occurred in No DCB tumors upon treatment. At C2D1, same levels of tumor infiltrating CD8+ T-cells were found in DCB & No DCB tumors. This tumor T-cell recruitment occurred in a context of high blood and tumor CXCL9 increases upon [N]+[P] treatment. However, No DCB patients presented more IL6, VEGF, IL8, in their blood & tumor secretome and they increased their tumor content in CD4+ CD25+ Tregs compared to DCB patients upon treatment. Tumor somatic copy number alterations (SCNA) were higher in tumors with no DCB and correlated with higher plasmatic IL-6 rate levels in MM patients. MM primary resistance to anti-PD1+antiangiogenics is not due to a lack of CD8+ T-cell tumor response but to an adaptive secretion of IL6 by tumors, driven by genomic SCNA, leading to VEGF and IL8 secretion, and recruitment of tumor infiltrating Tregs.

CXCL8 expression is associated with advanced stage, right sidedness, and distinct histological features of colorectal cancer.

CXCL8 is an inflammatory chemokine elevated in the colorectal cancer (CRC) tumour microenvironment. CXCR2, the major receptor for CXCL8, is predominantly expressed by neutrophils. In the cancer setting, CXCL8 plays important roles in neutrophil chemotaxis, facilitating angiogenesis, invasion, and metastasis. This study aimed to assess the spatial distribution of CXCL8 mRNA expression in CRC specimens, explore associations with clinical characteristics, and investigate the underlying biology of aberrant CXCL8 levels. CXCR2 expression was also assessed in a second cohort of unique CRC primary tumours and synchronously resected matched liver metastases. A previously constructed tissue microarray consisting of a cohort of stage I–IV CRC patients undergoing surgical resection with curative intent (n = 438) was probed for CXCL8 via RNAscope®. Analysis was performed using HALO® digital pathology software to quantify expression in the tumour and stromal compartments. Scores were assessed for association with clinical characteristics. Mutational analyses were performed on a subset of these patients to determine genomic differences in patients with high CXCL8 expression. A second cohort of stage IV CRC patients with primary and matched metastatic liver tumours was stained via immunohistochemistry for CXCR2, and scores were assessed for clinical significance. CXCL8 expression within the stromal compartment was associated with reduced cancer‐specific survival in the first cohort (p = 0.035), and this relationship was potentiated in right‐sided colon cancer cases (p = 0.009). High CXCL8 within the stroma was associated with driving a more stromal‐rich phenotype and the presence of metastases. When stromal CXCL8 scores were combined with tumour‐infiltrating macrophage counts or systemic neutrophil counts, patients classified as high for both markers had significantly poorer prognosis. CXCR2+ immune cell infiltration was associated with increased stromal invasion in liver metastases (p = 0.037). These data indicate a role for CXCL8 in driving unfavourable tumour histological features and promoting metastases. This study suggests that inhibiting CXCL8/CXCR2 should be investigated in patients with right‐sided colonic disease and stroma‐rich tumours.

Circulating CXCL13 could be serving as a biomarker for chronic lymphocytic leukemia severity.

Recent reports indicated the importance of chemotractant CXCL-13 in solid tumors and lymphoid malignancies. However, the prognostic value of the mentioned cytokines as biomarkers in chronic lymphocytic leukemia patient's remains to be identified. Therefore; this study was designed in order to address the relation between CXCL-13 concentrations levels and markers of severity in CLL patients. Our study included 150 CLL patients and 20 controls. Serum CXCL-13 was determined by ELISA for CLL patients at diagnosis as well as controls. The serum CXCL-13 levels were significantly higher in CLL patients as compared to controls. The high CXCL-13 concentration levels was significantly associated with high number of smudge cells; high LDH; high grade of Rai stage, short time to first treatment (TTT). Cox regression analysis was conducted for prediction of TTT, using age, gender, WBCs, smudge cells, CXCL-13, LDH, ZAP70, CD38, β2-microglobulin, Rai staging as covariates. High LDH, CXCL-13 and CD38% were significantly independent predictor for shorter TTT. High CXCL-13 serum levels at CLL diagnosis is correlated with other markers of disease activity; and could be served as biomarkers that predict CLL patient's outcome.

Biological Role of Tumor/Stromal CXCR4-CXCL12-CXCR7 in MITO16A/MaNGO-OV2 Advanced Ovarian Cancer Patients.

Simple SummaryDespite rapid progress in the research on epithelial ovarian cancer (EOC), it is usually diagnosed during the advanced stage with only 30% of patients surviving longer than 5 years. This is the first study in which the whole CXCR4-CXCL12-CXCR7 axis was systematically evaluated in tumor and stromal cells, through rigorous statistical methods in a prospective clinical trial. CXCL12 expression in cancer cells is associated with worse progression-free survival in stage III EOC patients, and deserves further attention as a potential prognostic and therapeutic target.This study investigated the prognostic role of the CXCR4-CXCL12-CXCR7 axis in advanced epithelial ovarian cancer (EOC) patients receiving first-line treatment within the MITO16A/MaNGO-OV2 phase-IV trial. CXCR4-CXCL12-CXCR7 expression was evaluated in the epithelial and stromal component of 308 EOC IHC-stained tumor samples. The statistical analysis focused on biomarkers’ expression, their association with other variables and prognostic value. Zero-inflated tests, shrinkage, bootstrap procedures, and multivariable models were applied. The majority of EOC (75.0%) expressed CXCR4 and CXCR7, 56.5% expressed the entire CXCR4-CXCL12-CXCR7 axis, while only 4.6% were negative for CXCL12 and its cognate receptors, in regard to the epithelial component. Stromal CXCL12 and CXCR7, expressed in 11.2% and 65.5%, respectively, were associated with the FIGO stage. High CXCL12 in epithelial cancer cells was associated with shorter progression-free and overall survival. However, after adjusting for overfitting due to best cut-off multiplicity testing, the significance was lost. This is a wide-ranging, prospective study in which CXCR4-CXCL12-CXCR7 were systematically evaluated in epithelial and stromal components, in selected stage III-IV EOC. Although CXCL12 was not prognostic, epithelial expression identified high-risk FIGO stage III patients for PFS. These data suggest that it might be worth studying the CXCL12 axis as a therapeutic target to improve treatment efficacy in EOC patients.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

RETRACTED ARTICLE: Pharmacological perturbation of CXCL1 signaling alleviates neuropathogenesis in a model of HEVA71 infection

Hand, foot and mouth disease (HFMD) caused by Human Enterovirus A71 (HEVA71) infection is typically a benign infection. However, in minority of cases, children can develop severe neuropathology that culminate in fatality. Approximately 36.9% of HEVA71-related hospitalizations develop neurological complications, of which 10.5% are fatal. Yet, the mechanism by which HEVA71 induces these neurological deficits remain unclear. Here, we show that HEVA71-infected astrocytes release CXCL1 which supports viral replication in neurons by activating the CXCR2 receptor-associated ERK1/2 signaling pathway. Elevated CXCL1 levels correlates with disease severity in a HEVA71-infected mice model. In humans infected with HEVA71, high CXCL1 levels are only present in patients presenting neurological complications. CXCL1 release is specifically triggered by VP4 synthesis in HEVA71-infected astrocytes, which then acts via its receptor CXCR2 to enhance viral replication in neurons. Perturbing CXCL1 signaling or VP4 myristylation strongly attenuates viral replication. Treatment with AZD5069, a CXCL1-specific competitor, improves survival and lessens disease severity in infected animals. Collectively, these results highlight the CXCL1-CXCR2 signaling pathway as a potential target against HFMD neuropathogenesis.

Identification of CXCL5 expression as a predictive biomarker associated with response and prognosis of immunotherapy in patients with non-small cell lung cancer.

BackgroundThe breakthrough of immunotherapy has revolutionized the treatment of non‐small cell lung cancer (NSCLC). However, only a limited part of patients could derive clinical benefits. To study how immune microenvironment (IME) of patients could influence the therapeutic efficacy of immunotherapy, we evaluated the response patterns of NSCLC patients treated with PD‐1 inhibitors and analyzed the molecules related to prognosis and efficacy of immunotherapy.MethodsTumor samples were collected from 47 NSCLC patients treated with PD‐1 inhibitors. RNA expressions of tumor immune‐related 289 genes were analyzed using NanoString nCounter. Immune infiltration and correlation between clinical information and expression of immune‐related genes were assessed.ResultsUnsupervised clustering analysis revealed two groups infiltrated with different immune cells and differentially expressed genes (DEGs) including CXCL5, CXCL9, IDO1, and LAG3 were found between groups. Stratification based on DEGs indicated that the group with high expression of CXCL5 was characterized by neutrophils. Univariate and multivariate Cox analysis further demonstrated that CXCL5 mRNA expression was positively associated with worse progression free survival (PFS). Logistic analyses indicated high CXCL5 was associated with worse response to immunotherapy.ConclusionsCXCL5 may be a potential biomarker for prognosis and responsiveness to immunotherapy and may be a novel preventive and therapeutic target for NSCLC.

Tertiary Lymphoid Structures and Chemokine Landscape in Virus-Positive and Virus-Negative Merkel Cell Carcinoma.

Tertiary lymphoid structures (TLSs) are used as biomarkers in many cancers for predicting the prognosis and assessing the response to immunotherapy. In Merkel cell carcinoma (MCC), TLSs have only been examined in MCPyV-positive cases. Here, we examined the prognostic value of the presence or absence of TLSs in 61 patients with MCC, including MCPyV-positive and MCPyV-negative cases. TLS-positive samples had a significantly better prognosis than TLS-negative samples. MCPyV-positive samples had a good prognosis with or without TLSs, and MCPyV-negative/TLS-positive samples had a similarly good prognosis as MCPyV-positive samples. Only MCPyV-negative/TLS-negative samples had a significantly poor prognosis. All cases with spontaneous regression were MCPyV-positive/TLS-positive. We also performed a comprehensive analysis of the chemokines associated with TLS formation using next-generation sequencing (NGS). The RNA sequencing results revealed 5 chemokine genes, CCL5, CCR2, CCR7, CXCL9, and CXCL13, with significantly high expression in TLS-positive samples compared with TLS-negative samples in both MCPyV-positive and MCPyV-negative samples. Only 2 chemokine genes, CXCL10 and CX3CR1, had significantly different expression levels in the presence or absence of MCPyV infection in TLS-negative samples. Patients with high CXCL13 or CCL5 expression have a significantly better prognosis than those with low expression. In conclusion, the presence of TLSs can be a potential prognostic marker even in cohorts that include MCPyV-negative cases. Chemokine profiles may help us understand the tumor microenvironment in patients with MCPyV-positive or MCPyV-negative MCC and may be a useful prognostic marker in their own right.

The allograft injury marker CXCL9 determines prognosis of anti-HLA antibodies after lung transplantation

Despite the common detection of non-donor specific anti-HLA antibodies (non-DSAs) after lung transplantation, their clinical significance remains unclear. In this retrospective single-center cohort study of 325lung transplant recipients, we evaluated the association between donor-specific HLA antibodies (DSAs) and non-DSAs with subsequent CLAD development. DSAs were detected in 30% of recipients and were associated with increased CLAD risk, with higher HRs for both de novo and high MFI (>5000) DSAs. Non-DSAs were detected in 56% of recipients, and 85% of DSA positive tests had concurrent non-DSAs. In general, non-DSAs did not increase CLAD risk in multivariable models accounting for DSAs. However, non-DSAs in conjunction with high BAL CXCL9levels were associated with increased CLAD risk. Multivariable proportional hazards models demonstrate the importance of the HLA antibody-CXCL9 interaction: CLAD risk increases when HLA antibodies (both DSAs and non-DSAs) are detected in conjunction with high CXCL9. Conversely, CLAD risk is not increased when HLA antibodies are detected with low CXCL9. This study supports the potential utility of BAL CXCL9measurement as a biomarker to risk stratify HLA antibodies for future CLAD. The ability to discriminate between high versus low-risk HLA antibodies may improve management by allowing for guided treatment decisions.

Clinical significance and role of CXCL16 in anti-neutrophil cytoplasmic autoantibody-associated vasculitis

Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a group of potentially life-threatening autoimmune diseases. The kidney and lung are the most common and most severely affected organs. Previous studies have shown that the chemokine ligand CXCL16 and its receptor CXCR6 play an important role in kidney disease. However, whether CXCL16/CXCR6 is involved in the pathogenesis of AAV remains elusive. In this study, the levels of CXCL16 and its specific receptor CXCR6 were investigated. According to kidney outcome, patients were divided into two groups, specifically one with high CXCL16 levels and one with low CXCL16 levels, by cut-off values using receiver operating characteristic (ROC) curves. The clinical parameters and histological features were further compared between the two groups. The ability of CXCL16 to induce neutrophil chemotaxis was analysed using a Transwell migration assay in a coculture system of conditional immortalized human glomerular endothelial cells (ciGEnCs) and neutrophils. We observed that the levels of CXCL16 were significantly increased in the circulation, along with the expression in renal tissue of AAV patients compared to healthy controls (HCs). CXCR6 expression on neutrophils was significantly higher in patients with AAV than in HCs. There were positive correlations between the levels of CXCL16 and serum creatinine, IL-6, CRP, and TNF-α and negative correlations with eGFR. The serum levels of CXCL16 could act as a predictive biomarker of renal outcome in AAV. CXCL16 secretion was upregulated in ciGEnCs treated with AAV serum. CXCL16 released from ciGEnCs contributed to neutrophil migration. Furthermore, neutrophil migration was attenuated by silencing CXCL16 expression via transfection with short hairpin RNA (shRNA) sequences and lentivirus. Taken together, these data suggest that the inhibition of the CXCL16/CXCR6 axis may provide new therapeutic strategies targeting AAV.

Abstract P042: Chemokine dysregulation creates the immunosuppressive tumor microenvironment and promotes human papillomavirus-associated head and neck cancer

Abstract Human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC) is a fast-growing health problem in the United States. By analyzing the transcriptomes of HPV-positive (HPV+) and HPV-negative (HPV-) HNSCC patient tissues, we previously revealed that expression of CXCL14, constitutively expressed in basal epithelial cells, is downregulated in HPV-positive HNSCC. In contrast, the expression of proinflammatory chemokines, particularly CXCL1, CXCL2, and CXCL8, are significantly upregulated in both HPV+ and HPV- HNSCCs. We further showed that restored expression of CXCL14 in HPV+ HNSCC cells leads to tumor-free survival in vivo by increasing MHC-I antigen presentation on the cancer cell surface and CD8+ T cell activation. Interestingly, high CXCL1, CXCL2, and CXCL8 expression levels significantly correlate to poor HNSCC patient survival, showing an exact opposite pattern of the clinical outcomes associated with the CXCL14 expression levels. To determine the roles of these chemokines in HPV+ HNSCC development, CXCL1 and CXCL2 genes (no murine CXCL8 ortholog) were deleted using a CRISPR-Cas9 knockout system in HPV+ murine HNSCC cells. Using these stably engineered HNSCC cells, we investigated tumor growth, immune cell infiltration, and metastasis in immunocompetent syngeneic mice. Interestingly, the deletion of CXCL1 and CXCL2 significantly delays in vivo tumor growth, decreases myeloid-derived suppressor cell (MDSC) infiltration in the tumor, and decreases lung metastasis. However, the numbers of natural killer (NK), CD4+, and CD8+ T cells in the tumor microenvironment were not affected by CXCL1 and CXCL2 knockout. Next, mice injected with the murine HNSCC cells were treated twice daily with AZD5068, a selective inhibitor of CXCR2, which is a common receptor of CXCL1, CXCL2, and CXCL8 expressed on myeloid cells. Our result showed that the treatment of CXCR2 suppresses in vivo tumor growth in a similar pattern with CXCL1 and CXCL2 knockout. These results suggest that the dysregulation of chemokine expression in HPV-infected cells plays an important role in regulating tumor growth, antitumor immune responses, and metastasis of HPV+ HNSCC. Our findings may provide promising strategies to develop novel immunotherapies for HPV+ HNSCC patients which can likely be extended to other non-viral cancers with immunosuppressive phenotypes. Citation Format: Daniel Vermeer, Joseph Westrich, Paul Colbert, Craig Welbon, John H. Lee, David Raben, William C. Spanos, Dohun Pyeon. Chemokine dysregulation creates the immunosuppressive tumor microenvironment and promotes human papillomavirus-associated head and neck cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P042.

Kynurenine pathway activation and deviation to anthranilic and kynurenic acid in fibrosing chronic graft-versus-host disease

SummaryFibrosing chronic graft-versus-host disease (cGVHD) is a debilitating complication of allogeneic stem cell transplantation (alloSCT). A driver of fibrosis is the kynurenine (Kyn) pathway, and Kyn metabolism patterns and cytokines may influence cGVHD severity and manifestation (fibrosing versus gastrointestinal [GI] cGVHD). Using a liquid chromatography-tandem mass spectrometry approach on sera obtained from 425 patients with allografts, we identified high CXCL9, high indoleamine-2,3-dioxygenase (IDO) activity, and an activated Kyn pathway as common characteristics in all cGVHD subtypes. Specific Kyn metabolism patterns could be identified for non-severe cGVHD, severe GI cGVHD, and fibrosing cGVHD, respectively. Specifically, fibrosing cGVHD was associated with a distinct pathway shift toward anthranilic and kynurenic acid, correlating with reduced activity of the vitamin-B2-dependent kynurenine monooxygenase, low vitamin B6, and increased interleukin-18. The Kyn metabolite signature is a candidate biomarker for severe fibrosing cGVHD and provides a rationale for translational trials on prophylactic vitamin B2/B6 supplementation for cGVHD prevention.