Chronic subdural hematoma (CSDH) is a relatively common disorder in neurosurgery on elderly patients, though the mechanism that causes the disease remains unclear. Studies have suggested that local anticoagulation and inflammatory changes may be important in its pathogenesis. Most studies have used a basic bivariate statistical analysis to assess complex immunological responses in patients with this disorder, hence a more sophisticated multivariate statistical approach might be warranted. Our objective was to assess the association and correlation between the pro- and anti-inflammatory responses in a cohort of patients with chronic subdural hematoma (n=57) using an exploratory and confirmatory factor analysis. Thirteen assigned pro-inflammatory (TNF-α, IL-1β, IL-2, IL-2R, IL-6, IL-7, IL-12, IL-15, IL-17, CCL2, CXCL8, CXCL9 and CXCL10) and five assigned anti-inflammatory (IL-1RA, IL-4, IL-5, IL-10 and IL-13) cytokines from blood and hematoma fluid samples were examined. Exploratory factor analysis indicated two major underlying immunological processes expressed by the cytokines in both blood and hematoma fluid, but with a different pattern and particularly regarding the cytokines IL-13, IL-6, IL-4 and TNF-α. Scores from confirmatory factor analysis models exhibited a higher correlation between pro- and anti-inflammatory activities in blood (r=0.98) than in hematoma fluid samples (r=0.92). However, correlations of inflammatory processes between blood and hematoma fluid samples were lower and non-significant. A structural equation model showed a significant association between increased anti-inflammatory activity in hematoma fluid samples and a lower risk of recurrence, but this relationship was not statistically significant in venous blood samples. Moreover, these findings indicate that anti-inflammatory activities in the hematoma may play a role in the risk of a recurrence of CSDH.

Zika virus (ZIKV) infection during pregnancy is associated with microcephaly, a congenital malformation resulting from neuroinflammation and direct effects of virus replication on the developing central nervous system (CNS). However, the exact changes in the affected CNS remain unknown. Here, we show by transcriptome analysis (at 48 h post-infection) and multiplex immune profiling that human induced-neuroprogenitor stem cells (hiNPCs) respond to ZIKV infection with a strong induction of type-I interferons (IFNs) and several type-I IFNs stimulated genes (ISGs), notably cytokines and the pro-apoptotic chemokines CXCL9 and CXCL10. By comparing the inflammatory profile induced by a ZIKV Brazilian strain with an ancestral strain isolated from Cambodia in 2010, we observed that the response magnitude differs among them. Compared to ZIKV/Cambodia, the experimental infection of hiNPCs with ZIKV/Brazil resulted in a diminished induction of ISGs and lower induction of several cytokines (IFN-α, IL-1α/β, IL-6, IL-8, and IL-15), consequently favoring virus replication. From ZIKV-confirmed infant microcephaly cases, we detected a similar profile characterized by the presence of IFN-α, CXCL10, and CXCL9 in cerebrospinal fluid (CSF) samples collected after birth, evidencing a sustained CNS inflammation. Altogether, our data suggest that the CNS may be directly affected due to an unbalanced and chronic local inflammatory response, elicited by ZIKV infection, which contributes to damage to the fetal brain.

Several studies have shown that exposure to bacterial lipopolysaccharide (LPS) can either prevent or inhibit asthma in humans and laboratory rodents. Much emphasis has been placed on the role of cytokines and chemokines in the establishment and maintenance of allergic airway disease. Therefore, it is of interest to study the role of LPS in affecting airway pathology and lung cytokine and chemokine responses in the maintenance phase of asthma. Increasing doses of LPS were administered into the airways of mice presensitized with cockroach allergen (CRAg), then allergic airway disease parameters were assessed after CRAg challenge. Airway hyperresponsiveness after antigen challenge decreased at the highest dose of LPS tested, which was accompanied by a decrease in airway and lung eosinophils. However, a dramatic increase in lung inflammation because of neutrophil influx was observed. Measurement of cytokines in lungs of LPS-treated, CRAg-sensitized mice indicated that interleukin (IL)-12 levels were increased by LPS treatment in a dose-dependent manner, as were levels of several inflammatory chemokines. In contrast, levels of IL-4, IL-13, IL-5, and IL-10 were reduced in whole lung homogenates only of high-dose LPS-treated mice. Intranasal administration of neutralizing anti-IL-12 at the time of high-dose LPS challenge reduced lung IL-12, interferon-gamma, CXCL9, and CXCL10 but did not affect levels of the other chemokines or Th2-type cytokines, and did not restore AHR. These findings suggest that the amelioration of airway hyperresponsiveness observed in LPS-treated, CRAg-sensitized mice is coincident with an immune deviation of the lung inflammatory response, independent of IL-12.

Purpose: Infused autologous tumor-infiltrating lymphocytes (TIL) and tumor-targeted chimeric antigen receptor (CAR) T cells typically surround malignant lesions or penetrate small tumor nodules but fail to penetrate large solid tumors, significantly compromising their antitumor impact. Strategies to overcome this primary challenge are largely required.Experimental Design: We tested the effects of IL12 plus doxorubicin on T-cell penetration and efficacy in solid tumors in a murine lung cancer model, a murine breast carcinoma lung metastasis model, and two human xenograft tumor models bearing large tumors (>10 mm).Results: Intriguingly, this simple approach increased the numbers, the distribution, and the depth of penetration of infused CD8+ T cells in these tumors, including both TILs and CAR T cells. This combined treatment halted tumor progression and significantly extended survival time. Studies of the underlying mechanism revealed multiple effects. First, the combined treatment maintained the high ratios of immune-stimulatory receptors to immune-inhibitory receptors on infiltrated CD8+ T cells, reduced the accumulation of immunosuppressive regulatory T cells, and enhanced the numbers of T-bet+ effector T cells in the tumors. Second, doxorubicin induced chemokines CXCL9 and CXCL10, which may attract NKG2D+CD8+ T cells to tumors, and this effect was boosted by IL12-induced IFNγ accumulation in tumors, promoting the penetration of NKG2D+CD8+ T cells.Conclusions: The deep penetration of infused T cells associated with combined IL12 plus doxorubicin yielded striking therapeutic effects in murine and human xenograft solid tumors. This approach might broaden the application of T-cell therapy to a wider range of solid tumors. Clin Cancer Res; 24(12); 2920-34. ©2018 AACRSee related commentary by Berraondo et al., p. 2716.

BACKGROUND

Although antibodies to aquaporin-4(AQP4) are strongly associated with Neuromyelitis optica (NMO), the sole transfer of these antibodies is not sufficient to induce an NMO-like disease in experimental animals and T-cells and complement are also needed. Initial data indicating the presence of T-cell responses to AQP4 in patients with NMO, have beeen recently reported.

OBJECTIVE

To evaluate the T-cell responses to specific AQP4 peptides/epitopes in patients with NMO and multiple sclerosis (MS).

METHODS

Peripheral blood mononuclear cells (PBMCs) were obtained from 14 patients fulfilling the criteria for definite NMO and the proliferation responses to one of 15 distinct pentadecapeptides of AQP4, spanning the whole protein (except of its transmembrane parts) were tested by a standard [H3]-thymidine uptake assay and compared with those of 9 healthy controls and 7 MS patients. A cytometric bead array assay (CBA) and flow cytometry were used to evaluate cytokine (IFNγ, IL17, IL2, IL4, IL5, IL10 and TNFα) and chemokine (CXCL8, CCL5, CXCL10, CXCL9, CCL2) secretion by PHA-stimulated PBMCs and AQP4-specific T-cell lines.

RESULTS

Four main immunodominant epitopes of the AQP4 protein (p137-151, p222-236, p217-231 and the p269-283) were identified in the NMO group. The first two epitopes (assigned as peptides 3 and 9) showed the highest sensitivity (~60% positivity), whereas the latter two (assigned as peptides 8 and 11), the higher specificity. Longitudinal follow up of 5 patients revealed changes in the epitope-specificities during the course of NMO. T-cell lines specific for the AQP4 peptides, produced from NMO patients (but not healthy donors) secreted mainly IL-17 and IL-10 and less IFNγ.

CONCLUSIONS

Our findings indicate that T-cells bearing characteristics of both Th1 and Th17 T-cells and targeting specific immunodominant epitopes of the AQP4 protein might be involved in the pathogenesis of NMO.

Finegoldia magna is an anaerobic Gram-positive bacterium and commensal, which is also associated with clinically important conditions such as skin and soft tissue infections. This study describes a novel subtilisin-like extracellular serine proteinase of F. magna, denoted SufA (subtilase of Finegoldia magna), which is believed to be the first subtilase described among Gram-positive anaerobic cocci. SufA is associated with the bacterial cell surface, but is also released in substantial amounts during bacterial growth. Papain was used to release SufA from the surface of F. magna and the enzyme was purified by ion-exchange chromatography and gel filtration. A protein band on SDS-PAGE corresponding to the dominating proteolytic activity on gelatin zymography was analysed by MS/MS. Based on the peptide sequences obtained, the sufA gene was sequenced. The gene comprises 3466 bp corresponding to a preprotein of 127 kDa. Like other members of the subtilase family, SufA contains the catalytic triad of aspartic acid, histidine and serine with surrounding conserved residues. A SufA homologue was identified in 33 of 34 investigated isolates of F. magna, as revealed by PCR and immunoprinting. The enzyme forms dimers, which are more proteolytically active than the monomeric protein. SufA was found to efficiently cleave and inactivate the antibacterial peptide LL-37 and the CXC chemokine MIG/CXCL9, indicating that the enzyme promotes F. magna survival and colonization.

IFN-γ-induced PD-L1 expression represents the existence of tumor-specific T cells, which predicts high-response rate to anti-PD-1/L1 therapy, but loss-of-function of IFN signals (e.g., JAK mutation) induces adaptive immune resistance in patients with low-response rate. Interferon regulatory factors (IRF) are frequently epigenetic silenced in carcinogenesis, while the role of methylation in anti-PD-1/L1 therapy remains unclear. We here investigated the methylation status of IFN-γ related genes IRF1/8 and IFN-α/β-related genes IRF3/7 in lung cancer tissues and found that only highly methylated IRF1 and 7 negatively correlated to cd274 (coding PD-L1) expression, similar to JAK mutation. Interestingly, decitibine (DAC) as methylation inhibitor could hypomethylate IRF1/7 to restore PD-L1 level. Meanwhile, IRF7 enhanced constitutive PD-L1 expression, which was independent of IFN-γ though directly promote transcription of PD-L1, leading to abrogating cytotoxic T lymphocytes (CTLs) generation which could be restored by anti-PD-L1 antibody, or siRNA-IRF7. The supplement of DAC to anti-PD-1 therapy in vivo improve the efficiency of anti-tumor with less methylated IRF1/7, more interferon-related genes expression (e.g., CXCL9) and IFN-γ/CD8+ T-cells infiltrations, suggesting that additional treatment of DAC could rescue the ability to response to IFN in lung cancer patients with anti-PD-1/L1 therapy resistance.

OBJECTIVE

The chemokine receptor CXCR3 is a GPCR found predominantly on activated T cells. CXCR3 is activated by three endogenous peptides; CXCL9, CXCL10 and CXCL11. Recently, a small-molecule agonist, VUF10661, has been reported in the literature and synthesized in our laboratory. The aim of the present study was to provide a detailed pharmacological characterization of VUF10661 by comparing its effects with those of CXCL11.

METHODS

Agonistic properties of VUF10661 were assessed in a chemotaxis assay with murine L1.2 cells transiently transfected with cDNA encoding the human CXCR3 receptor and in binding studies, with [(125)I]-CXCL10 and [(125)I]-CXCL11, on membrane preparations from HEK293 cells stably expressing CXCR3. [(35)S]-GTPγS binding was used to determine its potency to induce CXCR3-mediated G protein activation and BRET-based assays to investigate its effects on intracellular cAMP levels and β-arrestin recruitment.

RESULTS

VUF10661 acted as a partial agonist in CXCR3-mediated chemotaxis, bound to CXCR3 in an allosteric fashion in ligand binding assays and activated G(i) proteins with the same efficacy as CXCL11 in the [(35)S]-GTPγS binding and cAMP assay, while it recruited more β-arrestin1 and β-arrestin2 to CXCR3 receptors than the chemokine.

CONCLUSIONS

VUF10661, like CXCL11, activates both G protein-dependent and -independent signalling via the CXCR3 receptor, but probably exerts its effects from an allosteric binding site that is different from that for CXCL11. It could stabilize different receptor and/or β-arrestin conformations leading to differences in functional output. Such ligand-biased signalling might offer interesting options for the therapeutic use of CXCR3 agonists.

To gain further insight into the immunopathogenesis of bovine tuberculosis (bTB), the cytokine and chemokine expression of cattle experimentally infected with Mycobacterium bovis was analysed in TB granulomas, using immunohistochemistry (IHC) and laser capture microdissection (LCM) followed by qPCR. Immunohistochemistry was conducted for cell types using labelling for CD68, CD3, CD4, CD8, WC1 and CD79a and for the cytokines IFN-γ, TNF-α and TGF-β as well as inducible form of nitric oxide synthase (iNOS). qPCR was conducted for mRNA expression of IFN-γ, TNF-α, TGF-β, IL-17A, IL-22, IL-2, granzyme A and the chemokines CXCL9 and CXCL10. Early stages of granuloma were primarily comprised of epithelioid MΦs expressing high levels of IFN-γ and iNOS, with significantly upregulated expression of CXCL9 and CXCL10 when compared with control tissue. These chemokines displayed a trend of decreasing mRNA expression as lesion progressed, suggesting a higher level of importance during the early stages of the immune response to mycobacterial infection. IL-22 levels showed a strong trend of decrease through granuloma development, and IL-17A was shown to be upregulated, supporting its investigation as a potential biomarker of bTB. The use of LCM and qPCR may prove especially useful for the study of IL-17A as previous attempts to analyse its expression using IHC and in situ hybridization proved unsuccessful.

Malaria remains one of the world's most significant human infectious diseases and cerebral malaria (CM) is its most deadly complication. CM pathogenesis remains incompletely understood, hindering the development of therapeutics to prevent this lethal complication. Elevated levels of the chemokine CXCL10 are a biomarker for CM, and CXCL10 and its receptor CXCR3 are required for experimental CM (ECM) in mice, but their role has remained unclear. Using multiphoton intravital microscopy, CXCR3 receptor- and ligand-deficient mice and bone marrow chimeric mice, we demonstrate a key role for endothelial cell-produced CXCL10 in inducing the firm adhesion of T cells and preventing their cell detachment from the brain vasculature. Using a CXCL9 and CXCL10 dual-CXCR3-ligand reporter mouse, we found that CXCL10 was strongly induced in the brain endothelium as early as 4 days after infection, while CXCL9 and CXCL10 expression was found in inflammatory monocytes and monocyte-derived DCs within the blood vasculature on day 8. The induction of both CXCL9 and CXCL10 was completely dependent on IFN-γ receptor signaling. These data demonstrate that IFN-γ-induced, endothelium-derived CXCL10 plays a critical role in mediating the T cell-endothelial cell adhesive events that initiate the inflammatory cascade that injures the endothelium and induces the development of ECM.

Monocytic cells represent important cellular elements of the innate and adaptive immune responses in viral infections. We assessed the role of Fas/FasL in promoting monocyte apoptosis during HSV-2 infection by using an in vitro model based on the murine RAW 264.7 monocytic cell line and an in vivo murine model of HSV-2 infection applied to C57BL6, MRL-Fas(lpr)/J (Fas-/-) and C3-Fasl(gld)/J (FasL-/-) mice. HSV-2 infection of the monocytic cell line led to early induction of apoptosis, with no protective expression of anti-apoptotic Bcl-2. HSV-2 infected monocytes up-regulated Fas and FasL expression early during in vitro infection but were susceptible to Fas induced apoptosis. The vaginal monocytes in the HSV-2 murine model of infection up-regulated FasL expression and were susceptible to Fas induced apoptosis. HSV-2 infection of Fas and FasL- deficient mice led to decreased apoptosis of monocytes and impaired recruitment of NK, CD4+ and CD8+ T cells within the infection sites. The vaginal lavages of HSV-2 infected Fas and FasL- deficient showed decreased production of CXCL9, CXCL10 and TNF-α in comparison to HSV-2 infected wild-type mice strain. The decreased recruitment of immune competent cells was accompanied by delayed virus clearance from the infected tissue. Triggering of the Fas receptor on HSV-2 infected monocytes in vitro up-regulated the expression of CXCL9 chemokines and the cytokine TNF-α. Our study provides novel insights on the role of Fas/FasL pathway not only in apoptosis of monocytes but also in regulating local immune response by monocytes during HSV-2 infection.

BACKGROUND

Studies in mice have shown that PPARα is an important regulator of lipid metabolism in liver and key transcription factor involved in the adaptive response to fasting. However, much less is known about the role of PPARα in human liver.

METHODS

Here we set out to study the function of PPARα in human liver via analysis of whole genome gene regulation in human liver slices treated with the PPARα agonist Wy14643.

RESULTS

Quantitative PCR indicated that PPARα is well expressed in human liver and human liver slices and that the classical PPARα targets PLIN2, VLDLR, ANGPTL4, CPT1A and PDK4 are robustly induced by PPARα activation. Transcriptomics analysis indicated that 617 genes were upregulated and 665 genes were downregulated by PPARα activation (q value < 0.05). Many genes induced by PPARα activation were involved in lipid metabolism (ACSL5, AGPAT9, FADS1, SLC27A4), xenobiotic metabolism (POR, ABCC2, CYP3A5) or the unfolded protein response, whereas most of the downregulated genes were involved in immune-related pathways. Among the most highly repressed genes upon PPARα activation were several chemokines (e.g. CXCL9-11, CCL8, CX3CL1, CXCL6), interferon γ-induced genes (e.g. IFITM1, IFIT1, IFIT2, IFIT3) and numerous other immune-related genes (e.g. TLR3, NOS2, and LCN2). Comparative analysis of gene regulation by Wy14643 between human liver slices and primary human hepatocytes showed that down-regulation of gene expression by PPARα is much better captured by liver slices as compared to primary hepatocytes. In particular, PPARα activation markedly suppressed immunity/inflammation-related genes in human liver slices but not in primary hepatocytes. Finally, several putative new target genes of PPARα were identified that were commonly induced by PPARα activation in the two human liver model systems, including TSKU, RHOF, CA12 and VSIG10L.

CONCLUSIONS

Our paper demonstrates the suitability and superiority of human liver slices over primary hepatocytes for studying the functional role of PPARα in human liver. Our data underscore the major role of PPARα in regulation of hepatic lipid and xenobiotic metabolism in human liver and reveal a marked immuno-suppressive/anti-inflammatory effect of PPARα in human liver slices that may be therapeutically relevant for non-alcoholic fatty liver disease.

Allergic airway disease is characterized by a robust lymphocytic infiltrate, elaboration of Th2-type inflammatory mediators, pulmonary eosinophil accumulation, and airway hyperreactivity. The CXCR3 ligands, CXCL9 (monokine induced by IFN-gamma) and CXCL10 (IFN-inducible protein, 10 kDa), are IFN-gamma-inducible, Th1-type chemokines. As CXCL10 has been previously shown to participate in the modulation of allergic inflammation, we were interested in investigating the possible role that CXCL9 may play in this inflammatory response. Expression of CXCL9 was primarily identified in airway epithelial cells by immunohistochemical staining. Airway neutralization of CXCL9 at the time of allergen challenge significantly increased airway hyperreactivity, airway eosinophil accumulation, and IL-4 levels in the bronchoalveolar lavage while significantly decreasing airway levels of IL-12. In contrast, introduction of exogenous CXCL9 into the airway at the time of allergen challenge dramatically reduced airway hyper-reactivity and eosinophil accumulation. Moreover, pulmonary levels of IL-4 were significantly reduced, whereas levels of IL-12 were significantly increased, with exogenous CXCL9 treatment. In lymphocytes restimulated with CXCL9 and allergen in vitro, CXCL9 down-regulated IL-4 expression and up-regulated IFN-gamma expression, suggesting that CXCL9 is able to direct activated lymphocytes toward a Th1-type phenotype. Additionally, CXCL9 was shown to inhibit CC chemokine ligand 11-induced eosinophil chemotaxis in in vitro assays. Taken together, our results demonstrate that the CXCR3 ligand CXCL9 is involved in regulation of the allergic response in the lung by regulation of lymphocyte activation and eosinophil recruitment.

Background: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC.

Patients and methods: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies.

Results: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years.

Conclusion: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Keywords: formalin-fixed paraffin-embedded; gene expression; high-grade serous ovarian cancer; overall survival; prognosis.

- Macrolides can ameliorate inflammation in respiratory diseases, providing clinical benefits. Data in influenza is lacking.

- A randomized, open-label, multicenter trial among adults hospitalized for laboratory-confirmed influenza was conducted. Study treatments of oseltamivir and azithromycin (500 mg/day), or oseltamivir alone, both for 5 days, were allocated at 1:1 ratio. The primary outcome was plasma cytokine/chemokine concentration change over time (Day 0-10); secondary outcomes were viral load and symptom score changes. Generalized Estimating Equation (GEE) models were used to analyze longitudinal data.

- Fifty patients were randomized to the oseltamivir-azithromycin or oseltamivir groups, with comparable baseline characteristics (age, 57 ± 18 years; A/H3N2, 70%), complications (72%), and viral load. Pro-inflammatory cytokines IL-6 (GEE: β -0.037, 95%CI-0.067,-0.007, P = 0.016; reduction from baseline -83.4% vs -59.5%), CXCL8/IL-8 (β -0.018, 95%CI-0.037,0.000, P = 0.056; -80.5% vs -58.0%), IL-17 (β -0.064, 95%CI-0.117,-0.012, P = 0.015; -74.0% vs -34.3%), CXCL9/MIG (β -0.010, 95%CI-0.020,0.000, P = 0.043; -71.3% vs -56.0%), sTNFR-1, IL-18, and CRP declined faster in the oseltamivir-azithromycin group. There was a trend toward faster symptom resolution (β -0.463, 95%CI-1.297,0.371). Viral RNA decline (P = 0.777) and culture-negativity rates were unaffected. Additional ex vivo studies confirmed reduced induction of IL-6 (P = 0.017) and CXCL8/IL-8 (P = 0.005) with azithromycin.

- We found significant anti-inflammatory effects with adjunctive macrolide treatment in adults with severe influenza infections. Virus control was unimpaired. Clinical benefits of a macrolide-containing regimen deserve further study. [ClinicalTrials.gov NCT01779570].

Ovarian cancer (OC) has the highest mortality rate among gynecological malignancies. Because chemokine network is involved in OC progression, we evaluated associations between chemokine expression and survival in tumor suppressor protein p53 (TP53) wild-type (TP53WT) and mutant (TP53m) OC datasets. TP53 was highly mutated in OC compared to other cancer types. Among OC subtypes, CXCL14 was predominantly expressed in clear cell OC, and CCL15 and CCL20 in mucinous OC. TP53WT endometrioid OC highly expressed CXCL14 compared to TP53m, showing better progression-free survival but no difference in overall survival (OS). TP53m serous OC highly expressed CCL8, CCL20, CXCL10 and CXCL11 compared to TP53WT. CXCL12 and CCL21 were associated with poor OS in TP53WT serous OC. CXCR2 was associated with poor OS in TP53m serous OC, while CXCL9, CCL5, CXCR4, CXCL11, and CXCL13 were associated with better OS. Taken together, specific chemokine signatures may differentially influence OS in TP53WT and TP53m OC.

Chemokines are a family of small, structurally related molecules that regulate cell trafficking of various types of leukocytes through interactions with their seven-transmembrane, G protein-coupled receptors. Their major function is the recruitment of leukocytes to inflammation sites, but they also play roles in tumor growth, angiogenesis, organ sclerosis, and autoimmunity. A variety of evidence has accumulated to support the concept that thyroid follicular cells as well as intrathyroidal lymphocytes are able to produce CC and CXC chemokines, which, in turn, promote the initiation and maintenance of an inflammatory process resulting in autoimmune thyroid diseases (AITD). Overexpression of several chemokines in AITD has been demonstrated. Moreover, alterations of CCL2, CCL5, CXCL9, and CXCL10 have been shown in circulation of many patients with AITD. In subjects with Graves' disease, antithyroid drug treatment, radioactive iodine ablation, and thyroidectomy can significantly reduce CXCL10 levels. The measurement of chemokines in serum of AITD patients might provide a useful parameter for the evaluation and prediction of disease activity and progression. Further experimental and clinical studies will expand our understanding of the clinical implications of chemokine detection and the effects of chemokines on the pathogenesis of AITD.

Although adoptive T-cell therapy has shown remarkable clinical efficacy in haematological malignancies, its success in combating solid tumours has been limited. Here, we report that PTPN2 deletion in T cells enhances cancer immunosurveillance and the efficacy of adoptively transferred tumour-specific T cells. T-cell-specific PTPN2 deficiency prevented tumours forming in aged mice heterozygous for the tumour suppressor p53. Adoptive transfer of PTPN2-deficient CD8⁺ T cells markedly repressed tumour formation in mice bearing mammary tumours. Moreover, PTPN2 deletion in T cells expressing a chimeric antigen receptor (CAR) specific for the oncoprotein HER-2 increased the activation of the Src family kinase LCK and cytokine-induced STAT-5 signalling, thereby enhancing both CAR T-cell activation and homing to CXCL9/10-expressing tumours to eradicate HER-2⁺ mammary tumours in vivo. Our findings define PTPN2 as a target for bolstering T-cell-mediated anti-tumour immunity and CAR T-cell therapy against solid tumours.

BACKGROUND

Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells specialized in the production of type I interferon (IFN-α/β) and involved in various cutaneous inflammatory and autoimmune disorders, such as cutaneous lupus erythematosus (CLE) and vitiligo. Heat shock proteins (HSPs) are molecular chaperones essential for maintaining cellular functions, but they can act as a danger signal during inflammation.

OBJECTIVE

To decipher the role of HSP70 in the production of IFN-α by pDCs in CLE and vitiligo.

METHODS

Expression of HSP70 and CD123+ pDCs was analysed by immunohistochemistry or immunofluorescence in CLE and vitiligo skin samples. Flow cytometry was performed to analyse expression of HSP70 receptors, activation markers on pDCs and DNA uptake by pDCs in the presence of HSP70. The impact of HSP70 on DNA-induced IFN-α secretion by pDCs was evaluated by enzyme-linked immunosorbent assay (ELISA). The effect of IFN-α on chemokine (C-X-C motif) ligand 9 (CXCL9)/10 gene and protein expression by keratinocytes was determined by real-time polymerase chain reaction and ELISA.

RESULTS

Infiltration of pDCs in CLE and progressive vitiligo was primarily located in the epidermis, close to keratinocytes expressing HSP70. In vitro experiments revealed that the pDCs expressing HSP70 receptor Lox-1 (lectin-like oxidized low-density lipoprotein-receptor-1) were able to aggregate HSP70. Exogenous HSP70 induced activation of pDCs and increased the uptake of exogenous DNA. Furthermore, HSP70 potentiated DNA-induced IFN-α production by pDCs. Finally, IFN-α induced expression of CXCL9 and CXCL10 by keratinocytes.

CONCLUSIONS

These data demonstrate that interaction between HSP70 and pDCs in CLE and vitiligo is a prerequisite for the enhancement of IFN-α production, and could be an interesting target.

High-grade serous ovarian carcinoma (HGSC) accounts for 70% of all epithelial ovarian cancers but clinical management is challenged by a lack of accurate prognostic and predictive biomarkers of chemotherapy response. This study evaluated the role of Signal Transducer and Activator of Transcription 1 (STAT1) as an independent prognostic and predictive biomarker and its correlation with intratumoural CD8+ T cells in a second independent biomarker validation study. Tumour STAT1 expression and intratumoural CD8+ T cell infiltration were assessed by immunohistochemistry as a multicentre validation study conducted on 734 chemotherapy-naïve HGSCs. NanoString-based profiling was performed to correlate expression of STAT1 target genes CXCL9, CXCL10 and CXCL11 with CD8A transcript expression in 143 primary tumours. Multiplexed cytokine analysis of pre-treatment plasma from resistant and sensitive patients was performed to assess systemic levels of STAT1-induced cytokines. STAT1 was validated as a prognostic and predictive biomarker in both univariate and multivariate models and its expression correlated significantly with intra-epithelial CD8+ T cell infiltration in HGSC. STAT1 levels increased the prognostic and predictive value of intratumoural CD8+ T cells, confirming their synergistic role as biomarkers in HGSC. In addition, expression of STAT1 target genes (CXCL9, CXCL10 and CXCL11) correlated significantly with levels of, and CD8A transcripts from intratumoural CD8+ T cells within the resistant and sensitive tumours. Our findings provide compelling evidence that high levels of STAT1, STAT1-induced chemokines and CD8+ T cells correlate with improved chemotherapy response in HGSC. These results identify STAT1 and its target genes as novel biomarkers of chemosensitivity in HGSC. These findings provide new translational opportunities for patient stratification for immunotherapies based on emerging biomarkers of inflammation in HGSC. An improved understanding of the role of interferon-inducible genes will be foundational for developing immunomodulatory therapies in ovarian cancer.

Interleukin-27 (IL-27) is a novel IL-12 family member that plays a critical role in the regulation of T-cell responses. Its immunoregulatory effects on endothelial cells (EC) remain unexplored. Here we show a role for IL-27 in the induction of major histocompatibility complex (MHC) expression in primary human EC. Stimulation of human umbilical vein ECs by IL-27 rapidly induces IFN regulatory factor-1 and dramatically increases the expression of major histocompatibility class II transactivator (CIITA) isoform IV. Expression of this transactivator correlates with increased MHC class II gene expression. IL-27 also enhances expression of MHC class I molecules. Furthermore expression of beta2-microglobulin and transporter associated with antigen processing-1 transcripts increases in response to IL-27. Additional microarray analysis demonstrates that IL-27 significantly upregulates a panel of genes that correlates with immune regulation, including the chemokines CXCL9, CXCL10, and CX3CL1 in human umbilical vein ECs. This first demonstration that both MHC II and I expression are increased in EC after IL-27 stimulation suggests that IL-27 may be important in conferring immune function on vascular endothelium.

The ability to analyse expression of genes rapidly in small samples of tissue is essential for the clinical assessment of many conditions, including the onset of rejection after transplantation. Chemokines have been shown to play a critical role in leukocyte recruitment to transplanted organs and in leukocyte localisation within tissues and antagonism of certain chemokines or chemokine receptors, identified as being up-regulated during allograft rejection, it has been shown to delay leukocyte infiltration into the graft and to prolong graft survival. The analysis of chemokine and chemokine receptor expression in allografts after transplantation may therefore be a useful early indicator of the onset of rejection. RT-PCR techniques are the most sensitive for the detection of low abundance mRNA when the amount of tissue sample is limited. Here we compared competitive-quantitative RT-PCR (CQ-PCR) with real-time PCR for the sequential quantification of chemokine transcripts after transplantation of a fully MHC mismatched mouse cardiac allograft. Although CQ-PCR was found to be an accurate and sensitive technique, real-time PCR was more sensitive and reproducible. Despite the reproducibility, differences in sensitivity between the two techniques were high. Real-time PCR avoids hazardous post-PCR manipulations thereby decreasing the potential risk of sample contamination, and offers the advantage that several genes can be analysed from small tissue samples in a shorter period of time, a key parameter for graft biopsy samples. Real-time PCR was therefore used to extend the analysis of intragraft mRNA chemokine expression levels. Expression of CXCL5 and CCL2 was found to be independent of T cell infiltration while intragraft expression of CCL3, CCL4, CCL5, CXCL9, CXCL10, XCL1 and CCL1 was clearly T cell dependent and increased significantly with time after transplantation. Overall, real-time PCR analysis showed that chemokine gene expression during rejection is clearly distinct from that in non-rejecting syngeneic grafts and is altered by the onset of infiltration of alloantigen-reactive T cells into the graft.

OBJECTIVE

Despite the presence of lymphocyte infiltration, human hepatocellular carcinoma (HCC) is typically a rapidly progressive disease. The mechanism of regulation of lymphocyte migration is poorly understood. In this study, we investigated various factors regulating T cell migration in HCC patients. We examined serum CXC chemokine levels in HCC patients and demonstrated the production of CXC chemokines by HCC cell lines. We determined the effect of both HCC patient serum and tumor cell conditioned supernatant upon lymphocyte expression of chemokine receptor CXCR3 as well as lymphocyte migration. Lastly, we examined the chemotactic responses of lymphocytes derived from HCC patients.

METHODS

The serum chemokines IP-10 (CXCL10) and Mig (CXCL9) levels were measured by cytometric bead array (CBA) and the tumor tissue IP-10 concentration was measured by ELISA. The surface expression of CXCR3 on lymphocytes was determined by flow cytometry. The migratory function of lymphocytes to the corresponding chemokines was assessed using an in vitro chemotactic assay. Phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis.

RESULTS

Increased levels of IP-10 and Mig were detected in HCC patient serum and culture supernatants of HCC cell lines. The IP-10 concentration in the tumor was significantly higher than that in the non-involved adjacent liver tissues. HCC cell lines secreted functional chemokines that induced a CXCR3-specific chemotactic response of lymphocytes. Furthermore, tumor-cell-derived chemokines induced initial rapid phosphorylation of lymphocyte ERK followed by later inhibition of ERK phosphorylation. The culture of normal lymphocytes with HCC cell line supernatants or medium containing serum from HCC patients resulted in a significant reduction in the proportion of lymphocytes exhibiting surface expression of CXCR3. The reduction in T cell expression of CXCR3 resulted in reduced migration toward the ligand IP-10, and both CD4+ and CD8+ T cells from HCC patients exhibited diminished chemotactic responses to IP-10 in vitro compared to T cells from healthy control subjects.

CONCLUSIONS

This study demonstrates functional desensitization of the chemokine receptor CXCR3 in lymphocytes from HCC patients by CXCR3 ligands secreted by tumor cells. This may cause lymphocyte dysfunction and subsequently impaired immune defense against the tumor.

Malaria is second only to tuberculosis as the leading cause of morbidity and mortality as a consequence of a single infectious agent. Much of the pathology of malaria arises from the inappropriate or excessive immune response mounted by the host in an attempt to eliminate the parasite. We here report the inflammatory changes observed in the cerebral microvasculature of C57BL/6 and BALB/c mice that had been inoculated with Plasmodium berghei NK65, a lethal strain of rodent malaria. Although no neurological signs were observed in experimentally infected mice, inflammation of the cerebral microvasculature was clearly evident. Histopathological analysis demonstrated that alterations in cerebral tissue were more intense in infected C57Bl/6 mice than in infected BALB/c animals. Intravital microscopic examination of the cerebral microvasculature revealed increased leukocyte rolling and adhesion in pial venules of infected mice compared with non-infected animals. The extravasation of Evans blue dye into the cerebral parenchyma was also elevated in infected mice in comparison with their non-infected counterparts. Additionally, protein levels of TNF-α, MIG/CXCL9, MCP-1/CCL2, MIP-1α/CCL3 and RANTES/CCL5 were up-regulated in brain samples derived from infected C57Bl/6 mice. Taken together, the data reported here illustrate the complex strain-dependent relationships between leukocyte recruitment, blood brain barrier permeability and chemokine production.