A combination of local inflammation and central memory T cells potentiates immunotherapy in the skin

Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro - derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8+ T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation. Copyright © 2012 by The American Association of Immunologists, Inc.

Distinct subpopulations of gamma delta T cells are present in normal and tumor-bearing human liver

Gamma delta T cells are thought to mediate immune responses at epithelial surfaces. We have quantified and characterized hepatic and peripheral blood gamma delta T cells from 11 normal and 13 unresolved tumor-bearing human liver specimens. gamma delta T cells are enriched in normal liver (6.6% of T cells) relative to matched blood (0.9%; P = 0.008). The majority express CD4(-)CD8(-) phenotypes and many express CD56 and/or CD161. In vitro, hepatic gamma delta T cells can be induced to kill tumor cell lines and release interferon-gamma, tumor necrosis factor-alpha, interleukin-2 and interleukin-4. Analysis of V gamma and V delta chain usage indicated that V delta 3(+) cells are expanded in normal livers (21.2% of gamma delta T cells) compared to blood (0.5%; P = 0.001). Tumor-bearing livers had significant expansions and depletions of gamma delta T cell subsets but normal cytolytic activity. This study identifies novel populations of liver T cells that may play a role in immunity against tumors.

A new regulatory variant in the interleukin-6 receptor gene associates with asthma risk

The main genetic determinant of soluble interleukin 6 receptor (sIL-6R) levels is the missense variant rs2228145 that maps to the cleavage site of IL-6R. For each Ala allele, sIL-6R serum levels increase by ∼20 ng ml -1 and asthma risk by 1.09-fold. However, this variant does not explain the total heritability for sIL-6R levels. Additional independent variants in IL6R may therefore contribute to variation in sIL-6R levels and influence asthma risk. We imputed 471 variants in IL6R and tested these for association with sIL-6R serum levels in 360 individuals. An intronic variant (rs12083537) was associated with sIL-6R levels independently of rs4129267 (P=0.0005), a proxy single-nucleotide polymorphism for rs2228145. A significant and consistent association for rs12083537 was observed in a replication panel of 354 individuals (P=0.033). Each rs12083537:A allele increased sIL-6R serum levels by 2.4 ng ml -1. Analysis of mRNA levels in two cohorts did not identify significant associations between rs12083537 and IL6R transcription levels. On the other hand, results from 16 705 asthmatics and 30 809 controls showed that the rs12083537:A allele increased asthma risk by 1.04-fold (P=0.0419). Genetic risk scores based on IL6R regulatory variants may prove useful in explaining variation in clinical response to tocilizumab, an anti-IL-6R monoclonal antibody.

Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product P16INK4A in ovarian cancer is associated with progression and unfavourable prognosis

Paraffin sections from 190 epithelial ovarian tumours, including 159 malignant and 31 benign epithelial tumours, were analysed immunohistochemically for expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product p16INK4A (p16). Most benign tumours showed no p16 expression in the tumour cells, whereas only 11% of malignant cancers were p16 negative. A high proportion of p16-positive tumour cells was associated with advanced stage and grade, and with poor prognosis in cancer patients. For FIGO stage 1 tumours, a high proportion of p16-positive tumour cells was associated with poorer survival, suggesting that accumulation of p16 is an early event of ovarian tumorigenesis. In contrast to tumour cells, high expression of p16 in the surrounding stromal cells was not associated with the stage and grade, but was associated with longer survival. When all parameters were combined in multivariate analysis, high p16 expression in stromal cells was not an independent predictor for survival, indicating that low p16 expression in stromal cells is associated with other markers of tumour progression. High expression of p16 survival in the stromal cells of tumours from long-term survivors suggests that tumour growth is limited to some extent by factors associated with p16 expression in the matrix.

A novel immunodeficiency disorder characterized by genetic amplification of interleukin 25

Many primary immunodeficiency disorders of differing etiologies have been well characterized, and much understanding of immunological processes has been gained by investigating the mechanisms of disease. Here, we have used a whole-genome approach, employing single-nucleotide polymorphism and gene expression microarrays, to provide insight into the molecular etiology of a novel immunodeficiency disorder. Using DNA copy number profiling, we define a hyperploid region on 14q11.2 in the immunodeficiency case associated with the interleukin (IL)-25 locus. This alteration was associated with significantly heightened expression of IL25 following T-cell activation. An associated dominant type 2 helper T cell bias in the immunodeficiency case provides a mechanistic explanation for recurrence of infections by pathogens met by Th1-driven responses. Furthermore, this highlights the capacity of IL25 to alter normal human immune responses.

Interleukin-6 is a potent inducer of S100P, which is up-regulated in androgen-refractory and metastatic prostate cancer

Elevated circulating interleukin-6 (IL6) and up-regulated S100P in prostate cancer (PCa) specimens correlate independently with progression to androgen-independent and metastatic PCa. The cause of up-regulated S100P levels in advanced PCa remains to be determined. We investigated the possibility that IL6 is an inducer of S100P. Determination of mRNA and protein levels by real-time PCR and Western blotting revealed that IL6 is a more potent inducer of S100P than the synthetic androgen, R1881, in the LNCaP/C4-2B model of PCa progression. IL6 did not require androgen to induce S100P in these cells, which express a functional androgen receptor (AR). Like R1881, IL6 was unable to induce S100P in PC3 cells that lack a functional AR. IL6 did not strongly induce the AR-dependent genes PSA and KLK2 and, contrary to R1881, down-regulated Cyr61/CCN1, a potential marker that is down-regulated in PCa. Epidermal growth factor (EGF), which like IL6 is a non-androgen activator of the AR, did not induce S100P. The data identifies a unique gene-induction profile for IL6 and suggests that IL6 may require a functional AR for S100P induction. A link between elevated IL6 and up-regulated S100P in androgen-refractory and metastatic PCa is postulated.

Controlled systemic release of interleukin-12 after gene electrotransfer to muscle for cancer gene therapy alone or in combination with ionizing radiation in murine sarcomas

Background: The present study aimed to evaluate the antitumor effectiveness of systemic interleukin (IL)-12 gene therapy in murine sarcoma models, and to evaluate its interaction with the irradiation of tumors and metastases. To avoid toxic side-effects of IL-12 gene therapy, the objective was to achieve the controlled release of IL-12 after intramuscular gene electrotransfer. Methods: Gene electrotransfer of the plasmid pORF-mIL12 was performed into the tibialis cranialis in A/J and C57BL/6 mice. Systemic release of the IL-12 was monitored in the serum of mice after carrying out two sets of intramuscular IL-12 gene electrotransfer of two different doses of plasmid DNA. The antitumor effectiveness of IL-12 gene electrotransfer alone or in combination with local tumor or lung irradiation with X-rays, was evaluated on subcutaneous SA-1 and LPB tumors, as well as on lung metastases. Results: A synergistic antitumor effect of intramuscular gene electrotransfer combined with local tumor irradiation was observed as a result of the systemic distribution of IL-12. The gene electrotransfer resulted in up to 28% of complete responses of tumors. In combination with local tumor irradiation, the curability was increased by up to 100%. The same effect was observed for lung metastases, where a potentiating factor of 1.3-fold was determined. The amount of circulating IL-12 was controlled by the number of repeats of gene electrotransfer and by the amount of the injected plasmid. Conclusions: The present study demonstrates the feasibility of treatment by IL-12 gene electrotransfer combined with local tumor or lung metastases irradiation on sarcoma tumors for translation into the clinical setting. Copyright © 2009 John Wiley & Sons, Ltd.

Evidence of associations between cytokine gene polymorphisms and quality of life in patients with cancer and their family caregivers

PURPOSE/OBJECTIVES: To identify latent classes of individuals with distinct quality-of-life (QOL) trajectories, to evaluate for differences in demographic characteristics between the latent classes, and to evaluate for variations in pro- and anti-inflammatory cytokine genes between the latent classes. DESIGN: Descriptive, longitudinal study. SETTING: Two radiation therapy departments located in a comprehensive cancer center and a community-based oncology program in northern California. SAMPLE: 168 outpatients with prostate, breast, brain, or lung cancer and 85 of their family caregivers (FCs). METHODS: Growth mixture modeling (GMM) was employed to identify latent classes of individuals based on QOL scores measured prior to, during, and for four months following completion of radiation therapy. Single nucleotide polymorphisms (SNPs) and haplotypes in 16 candidate cytokine genes were tested between the latent classes. Logistic regression was used to evaluate the relationships among genotypic and phenotypic characteristics and QOL GMM group membership. MAIN RESEARCH VARIABLES: QOL latent class membership and variations in cytokine genes. FINDINGS: Two latent QOL classes were found: higher and lower. Patients and FCs who were younger, identified with an ethnic minority group, had poorer functional status, or had children living at home were more likely to belong to the lower QOL class. After controlling for significant covariates, between-group differences were found in SNPs in interleukin 1 receptor 2 (IL1R2) and nuclear factor kappa beta 2 (NFKB2). For IL1R2, carrying one or two doses of the rare C allele was associated with decreased odds of belonging to the lower QOL class. For NFKB2, carriers with two doses of the rare G allele were more likely to belong to the lower QOL class. CONCLUSIONS: Unique genetic markers in cytokine genes may partially explain interindividual variability in QOL. IMPLICATIONS FOR NURSING: Determination of high-risk characteristics and unique genetic markers would allow for earlier identification of patients with cancer and FCs at higher risk for poorer QOL. Knowledge of these risk factors could assist in the development of more targeted clinical or supportive care interventions for those identified.

Conserved gene structure and function of interleukin-10 in teleost fish

Interleukin-10 (IL-10) is an important immunoregulatory cytokine produced by various types of cells. Researchers describe here the isolation and characterization of olive flounder IL-10 (ofIL-10) cDNA and genomic organization. The ofIL-10 gene encodes a 187 amino acid protein and is composed of a five exon/four intron structure, similar to other known IL-10 genes. The ofIL-10 promoter sequence analysis shows a high level of homology in putative binding sites for transcription factors which are sufficient for transcriptional regulation ofIL-10. Important structural residues are maintained in the ofIL-10 protein including the four cysteines responsible for the two intra-chain disulfide bridges reported for human IL-10 and two extra cysteine residues that exist only in fish species. The phylogenetic analysis clustered ofIL-10 with other fish IL-10s and apart from mammalian IL-10 molecules. Quantitative real-time Polymerase Chain Reaction (PCR) analysis demonstrated ubiquitous ofIL-10 gene expression in the 13 tissues examined. Additionally, the induction of ofIL-10 gene expression was observed in the kidney tissue from olive flounder infected with bacteria (Edawardsiella tarda) or virus (Viral Hemorrhagic Septicemia Virus; VHSV). These data indicate that IL-10 is an important immune regulator that is conserved strictly genomic organization and function during the evolution of vertebrate immunity.

Replication of association of IL1 gene complex members with ankylosing spondylitis in Taiwanese Chinese

Objective: To test the association of interleukin 1 (IL1) gene family members with ankylosing spondylitis (AS), previously reported in Europid subjects, in an ethnically remote population. Methods: 200 Taiwanese Chinese AS patients and 200 ethnically matched healthy controls were genotyped for five single nucleotide polymorphisms (SNPs) and the IL1RN.VNTR, markers previously associated with AS. Allele, genotype, and haplotype frequencies were compared between cases and controls. Results: Association of alleles and genotypes of the markers IL1F10.3, IL1RN.4, and IL1RN.VNTR was observed with AS (p<0.05). Haplotypes of pairs of these markers and of the markers IL1RN.6/1 and IL1RN.6/2 were also significantly associated with AS. The strongest associations observed were with the marker IL1RN.4, and with the two-marker haplotype IL1RN.4-IL1RN.VNTR (both p = 0.004). Strong linkage disequilibrium was observed between all marker pairs except those involving IL1B-511 (D′ 0.4 to 0.9, p<0.01). Conclusions: The IL1 gene cluster is associated with AS in Taiwanese Chinese. This finding provides strong statistical support that the previously observed association of this gene cluster with AS is a true positive finding.

Interleukin 17A is an immune marker for chlamydial disease severity and pathogenesis in the koala (Phascolarctos cinereus)

The koala (Phascolarctos cinereus) is an iconic Australian marsupial species that is facing many threats to its survival. Chlamydia pecorum infections are a significant contributor to this ongoing decline. A major limiting factor in our ability to manage and control chlamydial disease in koalas is a limited understanding of the koala’s cell-mediated immune response to infections by this bacterial pathogen. To identify immunological markers associated with chlamydial infection and disease in koalas, we used koala-specific Quantitative Real Time PCR (qrtPCR) assays to profile the cytokine responses of Peripheral Blood Mononuclear Cells (PBMCs) collected from 41 koalas with different stages of chlamydial disease. Target cytokines included the principal Th1 (Interferon gamma; IFNγ), Th2 (Interleukin 10; IL10), and pro-inflammatory cytokines (Tumor Necrosis Factor alpha; TNFα). A novel koala-specific IL17A qrtPCR assay was also developed as part of this study to quantitate the gene expression of this Th17 cytokine in koalas. A statistically significant higher IL17A gene expression was observed in animals with current chlamydial disease compared to animals with asymptomatic chlamydial infection. A modest up-regulation of pro-inflammatory cytokines, such as TNFα and IFNγ, was also observed in these animals with signs of current chlamydial disease. IL10 gene expression was not evident in the majority of animals from both groups. Future longitudinal studies are now required to confirm the role played by cytokines in pathology and/or protection against C. pecorum infection in the koala.

Association of a disintegrin and metalloprotease 33 (ADAM33) gene polymorphisms with the risk of COPD: An updated meta-analysis of 2,644 cases and 4,804 controls

A series of observational studies have been made to investigate the association of the ADAM33 gene polymorphisms with the risk of COPD, but their results were conflicting. Therefore, we performed an updated meta-analysis to quantitatively summarize the associations of ADAM33 gene polymorphisms with the risk of COPD. Thirteen case–control studies referring to nine SNPs were identified: V4 (rs2787094), T+1 (rs2280089), T2 (rs2280090), T1 (rs2280091), S2 (rs528557), S1 (rs3918396), Q−1 (rs612709), F+1 (rs511898) and ST+5 (rs597980). A dominant model (AA+Aa vs. aa), recessive model (AA vs. Aa+aa), additive model (AA vs. aa) and allelic model (A vs. a) were used to evaluate the association of ADAM33 polymorphism with the risk of COPD. The results indicated that significant associations were found for ADAM33 T1, T2, S1, Q−1, F+1 and ST+5 polymorphisms associated with the risk of COPD in different populations. However, no significant associations were found for V4, T+1 and S2 polymorphisms with the risk of COPD in all genetic models, even in the subgroup analysis by ethnicity. This meta-analysis provided evidence that the ADAM33 T1, T2, S1, Q−1, F+1 and ST+5 six locus polymorphisms association with the risk of COPD. Furthermore, T2, Q−1 and ST+5 indicated an association with the risk of COPD in the European populations, whereas T1, T2, S1, F+1 and Q−1 indicated an association with the risk of COPD in the Asian populations.

Cardiometabolic effects of genetic upregulation of the interleukin 1 receptor antagonist: A Mendelian randomisation analysis

Background To investigate potential cardiovascular and other effects of long-term pharmacological interleukin 1 (IL-1) inhibition, we studied genetic variants that produce inhibition of IL-1, a master regulator of inflammation. Methods We created a genetic score combining the effects of alleles of two common variants (rs6743376 and rs1542176) that are located upstream of IL1RN, the gene encoding the IL-1 receptor antagonist (IL-1Ra; an endogenous inhibitor of both IL-1α and IL-1β); both alleles increase soluble IL-1Ra protein concentration. We compared effects on inflammation biomarkers of this genetic score with those of anakinra, the recombinant form of IL-1Ra, which has previously been studied in randomised trials of rheumatoid arthritis and other inflammatory disorders. In primary analyses, we investigated the score in relation to rheumatoid arthritis and four cardiometabolic diseases (type 2 diabetes, coronary heart disease, ischaemic stroke, and abdominal aortic aneurysm; 453 411 total participants). In exploratory analyses, we studied the relation of the score to many disease traits and to 24 other disorders of proposed relevance to IL-1 signalling (746 171 total participants). Findings For each IL1RN minor allele inherited, serum concentrations of IL-1Ra increased by 0·22 SD (95% CI 0·18–0·25; 12·5%; p=9·3 × 10−33), concentrations of interleukin 6 decreased by 0·02 SD (−0·04 to −0·01; −1·7%; p=3·5 × 10−3), and concentrations of C-reactive protein decreased by 0·03 SD (−0·04 to −0·02; −3·4%; p=7·7 × 10−14). We noted the effects of the genetic score on these inflammation biomarkers to be directionally concordant with those of anakinra. The allele count of the genetic score had roughly log-linear, dose-dependent associations with both IL-1Ra concentration and risk of coronary heart disease. For people who carried four IL-1Ra-raising alleles, the odds ratio for coronary heart disease was 1·15 (1·08–1·22; p=1·8 × 10−6) compared with people who carried no IL-1Ra-raising alleles; the per-allele odds ratio for coronary heart disease was 1·03 (1·02–1·04; p=3·9 × 10−10). Per-allele odds ratios were 0·97 (0·95–0·99; p=9·9 × 10−4) for rheumatoid arthritis, 0·99 (0·97–1·01; p=0·47) for type 2 diabetes, 1·00 (0·98–1·02; p=0·92) for ischaemic stroke, and 1·08 (1·04–1·12; p=1·8 × 10−5) for abdominal aortic aneurysm. In exploratory analyses, we observed per-allele increases in concentrations of proatherogenic lipids, including LDL-cholesterol, but no clear evidence of association for blood pressure, glycaemic traits, or any of the 24 other disorders studied. Modelling suggested that the observed increase in LDL-cholesterol could account for about a third of the association observed between the genetic score and increased coronary risk. Interpretation Human genetic data suggest that long-term dual IL-1α/β inhibition could increase cardiovascular risk and, conversely, reduce the risk of development of rheumatoid arthritis. The cardiovascular risk might, in part, be mediated through an increase in proatherogenic lipid concentrations. Funding UK Medical Research Council, British Heart Foundation, UK National Institute for Health Research, National Institute for Health Research Cambridge Biomedical Research Centre, European Research Council, and European Commission Framework Programme 7.

Association between the interleukin 23 receptor and ankylosing spondylitis is confirmed by a new UK case-control study and meta-analysis of published series

Objectives. It has been shown previously that IL-23R variants are associated with AS. We conducted an extended analysis in the UK population and a meta-analysis with the previously published studies, in order to refine these IL-23R associations with AS. Methods. The UK case-control study included 730 new cases and 1331 healthy controls. In the extended study, the 730 cases were combined with 1088 published cases. Allelic associations were analysed using contingency tables. In the meta-analysis, 3482 cases and 3150 controls from four different published studies and the new UK cases were combined. DerSimonian-Laird test was used to calculate random effects pooled odds ratios (ORs). Results. In the UK case-control study with new cases, four of the eight SNPs showed significant associations, whereas in the extended UK study, seven of the eight IL-23R SNPs showed significant associations (P < 0.05) with AS, maximal with rs11209032 (P < 10-5, OR 1.3), when cases with IBD and/or psoriasis were excluded. The meta-analysis showed significant associations with all eight SNPs; the strongest associations were again seen not only with rs11209032 (P = 4.06 × 10-9, OR ∼1.2) but also with rs11209026 (P < 10-10, OR ∼0.6). Conclusions. IL-23R polymorphisms are clearly associated with AS, but the primary causal association(s) is(are) still not established. These polymorphisms could contribute either increased or decreased susceptibility to AS; functional studies will be required for their full evaluation. Additionally, observed stronger associations with SNPs rs11209026 and rs11465804 upon exclusion of IBD and/or psoriasis cases may represent an independent association with AS. © The Author 2009. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.

Association between endometriosis and the interleukin 1A (IL1A) locus

STUDY QUESTION Are single-nucleotide polymorphisms (SNPs) at the interleukin 1A (IL1A) gene locus associated with endometriosis risk? SUMMARY ANSWER We found evidence for strong association between IL1A SNPs and endometriosis risk. WHAT IS KNOWN ALREADY Genetic factors contribute substantially to the complex aetiology of endometriosis and the disease has an estimated heritability of ∼51%. We, and others, have conducted genome-wide association (GWA) studies for endometriosis, which identified a total of nine independent risk loci. Recently, two small Japanese studies reported eight SNPs (rs6542095, rs11677416, rs3783550, rs3783525, rs3783553, rs2856836, rs1304037 and rs17561) at the IL1A gene locus as suggestively associated with endometriosis risk. There is also evidence of a link between inflammation and endometriosis. STUDY DESIGN, SIZE, DURATION We sought to further investigate the eight IL1A SNPs for association with endometriosis using an independent sample of 3908 endometriosis cases and 8568 controls of European and Japanese ancestry. The study was conducted between October 2013 and July 2014. PARTICIPANTS/MATERIALS, SETTING, METHODS By leveraging GWA data from our previous multi-ethnic GWA meta-analysis for endometriosis, we imputed variants in the IL1A region, using a recent 1000 Genomes reference panel. After combining summary statistics for the eight SNPs from our European and Japanese imputed data with the published results, a fixed-effect meta-analysis was performed. An additional meta-analysis restricted to endometriosis cases with moderate-to-severe (revised American Fertility Society stage 3 or 4) disease versus controls was also performed. MAIN RESULTS AND THE ROLE OF CHANCE All eight IL1A SNPs successfully replicated at P < 0.014 in the European imputed data with concordant direction and similar size to the effects reported in the original Japanese studies. Of these, three SNPs (rs6542095, rs3783550 and rs3783525) also showed association with endometriosis at a nominal P < 0.05 in our independent Japanese sample. Fixed-effect meta-analysis of the eight SNPs for moderate-to-severe endometriosis produced a genome-wide significant association for rs6542095 (odds ratio = 1.21; 95% confidence interval = 1.13–1.29; P = 3.43 × 10−8). LIMITATIONS, REASONS FOR CAUTION The meta-analysis for moderate-to-severe endometriosis included results of moderate-to-severe endometriosis cases from our European data sets and all endometriosis cases from the Japanese data sets, as disease stage information was not available for endometriosis cases in the Japanese data sets. WIDER IMPLICATIONS OF THE FINDINGS SNP rs6542095 is located ∼2.3 kb downstream of the IL1A gene and ∼6.9 kb upstream of cytoskeleton-associated protein 2-like (CKAP2L) gene. The IL1A gene encodes the IL1a protein, a member of the interleukin 1 cytokine family which is involved in various immune responses and inflammatory processes. These results provide important replication in an independent Japanese sample and, for the first time, association of the IL1A locus in endometriosis patients of European ancestry. SNPs within the IL1A locus may regulate other genes, but if IL1A is the target, our results provide supporting evidence for a link between inflammatory responses and the pathogenesis of endometriosis. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.