HLA-G SNPS or haplotypes generally associated with high levels of soluble HLA-G in the healthy population have a low production profile in scleroderma: epigenetic mechanisms of regulation?
J. Di Cristofaro (Marseille cedex 05, FR)
Women with scleroderma (SSc) have lower quantities of soluble HLA-G (sHLA-G) in plasma compared to healthy women (p<0.0001, submitted data). HLA-G production is controlled by several single nucleotide polymorphisms (SNPs), both in the 5’ upstream regulatory region (5’URR) and the 3’ untranslated region (3’ UTR), modifying the affinity of gene targeted sequences for transcriptional or post-transcriptional factors, respectively. Some SNPs are associated with low sHLA-G levels (i.e. 14bp insertion in exon 8, -201A, +3142G) while others are associated with high levels (i.e. 14bp deletion, -201G, +3142C). We hypothesized that patients with SSc have increased frequencies of HLA-G SNPs generally associated with low secretor profiles.
HLA-G polymorphism typing was performed for the 7 most common SNPs having an influence on sHLA-G levels on DNA samples from 86 healthy women, 94 patients with SSc and was correlated with plasmatic sHLA-G levels measured by Biovendor ELISA. Contrary to expectations, SNPs associated with low levels of sHLA-G were not at increased frequency in SSc compared to healthy controls. The most commonly described “low secretor profile” 14bp ins allele was present at a frequency of 39% in patients with SSc versus 48% in healthy controls. On the other hand, while levels of sHLA-G were low and comparable in patients and controls who carried the 14bp ins/ins genotype (106 vs 111 UI/mL), striking differences were observed in patients who carried the 14 bp del/del or ins/del genotypes compared to matched healthy controls (70 vs 121 UI/mL, p<0.0001). This lower secretor profile was systematically observed in patients carrying SNPs generally expected to correlate with high sHLA-G levels (-716G, -201G, +3142C, +3187G and +3196C). These results may indicate that the decreased production of sHLA-G observed in SSc is not genetically determined, but rather the result of epigenetic mechanisms. This opens a new field of investigation for sHLA-G regulation in SSc.
A previous miscarriage and a previous successful pregnancy have a different impact on HLA antibody formation during a subsequent successful pregnancy
K. Geneugelijk (Utrecht, NL)
Inherited paternal HLA antigens from the semi-allogeneic fetus may trigger maternal immune responses during pregnancy, leading to the production of child-specific HLA antibodies. The prevalence of these HLA antibodies increases with the number of successful pregnancies. In the present study we investigated the effect of a single prior miscarriage on HLA antibody formation during a subsequent successful pregnancy. Women with a successful pregnancy with one or more prior miscarriages (n=58) and women with a successful pregnancy without a prior miscarriage (n=229) and their children were HLA typed. HLA-antibody analyses were performed in these women to identify whether HLA antibodies were formed against mismatched HLA class I antigens of the last child. The percentage of immunogenic antigens was significantly lower after a single successful pregnancy that was preceded by a single miscarriage (n=18 women) compared to a successful pregnancy that was preceded by a first successful pregnancy (n=62 women). Thus, our data suggest that a previous miscarriage has a different impact on child-specific HLA antibody formation during a subsequent successful pregnancy than a previous successful pregnancy. The lower immunogenicity in these women cannot be explained by reduced numbers of immunogenic B-cell and T-cell epitopes. In conclusion, our observations indicate that increasing gravidity is not related to an increased prevalence of HLA antibodies in a single successful pregnancy that was preceded by a single prior miscarriage.
Modulation of anti-myeloperoxidase antibodies mediated neutrophil activation in vasculitis
N. Svetlicky (Ramat Gan, IL)
Anti-neutrophil-cytoplasmic antibody (ANCA)-associated vasculitidies (AAV) are a group of autoimmune diseases characterized by inflammation and necrosis of small blood vessel walls. The origin of the ANCA-myeloperoxidase (MPO) autoimmune response is unknown, but appears to involve HLA-DQ genes and environmental factors such as microbe-derived molecular mimicry cross-reaction. There is circumstantial evidence for a pathogenic role of neutrophil activation by ANCA-MPO antibodies in the development of vasculitic symptoms. We have studied the ANCA-MPO modulated neutrophils activity by immunodominanat epitope identification. We developed synthetic peptide SF-12 using a peptide phage display library and RL-14 peptide that was predicted as antigen binding site on MPO molecule in-silico. Mice immunized with MPO-related peptides developed ANCA-MPO pathogenic antibodies. Different immunomodulatory effects of the peptides on neutrophil activation by ANCA-MPO antibodies were observed. SF-12 considerably attenuated the neutrophil superoxide production (p<0.001) and induced anti-inflammatory cytokines production by splenocytes of mice in-vitro. RL-14 significantly accelerated neutrophil oxidative burst (p<0.001) and even caused a vasculitic sign in the lungs of immunized mice. Furthermore, we demonstrated the homology of amino acid sequence of RL-14 peptide to Staphyloccocus aureus (S. aureus) membrane protein. Mice immunized with membrane proteins of S. aureus developed significant levels of anti-RL-14 and ANCA-MPO antibodies. Our findings with MPO-peptides can form a basis for the development of innovative tolerogenic peptide therapy in AAV. In addition, we propose a potential role for S. aureus infection in the origin of pathogenic ANCA-MPO in the autoimmunity of AAV.
New HLA-G-related biomarkers for staging type 1 diabetes
S. Gregori (Milan, IT)
In the field of Type 1 diabetes (T1D) no predictive biomarkers are available for appropriate disease staging other than auto-antibodies. HLA-G is an immune-modulatory molecule, involved in promoting/maintaining tolerance. Genetic variations of the HLA-G locus within the 5’ upstream regulatory region (URR) and the 3’ Untranslated region (UTR) finely tune HLA-G expression and have been associated with susceptibility to autoimmunity, including T1D. DC-10 is a subset of HLA-G-expressing dendritic cells that promote tolerance. DC-10 tolerogenic activity relies on the high HLA-G expression and is associated with HLA-G polymorphisms. We assess if DC-10 and HLA-G polymorphisms can be used as biomarkers for staging T1D.
We defined the DC-10 frequency in peripheral blood from healthy controls (HC) and T1D patients at different stages of disease by FACS. Using specific PCRs we amplified the 5’URR, the coding region, and the 3’UTR of HLA-G, and we analyzed PCR products to infer haplotypes and genotypes.
We showed that DC-10 frequency was comparable between HC and T1D patients, progressively decreased in first-degree relatives (FDRs) according to the risk of developing T1D, and that HLA-G+DC-10 were less frequent in FDRs with low risk of developing T1D compared to other groups. By 3’UTR genetic analysis we showed: a lower frequency of the 14 bp insertion in FDRs and T1D patients compared to HC; a significantly higher frequency of insG/delC genotype in T1D patients compared to HC; a significantly higher frequency of the UTR-1 haplotype in high risk FDRs compared to HC; and a significantly higher frequency of UTR-3 haplotype in low risk FDRs compared to HC. This is the first demonstration that DC-10 frequency progressively decrease in FDRs according to the risk to develop T1D and our data support that HLA-G genotype may be associated with T1D. Further studies are needed to confirm these findings and to define whether HLA-G genotypes are associated with HLA-G expression on DC-10.
Pre-existing clonotypes dominate the CD4 T cell response to gluten antigen re-exposure in celiac disease
S. Dahal-Koirala (Oslo, NO)
Clonal expansion of CD4 T cells in response to antigen is the underlying cause of immunological memory, a hallmark of adaptive immunity. However, whether the memory CD4 T-cell pool remains stable or is replenished from naive cells during antigen re-exposure is unclear. Here, we addressed this issue by studying gluten-specific T-cell response during a 14-day gluten challenge in celiac disease patients in remission on gluten-free diet. We performed high-throughput sequencing of T-cell receptor (TCR) alpha and beta-genes of single and bulk populations of cells isolated by use of HLA-DQ: gluten tetramers. This allowed us to analyze the repertoire of the T-cell response at a detailed level. We demonstrate that the T-cell response to gluten is dominated by clonally expanded T cells, and that the majority of expanded clonotypes at peak response is present prior to challenge suggesting a pre-determined TCR repertoire that does not change upon antigen re-exposure. Moreover, the majority of T-cell clonotypes observed in the gut, the primary disease site, following challenge is identical to those circulating in blood in peak response suggesting that these clonotypes play a major role in recall response. Overall, our data suggests that the memory T-cell repertoire remains stable during antigen re-exposure.
Genetic variations in classes I and III of MHC associate with acute coronary syndrome in Finnish population
M. Marchesani (Helsinki, FI)
The HLA-DRB1*01 allele has been associated as a risk factor for acute coronary syndrome (ACS) in several studies. We have identified a novel risk haplotype for the ACS containing single nucleotide polymorphisms (SNPs) from BTNL2 and HLA-DRA genes and the HLA-DRB1*01. In this study we investigated ACS associations with SNPs on class I and III of human major histocompatibility complex (MHC) in subjects positive for HLA-DRB1*01. Case–control studies comprised 3594 ACS cases from three different parts of Finland. The discovery study included 2090, the first replication study 742 and the second replication study 762 ACS cases. Control subjects for three studies were selected from Finnish FINRISK studies from the same geographical region as the cases using risk set sampling. We revealed nine SNPs in the region of MICB-MCCD1-ATP6V1G2-DDX39B-LTA genes associated with the risk of ACS. Three SNPs localized in the MICB-MCCD1 intergenic region, and in ATP6V1G2-DDX39B (BAT1) and LTA introns constituted the risk haplotype with p-value of 2.0E-26 (OR=3.42) and r2 values of ≥0.68. Five SNPs in MCCD1 and ATP6V1G2-DDX39B genes constituted another risk haplotype with the p-value of 1.2E-10 (OR=1.91) and r2 values of ≥0.99. The SNP rs2229094 in exon 2 of lymphotoxin-α (LTA) with p-value of 9.73E-15 (OR=2.57) causing amino acid change from cysteine to arginine was not in linkage with other eight SNPs. Our results indicate that we have identified novel and confirmed previously known ACS risk variants of MHC genes. The genes are related to inflammatory and antiviral responses, coding of a mitochondrial protein and platelet aggregation in patients with acute myocardial infarction, as well as to non-coding RNA regions. The finding may help to understand the gene interactions causing high risk of atherosclerosis.
SNPs correlating with functional HLA region epitopes and supertypes offer Insight into GWAS
M. Dorak (Liverpool, GB)
Single-nucleotide polymorphisms (SNPs) across the HLA region are often significant in genome-wide association studies (GWAS), but most of these associated SNPs do not correspond to classical HLA alleles, even in immune-mediated disorders. Another level of functional HLA region variation (epitopes/supertypes) is relevant in disease associations, but has not attracted much attention in GWAS. To initiate a systematic survey of their involvement with GWAS findings, we examined correlations between SNPs and common HLA epitopes (Bw4/Bw6; C1/C2) / genetic supertypes (DR52/DR53) in a panel of 95 HLA-typed cell lines using ImmunoChip genotyping. We examined univariate correlations between 8,045 SNPs that passed quality control procedures and HLA epitopes/supertypes. We examined the reported GWAS associations of SNPs that were most significantly correlated with HLA epitopes/supertypes on GRASP and PhenoScanner. We found proxy SNPs for each epitope/supertype with r>0.50 (P<0.0001). The HLA-C region SNP rs9264942 correlated with Bw4. This SNP is the top GWAS hit for HIV-1 control (P=3E-35) and implicated in HLA-C expression levels. Our result offers an alternative explanation that rs9264942 is a proxy for Bw4 (a known marker for HIV-1 control). rs9264942 is also the top GWAS marker for Crohns disease in the HLA class I region. The strongest proxy for C1/C2, rs12211087, is the top genome-wide risk marker for psoriasis via its proxy (r2=1) rs4406273 (P=5E-723). Four SNPs showed strong correlations (r>0.97) with DR53 (rs2133035, rs9271574, rs9271488, rs3104389) and were among the top GWAS hits for rheumatoid arthritis, ulcerative colitis and sarcoidosis, and strongest eQTLs for HLA-DQA2, -DQA1, -DRB6 and -DRB1. We identified three ImmunoChip SNPs (rs2097432, rs9271613, rs9271850) as DR52 proxies, and their GWAS associations included the top genome-wide risk marker for systemic sclerosis (via proxy rs3129763; P=9E-187). The proxies for DR52 were the strongest eQTLs for HLA class II genes (P=3E-189 for HLA-DQA1). Using two SNPs together increased the strength of the correlation with DR52 (for rs3129887-A - rs2097432-G, r=0.94, P=1E-38). Our results suggest that the interpretation of HLA region SNP associations can be improved by taking into account additional levels of functional variation within the HLA region.
Colorectal cancer: new biomarkers system in prevention and treatment.
A. Aureli (L'Aquila, IT)
Colorectal cancer (CRC) is the third most common type of cancer worldwide and the discovery and validation of new biomarkers is a continuing challenge of the scientists in this field. It is known that oxidative state and inflammatory cytokines are regulators of tumour-associated inflammation and tumourigenesis, and the genetic distribution of killer immunoglobulin-like receptors (KIRs) and different binding affinity of the Fcγ receptor IIIA (CD16A), Fcy receptor IIa (CD32A) may affect the CRC pathogenesis. Our previous data show that the systems of Thioredoxin 1 (Trx1, a redox molecule) and the CD30 (Trx1 receptor on immune cells, including NK and dendritic cells) represent a double target/biomarker and testing the effect of this system with cytokines, KIRs, CD16A and CD32A genotypes on the susceptibility to develop CRC has represented our goal. So, we hypothesized that serum levels of cytokines, sCD30 and Trx1/RTrx1 combined with KIR, CD16A and CD32A genic polymorphisms could be clinical indicators in CRC. To do this, we evaluated, through biological system studies, serum levels of sCD30, Trx1/RTrx1 and cytokines (ELISA test) and KIR and CD16 and CD32A polymorphisms (SSP and SBT genotyping) in 146 CRC patients and 219 healthy subjects. Statistical analysis was done using a multivariate approach. Our results were as follows: a) CRC progression is associated to an increase of sera levels of IL6 (p=0.0001), IL10 (p=0.00001) and TGFbeta (p=0.0007) and a decrease of IL2 level (p=0.0003); b) Trx1/RTrx1 and sCD30 are positively associated to CRC progression (p=0.003 and 0.001 respectively); c) the presence of >2 aKIR, FcyRIIa-131H/H and FcyRIIIa-158V/V seems protective of disease and index of therapeutic benefit, while the presence of <3 aKIR genes and of FcyRIIa-131R/R and FcyRIIIa-158F/F genotypes increases susceptibility to CRC. In conclusion, we propose a new model of prognostic biomarkers that could be suitable as risk indicator of CRC and for a personalized clinic/therapeutic approach.