T-cell infiltration and macrophage polarization correlates with HLA class I expression in non-small cell lung cancer
F. Garrido (Granada, ES)
The main objective of this work was to investigate whether tumor HLA class I (HLA-I) expression influences immune infiltration. Tumor tissues obtained from 40 patients diagnosed with lung carcinomas were analyzed for HLA-I expression and lymphocyte infiltration using immunohistochemistry and flow cytometry. 22 patients out of the 40 were completely negative for HLA-I expression or showed down-regulation of HLA-A, -B and -C molecules (55%). We quantified tumor infiltration with CD8+ T lymphocytes, and M1 and M2 macrophages. We found that HLA-I positive tumors have statistically significant increases in tumor T lymphocyte infiltration compared to HLA-I negative tumors. M1 and M2 infiltration was analyzed in order to determine if they are associated with tumor HLA-I expression. In addition, we observed that tumors with HLA-I positive expression have higher M1/M2 ratio than HLA-I negative tumors (also statistically significant). These data suggest that there is a phase during tumor development when anti-tumor responses mediated by CD8+ T-lymphocytes and pro-inflammatory M1 macrophages prevail. In contrast, noticeably reduced presence of T-CD8 lymphocytes and polarization of macrophages to M2 phenotype in HLA class I negative tumors may favor the immune escape. Additionally, using flow cytometry analysis of the tumor infiltrate we noted a positive correlation between the total percentage of lymphocytes and the proportion of Treg cells. In conclusion, this study revealed that HLA-I-positive and -negative tumors show striking differences in the composition of leukocyte infiltration and that greater tumor infiltration with lymphocytes correlates with increased presence of Treg cells, which is a feature of immunosuppressive tumor microenvironment.
The absence of HLA class I expression in non-small cell lung cancer correlates with the tumor tissue structure and the pattern of T cell infiltration
F. Garrido (Granada, ES)
We wanted to analyze whether tumor HLA class I (HLA-I) expression influences the pattern of the immune cell infiltration and stromal cell reaction in the tumor microenvironment. Tumor tissues obtained from 57 patients diagnosed with lung carcinomas were analyzed for HLA expression and leukocyte infiltration. 28 patients out of the 57 were completely negative for HLA-I expression (49.1%) or showed a selective HLA-A locus down-regulation (three patients, 5.2%). In 26 out of 57 tumors (47.8%) we detected a positive HLA-I expression but with a percentage of HLA-I negative cells between 10 and 25%. The HLA-I negative phenotype was produced by a combination of HLA haplotype loss and a transcriptional down-regulation of beta 2-microglobulin (b2-m) and LMP2 and LMP7 antigen presentation machinery genes. The analysis and localization of different immune cell populations revealed the presence of two major and reproducible patterns. One pattern, which we designated “immune-permissive tumor microenvironment" (TME), was characterized by positive tumor HLA-I expression, intratumoral infiltration with cytotoxic T-CD8+ cells, M1-inflammatory type macrophages, and a diffuse pattern of FAP+ cancer-associated fibroblasts. In contrast, another pattern defined as “non-immune-permissive TME” was found in HLA-I negative tumors with strong stromal-matrix interaction, T-CD8+ cells surrounding tumor nests, a dense layer of FAP+ fibroblasts and M2/ repair-type macrophages. In conclusion, this study revealed marked differences between HLA class I-positive and negative tumors related to tissue structure, the composition of leukocyte infiltration and stromal response in the tumor microenvironment.
Associations of polymorphisms within toll-like receptors and nuclear factor kappa genes with progression of rheumatoid arthritis
B. Wysoczanska (Poland, PL)
Rheumatoid arthritis (RA) is one of the most common autoimmune diseases. The signal cascade mediated by Toll-Like Receptors (TLRs) and the nuclear factor kappa B (NF-κB) results in an immune response via rapidly increased production of cytokines, such as TNF-alpha, and chemokines, that may lead to inflammatory and joint destructive process. Genetic polymorphisms of TLR and NF-kB1 genes were investigated in 303 RA patients (237 females / 66 males) and in 162 healthy individuals. TLR2 (rs111200466, -196/-174 del/ins), TLR3 (rs3775391; Leu412Phe G>A), and NF-kB1 (rs28362491; -94ins/del ATTG) polymorphisms were assessed by real-time PCR (using LightSNiP assays) and capillary electrophoresis. Patients and controls did not differ with respect to genotype/allele distributions. More effective EULAR response to treatment with TNF-inhibitors was observed after 24 weeks as compared to 12 weeks of treatment (p=0.001). Men had a higher activity of the disease (DAS28) before treatment compared to women (p=0.05) and the response to treatment was better in female than in male patients after 24 weeks (p=0.05). Remission rate was higher in women compared to men (p=0.04). Rheumatoid factor (RF)-positive patients presented with more severe disease as compared to RF-negative patients (p=0.01). This relationship was especially seen in women (p=0.001). Moreover, RF-positive patients were more carrying the ins+ NFkB1 allele (p=0.07). TLR2 del+ positive patients were more frequently characterized with lower stage of disease (0-II) as compared to ins/ins homozygotes (p=0.05) and lacked RF (p=0.05). In conclusion, our results imply that TLRs and NFkB1 may play an important role in chronic inflammation in rheumatoid arthritis and their polymorphisms may potentially affect the disease outcomes in RA patients.
Combined effect of glutamine at position 70 of HLA-DRB1 and alanine at position 57 of HLA-DQB1 in type 1 diabetes: an epitope analysis
P. Gerasimou (Nicosia, CY)
The contribution of specific HLA Class II alleles in type 1 diabetes is determined by polymorphic amino acid epitopes that direct antigen binding. Therefore, along with conventional allele frequency analysis, epitope analysis can provide important insights into disease susceptibility. Within our highly genetically heterogeneous patient cohort we identified a subgroup that did not carry the DRB1*03:01-DQA1*05:01-DQB1*02:01 and DRB1*04:xx-DQA1*03:01-DQB1*03:02 risk haplotypes but a novel recombinant one, DRB1*04:XX-DQA1*03:01-DQB1*02:01 designated DR4-DQ2.3. Through epitope analysis we identified established susceptibility (DQB1 A57, DRB1 H13) and resistance (DQB1 D57) epitopes as well as other novel susceptibility epitopes DRB1 Q70, DQB1 L26 and resistance epitopes DRB1 D70, R70 and DQB1 Y47. Prevalence of susceptibility epitopes was higher in patients and was not exclusively a result of linkage disequilibrium. Epitopes DRB1 Q70, DQB1 L26 and A57 and a 10 amino acid epitope of DQA1 were the most significant in discriminating risk alleles. An extended haplotype containing these epitopes was carried by 92% of our patient cohort. Sharing of susceptibility epitopes could also explain the absence of risk haplotypes in patients. Finally, many significant epitopes were non-pocket residues suggesting that critical immune functions exist spanning further from the binding pockets.
The role of HLA-DRB1 alleles and the shared epitope hypothesis in the susceptibility to seronegative and seropositive rheumatoid arthritis in patients from northern Spain (Cantabria)
J. Irure-Ventura (Santander, ES)
Rheumatoid arthritis (RA) is a complex, multifactorial disease in which HLA, mainly HLA-DRB1 has been shown to play an important role, but its exact contribution in the pathogenesis remains unclear. According to different populations, a lot of studies focused on finding the associated specific alleles have been performed. Alleles from the HLA-DRB1*01, *04, *10 and *14 have been found in association with RA. All these alleles share a RAA motif in the 72-74 position of the amino acid molecule which is known as the shared epitope hypothesis. In 2005, du Montcel ST et al proposed a new classification including the amino acids at positions 70 and 71 because of their role in modulating the susceptibility to RA. Since then, few studies taking into account this additional residues have been published. 331 seropositive and seronegative RA patients (presence / absence of anti citrullinated protein antibodies) and 252 age and sex-matched healthy controls were analysed for the HLA-DRB1 allelic distribution. All of them, belong to a well conserved region from northern Spain (Cantabria). Our results showed that HLA-DRB1*01:01 (QRRAA) and *04:01 (QKRAA) were significantly more frequent in RA patients and HLA-DRB1*07:01 (DRRGQ) and *13 (DERAA / DKRAA) in healthy controls. Furthermore, when we compared seropositive and seronegative patients we found that HLA-DRB1*01:01, *04:01 and *04:08 were significant associated with seropositive RA whereas HLA-DRB1*03:01, *13:01 and *13:02 were associated with seronegative RA. Distribution of HLA-DRB1 alleles were also analysed in men and women and we found that HLA-DRB1*13:01 and *15:01 were more frequent in men with RA. Finally, genotype distribution according the new shared epitope classification was studied. In conclusion, our findings regarding HLA-DRB1 allele distribution partially agree with previously reported data. Moreover, when we considered the presence or absence of anti citrullinated protein antibodies compared with HLA-DRB1 alleles distribution, we found different results than other published studies.
HLA-DRB1 and HLA-DQB1 antigens in Bulgarian patients with primary and secondary antiphospholipid syndrome
T. Lukanov (Sofia, BG)
Antiphospholipid syndrome (APS) is a disease with multifactorial and polygenic pathogenesis. Recently, genetic predisposition to APS has been subjected to wide discussion. The aim of this study is to determine the prevalence of HLA-DRB1 and -DQB1 loci in a Bulgarian population of healthy persons and patients with primary (PAPS) and secondary (SAPS) APS.
We investigated the prevalence of DRB1 and DQB1 in 204 healthy volunteers and 127 patients tested for antiphospholipid antibodies (aPL), divided into five groups: I-29 patients with systemic lupus erythematosus (SLE) with SAPS, II-35 patients with PAPS, III-32 women with spontaneous abortions without aPL, IV- patients with different thrombosis without clinical and laboratory data for APS, and V-16 SLE patients without clinical and laboratory data for APS. HLA-DRB1 and DQB1 alleles were determined with a PCR-SSP/SSOP technique. Chi square, and Fischer’s exact test were used to evaluate differences between groups. Maximum-likelihood and descriptive statistic were also calculated. Patients from group I had more frequently DRB1*03 than the control group (p<0.0001; O.R. 4.04), than group II (p=0.025; O.R. 0.32) and those from group III (p=0.02; O.R. 0.3); and more frequently DQB1*02 compared to control group (p=0.002; O.R. 2.88) and patients from group II (p=0.029; O.R. 0.35), but more rarely DRB1*11 compared to control group (p=0.014; O.R. 0.29), patients from group II (p=0.03; O.R. 3.82) and those from group III (p=0.012; O.R. 4.5). Additionally, HLA-DQB1*03 was at significantly lower frequency in patients from group I (0.207) compared to control group (0.386; p=0.01; O.R. 0.42), group II (0.382; p=0.05) and group III (0.406; p=0.028). There were no statistically significant differences between other groups. HLA-DRB1*03 and DQB1*02 antigens have a strong association with SLE patients with APS, whereas HLA-DRB1*11 and DQB1*03 seems to have a “protective” role. Patients with PAPS and those with spontaneous abortions have DRB1 and DQB1 alleles similar to a healthy population. Our results show the need for further investigations in order to identify the exact alleles associated with this complex autoimmune disorder with high socioeconomic status.
Association of HLA-DR4/HLA-DRB1*04 with Vogt-Koyanagi-Harada disease
R. D'Auria (Avellino, IT)
Vogt-Koyanagi-Harada disease is characterized by chronic granulomatous panuveiti and diffuse bilateral etiology, whose pathogenesis has been related to immunological disorders affecting the melanocytes and involving mediated cytotoxicity by T lymphocytes and apoptosis. It develops towards the detachment of the exudative retina, often associated with immunological abnormalities. The incidence is estimated at 1 / 400,000 cases a year. The disease affects mainly female individuals and those with darker skin pigmentation from Asian, Hispanic, Middle Eastern and Native American populations. The disease can be divided into four clinical stages. The pro-dromal phase is characterized by a non-specific symptomatology. The next phase shows neurological symptoms, while the convalescent phase that occurs within three months of the onset of the disease is characterized by cutaneous signs such as poliosis of eyelashes, eyebrows and hair. Recurrent uveitis and ocular complications characterize the chronic recurrent the last phase. In one case, a 56 year-old woman was at the prodromal stage, with non-specific symptoms such as fever, headache, dizziness (vertigo) and nausea, then after a few days progressed to the ophthalmologic stage. The patient complained of blurred vision, orbital pain, and bilateral central scotoma. HLA class II typing identified DRB1*04:05 in this patient. Confirming the diagnosis, the patient was treatment with high-dose corticosteroids and after being under observation for 10 months, the patient has had no recurrences of disease.
Discovery of hybrid peptides in autoimmune disease
K. Haskins (Denver, US)
We recently discovered hybrid insulin peptides (HIPs) as a new class of antigens targeted by autoreactive T cells in Type 1 Diabetes (T1D). These peptides form in pancreatic beta cells as a result of the post-translational crosslinking of insulin fragments to other naturally occurring peptides that are present in the secretory granules of beta cells. At the junction between two peptide binding partners HIPs contain entirely new amino acid sequences, which provide plausible explanations on how immune self-tolerance may be broken in T1D. We are currently developing proteomic strategies to characterize the hybrid peptide landscape in beta cells, setting the stage for the identification of hybrid peptide antigens in other autoimmune diseases. The amino acid sequences of hybrid peptides are not represented in traditional protein databases used for mass spectrometric analyses of biological samples. Consequently, we are working on the generation of new databases, which can be used for the mass spectrometric identification of hybrid peptide sequences. For this we are using biochemical strategies to identify potential hybrid peptide binding partners in beta cell extracts, which can be used to generate hybrid peptide databases. The identification of hybrid peptides in tissue targeted by autoimmune attacks may provide tools to predict, prevent or reverse various autoimmune diseases.
Autoimmune cytopenias in primary immunodeficiency diseases: single center experience
T. Patiroglu (Kayseri, TR)
Primary immunodeficiency diseases (PIDs) are associated with hematologic complications such autoimmune hemolytic anemia (AIHA) and thrombocytopenia (ITP). The most common autoimmune cytopenia is ITP. Although ITP is observed in 7.6% of patients with PID, AIHA is seen at 4.8%. Also, we aimed to present the patients who had autoimmune cytopenias and PID.
Fifty six PID patients who were followed at the Pediatric Immunology Department of Erciyes University Medical Faculty (analyzed genetically) were evaluated retrospectively. Autoimmune cytopenias such as ITP and AIHA were detected in 9 (16.07%) of the patients (combined immunodeficiency: 4 patients, common variable immunodeficiency: 2 patients, hyper-immunoglobulin E syndrome: 1 patient, X-linked lymphoproliferative: 1 patient, chronic granulomatous disease: 1 patient). ITP was detected in 8 of 9 patients and AIHA was also detected in 6 patients. In four patients (LRBA deficiency: 2 patients, hyper IgE syndrome:1 patient and CGD:1patient), both ITP and AIHA were observed. Immunosuppressive therapy with steroid, cyclosporine, mycophenolate mofetyl and intravenous immunoglobulin were given to all patients. Bone marrow transplantation was performed to the four patients. However, five patients died because of immunodeficiency.
There is a paradoxal situation between PID and autoimmunity. The reduction of central and peripheral tolerance is held responsible for autoimmunity in PID. As a conclusion, we wanted to point out autoimmune cytopenias in patients with PID and the requirement of multidisciplinary approach for treatment.
HLA-DRB1, known to be associated with different autoimmune disorders, in pregnancies complicated by autoimmune rheumatic diseases.
A. Pasi (Pavia, IT)
Rheumatic diseases are chronic systemic diseases often affecting young women during reproductive ages, so pregnancy is a major issue in their management. The fetus represents a natural allograft that is not normally rejected. While the maternal immune system preserves the ability to respond to foreign antigens, tolerance mechanisms are up-regulated to protect the fetus from immunologic attacks by the mother. This deep immunologic adaptation influences maternal rheumatic diseases in several ways. Associations between HLA molecules and systemic autoimmune diseases have been reported and vary among ethnic groups and diseases. This is a case-control study including 30 pregnancies in women with preexisting rheumatic diseases and 177 physiological pregnancies. The rheumatic diseases included: 22 undifferentiated connective tissue disease cases, five of primary anti-phospholipid syndrome, one systemic sclerosis, one Sjögren’s syndrome, and one fibromyalgia case. PCR-SSP/-SSO techniques were used to define HLA-DRB1 allelic polymorphisms. The frequency of the HLA-DRB1*01,*04,*10 rheumatoid arthritis shared epitope was similar in patients and controls (10% vs 11%). The same was observed for DRB1*15, described as associated with Sjögren’s syndrome (3.3% vs 4.5%). The allele frequency of HLA-DRB1*03, significantly associated with SLE, was slightly increased in patients with respect to controls (10% vs 6.2% p=n.s. OR=1.68). Only HLA-DRB1*07 showed a significantly higher frequency among patients with respect to controls (18.33% vs 8.76% p=0.023). Considering this study as a genetic counseling, it may be useful to predict the evolution of undifferentiated connective tissue diseases to overt rheumatoid arthritis. We found six rheumatic pregnant women carriers of the HLA-DRB1 shared epitope peculiar to rheumatoid arthritis and we informed the clinicians in order to properly follow them up. The high frequency of HLA-DRB1* 03 and DRB1*07 in rheumatic patients might be indicative of a silent celiac disease and suggests a need to perform a search for anti-tissue tranglutaminase antibodies.
Relationship between HLA-DQA1, HLA-DQB1 and HLA-DRB1 alleles in immunoglobulin A nephropathy
s. akgul (Istanbul, TR)
Immunoglobulin A nephropathy (IgAN) is the most prevalent primary glomerular disease worldwide and an important cause of end stage renal disease. Data obtained in recent years have shown the ethnic-specific association between certain human leukocyte antigen (HLA) alleles and IgAN susceptibility. This study was designed to explore the relationship between HLA-DQA1, HLA-DQB1 and HLA-DRB1 alleles and disease susceptibility and clinical manifestations of patients with IgAN in Turkish population. A PCR-SSO Luminex typing technique was used to detect HLA-DQA1, HLA-DQB1 and HLA-DRB1 alleles in 265 IgAN patients and 232 healthy subjects. Clinical data were collected from each patient at the time of renal biopsy. Twenty-one HLA-DQA1 alleles, twenty-seven HLA-DQB1 alleles, sixty-one HLA-DRB1 alleles were detected in IgAN patients and healthy subjects. A high frequency of DQA1*01:01 [odds ratio (OR) = 2.22, Pc = 0.0021], DQB1*05:07 (OR = 45.2, Pc = 0.0027) and low frequency of DQA1*01:02 (OR = 0.45, Pc = 0.006), *01:04 (OR = 0.05, Pc = 0.004), *03:03 (OR = 0.04, Pc = 0.002) and DRB1*16:01 (OR = 0.3, Pc = 0.018) were observed in IgAN patients compared with healthy controls. Further stratification analysis revealed that the frequency of DRB1*14:01/54 was higher in patients with urine protein ≥ 1.0 g/24 h than in patients with urine protein < 1.0 g/24 h. But this did not remain significant after application of the Bonferroni correction. ur study indicated that HLA-DQA1*01:01 and DQB1*05:07 alleles may be a potential predictor of high-risk IgAN susceptibility in Turkish populations.
Analysis of HLA-DRB1 and TLR polymorphisms in Spanish Q fever patients
A. Torío Ruiz (Albacete, ES)
Q fever is a bacterial infection caused by Coxiella burnetii that can be spread to humans by infected animals, mainly sheep, cattle and goats. Variations in human immune response genes are well recognized to influence the course of infection, particularly by obligate intracellular pathogens which have a similar cell dependency to that of Coxiella burnetii. The relation between immune response genes variations and disease outcomes may be clarified by the analysis of polymorphic variations in individual candidate genes deduced to be of direct importance in disease pathogenesis. The aim of this study was to determine the possible association of Q fever with 3 polymorphic immune genes (TLR2, TLR4 and HLA-DRB1) in our population. Low resolution HLA-DRB1 typing, TLR2 (Arg753Gln) and TLR4 (Asp299Gly, Thr399Ile) polymorphisms were genotyped in 38 patients with acute Q fever and 38 healthy controls (relatives and patients co-workers). In addition, HLA typing results were compared with a representative sample of 121 donors from the same geographical area (Gran Canaria, Spain). No differences were observed in the three TLR polymorphisms studied. However, when the results of HLA-DRB1 frequencies of the patients were compared with their control group, an increase in the frequency of HLA-DRB1*04 (26.68% vs. 14.67%; p=0.049, pc=ns) and HLA-DRB1*16 (3.5% vs. 0%; p=0.038, pc=ns), as well as a decrease in the frequency of HLA-DRB1*08 (1.32% vs. 6.67%; p=0.044; pc=ns) was observed. Also, when HLA-DRB1 patients frequencies were compared to the general population, a significant association was observed between HLA-DRB1*04 alleles and the presence of Q fever infection (26.68% vs. 12.41%; pc=0.014). In conclusion, HLA-DRB1*04 allele might be associated with an increase in susceptibility to the development of Q fever in our area.