Variation In HLA Class Research Articles

Prediction of differential Gag versus Env responses to a mosaic HIV-1 vaccine regimen by HLA class I alleles.

HLA class I variation has the strongest effect genome-wide on outcome after HIV infection, and as such, an understanding of the impact of HLA polymorphism on response to HIV vaccination may inform vaccine design. We sought HLA associations with HIV-directed immunogenicity in the phase 1/2a APPROACH vaccine trial, which tested vaccine regimens containing mosaic inserts in Ad26 and MVA vectors, with or without a trimeric gp140 protein. While there were no HLA allelic associations with the overall cellular immune response to the vaccine assessed by ELISpot (Gag, Pol, and Env combined), significant associations with differential response to Gag compared to Env antigens were observed. Notably, HLA class I alleles known to associate with disease susceptibility in HIV natural history cohorts are associated with stronger Env-directed responses, whereas protective alleles are associated with stronger Gag-directed responses. Mean viral loads determined for each HLA allele in untreated individuals correlated negatively with the strength of the Gag response minus the Env response in Black vaccinees based on both ELISpot and CD8+ T cell ICS responses. As the association of T cell responses to conserved Gag epitopes with lower viral load in untreated individuals is well established, our data raise the possibility that the Ad26.Mos.HIV vaccine may induce more effective cellular responses in those with HLA alleles that confer improved virologic control in untreated HIV infection.IMPORTANCENo vaccine tested to date has shown sufficient efficacy against HIV infection. A vaccine that induces robust responses in one individual may fail to do so in another individual due to variation in HLA class I genes, loci central to the immune response. Extensive data have shown the strong effect of HLA variation on outcome after HIV infection, but very little is known about the effect of such variation on HIV vaccine success. Here, we identify a link between the effect of HLA variation on HIV disease outcome and immune responses to an HIV vaccine. HLA variants associated with better HIV control after infection also induce stronger responses against the HIV Gag protein relative to the Env protein after vaccination. Given the virologic control conferred by responses to Gag in natural history of HIV infection, these data suggest that HLA alleles conferring protection after HIV infection may also support a more effective cellular response to HIV vaccination.

Association between alleles, haplotypes, and amino acid variations in HLA class II genes and type 1 diabetes in Kuwaiti children.

Type 1 diabetes (T1D) is a complex autoimmune disorder that is highly prevalent globally. The interactions between genetic and environmental factors may trigger T1D in susceptible individuals. HLA genes play a significant role in T1D pathogenesis, and specific haplotypes are associated with an increased risk of developing the disease. Identifying risk haplotypes can greatly improve the genetic scoring for early diagnosis of T1D in difficult to rank subgroups. This study employed next-generation sequencing to evaluate the association between HLA class II alleles, haplotypes, and amino acids and T1D, by recruiting 95 children with T1D and 150 controls in the Kuwaiti population. Significant associations were identified for alleles at the HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci, including DRB1*03:01:01, DQA1*05:01:01, and DQB1*02:01:01, which conferred high risk, and DRB1*11:04:01, DQA1*05:05:01, and DQB1*03:01:01, which were protective. The DRB1*03:01:01~DQA1*05:01:01~DQB1*02:01:01 haplotype was most strongly associated with the risk of developing T1D, while DRB1*11:04-DQA1*05:05-DQB1*03:01 was the only haplotype that rendered protection against T1D. We also identified 66 amino acid positions across the HLA-DRB1, HLA-DQA1, and HLA-DQB1 genes that were significantly associated with T1D, including novel associations. These results validate and extend our knowledge on the associations between HLA genes and T1D in Kuwaiti children. The identified risk alleles, haplotypes, and amino acid variations may influence disease development through effects on HLA structure and function and may allow early intervention via population-based screening efforts.

Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires

AbstractThe functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex–deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.

P696 Genetic predisposition to infliximab immunogenicity in patients with immune-mediated inflammatory diseases – secondary analyses from a randomised clinical trial

Abstract Background Immunogenicity is a leading cause of treatment failure to TNF inhibitors. Genetic variations in HLA class II genes have been suggested to predispose to anti-drug antibody formation, but studies using high-resolution HLA typing as well as characterisation of biologically relevant haplotypes are currently lacking. The aim of this study was to assess associations between HLA loci and immunogenicity to infliximab. Methods Patients with immune-mediated inflammatory diseases (IMID) treated with infliximab (N=612) (120 rheumatoid arthritis, 181 spondyloarthritis, 72 psoriatic arthritis, 114 ulcerative colitis, 80 Crohn disease, and 45 psoriasis) were included in the randomised, Norwegian Drug Monitoring (NOR-DRUM) trial. Neutralising anti-drug antibodies were detected with an automated fluorescence assay. Next generation HLA sequencing at G group resolution were performed and trimmed to 2nd field. Haplotype analyses including the most significant HLA loci were performed, and epitope predictions for the infliximab light- and heavy-chain were assessed. Results Anti-drug antibodies to infliximab were detected in 147 (24 %) of patients. The most significant associations with immunogenicity were with the genes HLA-DQB1 (P=1.4x10-6), -DRB1 (P= 2.7x10-5), -DQA1 (P=0.00017) and -B (P=0.0009). Conditional analyses indicated that HLA-DQB1 represents the primary association(Table 1). Haplotype analyses showed that there was significantly increased risk of immnogenicity in patients carrying the HLA-DQ2 haplotypes DQB1*02:01~DQA1*05:01 (P=1.1x10-6) and DQB1*02:02~DQA1*02:01 (P=0.008), while one protective haplotype DQB1*05:01~DQA1*01:01 (P=0.0005) was identified (Figure 1a-c). Presence of HLA-DQ2 more than doubled the risk of anti-drug antibody formation (HR 2.54, 95%CI 1.82–3.56) (Figure 1d), in a model including the covariates age, sex, diagnosis, and concomitant immunosuppressive therapy. Association tests of each level of HLA analyses showed that HLA-DQ2 represents the strongest association(Figure 2). Epitope predictions revealed that the HLA-DQ2 haplotypes DQB1*02:01-DQA1*05:01 and DQB1*02:02-DQA1*02:01 share nine strong binder peptides, originating from the infliximab light chain, encompassing the amino acid core-peptide sequences INTVESEDI and VYACEVTHQ. Conclusion Using high-resolution HLA sequencing, we here demonstrate a genetic association between HLA-DQ2 and infliximab immunogenicity in IMID patients. The presence of either HLA-DQ2 risk haplotypes more than doubled the risk of anti-drug antibody formation across all diagnoses. Testing for HLA-DQ2 could be of clinical value in identifying patients at risk for immunogenicity and facilitate treatment decisions. Whether these findings apply to other TNF inhibitors should be further explored.

HLA-A*32:01 is strongly associated with vancomycin-induced drug reaction with eosinophilia and systemic symptoms

Vancomycin is a prevalent cause of the severe hypersensitivity syndrome drug reaction with eosinophilia and systemic symptoms (DRESS), which leads to significant morbidity and mortality and commonly occurs in the setting of combination antibiotic therapy, affecting future treatment choices. Variations in HLA class I in particular have been associated with serious T cell-mediated adverse drug reactions, which has led to preventive screening strategies for some drugs. We sought to determine whether variation in the HLA region is associated with vancomycin-induced DRESS. Probable vancomycin-induced DRESS cases were matched 1:2 with tolerant control subjects based on sex, race, and age by using BioVU, Vanderbilt's deidentified electronic health record database. Associations between DRESS and carriage of HLA class I and II alleles were assessed by means of conditional logistic regression. An extended sample set from BioVU was used to conduct a time-to-event analysis of those exposed to vancomycin with and without the identified HLA risk allele. Twenty-three subjects met the inclusion criteria for vancomycin-associated DRESS. Nineteen (82.6%) of 23 cases carried HLA-A*32:01 compared with 0 (0%) of 46 of the matched vancomycin-tolerant control subjects (P=1×10-8) and 6.3% of the BioVU population (n=54,249, P=2×10-16). Time-to-event analysis of DRESS development during vancomycin treatment among the HLA-A*32:01-positive group indicated that 19.2% had DRESS and did so within 4weeks. HLA-A*32:01 is strongly associated with vancomycin-induced DRESS in a population of predominantly European ancestry. HLA-A*32:01 testing could improve antibiotic safety, help implicate vancomycin as the causal drug, and preserve future treatment options with coadministered antibiotics.

HLA and MuSK-positive myasthenia gravis: A systemic review and meta-analysis.

Myasthenia gravis (MG) represents a spectrum of clinical subtypes with differences in disease mechanisms and treatment response. MG with muscle-specific tyrosine kinase (MuSK) antibodies accounts for 1%-10% of all MG patients. We conducted a meta-analysis to evaluate the association between HLA genes and MuSK-MG susceptibility. Studies were searched in Pubmed, EMBASE database and other sources between 2001 and 2018. Genotype, allele and haplotype frequencies of HLA loci in MuSK-MG patients and healthy controls were extracted from each included study. The meta-analysis showed that HLA DQB1*05, DRB1*14 and DRB1*16 were strongly associated with an increased risk of MuSK-MG (P<.0001), whereas HLA DQB*03 was less frequent in MuSK patients compared with healthy controls (P<.05). Haplotype analysis showed that these DQB1 and DRB1 alleles were closely linked, forming both risk (DQ5-DR14, DQ5-DR16, P<.0001) and protective (DQ3-DR4, DQ3-DR11, P<.05) haplotypes. The distinct genetic patterns of MuSK-MG indicate that variation in HLA class II genes plays an important role in the pathogenesis of MuSK-MG patients.

ES 04.01 Novel Targetable Oncogenes in Lung Cancer

Aberrations of oncogenes, such as EGFR mutation; and ALK and ROS1 fusions, function as a driver in the development of lung adenocarcinoma (LADC) and are established therapeutic targets. We previously identified RET fusion present in 1-2% of LADC (Kohno, Ichikawa, Nat Med, 2012). Its activity as an oncogenic driver in lung carcinogenesis was validated by a study of transgenic mice expressing KIF5B-RET cDNA in their lungs (Saito, Carcinogenesis, 2014). RET fusion is likely to be another target of therapy using tyrosine kinase inhibitors, as represented by a high response rate of RET-fusion positive LADC to vandetanib (LURET study, Yoh et al., Lancet Resp Med, 2016). Our genome-wide sequencing study revealed that ALK, RET and ROS1 oncogene fusion-positive LADCs carry less numbers of mutations in cancer-related genes than others (Saito, Cancer Res, 2015), indicating a small mutation burden in the development of the formers. On the other hand, >30% of LADC and most of other types of lung cancers are negative for the oncogene aberrations above, therefore, other therapeutic targets are needed for precision lung cancer medicine. We have revealed frequent inactivation of chromatin-regulating genes, such as SMARCA4/BRG1 and CREBBP, in lung cancers negative for oncogene aberrations (Ogiwara et al, Cancer Discovery, 2016). We propose a synthetic lethal therapeutic method for chromatin regulator-deficient lung cancers based on inhibition of paralog proteins. LADC driven by somatic EGFR mutations is more prevalent in East Asians (30-50%) than in European/Americans (10-20%). We recently revealed that variations in HLA-class II loci underlie the risk of the disease, by conducting a genome-wide association study of 3,173 EGFR-LADC patients and 15,158 controls (Shiraishi et al., Nature Comm, 2016). The result indicates that LADC develops in vivo through interaction between somatic oncogene mutations and germline variations that modulate immune reaction. We would like to discuss here precision lung cancer medicine based on information on cancer and the host genomes. RET fusion, synthetic lethal therapy, lung cancer risk

Abstract 1432: Precision lung cancer medicine based on information on cancer and the host genomes

Abstract Aberrations of oncogene, such as EGFR mutation; and ALK, RET, and ROS1 fusions, function as a driver in the development of lung adenocarcinoma (LADC). They are a solid target of therapy using tyrosine kinase inhibitors, as represented by a high response rate of RET-fusion positive LADC to vandetanib (LURET study, Yoh et al., Lancet Resp Med, 2016). Our genome-wide sequencing study revealed that oncogene aberration positive LADCs carry less numbers of mutations in cancer-related genes than negative ones (Saito, Cancer Res, 2015), indicating a small mutation burden in the development of the formers. Consistently, a clinical trial of anti-PD-1 therapy indicated that EGFR-mutation positive cases do not benefit well from the therapy (Borghaei et al., N Engl J Med. 2015). LADC driven by somatic EGFR mutations is more prevalent in East Asians (30-50%) than in European/Americans (10-20%). We recently revealed that variations in HLA-class II loci underlie the risk of the disease, by conducting a genome-wide association study of 3,173 patients and 15,158 controls, combined with imputation of HLA alleles using a Japanese-specific panel (Shiraishi et al., Nature Comm, 2016). The result indicates that LADC develops in vivo through interaction between somatic mutations and germline variations that modulate immune reaction. Therefore, efficacy of immune checkpoint blockade targeting therapy might also be influenced by such germline variations. On the other hand, oncogene-negative LADCs show a high mutation burden associated with nonsynonymous mutations in chromatin remodeling and other cancer-related genes. Thus, these cases would respond well to immune checkpoint blockade targeting therapies. We would like to discuss here precision lung cancer medicine based on information on cancer and the host genomes. Citation Format: Takashi Kohno, Kouya Shiraishi, Kazushi Yoshida, Motonobu Saito, Yukinori Okada. Precision lung cancer medicine based on information on cancer and the host genomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1432. doi:10.1158/1538-7445.AM2017-1432

Abstract B25: Association of variations in HLA-class II and other loci with susceptibility to EGFR-mutated lung adenocarcinoma

Abstract Background: Lung adenocarcinoma (LADC) driven by somatic EGFR mutations is more prevalent in East Asians (30-50%) than in European/Americans (10-20%). Understanding the genetic factors underlying such LADC is required to elucidate disease etiology and to identify effective methods of prevention. Methods: We investigate genetic factors underlying the risk of this disease by conducting a genome-wide association study, followed by two validation studies, in 3,173 Japanese patients with EGFR mutation-positive lung adenocarcinoma and 15,158 controls. Results: Four loci, 5p15.33 (TERT), 6p21.3 (BTNL2, HLA-class II), 3q28 (TP63) and 17q24.2 (BPTF), previously shown to be strongly associated with overall lung adenocarcinoma risk in East Asians, were re-discovered as loci associated with a higher susceptibility to EGFR mutation-positive. Citation Format: Kouya Shiraishi, Yukinori Okada, Atsushi Takahashi, Yoichiro Kamatani, Kyota Ashikawa, Yukihide Momozawa, Hideo Kunitoh, Shingo Matsumoto, Atsushi Takano, Kimihiro Shimizu, Akiteru Goto, Koji Tsuta, Shun-ichi Watanabe, Yuichiro Ohe, Yukio Watanabe, Yasishi Goto, Hiroshi Nokihara, Koh Furuta, Akihiko Yoshida, Koichi Goto, Tomoyuki Hishida, Masahiro Tsuboi, Katsuya Tsuchihara, Yohei Miyagi, Haruhiko Nakamura, Tomoyuki Yokose, Katsuya Tanaka, Toshiteru Nagashima, Yoichi Ohtaki, Daichi Maeda, Kazuhiro Imai, Yoshihiro Minamiya, Hiromi Sakamoto, Akira Saito, Yoko Shimada, Kuniko Sunami, Motonobu Saito, Teruhiko Yoshida, Jun Yokota, Fumihiko Matsuda, Keitaro Matsuo, Yataro Daigo, Michiaki Kubo, Takashi Kohno. Association of variations in HLA-class II and other loci with susceptibility to EGFR-mutated lung adenocarcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr B25.

OA10.02 Association of Variations in HLA-Class II and Other Loci with Susceptibility to EGFR-Mutated Lung Adenocarcinoma

Lung adenocarcinoma (LADC) driven by somatic EGFR mutations is more prevalent in East Asians (30-50%) than in European/Americans (10-20%). Understanding the genetic factors underlying such LADC is required to elucidate disease etiology and to identify effective methods of prevention. We investigate genetic factors underlying the risk of this disease by conducting a genome-wide association study, followed by two validation studies, in 3,173 Japanese patients with EGFR mutation-positive lung adenocarcinoma and 15,158 controls. Four loci, 5p15.33 (TERT), 6p21.3 (BTNL2, HLA-class II), 3q28 (TP63) and 17q24.2 (BPTF), previously shown to be strongly associated with overall lung adenocarcinoma risk in East Asians, were re-discovered as loci associated with a higher susceptibility to EGFR mutation-positive lung adenocarcinoma. In addition, two additional loci, HLA-class II at 6p21.32 and 6p21.1 (FOXP4) were newly identified as loci associated with EGFR mutation-positive lung adenocarcinoma (Shiraishi et al., Nature Communications, 2016, in press). This study indicates that multiple genetic factors, including an immunologic one, underlie the risk of lung adenocarcinomas with EGFR mutations.

The Presence, Persistence and Functional Properties of Plasmodium vivax Duffy Binding Protein II Antibodies Are Influenced by HLA Class II Allelic Variants.

BackgroundThe human malaria parasite Plasmodium vivax infects red blood cells through a key pathway that requires interaction between Duffy binding protein II (DBPII) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). A high proportion of P. vivax-exposed individuals fail to develop antibodies that inhibit DBPII-DARC interaction, and genetic factors that modulate this humoral immune response are poorly characterized. Here, we investigate if DBPII responsiveness could be HLA class II-linked.Methodology/Principal FindingsA community-based open cohort study was carried out in an agricultural settlement of the Brazilian Amazon, in which 336 unrelated volunteers were genotyped for HLA class II (DRB1, DQA1 and DQB1 loci), and their DBPII immune responses were monitored over time (baseline, 6 and 12 months) by conventional serology (DBPII IgG ELISA-detected) and functional assays (inhibition of DBPII–erythrocyte binding). The results demonstrated an increased susceptibility of the DRB1*13:01 carriers to develop and sustain an anti-DBPII IgG response, while individuals with the haplotype DRB1*14:02-DQA1*05:03-DQB1*03:01 were persistent non-responders. HLA class II gene polymorphisms also influenced the functional properties of DBPII antibodies (BIAbs, binding inhibitory antibodies), with three alleles (DRB1*07:01, DQA1*02:01 and DQB1*02:02) comprising a single haplotype linked with the presence and persistence of the BIAbs response. Modelling the structural effects of the HLA-DRB1 variants revealed a number of differences in the peptide-binding groove, which is likely to lead to altered antigen binding and presentation profiles, and hence may explain the differences in subject responses.Conclusions/SignificanceThe current study confirms the heritability of the DBPII antibody response, with genetic variation in HLA class II genes influencing both the development and persistence of IgG antibody responses. Cellular studies to increase knowledge of the binding affinities of DBPII peptides for class II molecules linked with good or poor antibody responses might lead to the development of strategies for controlling the type of helper T cells activated in response to DBPII.

Association of variations in HLA class II and other loci with susceptibility to EGFR-mutated lung adenocarcinoma.

Lung adenocarcinoma driven by somatic EGFR mutations is more prevalent in East Asians (30–50%) than in European/Americans (10–20%). Here we investigate genetic factors underlying the risk of this disease by conducting a genome-wide association study, followed by two validation studies, in 3,173 Japanese patients with EGFR mutation-positive lung adenocarcinoma and 15,158 controls. Four loci, 5p15.33 (TERT), 6p21.3 (BTNL2), 3q28 (TP63) and 17q24.2 (BPTF), previously shown to be strongly associated with overall lung adenocarcinoma risk in East Asians, were re-discovered as loci associated with a higher susceptibility to EGFR mutation-positive lung adenocarcinoma. In addition, two additional loci, HLA class II at 6p21.32 (rs2179920; P =5.1 × 10−17, per-allele OR=1.36) and 6p21.1 (FOXP4) (rs2495239; P=3.9 × 10−9, per-allele OR=1.19) were newly identified as loci associated with EGFR mutation-positive lung adenocarcinoma. This study indicates that multiple genetic factors underlie the risk of lung adenocarcinomas with EGFR mutations.

Host genetic variants influencing the clinical course of hepatitis B virus infection.

The clinical course of hepatitis B virus (HBV) infection greatly differs in individuals. Various viral, host, and environmental factors influence the natural history of HBV infection. Recent genome-wide association studies identified several host genetic factors influencing the clinical course of HBV infection. Genetic variations in HLA class II loci were significantly associated with susceptibility to persistent HBV infection. Other polymorphisms in or near the genes EHMT2, TCF19, and HLA-C, located near HLA class II loci, and UBE2L3 were also associated with persistent HBV infection. Meanwhile, polymorphisms in KIF1B, GRIK1, and STAT4 were associated with HBV-related hepatocellular carcinoma (HCC). Interestingly, HLA class II genetic variations were strongly associated with not only persistent HBV infection, but also disease progression and HBV-related HCC in chronic hepatitis B. Understanding the various genetic factors associated with the clinical course of HBV infection is essential for personalized treatment and surveillance of disease progression and HCC.

Interactions between household air pollution and GWAS-identified lung cancer susceptibility markers in the Female Lung Cancer Consortium in Asia (FLCCA).

We previously carried out a multi-stage genome-wide association study (GWAS) on lung cancer among never smokers in the Female Lung Cancer Consortium in Asia (FLCCA) (6,609 cases, 7,457 controls) that identified novel susceptibility loci at 10q25.2, 6q22.2, and 6p21.32, and confirmed two previously identified loci at 5p15.33 and 3q28. Household air pollution (HAP) attributed to solid fuel burning for heating and cooking, is the leading cause of the overall disease burden in Southeast Asia, and is known to contain lung carcinogens. To evaluate the gene-HAP interactions associated with lung cancer in loci independent of smoking, we analyzed data from studies participating in FLCCA with fuel use information available (n = 3; 1,731 cases; 1,349 controls). Coal use was associated with a 30% increased risk of lung cancer (OR 1.3, 95% CI 1.0-1.6). Among the five a priori SNPs identified by our GWAS, two showed a significant interaction with coal use (HLA Class II rs2395185, p = 0.02; TP63 rs4488809 (rs4600802), p = 0.04). The risk of lung cancer associated with coal exposure varied with the respective alleles for these two SNPs. Our observations provide evidence that genetic variation in HLA Class II and TP63 may modify the association between HAP and lung cancer risk. The roles played in the cell cycle and inflammation pathways by the proteins encoded by these two genes provide biological plausibility for these interactions; however, additional replication studies are needed in other non-smoking populations.

HLA class I and KIR genes do not protect against HIV type 1 infection in highly exposed uninfected individuals with hemophilia A.

A recent genome-wide association study (GWAS) involving patients with hemophilia A who were exposed to but uninfected with human immunodeficiency virus type 1 (HIV-1) did not reveal genetic variants associated with resistance to HIV-1 infection, beyond homozygosity for CCR5-Δ32. Since variation in HLA class I and KIR genes is not well interrogated by standard GWAS techniques, we tested whether these 2 loci were involved in protection from HIV-1 infection in the same hemophilia cohort, using controls from the general population. Our data indicate that HLA class I alleles, presence or absence of KIR genes, and functionally relevant combinations of the HLA/KIR genotypes are not involved in resistance to parenterally transmitted HIV-1 infection.

6-P

Flow cytometric crossmatches (FCXM) are used to detect low-level donor-specific antibody and determine transplantation eligibility. We measured HLA class I and II surface expression using different lymphocyte sources (peripheral blood (PBL), spleen, or nodes) to investigate the impact of HLA expression on detecting low-level donor specific antibody. HLA expression was measured on a BD FACSCanto II using fluorescently conjugated antibodies specific for monomorphic determinants on HLA class I and II; measurements were converted to Molecules of Soluble Fluorescence (MESF) units. FCXM reactivity was determined using the ratio of test serum to negative control serum and MESF units. Compared to Live Donor (LD) PBLs (n=249), mean HLA class I expression on lymphocytes isolated from Deceased Donor (DD) (n=25) spleen and lymph nodes was lower (5.8% and 10.4%, respectively). The mean HLA class II expression was significantly lower on DD splenic (90.4%, p < 0.05) and lymph node (125.6%, p < 0.05) lymphocytes. We then compared lymphocytes isolated from the same DD (n=4) to better assess the variability of HLA expression on cells derived from different tissues. Compared to DD PBL, HLA class I expression was higher on splenic (54%) and lymph node lymphocytes (34%). HLA class II expression on DD PBLs was lower than that from spleen (63%), but higher than the lymph node-derived lymphocytes (99%). Variation in HLA class II expression on cells from different tissues also impacted crossmatch reactivity. HLA class II donor specific sera, expected to yield a positive FCXM, consistently showed the highest reactivity with splenic lymphocytes; intermediate reactivity with PBL; and the lowest reactivity with lymph node cells. HLA expression may not be routinely analyzed; however, it can substantially impact crossmatch reactivity. Laboratories using a single positive threshold for all FCXMs should consider that splenic lymphocytes may yield reactivity most comparable to that of PBLs from LDs.

HLA class II susceptibility to cervical cancer among Tunisian women

The variability in host immunogenetic background, especially in human major histocompatibilty genes, has been shown to influence the susceptibility to human papillomavirus (HPV) infection and cervical neoplasia. Here, we conducted a case-control study in Tunisian women to examine the effect of genetic variation in HLA class II DRB1 and DQB1 genes on invasive cervical cancer (ICC) and squamous cell carcinoma (SCC). HLA genotyping was performed by PCR sequence-specific primers technique. The data revealed significant positive and negative associations, suggesting either predisposing or protective effects of these genes in the disease outcome. DRB1*15, alone or linked to DQB1*06, was associated with a 2.7- and 3.5-fold increase in risk for ICC, respectively. DRB1*13-DQB1*03 showed a similar 3.5 risk effect. Concerning SCC, we observed a relatively higher, about 1.2 times more, risk effect for these genetic markers. In contrast, only one haplotype – DRB1*13-DQB1*06 – provides evidence for a weak protection (about 0.3-fold reduction) of ICC and SCC. In conclusion, we suggest that HLA class II polymorphisms are involved in the genetic susceptibility to cervical cancer in Tunisian women.

Variation in HLA Class I Antigen‐Processing Genes and Susceptibility to Human Papillomavirus Type 16–Associated Cervical Cancer

Persistent infection with human papillomavirus type 16 (HPV16) is a primary etiological factor for the development of cervical cancer. Genes involved in antigen processing influence both the repertoire of antigens presented by HPV16-infected cells and the nature of HPV16-specific immune responses. Genetic variation in these genes may affect protein structure and function and, consequently, the ability of an individual to clear HPV infection. Thirty-five single-nucleotide polymorphisms (SNPs) in 5 genes (LMP2, TAP1, LMP7, TAP2, and Tapasin) were investigated for association with susceptibility to HPV16-associated cervical cancer. Sequencing of these genes resulted in the discovery of 15 previously unreported SNPs. Microsphere-array flow cytometry-based genotyping was conducted on 787 samples from Hispanic and non-Hispanic white women (241 randomly selected control subjects, 205 HPV16-positive control subjects, and 341 HPV16-positive case subjects with cervical cancer). For 9 SNPs, 8 of which had not previously been reported in the context of cervical cancer, there were statistically significant differences between the genotype distribution in case subjects and that in control subjects. Haplotype analysis of 3 haplotype blocks revealed 3 haplotypes with significant differences in frequency in case-control comparisons. Both HPV16-specific and non-type-specific differences in genotype distribution were seen. Genes involved in antigen processing for HLA class I presentation may contribute to susceptibility to cervical cancer.

DRB4 promoter polymorphism in DR7 individuals: correlation with DRB4 pre-mRNA and mRNA levels.

DRB genes encode proteins which play an important role in the immune response, and variation in their expression levels may be of functional immunological significance. To date, HLA class II gene promoter polymorphism has been analyzed almost exclusively in Epstein-Barr virus (EBV)-transformed B-cell lines. While previous studies have established important paradigms with regard to the mechanisms in DRB gene expression, the role of DRB4 promoter polymorphism in the variation of DRB4 expression levels, if any, is still unclear. We analyzed the effects of a previously described polymorphism in the promoter and 5' untranslated region of the DRB4 locus in peripheral blood mononuclear cells (PBMCs) from nine healthy DR7 individuals using the RNase protection assay. The RNase protection analyses showed that the location of transcription initiation is not affected. The DRB4 alleles show a threefold difference in pre-mRNA and a corresponding fivefold difference in mRNA levels in PBMCs. These data show that the DRB4 promoter polymorphism is associated with differential gene expression of this locus. The difference in pre-mRNA levels is directly affected by this DRB4 promoter polymorphism. The additional difference in mRNA levels is the result of post-transcriptional regulation. Therefore, the DRB4 promoter polymorphism may explain the previously observed variation in HLA class II DRB4 gene expression levels among different healthy DR7 individuals.

O38 - Variations in HLA class I promoter sequences and differential regulation of HLA-A, -B, and -C alleles

O38 - Variations in HLA class I promoter sequences and differential regulation of HLA-A, -B, and -C alleles