ĐA HÌNH ĐƠN NUCLEOTIDE RS2856718 CỦA GEN HLA-DQ TRÊN BỆNH NHÂN UNG THƯ BIỂU MÔ TẾ BÀO GAN

Ung thư gan nguyên phát là bệnh ung thư phổ biến và có tỉ lệ tử vong cao. Gen HLA-DQ mã hóa các chuỗi polypeptid của phân tử MHC lớp II tham gia hoạt hóa tế bào trình diện kháng nguyên trong đáp ứng miễn dịch. Đa hình đơn nucleotide (SNP) rs2856718 của gen HLA-DQ có thể liên quan tới nhiễm virus viêm gan B mãn tính và nguy cơ ung thư biểu mô tế bào gan. Nghiên cứu này được thực hiện nhằm mục tiêu xác định sự phân bố của SNP rs2856718, đồng thời đánh giá mối liên quan giữa SNP này với nguy cơ mắc ung thư biểu mô tế bào gan. Kỹ thuật RT-PCR được sử dụng để xác định kiểu gen SNP rs2856718 trên 93 bệnh nhân ung thư biểu mô tế bào gan và 93 người khỏe mạnh. Kết quả đã xác định được tỷ lệ kiểu gen GG, AG, AA ở nhóm bệnh là 39,8%, 46,2%, 14% và nhóm chứng là 30,1%, 53,8%, 16,1%. Chưa tìm thấy mối liên quan giữa SNP rs2856718 với nguy cơ ung thư biểu mô tế bào gan cũng như với một số yếu tố nguy cơ khác.

HLA-DQ-Specific Recombinant Human Monoclonal Antibodies Allow for In-Depth Analysis of HLA-DQ Epitopes

HLA-DQ donor-specific antibodies (DSA) are the most prevalent type of DSA after renal transplantation and have been associated with eplet mismatches between donor and recipient HLA. Eplets are theoretically defined configurations of surface exposed amino acids on HLA molecules that require verification to confirm that they can be recognized by alloantibodies and are therefore clinically relevant. In this study, we isolated HLA-DQ specific memory B cells from immunized individuals by using biotinylated HLA-DQ monomers to generate 15 recombinant human HLA-DQ specific monoclonal antibodies (mAb) with six distinct specificities. Single antigen bead reactivity patterns were analyzed with HLA-EMMA to identify amino acids that were uniquely shared by the reactive HLA alleles to define functional epitopes which were mapped to known eplets. The HLA-DQB1*03:01-specific mAb LB_DQB0301_A and the HLA-DQB1*03-specific mAb LB_DQB0303_C supported the antibody-verification of eplets 45EV and 55PP respectively, while mAbs LB_DQB0402_A and LB_DQB0602_B verified eplet 55R on HLA-DQB1*04/05/06. For three mAbs, multiple uniquely shared amino acid configurations were identified, warranting further studies to define the inducing functional epitope and corresponding eplet. Our unique set of HLA-DQ specific mAbs will be further expanded and will facilitate the in-depth analysis of HLA-DQ epitopes, which is relevant for further studies of HLA-DQ alloantibody pathogenicity in transplantation.

High cell surface expression and peptide binding affinity of HLA-DQA1*05:03, a susceptible allele of neuromyelitis optica spectrum disorders (NMOSD)

Neuromyelitis optica spectrum disorder (NMOSD) is a relapsing autoimmune disease characterized by the presence of pathogenic autoantibodies, anti-aquaporin 4 (AQP4) antibodies. Recently, HLA-DQA1*05:03 was shown to be significantly associated with NMOSD in a Japanese patient cohort. However, the specific mechanism by which HLA-DQA1*05:03 is associated with the development of NMOSD has yet to be elucidated. In the current study, we revealed that HLA-DQA1*05:03 exhibited significantly higher cell surface expression levels compared to other various DQA1 alleles, and that its expression strongly depended on the amino acid sequence of the α1 domain, with a preference for leucine at position 75. Moreover, in silico analysis indicated that the HLA-DQ encoded by HLA-DQA1*05:03 preferentially presents immunodominant AQP4 peptides, and that the peptide major histocompatibility complexes (pMHCs) are more energetically stable in the presence of HLA-DQA1*05:03 than other HLA-DQA1 alleles. In silico 3D structural models were also applied to investigate the validity of the energetic stability of pMHCs. Taken together, our findings indicate that HLA-DQA1*05:03 possesses a distinct property to play a pathogenic role in the development of NMOSD.

Comprehensive Analysis of CD4+ T Cell Response Cross-Reactive to SARS-CoV-2 Antigens at the Single Allele Level of HLA Class II

Common human coronaviruses have been circulating undiagnosed worldwide. These common human coronaviruses share partial sequence homology with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); therefore, T cells specific to human coronaviruses are also cross-reactive with SARS-CoV-2 antigens. Herein, we defined CD4+ T cell responses that were cross-reactive with SARS-CoV-2 antigens in blood collected in 2016–2018 from healthy donors at the single allele level using artificial antigen-presenting cells (aAPC) expressing a single HLA class II allotype. We assessed the allotype-restricted responses in the 42 individuals using the aAPCs matched 22 HLA-DR alleles, 19 HLA-DQ alleles, and 13 HLA-DP alleles. The response restricted by the HLA-DR locus showed the highest magnitude, and that by HLA-DP locus was higher than that by HLA-DQ locus. Since two alleles of HLA-DR, -DQ, and -DP loci are expressed co-dominantly in an individual, six different HLA class II allotypes can be used to the cross-reactive T cell response. Of the 16 individuals who showed a dominant T cell response, five, one, and ten showed a dominant response by a single allotype of HLA-DR, -DQ, and -DP, respectively. The single allotype-restricted T cells responded to only one antigen in the five individuals and all the spike, membrane, and nucleocapsid proteins in the six individuals. In individuals heterozygous for the HLA-DPA and HLA-DPB loci, four combinations of HLA-DP can be expressed, but only one combination showed a dominant response. These findings demonstrate that cross-reactive T cells to SARS-CoV-2 respond with single-allotype dominance.

Epitope Based Vaccine Peptide Predictions for fimH gene of Acinetobacter baumannii

Background: Acinetobacter baumannii is an opportunistic bacterial pathogen that is primarily associated with hospital-acquired infections. Recently, there is a dramatic increase in the incidence of multidrug-resistant (MDR) strains, which has significantly raised the profile of this emerging opportunistic pathogen. MDR is often associated with the formation of biofilms and various other virulence factors. Amidst all the genes, fimH gene is our area of interest in this research, because it is an important virulence factor in A. baumannii which encodes the Type 1 fimbriae, that helps bacteria bind to the surface of host cells initiating further infection. Aim: The aim of this study was to assess the epitope based vaccine epitope peptide predictions for the fimH protein of A. baumannii. Materials and Methods: The fimH gene for epitope prediction was selected. Druggability and physico-chemical properties were analysed. Antigenicity was predicted. Epitope mapping of T-cell MHC class 1, Class 1 immunogenicity, conservancy and toxicity analysis was done. T-cell class II epitopes were further mapped together with the immuno-dominant B-cell epitopes. Results: From the selected 20 epitopes, 2 best epitopes (AALVASVCL and YSSGANAFT) were selected after analysing their antigenicity and allergenicity. The epitope YSSGANAFT showed better values in association with HLA alleles - HLA-DP, HLA-DQ, HLA-DR and TLR-2. Conclusion: The finding of the study documents a single immunodominant peptide (sequence) as a promising vaccine candidate to treat infections caused by A. baumannii. However further experimental analysis must be performed to assess the immunological memory and response of the peptide in both in-vitro and in-vivo studies.

BpOmpW Antigen Stimulates the Necessary Protective T-Cell Responses Against Melioidosis

Melioidosis is a potentially fatal bacterial disease caused by Burkholderia pseudomallei and is estimated to cause 89,000 deaths per year in endemic areas of Southeast Asia and Northern Australia. People with diabetes mellitus are most at risk of melioidosis, with a 12-fold increased susceptibility for severe disease. Interferon gamma (IFN-γ) responses from CD4 and CD8 T cells, but also from natural killer (NK) and natural killer T (NKT) cells, are necessary to eliminate the pathogen. We previously reported that immunization with B. pseudomallei OmpW (BpOmpW antigen) protected mice from lethal B. pseudomallei challenge for up to 81 days. Elucidating the immune correlates of protection of the protective BpOmpW vaccine is an essential step prior to clinical trials. Thus, we immunized either non-insulin-resistant C57BL/6J mice or an insulin-resistant C57BL/6J mouse model of type 2 diabetes (T2D) with a single dose of BpOmpW. BpOmpW induced strong antibody responses, stimulated effector CD4+ and CD8+ T cells and CD4+ CD25+ Foxp3+ regulatory T cells, and produced higher IFN-γ responses in CD4+, CD8+, NK, and NKT cells in non-insulin-resistant mice. The T-cell responses of insulin-resistant mice to BpOmpW were comparable to those of non-insulin-resistant mice. In addition, as a precursor to its evaluation in human studies, humanized HLA-DR and HLA-DQ (human leukocyte antigen DR and DQ isotypes, respectively) transgenic mice elicited IFN-γ recall responses in an enzyme-linked immune absorbent spot (ELISpot)-based study. Moreover, human donor peripheral blood mononuclear cells (PBMCs) exposed to BpOmpW for 7 days showed T-cell proliferation. Finally, plasma from melioidosis survivors with diabetes recognized our BpOmpW vaccine antigen. Overall, the range of approaches used strongly indicated that BpOmpW elicits the necessary immune responses to combat melioidosis and bring this vaccine closer to clinical trials.

Sudomotor dysfunction in patients with gluten neuropathy

Background and aim Gluten neuropathy (GN) is a common neurological manifestation of gluten sensitivity (GS), characterized by serological evidence of GS, while other risk factors for developing neuropathy are absent. The degree of small fiber dysfunction in GN has not been studied in depth to date. Small fiber involvement may lead to pain, thermal perception abnormalities, and sweat gland dysfunction. Sudomotor innervation refers to the cholinergic innervation of the sympathetic nervous system through small fibers in the sweat glands. The aim of our study was to assess the sudomotor function of GN patients. Methods Patients with GN were recruited. Clinical and neurophysiological data were obtained. HLA-DQ genotyping was performed. The skin electrochemical conductance (ESC) was measured with SUDOSCANTM. Results Thirty-two patients (25 males, mean age 69.5±10.2 years) were recruited. Thirteen patients (40.6%) had abnormal sudomotor function of the hands. Sixteen patients (50%) had abnormal sudomotor function of the feet. Twenty-one patients (65.6%) had abnormal sudomotor function of either the hands or feet. Sudomotor dysfunction did not correlate with the type of neuropathy (length-dependent neuropathy or sensory ganglionopathy), gluten-free diet adherence, severity of neuropathy, and duration of disease or HLA-DQ genotype. No differences in the ESC were found between patients with painful and patients with painless GN. Conclusion Sudomotor dysfunction affects two-thirds of patients with GN. The lack of correlation between pain and sudomotor dysfunction suggests different patterns of small fiber involvement in patients with GN.

The Impact of Hypomethylating Agents and BCL-2 Inhibitor on HLA Expression on THP-1 Cells

Note: BP, MV and LG, KG contributed equally Background Relapsed acute myeloid leukemia (AML) remains the most common reason for allogeneic hematopoietic cell transplant (HCT) failure. Thus, understanding AML immune escape mechanism is important for improving the odds of curing HCT patients with AML. Downregulation of HLA Class I and II expression by AML is one of the potential immune escape mechanisms. Therefore, treatment to restore HLA surface expression is crucial to prevent and treat relapse. Endogenous cytokines, such as IFN-γ, have been shown to stimulate HLA expression but are poorly tolerated by patients. However, two hypomethylating agents (HMA), decitabine (Dec) and azacitadine (Aza), that are routinely used in AML treatment are known to augment HLA expression. For AML, HMAs are often combined with venetoclax (Ven), a drug that blocks the anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein. Thus, while HMAs have been reported to increase HLA expression, what is unknown is whether these agents impact individual HLA loci differently and whether Ven has any impact on HLA expression. To address these questions, we treated the THP-1 cell line with Dec, Aza or Ven and measured changes in cell-surface expression of HLA proteins by flow cytometry using locus-specific HLA mAbs. Methods THP-1 cells were incubated with IFN-γ (500 U/mL), Aza (2µM), Dec (5µM), or Ven (30nM) for 48 hours (drug concentrations were determined by earlier titration experiments). THP-1 cells are a monocytic cell line, derived from the peripheral blood of a childhood case of acute monocytic leukemia (M5 subtype), that express HLA Class I and HLA-DR but not HLA-DQ or -DP under basal conditions, although they are inducible by IFN-γ. Thus, the induction of HLA Class II expression by IFN-γ serves as a positive control. Isotype controls were included to measure background. Data is presented as the difference in MFI (delta MFI) between cells treated with a drug and those treated with diluent only. Results Treatment of THP-1 cells with either IFN-γ or Dec led to increases in Class I HLA-A, -B & -C (Figure 1) compared to untreated cells (a mean fold increase of 1.4 and 1.2, respectively). Notably, Aza did not stimulate additional HLA-C expression and induced less of an increase in HLA-A & -B expression (an increase of 1.1-fold) than IFN-γ or Dec. Treatment of THP-1 cells by Ven did not induce a change in HLA Class I expression. For Class II, IFN-γ or Dec increased HLA-DR, -DQ and -DP expression in comparison to untreated cells (Figure 1). IFN-γ induced greater HLA-DR expression compared to Dec (an increase of 2.3-fold and 1.5-fold, respectively), and both stimulated similar increases in HLA-DQ (increases of 1.5-fold and 1.4-fold, respectively) & -DP (increases of 1.9-fold and 1.5-fold, respectively). However, treatment of cells with either Aza or Ven did not lead to changes in HLA Class II expression. Discussion Previous studies have illustrated the ability of IFN-γ to induce HLA Class II expression in THP-1 cells, however, data for Dec to induce HLA Class II expression was unconfirmed. We report differences in the degree to which IFN-γ and Dec are capable of stimulating HLA-DR with IFN-γ being more potent. The inability of Aza to induce HLA Class II expression in THP-1 cells may be related to the differing drug activating pathways of the two HMAs. Indeed, there are conflicting reports as to whether Aza can stimulate HLA Class II expression. Though Ven treatment of THP-1 cells did not impact HLA expression, because it is given with HMAs, it remains to be seen what effect these drugs may have on HLA expression when administered together. Additional studies to confirm these observations in patient-derived AML blasts are ongoing. Conclusion We report that HMAs increased expression of HLA-A, -B, & -C loci and Dec but not Aza stimulated HLA-DR, -DQ, and -DP expression in THP-1 cells. Given these data, Dec may be superior in increasing HLA Class II expression post-HCT. Figure 1 Figure 1. Disclosures Marcucci: Abbvie: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Novartis: Other: Speaker and advisory scientific board meetings. Al Malki: Neximmune: Consultancy; CareDx: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy.

Prevalence and Risk Factors of Antibodies to Class I and II Human Leukocyte Antigens in Haploidentical Allograft Candidates: A Prospective Study on 3805 Subjects

The aim of this study is to investigate the prevalence and risk factors of anti-human leukocyte antigen (HLA) antibodies in haploidentical candidates. This study was completed at Peking University People's Hospital, Beijing China. We performed a prospective analysis of patients with hematological diseases concerning the prevalence and risk factors of anti-HLA antibodies. Patients were enrolled between July 2015 - December 2019. Serum was collected for PRAs test within 1 month before haploidentical transplantation. The risk factors, such as age, sex, total transfusion, red blood cell (RBC) transfusion, platelet (PLT) transfusion, pregnancy, disease duration and diagnosis were collected. Univariate and multivariate logistic regression analyses were performed to evaluate the risk factors of anti-HLA antibodies. Six hundred and eighty (17.9%) patients were positive for panel reactive antibodies (PRA)-class I, 360 (9.5%) for class II, 768 (20.2%) class I or II, and 272 (7.1%) positive for class I and II both. Multivariate analysis indicated that female was related to higher risk of having PRAs for class I (P = 0.011), class I or II (P = 0.009), anti-HLA-A (P = 0.015), anti-HLA-DP (P = 0.048) and also for having higher mean fluorescence intensity (MFI) (2000 or more) of PRAs in class I (P = 0.020) and class I or II (P = 0.005). Compared to patients with myelodysplastic syndrome (MDS), patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), aplastic anemia (AA) had a lower incidence for PRAs in class I, class II, class I or II, class I and II, anti-HLA-A. anti-HLA-B, anti-HLA-C, anti-HLA-DQ, anti-HLA-DR, anti-HLA-DP (Table 1). Prior pregnancy was a risk factor for PRAs (P < 0.001), and no previous pregnancy group having lower MFI of PRAs in class I (P = 0.001) and class I or II (P = 0.004). PLT transfusion (more than 4 times) rleted with a higher prevalence of PRAs (P < 0.001), and also had a higher MFI of PRAs in class II (P < 0.001), class I and II (P < 0.001). Patients with RBC transfusion (more than 3 times) had a higher prevalence of PRAs in class I (P = 0.001), class II (P = 0.029), class I or II (P < 0.001), anti-HLA-A (P = 0.001), anti-HLA-B (P < 0.001), anti-HLA-C (P = 0.007), anti-HLA-DQ (P < 0.001) and anti-HLA-DR (P = 0.011). In addition, diseases duration (8 months or more) was also associated with higher MFI of PRAs in class I (P = 0.023) and class I or II (P = 0.004). Subgroup analysis showed that 11.7% of pediatric patients were positive for PRAs in class I; 19.2% of adults, 17.9% of elder patients; 12.4% of males; 26.1% of females; 21.0% of patients with AML; 10.5% of patients with acute lymphoblastic leukemia (ALL); 18.9% of patients with AA; 30.3% of patients with MDS; 16.6% of patients with other hematological diseases. The positive rate of class II PRAs in children was 4.3%; 11.1% for adults; 9.5% for elder patients; 5.5% for males; 15.4% for females; 11.4% for patients with AML; 5.2% for patients with ALL; 10.3% for patients with AA; 17.2% for patients with MDS; 6.6% of patients with other hematological diseases. Multivariate analysis showed that, in children, PLT transfusion and diagnosis were the two main risk factors of PRAs in class I and class II (P < 0.001, P = 0.017). In adults, diagnosis (P = 0.003), transfusion (P < 0.001) and pregnancy (P < 0.001) were the three main factors associated with PRAs in class I and transfusion (P < 0.001) and pregnancy (P < 0.001) were the two main factors associated with PRAs in class II. In males, PLT transfusion (P < 0.001) and diagnosis (P < 0.001) were the two main factors associated with PRAs in class I and class II. In ALL subgroup, gender (P = 0.026, P = 0.048), pregnancy (P < 0.001) and transfusion (P < 0.001) were the three main factors associated with PRAs in class I and II. In AA subgroup, gender (P = 0.004) and PLT transfusion (P < 0.001) were risk factors for class I PRAs, pregnancy (P = 0.008) and PLT transfusion (P = 0.003) were risk factors for class II PRAs. In elder patients, females, AML, MDS and other diseases subgroup, transfusion and pregnancy were the two main factors associated with PRAs in class I and class II. Our results indicated that female sex, diagnosis, pregnancy, transfusion, disease duration were independent risk factors of anti-HLA antibodies in haploidentical allograft candidates, which provided evidence for best haploidentical donor selection. The risk factors of anti-HLA antibodies were different among total patients and those of cases in different subgroups. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.