Observations on the HLA-A2403 specificity

We found HLA-A*2423 in a subject possessing the HLA-A2403 specificity. Consequently we delineated the A*24 alleles of 41 subjects previously assigned as 'A2403' by serology and/or 'low-resolution' PCR-SSP (allele group: A*2403/10/18/22/23/33) and found that 36 were A*2403, 3 were A*2410 and 2 were A*2433. The A*2410 and A*2433 subjects also typed as A2403. Several likely A*2410/23/33-bearing haplotypes were identified and the frequency of the A*2403/10/18/22/23/33 alleles was determined in 26,826 Welsh blood donors. We suggest that HLA-A9/A24 antibodies against epitope(s) involving 166D and 167G will fail to react with all A*2403/10/18/22/23/33 products. As it is this 'short' A9/A24 serological reactivity that defines HLA-A2403 all these products will be serologically designated as HLA-A2403.     

Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation

Anti‐HLA donor‐specific antibodies are deleterious for organ transplant survival. Class I HLA donor‐specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti‐denatured HLA antibodies (anti‐dHLAs). Anti‐dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti‐dHLAs to be discriminated from anti‐nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid‐treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti‐dHLAs. However, some anti‐dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti‐nHLAs conserved significant reactivity toward acid‐treated LSAB. After depleting serum anti‐nHLA reactivity with HLA‐typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid‐treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti‐nHLAs and anti‐dHLAs, or anti‐nHLAs recognizing acid‐resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti‐HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti‐HLA antibodies in human serum.     

A new murine lymphocytotoxic monoclonal antibody recognizing HLA-A2, -A28 and -A9

Monoclonal antibodies recognizing polymorphic as well as monomorphic epitopes on HLA antigens are important tools for understanding the immuno-biology of HLA molecules. We immunized BALB/c mice with a HLA-A2 transfectant and screened for hybridomas which reacted with a HLA-A2 trans-fectant but not with a HLA-B75 transfectant. After subcloning by limiting dilution four times, a hybridoma secreting a monoclonal antibody (mAb) (IgG 2a, kappa) designated 1–145 was established. 1–145 reacted with Epstein-Barr virus transformed B lymphoblastoid cell lines (B cell lines) which expressed HLA-A2, -A28, -A23 and -A24. The titer of 1–145 in culture supernatant against HLA-A2 and -A28 antigens was similar and the titer against HLA-A23 was lower. 1–145 reacted with cells expressing HLA-A24 but the titer against HLA-A24 antigens was even lower than that againt HLA-A23 antigens. The HLA-A24 antigens on the peripheral blood lymphocytes were not detected by 1–145 possibly due to the lower expression compared to the B cell lines. These differences of the titers were reflected to microlymphocytotoxic-ity assay in which 1–145 culture supernatant lysed all PBLs expressing HLA-A2.-A28 and -A23 but did not lyse PBLs expressing HLA-A24. Published deduced amino acid sequence data of HLA class I molecules indicate that Lys in position 127 may be critical for 1–145 binding.     

Heart transplantation with donor-specific antibodies directed toward denatured HLA-A*02:01: a case report

The development of solid-phase assays for antibody detection has aided in the frequent detection of human leukocyte antigen (HLA) antibodies in nonalloimmunized males. Some scientists have reported that these HLA antibodies are produced to pathogens or allergens and the reactivity with HLA coated beads is the result of cross-reactive epitopes. These antibodies may also be directed toward cryptic epitopes exposed on the denatured beads. In this report, we describe the case of a heart transplanted patient who exhibited anti-HLA-A*02:01 donor-specific antibodies detected with a bead-based assay (Luminex) and undetected with the complement-dependent cytotoxicity (CDC) test. Posttransplant monitoring, carried out with CDC and with Luminex on sera from this patient collected at the 2nd, 4th, 8th, and 12th posttransplant weeks and at 1 year confirmed the presence of anti-HLA-A*02:01 in all serum samples. Additional tests carried out with denatured and intact HLA molecules using single antigen beads demonstrated that the antibody was directed toward a cryptic epitope. One year after transplantation the patient is doing well. No sign of antibody-mediated rejection was observed throughout the follow-up. A comprehensive evaluation of the anamnesis and of antibodies is critical to avoid needless exclusion of organ donors.     

丙型肝炎病毒CTL表位的HLA-A2限制性及其免疫学效应研究

目的 研究前期鉴定的5条丙型肝炎病毒(HCV)特异性细胞毒性T细胞(CTL)表位的HLA-A2限制性及其免疫学效应.方法 基于T2胞株,采用MHC-肽复合物稳定性试验研究前期鉴定的HCV特异性CTL表位与HLA-A2分子的亲和力;采用细胞内细胞因子染色(intracellular cy-tokine staining,ICS)和ELISPOT研究七述HLA-A2限制性CTL表位体外刺激HLA-A2刚性外周血单个核细胞(PBMC)产生肽特异性CTL的效应;采用CTL杀伤试验研究上述肽特异性CTL杀伤靶细胞(负载相同肽的T2细胞)的效应.结果 前期研究鉴定的5条CTL表位肽中,NS4b_78(SMMAF-SAAL)和NS5a_367(TVSSALAEL)与HLA-A2分子有高亲和力(FI1);ELISPOT结果显示NS4b_78(SMMAFSAAL)和NS5a_367(TVSSALAEL)可诱导高水平的分泌IFN-γ的效应细胞[分别为(60±6)SFC/10~4PBMC vs(4±1)SFC/10~4PBMC,P<0.001;(10±3)SFC/10~4PBMC vs(2±1)SFC/10~4PBMC,P<0.001];ICS结果证实这两条肽刺激HLA-A2阳性PBMC后,在CD8~+T细胞中产生了高百分比的CD8~+IFN-γ~+T[分别为(2.33±0.22)%vs(0.05±0.01)%,P<0.001;(0.36±0.06)%vs(0.03±0.01)%,P<0.001];并且,肽特异性CTL可特异性杀伤负载相同肽的T2细胞.结论 NS4b_78(SMMAFSAAL)和NS5a_367(TVSSALAEL)受HIA-A2限制,并具有较好的免疫原性.     

Properties of cryptic epitopes and their corresponding antibodies as indicated by the study of human and ovine growth hormones

Antibodies (Ab) directed to hidden antigenic determinants (cryptotopes) are undesirable because they are not neutralizing. Additionally, we have previously demonstrated a close association between the extent of Ab to cryptic determinants and the expression of autoantibodies (autoAb) under some experimental conditions. Thus, the first objective of this work was to establish the physicochemical characteristics of Ab to cryptotopes and the second one was to examine the structural features of cryptic epitopes themselves. Using human and ovine growth hormones (hGH and oGH) as antigenic models and competition ELISA under different conditions of temperature, pH or ionic strength, we did not find any difference between the binding properties of anti-cryptic epitope antibodies (Ab) and anti-native epitope Ab. Then, using synthetic peptides and tryptic digests and direct and competition ELISAs we studied the structures of cryptic hGH and oGH epitopes. Isolated peptides either in solution or adsorbed on microplates failed to react. Partially digested hGH was recognized only when insolubilized on microplates, and anti-oGH Ab only reacted with a large fragment of the hormone either in solution or insolubilized. These results indicate that, at least in the case of hGH and oGH, cryptic epitopes are not simple linear sequences, as commonly referred without any evidence, but new exposed conformational structures different from those found in the native antigen.     

First report on the antibody verification of HLA-DR,HLA-DQ and HLA-DP epitopes recorded in the HLA Epitope Registry

The International Registry of Antibody-Defined HLA Epitopes (http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to HLA mismatches. These epitopes can be structurally defined as eplets by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. A major goal is to identify HLA eplets that have been verified experimentally with informative antibodies. This report addresses class II epitopes encoded by genes in the HLA-D region. Our analysis included reviews of many publications about epitope specificity of class II reactive human and murine monoclonal antibodies and informative alloantibodies from HLA sensitized patients as well as our own antibody testing results. As of July 1, 2014, 24 HLA-DRB1/3/4/5, 15 DQB, 3 DQA and 8 DPB antibody-verified epitopes have been identified and recorded. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients.     

新等位基因HLA-A*240212的认定

目的发现和认定1个HLA新等位基因。方法应用PCR-SSO基因分型技术筛选可能的HLA新等位基因，PCR产物测序，确认与最同源HLA等位基因序列的差异。结果发现一个样品的HLA-A位点结果异常。其序列与已知所有HLA—A等位基因序列不一致，与同源性最高等位基因A＊24020101的差异只是在第2外显子区域中的230位碱基发生了C→T碱基的替换，但并未改变第77密码子编码的氨基酸。结论该等位基因为HLA-A位点的1个新等位基因，已被WHOHLA因子命名委员会于2006年8月23日正式命名为HLA-A＊240212。     

Recognition of HLA-A1 by murine monoclonal antibodies

Abstract: Balb/c mice were immunized with cells from the mouse mastocytoma line P815 transfected with an HLA-A1 gene. The splenocytes of the immunized mice were fused with cells from the murine myeloma NS-1. In an initial screening, supernatants of growing cultures were tested for their binding capacity to the immunizing P815/A1⁺ cells as well as to P815/A2⁺ cells. Three out of 756 hybrids produced antibodies which bound to P815/A1⁺ cells only. They were cloned and further analyzed for their binding reactivity to reference B-lymphoblastoid cells from the Tenth International Histocompatibility Workshop. One monoclonal antibody, designated 6B11, reacted only with HLA-A1⁺ cells, while the two other antibodies, 3G3 and 7F10, appeared to detect antigenic determinants shared by HLA-A1, A3, A11, A26, and A30 (3G3) and by HLA-A1, A3, A11, A26, A28 and A30 (7F10). Flow cytometric studies on B-lymphoblastoid cell lines as well as on a series of tumor cell lines, including melanoma and colon carcinoma lines, confirmed the specificity of these antibodies. Monoclonal antibodies 7F10 and 6B11 were found to be of the IgM class and 3G3 of the IgG1 class. By complement-dependent ⁵¹Cr release experiments it was further shown that the two IgM antibodies 7F10 and 6B11 were able to lyse all cell lines of the HLA-A1 haplotype tested.     

Distribution of the HLA-A9 antigen family (A23, A24 and A2403)

Characterization of 50 local anti-HLA-A9 (A23 and/or A24) sera against the HLA-A2403 antigen allowed the retrospective assignment of A2403 and the verification of A23 and A24 specificities in a panel of 9196 volunteer bone marrow donors. The six A2403 positive subjects identified were confirmed by PCR using four sequence-specific primer mixtures. Population analysis showed the distribution and HLA-A/B linkage disequilibrium of HLA-A23 and A24 to be typical of a Northern European Caucasoid population. HLA-A2403 has a phenotype frequency of 0.00065 (gene frequency - 0.00033) and is in linkage disequilibrium with HLA-B63 and possibly B35.     

Analysis of three HLA-A*3303 binding peptide anchors using an HLA-A*3303 stabilization assay

The affinity of 232 8- to 11-mer peptides carrying HLA-A*3303 anchor residues at position 2 (P2) (Ala, Ile, Leu, Val, Phe or Tyr) and the C-terminus (Arg) was analysed by a stabilization assay using RMA-S transfectants expressing HLA-A*3303 and human beta2-microglobulin. One hundred and nineteen of these peptides (51.3%) bound to HLA-A*3303, confirming that these residues are anchors for HLA-A*3303. Evaluation of P2 residues demonstrated that binding of peptides with Phe or Tyr at P2 is stronger than that of peptides with aliphatic hydrophobic residues at P2. This was confirmed by analysis of a panel of peptides mutated at P2. Analysis of the C-terminal mutant peptides showed that substitution of Lys for Arg had minimal influence on binding to HLA-A*3303. This implies that peptides carrying HLA-A*1101 anchor residues (Val, Ile, Phe or Tyr at P2 and Lys at the C-terminus) can bind to HLA-A*3303. However, such peptides showed lower binding for HLA-A*3303 than for HLA-A*1101. Thus, Arg at the C-terminus is much stronger anchor for HLA-A*3303 than Lys. The preference for Arg and Lys at the C-terminus by HLA-A*1101 and HLA-A*3303 respectively may be due to sequences of three residues (70, 97 and 114) forming the F-pocket of these HLA class I molecules. Statistical analysis of 232 peptides further showed a positive effect of negatively charged residues at P1 for peptide binding to HLA-A*3303. Thus, residues at P1, P2 and the C-terminus play an important role in peptide binding to HLA-A*3303.     

Differential tissue expression of epitopes of the tetraspanin CD151 recognised by monoclonal antibodies

CD151, a member of the tetraspanin family of cell membrane proteins, is widely expressed in epithelial, endothelial and muscle cells as well as platelets and megakaryocytes. Several monoclonal antibodies recognising CD151 in transfected cells and immunoprecipitating typical bands of 28 and 32 kDa from cell lysates have been produced. Surprisingly, these antibodies show different patterns of staining on tissue sections and on haemopoietic cells. Here we show that these differences are at least in part due to masking of certain epitopes in integrin/CD151 complexes. These data have important implications for the use of monoclonal antibodies in studies of the distribution and function of CD151. Of six monoclonal antibodies from four laboratories, 11B1 was found to be the most reliable for detection of CD151 in different cell and tissue contexts.     

Novel HLA-A*31 allele, A*3111 identified by sequence-based typing

In this report, we describe the identification of an HLA-A*31 nucleotide sequence variant, a new HLA-A*3111, in three members of a Korean family by using sequence-based typing (SBT). The new allele was detected during routine HLA typing by high-resolution SBT. Allele A*3111 showed one nucleotide difference with A*310102 at codon 165 (GTG-->CTG) resulting in an amino acid change from valine to leucine (V165L). Serologic reactivity was shorter than normally expected.     

肝素酶HLA-A2限制性CTL表位肽的筛选及其活性鉴定

目的 预测并鉴定新的人乙酰肝素酶(Hpa)抗原的HLA-A2限制性CTL表位,为恶性肿瘤多肽疫苗的免疫治疗提供依据. 方法 采用SYFPEITHI和BIMAS软件预测方法,对肝素酶HLA-A2限制性CTL表位进行预测,合成候选表位肽;利用T2细胞特点,对合成的候选肽与HLA-A2分子进行亲和力分析;利用乳酸脱氢酶释放试验检测待检肽特异性CTL诱导活性;利用ELISPOT检测T细胞活性. 结果 在所筛选的6条候选CTL表位肽中,Hpa(310～318)FLNPDVLDI与HLA-A2分子具有高亲和力,在体外可有效诱导肝素酶特异性CTL的产生,对肝素酶阳性且HLA-A2限制性的HCC-LM6肝癌细胞及SW-480结肠癌细胞具有明显的杀伤效应,且能有效诱导IFN-γ分泌,增强免疫活性. 结论 首次发现Hpa(310～318)FLNPDVLDI可能是肿瘤肝素酶抗原的HLA-A2限制性CTL表位.     

Novel HLA-A*11 allele, A*1120, identified by sequence-based typing

In this report, we describe the identification of a human leucocyte antigen-A*11 (HLA-A*11) nucleotide sequence variant, a new HLA-A*1120 by using sequence-based typing (SBT). The new allele was detected during routine HLA typing by high-resolution SBT. Allele A*1120 showed one nucleotide difference with A*110101 at codon 152 (GCG-->GAG) resulting in an amino acid change from alanine to glutamate. Residue 152 is located on alpha(2)-helix of HLA class I molecule and involved in peptide binding by constructing E pocket of peptide-binding groove, implying that the change of the residue 152 would affect the binding affinity of peptides to A*1120 allele.     

Identification of a novel allele HLA-A*1113 in a Japanese donor

Anew HLA-A*11 allele, A*1113, was identified in a healthy Japanese female. She was typed as HLA-A11?, A2, B46, B67, Cw1, Cw7 (Bw6) with unusual serological reactivity of A11, suggesting possible presence of a new A*11 allele. The novel A*1113 allele was identified by haplotypic group-specific allele amplification using A*11 allele-specific primer pairs and sequence-based typing. The A*1113 allele differs from A*11011 by one nucleotide substitution in exon 3 at position 503 (A --> G) which causes an amino acid change in the alfa2 domain at residue 144 (lysine : K --> arginine : R), thus resulting in the unusual serological reactivity.     

HLA epitopes – Empirically defined as conformational amino acids sequences of the HLA antigen and are likely to be part of the binding sites of anti-HLA antibodies

Antibodies against HLA antigens are ubiquitous in the sera of transplant patients. Analysis of anti-HLA antibodies specificity has gone through a long history of development using assays like agglutination and lymphocytotoxicity, which utilize lymphocytes, and flow cytometry, which utilize multiplex beads coupled with single antigens. Hundreds of HLA antigens are identified to date, and the realization that antibody reactivity against the antigens is multispecific presented difficulties in accurately defining antibody specificity. Although Cross Reacting Groups (CREG), describing cross reactivity among HLA antigens, were helpful with determining specificity, they proved to be inadequate for the highly sensitized patients. Amino acid sequencing and three-dimensional modeling of the HLA molecules significantly advanced our understating of the HLA antigens and their epitopes. Although sensitive assays for antibody testing advanced analysis, they unmasked additional specificities undetectable by traditional methods, and the presence of naturally occurring anti-HLA antibodies in sera further complicated analysis and underscored the need to understand antibody reactivity and their epitopes. Hundreds of HLA class-I and class-II epitopes were defined by the Tekasaki and Duquesnoy groups and their usefulness in organ transplants were further advanced by a great number of transplant centers. Alloantibody specificities, CREGs, and nondonor specific antigens (NDSA) are now explained by public epitopes     

Serological and nucleotide sequence analysis of HLA-A*6812

We describe a novel HLA-A allele, HLA-A*6812. HLA-typing of a patient was performed by serology and by DNA-based analyses. Cloning and sequencing of exons 2 and 3 revealed that the genotype consisted of a common HLA-A*0101 allele and the novel HLA-A*6812 allele. HLA*6812 is identical to HLA-A*68012 except for a single non-synonymous nucleotide substitution leading to an exchange of a threonine to an isoleucine residue at position 142 of the HLA-class I alpha chain. The allele was also found in the patient's mother. The novel HLA-A68 molecule is recognised by some but not all of our HLA-A28-specific sera. These results confirm that position 142 is important for the serological properties of the HLA-A molecule.