Identification of a novel HLA-Cw*08 variant allele, Cw*0824

A novel HLA-C allele, Cw*0824, which was identified from an individual of the Han Chinese, differs from Cw*080101 at codon 222 (GAG > AAG ) in exon 4, which results in an amino acid change Glu222Lys.
In recent years, many human leukocyte antigen (HLA)-C alleles have been identified. Up to date, 23 different Cw*08 alleles have been identified according to the IMGT/HLA Database release 2.25.2 May 2009 (1) . Here, we describe the identification of the novel allele HLA-Cw*0824 that was found during routine high resolution sequence-based typing (SBT) of a Chinese stem cell voluntary donor. The HLA alleles of the donor were typed as A*11, 24; B*15, 46; and DRB1*09, 12.     

Autologous cytolytic T lymphocytes recognize a MAGE-1 nonapeptide on melanomas expressing HLA-Cw* 1601

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, which codes for antigen MZ2-E which is presented by HLA-A1. Gene MAGE-1 is expressed in many tumors of several types but not in normal tissues except for testis. We show here that gene MAGE-1 directs the expression of another antigen recognized by CTL on the MZ2-MEL cells. This antigen, which was named MZ2-Bb, consists of MAGE-1-encoded peptide SAYGEPRKL bound to major histocompatibility molecule HLA-Cw* 1601. The HLA-Cw* 1601 allele was found to be expressed by 7 out of 99 individuals from a Caucasian population. Our results extend the range of tumor patients who could be eligible for immunization against MAGE antigens.     

The kinetics of peptide binding to HLA-A2 and the conformation of the peptide-A2 complex can be determined by amino acid side chains on the floor of the peptide binding groove

The ability of amino acid side chains in the floor of the peptide binding groove of HLA-A2 to affect the presentation of a viral peptide to peptide-specific cytotoxic T lymphocytes (CTL) has been examined. HLA-A2 molecules with naturally occurring single amino acid substitutions of Phe to Tyr at position 9 (HLA-A2.4a, Tyr9) and Tyr to Cys at position 99 (HLA-A2.4b, Cys99) and a site directed mutant with a Val to Leu substitution at position 95 (Leu95) were examined for their ability to present the influenza virus matrix M1 55-73 peptide and several sequence variants of the M1 peptide to a panel of 36 M1 55-73-specific HLA-A2.1-restricted CTL lines. The Leu95 molecule demonstrated enhanced kinetics of M1 peptide presentation and the ability to be sensitized by lower concentrations of the M1 peptide than the A2.1 molecule. The Tyr9 and Cys99 molecules exposed to M1 peptide were not recognized by 33 out of 36 CTL lines. The Tyr9 and Cys99 HLA-A2 molecules could bind the M1 55-73 peptide because at least one CTL line was found that could recognize each of these molecules that were exposed to the M1 peptide. CTL recognition patterns of variant M1 peptides presented by the Tyr9 molecule demonstrated that the amino acid at position 9 can be a critical determinant of the conformation of the peptide-A2 complex, and indicated that a particular peptide can bind in the HLA-A2 peptide binding groove in more than one conformation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired binding of a DQ2 and DQ8-binding HSV VP16 peptide to a DQA1*0501/DQB1*0302 trans class II heterodimer

DQalpha and DQbeta trans heterodimeric HLA-DQ molecules form in individuals heterozygous for the DQ2 and DQ8 specificities. Unique functions and disease associations have been postulated for such trans-dimers, which may be different from cis-encoded DQ molecules encoded by the corresponding haplotypes. We analyzed the ability of the trans-dimer encoded by HLA-DQA1*0501/DQB1*0302 to bind a peptide antigen which interacts with DQ molecules encoded by both parental haplotypes. Markedly impaired binding was observed, consistent with both the use of different anchor residues and with changes in levels of DQ cis-dimer availability for peptide binding interactions.     

A novel HLA-Cw*03 allele, Cw*033802, identified by sequence-based typing

New allele Cw*033802 showed one nucleotide difference with Cw*0338 at codon 99 (TAC-->TAT).     

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.     

HLA-Cw基因全长序列分子克隆及测序方法的建立

目的 建立可靠的HLA-Cw基因全长序列的分子克隆和测序技术.方法 设计合成HLA-Cw基因全长序列PCR引物和探索PCR反应体系,采用长距离PCR技术,扩增HLA-Cw基因非翻泽区(untranslated region,5'-UTR)区、8个外显子、7个内含子和3'-UTR区,全长约4.5 kb.PCR产物纯化后进行分子克隆,筛选阳性克隆,提取质粒DNA,采用自行设计的测序引物进行全长双向测序.12份已经AlleleSEQR HLA-Cw测序分型试剂盒进行PCR产物直接测序、基因型已知的样本,分别用TaKaRa LATaq酶和Stratagene Pfu Taq DNA聚合酶进行HLA-Cw基因全长扩增,以及PCR产物分子克隆和序列测定,克隆测序结果分别与PCR产物直接测序结果进行对比分析.结果 PCR扩增获得了特异性目的 片段,测序获得了HLA-Cw基因-962～3576位碱基全长序列.克隆测序结果的对比表明,Pfu酶保真性高于LA Taq酶.比较本文测定的Cw*010201与Cw*07020101等位基因序列,在5'上游-962～-284位碱基区域存在11个单核苷酸多态(single nucleotide polymorphisms,SNPs)和2个插入(或)缺失多态性位点;3'-UTR下游3067～3576位碱基区域存在11个SNPs和1个插入(或)缺失.结论 建立了HLA-Cw基因全长序列分子克隆及测序方法,在HLA-Cw基因全长序列分子多态性及表达调控等研究领域,具有广泛应用前景.     

Augmented induction of CD8+ cytotoxic T-cell response and antitumour resistance by T helper type 1-inducing peptide

The effector CD8(+) T cells recognize major histocompatibility complex (MHC) class I binding altered self-peptides expressed in tumour cells. Although the requirement for CD4(+) T helper type 1 (Th1) cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumour responses remain unclear. We examined whether a potent immunogenic peptide of Mycobacterium tuberculosis eliciting Th1 immunity contributes to the generation of CD8(+) T cells and to protective antitumour immune responses to unrelated tumour-specific antigens. Peptide-25, a major Th epitope of Ag85B from M. tuberculosis preferentially induced CD4(+) Th1 cells in C57BL/6 mice and had an augmenting effect on Th1 generation for coimmunized unrelated antigenic peptides. Coimmunization of mice with Peptide-25 and ovalbumin (OVA) or Peptide-25 and B16 melanoma peptide [tyrosinase-related protein-2 (TRP-2)] for MHC class I led to a profound increase in CD8(+) T cells specific for OVA and TRP-2 peptides, respectively. This heightened response depended on Peptide-25-specific CD4(+) T cells and interferon-gamma-producing T cells. In tumour protection assays, immunization with Peptide-25 and OVA resulted in the enhancement of CD8(+) cytotoxic cell generation specific for OVA and the growth inhibition of EL-4 thymoma expressing OVA peptide leading to the tumour rejection. These phenomena were not achieved by immunization with OVA alone. Peptide-25-reactive Th1 cells counteractivated dendritic cells in the presence of Peptide-25 leading them to activate and present OVA peptide to CD8(+) cytotoxic T cells.     

Processing of the DRB1*1103-restricted measles virus nucleoprotein determinant 185–199 in the endosomal compartment

MHC class II molecules present to CD4⁺ T cells protein fragments which mostly derive from the extracellular and from the endosomal compartments. Determinants of cytosolic proteins are, however, also displayed by MHC class II molecules following pathways which are still not yet fully characterized. Here we describe the isolation of DRB1*1103-restricted T cell clones specific for the measles virus (MV) nucleoprotein peptide 185–199 (N185). Experiments were then conducted to delineate how this determinant is assembled with DR molecules. In vitro binding analyses indicated that complexes between the N185 peptide and DRB1*1103 protein are optimally constituted at pH 4–4.5. In cellular experiments it was observed that chloroquine, leupeptin and emetine, which are classical inhibitors of presentation of MHC class II-restricted antigens, when added during infection of B cells with MV, prevent presentation of the N185 determinant. In addition, it was found that the N185 determinant is efficiently presented when the nucleoprotein is exogenously provided to B cells, either by blocking MV fusion with the peptide FFG or by the use of purified nucleoprotein. In contrast, it was observed that nucleoprotein recombinant vaccinia virus (vv-N)-infected B cells weakly stimulated N185-specific T cells, indicating that the restricted localization of the nucleoprotein in the cytosol resulted in a poor presentation of the N185 determinant. Taken together, these findings suggest that it is prior to delivery of the nucleoprotein into the cytosol that the N185 determinant is efficiently assembled with newly synthesized DR molecules in the acidic environment of the endosomal compartment.     

Hepatitis B surface antigen presentation and HLA-DRB1*- lessons from twins and peptide binding studies

The aim of this study was to investigate the underlying mechanisms of the genetic association between certain HLA-DRB1* alleles and the immune response to HBsAg vaccination. Therefore, HBsAg peptide binding to HLA-DR molecules was measured in vitro by peptide binding ELISAs. Additionally, HBsAg-specific T cell reaction and cytokine profile of immune response were analysed ex vivo in ELISPOT assays and DR-restriction of T-cell proliferative responses was investigated with HBsAg specific T cell clones. In addition, we compared HBsAg specific T cell responses of 24 monozygotic and 3 dizygotic twin pairs after HBsAg vaccination. Our results showed that the peptide binding assays did not reflect antigen presentation in vivo. DR alleles associated with vaccination failure like DRB1*0301 and 0701 efficiently presented HBsAg peptides. In 11 of 24 investigated monozygotic twin pairs we observed pronounced differences in the recognition of HBsAg peptides. This study indicates that HLA-DR associations with HBsAg vaccination response are not caused by differences in peptide binding or by a shift in the Th1/Th2 profile. Our findings strongly argue for differences in the T cell recognition of peptide/MHC complexes as the critical event in T cell responsiveness to HBsAg.     

Polarity of the P1 anchor residue determines peptide binding specificity between HLA-A*3101 and HLA-A*3303

A previous pool sequence analysis showed that HLA-A*3101 and HLA-A*3303 binding peptides have the same anchor residues at P2 and the C-terminus, the only difference being that HLA-A*3303 binding peptides have two additional P2 anchor residues. Using a stabilization assay with RMA-S transfectants expressing HLA-A*3101 and human beta2-microglobulin, we tested the binding of 232 8- to 11-mer peptides carrying HLA-A*3303 anchor residues to HLA-A*3101. One hundred of these peptides (43.1%) bound to HLA-A*3101, confirming that these residues are also anchors for HLA-A*3101. Although aromatic hydrophobic P2 residues were previously shown to be stronger anchors than aliphatic hydrophobic P2 residues in HLA-A*3303 binding peptides, we detected no significant difference in HLA-A*3101 binding affinity between peptides carrying aromatic or aliphatic hydrophobic P2 residues. Statistical analysis previously showed a positive effect of negatively charged P1 residues and a negative effect of positively charged P1 residues for peptide binding to HLA-A*3303. In contrast such analysis demonstrated a positive effect of positively charged P1 residues and a negative effect of negatively charged P1 residues for peptide binding to HLA-A*3101. Analysis using mutated peptides confirmed these results. The present study therefore demonstrates that peptide binding specificity between HLA-A*3101 and HLA-A*3303 is determined by the polarity of the P1 anchor residue.     

Real-time measurement of antigenic peptide binding to empty and preloaded single-chain major histocompatibility complex class I molecules

Cytotoxic T lymphocytes (CTL) recognize peptides in association with major histocompatibility complex (MHC) class I proteins, but how peptides bind to class I is not well understood. We used a fluorescence technique to measure antigenic peptide binding to a soluble, single-chain K^d (SC-K^d) molecule in which the K^d heavy chain was connected by a 15-residue link to β₂-microglobulin. Peptides were covalently labeled at their N terminus with dansyl, and binding of dansylated K^d-restricted peptides to SC-K^d resulted in significant fluorescence enhancement, which could be inhibited by unmodified K^d-restricted peptides. Real-time binding of a dansylated peptide could be followed by monitoring the fluorescence at 530 nm. The dansylated Plasmodium berghei circumsporozoite (PbCS) 263–260 peptide bound to “empty” SC-K^d with an association rate constant of 1140 M^?1s^?1, and the subsequent spontaneous dissociation of the SC-K^d-peptide complex was slow. The dissociation increased dramatically after addition of excess unlabeled PbCS 253–260 peptide, but with a slower association constant for unlabeled peptide, 77 M^?1s^?1. Thus, the K^d-peptide complex on the surface of antigen-presenting cells should be stable, but high concentrations of peptides in the endoplasmic reticulum (ER) lumen would allow for peptide exchange on K^d before export to the surface. The apparent activation energy for PbCS 253–260 peptide binding to SC-K^d was 6.78 ± 0.64 kcal/mole, similar to values previously reported for antigen-antibody interactions.     

Binding of peptides to HLA-DQ molecules: peptide binding properties of the disease-associated HLA-DQ({alpha}1*0501, {beta}1*0201) molecule

Peptide binding to DQ molecules has not previously been described.Here we report a biochemical peptlde-blndlng assay specificfor the BQ2 [I.e. DQ(1^*0501, ß1^*0201)] molecule. Thismolecule was chosen since It shows a strong association to diseasessuch as celiac disease and insulin-dependent diabetes mellitus.Initially we radlolabelled some selected peptides and testedthem for binding to affinity-purified DQ2 molecules. One ofthe peptides, a Mycobacterium bovis (MB) 65 kDa 243–255Ypeptide, displayed a good slgnal-to-noise ratio and was thuschosen as an indicator peptide in the DQ2 binding assay. TheMB 65 kDa 243–255Y peptide bound to DQ2 In a strictlypH-dependent fashion, with optimal binding around pH 5 and onlyweak binding at pH 7.4. The association of the MB 65 kDa 243–255Ypeptide to DQ2 was slow, but once formed, the peptide-HLA complexeswere very stable. The binding of peptides to DQ2 was specific,as shown in Inhibition experiments with a panel of 47 peptides,differing in length, sequence, and origin. The binding of peptidesto DR3 was tested in a similar assay with a Mycobacterium tuberculosis65 kDa 3–13 peptide as the binding indicator. DQ2 andDR3 molecules bound to different sets of peptides. However,the peptide binding to DQ2 and DR3 showed, In general, similarcharacteristics with respect to pH dependence and kinetic parameters,Indicating that the overall rules for peptide binding to DQmolecules are the same as those previously shown for human DRand murlne I-A and I-E molecules.     

A sensitive fluorometric assay for quantitatively measuring specific peptide binding to HLA class I and class II molecules

A sensitive, highly reproducible assay was developed for measuring binding of peptides to various HLA class I and II alleles. The assay is based on competition for binding to HLA between a peptide of interest and a fluorescent labelled standard peptide. This mixture is incubated with HLA to obtain equilibrium binding, and subsequently separated on an HPLC size-exclusion column in (i) a protein fraction containing HLA and bound peptide and (ii) a free peptide fraction. Each assay uses only 100 fmol labelled peptide and approximately 10 pmol of HLA. The analytical system contains an autosampler that samples from 96-well microtiter plates. Injections and data recording/evaluation is fully automated. Typical analysis time is 10–12 min per sample. The fluorescence in the HLA-bound peptide and free peptide containing fractions is measured on-line. The ratios of fluorescence signal in protein and peptide fractions at various concentrations of the peptide of interest are determined. IC₅₀ values are calculated from the binding curve as obtained by curve fitting of the data. Here we show results for peptide binding to HLA-DR1 and -DR17 molecules purified from detergent solubilized cell lysates, and for recombinant HLA-A^* 0201 and HLA-A^* 0301 expressed in E. coli.

The assay reported is sensitive and reproducible. It is non-radioactive and is non-labor intensive due to the high degree of automation.     

Human vascular endothelial cells process and present autoantigen to human T cell lines

The effectiveness of cultured human umbilical vein endothelialcells as accessory cells for T cell activation has been investigatedusing T cell clones and lines derived from patients with myasthenlagravis which were specific for different epitopes on the subunitof the human acetylcholine receptor. The endothelial cells wereinduced with IFN- to express HLA-DR and -DQ at high and lowlevels respectively. They could then efficiently present specificpeptides of the subunit to an HLA-DR- and an HLA-DQw5-restrictedT cell line. They could also process epitopes for both T celllines from the full-length recombinant a subunlt (r1—437)of the human acetylcholine receptor, where the known epitopesare 80 amino acid residues apart. The endothelial presentationof r1—437, but not of the peptides, was sensitive to chloroquineinhibition. Presentation appeared slightly less efficient (by1.5- to 3.0-fold) with endothelial cells than with presentingcells from peripheral blood. This may reflect differences inaccessory signalling since mAb blocking studies suggested thatligands for CD28 provided important accessory signalling byperipheral blood presenting cells while LFA-3 was used by endothelialcells.     

Detailed and atypical HLA-E peptide binding motifs revealed by a novel peptide exchange binding assay

Diverse SIV and HIV epitopes that bind the rhesus homolog of HLA-E, Mamu-E, have recently been identified in SIVvaccine studies using a recombinant Rhesus cytomegalovirus (RhCMV 68-1) vector, where unprecedented protection against SIV challenge was achieved. Additionally, several Mycobacterial peptides identified both algorithmically and following elution from infected cells, are presented to CD8⁺ T cells by HLA-E in humans. Yet, a comparative and comprehensive analysis of relative HLA-E peptide binding strength via a reliable, high throughput in vitro assay is currently lacking. To address this, we developed and optimized a novel, highly sensitive peptide exchange ELISA-based assay that relatively quantitates peptide binding to HLA-E. Using this approach, we screened multiple peptides, including peptide panels derived from HIV, SIV, and Mtb predicted to bind HLA-E. Our results indicate that although HLA-E preferentially accommodates canonical MHC class I leader peptides, many non-canonical, sequence diverse, pathogen-derived peptides also bind HLA-E, albeit generally with lower relative binding strength. Additionally, our screens demonstrate that the majority of peptides tested, including some key Mtb and SIV epitopes that have been shown to elicit strong Mamu-E-restricted T cell responses, either bind HLA-E extremely weakly or give signals that are indistinguishable from the negative, peptide-free controls.     

Functional analysis of HLA-DP polymorphism: a crucial role for DPbeta residues 9, 11, 35, 55, 56, 69 and 84-87 in T cell allorecognition and peptide binding

The information available on the specific function of HLA-DP and the structure-function relationships is very limited. Here, single amino acid substitutions of HLA-DPB1*02012 have been used to analyze the role of polymorphic residues of the DPbeta1 domain on DP-mediated T cell allorecognition and peptide binding. Using a panel of specific anti-HLA-DP mAb, we identified the HLA-DP residues involved in the recognition by these mAb, with a crucial role for DPbeta56 for most of the mAb assayed. Individual substitutions at residues 9, 11, 35, 55, 56 and 69 completely abrogated T cell recognition mediated by two different HLA-DPw2-allospecific T cell clones (8.3 and 8.9). Interestingly single changes at positions 9, 11, 35 and 55 of HLA-DPbeta also altered the binding of peptides AAII(12-27) and IIP(53-65), natural ligands of the HLA-DPB1*02012 molecule. Individual changes at residues located in pocket 1 (84, 85, 86 and 87 from HLA-DPbeta) led to a partial reduction in cytotoxic T lymphocyte-mediated lysis and also partially affected peptide binding. However, the simultaneous substitution of these positions completely abolished both T cell allorecognition and peptide binding, suggesting a major role for polymorphisms at pocket 1 in HLA-DP function. Molecular modeling, used to predict changes induced by amino acid substitutions, supported the functional data. Taken together, these results strongly suggest that polymorphic residues 84, 85, 86 and 87 at pocket 1, residues 9, 35 and 55 at pocket 9, and residues 11 and 69 at pockets 6 and 4 respectively play a key role in HLA-DP function, probably by modifying the way the peptide is bound within the groove of HLA-DP2 and determining changes in the conformation of the MHC-peptide complex recognized by the TCR.     

CT抗原KM-HN-1 HLA-A * 0201限制性表位预测及其与HLA-A * 0201结合强度分析

目的通过对CT抗原（cancer-testis antigen）KM-HN-1进行HLA-A＊0201限制性表位预测,并对候选表位肽与HLA-A＊0201分子结合亲和力及复合物稳定性进行分析,为探索基于KM-HN-1的免疫治疗奠定基础。方法利用基于蛋白酶体剪切位点特异性的算法PAProc及基于肽MHC-I结合的算法BIMAS和SYFPEITHI对KM-HN-1进行HLA-A＊0201限制性表位预测．合成KM-HN-1相关候选表位肽KM-HN-I321-329（KLLPFRETV）,KM-HN-I303-211,（FLPTAPPNV）,KM-HN-I629-637。（TLLQIIETV）,KM-HN-I87-95（ILNKSIIEV）,KM-HN-I538-596。（QMMEALDQL）及阳性对照肽HBVcAg18-27（FLPSDFFPSV）;对这些合成肽与HIA-A＊0201分子结合亲和力及其复合物稳定性根据文献报道的方法进行分析。结果KM-HN-I321-329（KLLPERETV）结合亲和力最低,KM-HN—I203-211（FLPTAPPNV）结合亲和力最高,其余3条肽结合亲和力介于2者之间;稳定性实验（DC50）结果显示：KM-HN-I538—546（QMMEALDQL）DC50小于2h,KM—HN-I321-329（KLLPERETV）的DC50介于2～4h之间,KM-HN-I87-95。（ILNKSIIEV）的DC50介于6～8h之间,KM-HN-I233-211（HLPTAPPNV）及KM-HN-I629—633（TLLQIIETV）的DC50均大于8h。结论基于蛋白酶体剪切位点特异性的算法及基于肽MHC-I结合的算法对KM-HN-1进行HLA-A＊0201限制性表位预测,结合候选表位肽与HLA-A＊0201分子结合的亲和力与复合物稳定性实验分析,为该抗原HLA-A＊0201限制性表位的鉴定奠定了基础。