Topographic map of the HLA-A2 CREG epitopes using human alloantibody probes.

The topographic architecture of the epitopes expressed on the HLA-A2 glycoprotein using murine monoclonal antibody (mAb) probes indicates at least two sterically distinct domains. Previously, we have demonstrated using human HLA alloantibodies (aAb) that multiple determinants are expressed on each HLA antigen: the highly polymorphic private epitopes and the public determinants that are shared within a family of crossreactive groups (CREG). Our objectives now focus on probing the antigenic structure of the HLA-A2-28-9-B17 CREG using highly specific aAb in conjunction with mAb that have previously been used for structural studies. Both mAb-mediated blockage of complement-dependent cytotoxic aAb and reciprocal antibody (Ab) binding inhibition assays with quantitation by fluorescence flow cytometry have been utilized. We have found that xenogeneic mAb directed against A2-69, A2-B17, and A2-28 crossblock aAb of the same serologic specificity, and vice versa, indicating that the epitopes they respectively recognize are at least in close steric proximity. However, additional HLA-A2, A28, and B17 aAb of private specificity and A2-28-9 aAb of public specificity, for which there are no known mAb counterparts, paint an additional complexity not previously known. We conclude that at least four different alloepitopes can be expressed by each serologically defined HLA antigen. Based on the primary sequence data, we have assigned the location and the amino acid substitutions which most likely account for these discrete epitopes. The unique private determinants are located on the alpha 1 domain together with the interlocus A2-B17 epitope while the public epitopes A2-69, A2-28-9 and A2-28 are located on the alpha 2 domain.     

A reanalysis of the HLA-B7 cross-reactive group

Cross-reactivity between antigens of the HLA-B7 cross-reactive group (B7 CREG) was investigated by the serological analysis of 60 "broad" cytotoxic HLA antisera produced by pregnancy alone, the HLA typing of the antiserum donors and the identification of their immunizing antigens. Thirty-five sera, made in response to B7, covered (as a group) HLA-B7, B27, Bw42, Bw48, Bw54, Bw55, Bw56, Bw60 and Bw61 — the B7 CREG antigens. Bidirectional cross-reactivity occurred between the B7 antigen and B27, Bw55, Bw56 and Bw60 antigens but not between the major B7 CREG antigens B27, Bw22 and B40. HLA-B27 stimulated antisera included B7, B13 and Bw47 within their reaction range and Bw55/Bw56 cross-reacted with Bw42, Bw54, Bw57, Bw58, Bw62 and Bw63. Bidirectional cross-reactivity was observed between Bw55 and Bw57. Fourteen responders possessed an antigen cross-reactive with their immunizing antigen. These findings are discussed in relation to the sharing of determinants by pairs and "families" of HLA antigens.     

Antigenicity of HLA-A2 and HLA-B7. Loss and gain of serologic determinants induced by site-specific mutagenesis at residues 62 to 80.

The contribution of the hypervariable region spanning amino acid residues 62 to 80 to the serologic determinants of HLA-A2 and HLA-B7 has been examined by site-directed mutagenesis. Three HLA-A2 mutants, having changes as in HLA-B7 at positions 62, 76, and at the complete 65-to-80 segment, respectively, were obtained and expressed on class I HLA-deficient human cells upon transfection. The reactivity of 19 monoclonal antibodies (mAbs) against both broad public and allospecific determinants on HLA-A2 and HLA-B7 was analyzed. The results indicate that: (1) the change at residue 62 abrogated recognition of the corresponding HLA-A2 mutant by mAb MA2.1 (anti-A2 + B17); (2) the change at residue 76 did not effect any of the determinants analyzed, although its side chain is easily accessible at the surface of the molecule; (3) the replacement of the whole 65-to-80 segment in HLA-A2 by that from HLA-B7 abrogated recognition by MA2.1 and by 108-2C5, a mAb recognizing a public determinant from the HLA-A locus. Such replacement led to gaining the determinants recognized by mAbs GS145.2 (anti-B7 + B27) and SFR8-B6 (anti-Bw6); and (4) the HLA-A2-reactive mAbs whose reactivity was known to be abrogated by changes in alpha 2 were unaffected by the changes introduced in alpha 1, underlining the frequent segregation of serologic determinants on class I antigens to single domains.     

HLA Alloantibodies and the Mechanism of the Antiglobulin-Augmented Lymphocytotoxicity Procedure

ABSTRACT: HLA Class I alloantigens express multiple epitopes which can be defined serologically using human HLA alloantibodies (aAb). We have shown that the vast majority of HLA antisera exhibit the CYNAP phenomenon (complement-dependent cytotoxicity (CDC) negative, adsorption positive) which can be identified by conversion to direct CDC positive reactivity with the addition of an antihuman immunoglobulin (Ig) light chain (AHG) reagent.

In this study, the immunochemical mechanisms responsible for the CYNAP phenomena and how AHG overrides CYNAP have been further characterized using affinity-purified HLA aAb, class-specific anti-IgH reagents and human C1q binding assays quantified by flow cytometry. We have found that CYNAP reactions are not the result of low affinity aAb or generally caused by non-complement fixing HLA aAb. Our experiments illustrate that only anti-human IgL AHG reagents can consistently augment CDC and override CYNAP; anti-IgH have not effective. Two noncompeting HLA aAb of different epitopic specificity or one aAb in conjunction with the AHG-augmenting reagent results in striking synergy with a 200 to 400% increase in binding of C1q.

We conclude from these and other experiments detailed in this article that an IgM aAb or either two adjacent, noncompeting IgG HLA aAb bound to spatially distinct epitopes on a single HLA molecule or a monospecific IgG HLA aAb in concert with the AHG binding to this HLA aAb, is required for efficient (bivalent) C1q binding and initiation of C-mediated lympholysis. In contrast, the CYNAP phenomenon usually occurs because monospecific HLA aAb directed against a single epitope cannot effect high affinity, bivalent interaction with C1q and activate complement that would ultimately lead to cytolysis.     

HLA antigens and Graves' disease in Black South Africans

This study concerns the frequencies with which 36 HLA-A, -B and -C antigens occurred in 84 Black Africans with Graves' disease and in 311 Black controls. In the hyperthyroid patients significant reductions were found in the frequencies of HLA-B7 ( P <0.001, relative risk (RR) 0.33), HLA-Bw42 ( P <0.001, RR 0.32) and the HLA-B7-Bw42 crossreactive group (CREG) ( P <0.0001, RR 0.27), and in the frequencies of the phenotypic combinations HLA-A1, B7 ( P <0.001) and Aw30, B7-Bw42 ( P <0.001). HLA-B8 was increased in frequency ( P <0.01, RR 2.84). In patients without circulating antithyroglobulin or antimitichondrial antibodies the frequencies of HLA-A2 and B17 were increased when compared to those with antibodies or to the controls. In patients with and without clinically evident infiltrative ophthalmopathy the frequencies of HLA antigens were similar. In 62 Caucasian patients with Graves' disease, no antigens or phenotypic combinations occurred with increased or decreased frequency when compared to 278 controls.
Analysis of the frequencies of 9 HLA antigens and phenotypic combinations common in Caucasians but rare in Blacks revealed that only two antigens (A2 and B8) occurred with increased frequency in Black patients, suggesting that a contribution of Caucasian genes to the Black thyrotoxic subjects was unlikely.
Similarly, only one common Black antigen (A28) of 8 common antigens and phenotypic combinations, occurred in Caucasian patients with a frequency similar to that of Black controls. Thus it is unlikely that Black genes contributed to the lack of a significant increase of HLA antigens in the Caucasian thyrotoxic patients. The possession of HLA-B7-Bw42 CREG or related genes may be a protection against Graves' disease in Black Africans.     

Low resolution DNA typing of the HLA-B5 cross-reactive group by nested PCR-SSP

Abstract: We have established a DNA typing system for the HLA-B5 serologically cross-reactive group (CREG) by means of a two-step PCR amplification with nested sequence-specific primers (nPCR-SSP). The present study provides a low resolution definition of the HLA-B5 CREG, i.e. identifying polymorphism equivalent to serology. Two different primer combinations allow group-specific amplification of all HLA-B5 CREG alleles and other related HLA class I alleles from genomic DNA. The amplified DNA is subjected to a second amplification step using eleven nested primer pairs. This assay permits the detection of the HLA-B5 CREG specificities B35, B51, B52, B53, and B7801 in all homozygous and heterozygous combinations. Sensitivity and specificity as judged by a blind quality control study investigating a reference panel (n=50) is 100%. Extension of this approach should allow rapid DNA typing of all serologically defined HLA-B specificities by nPCR-SSP.     

Evidence for a regulatory role of the T8 (CD8) antigen in antigen-specific and anti-T3-(CD3)-induced lytic activity of allospecific cytotoxic T lymphocyte clones

The functional role of the T8 antigen of human T cells was studied by inhibition with anti-T8 monoclonal antibodies (mAb) of the cytotoxic action of T8+ cytotoxic T lymphocyte clones (CTL). All clones were allospecific and directed against HLA-B7. The ability of seven different anti-T8 mAb to inhibit the cytotoxicity of these alloreactive CTL clones corresponded with their avidity for a particular target cell. The lysis of cross-reactive antigen-bearing target cells was more readily blocked by anti-T8 mAb than lysis of the specific B7 target cell against which a clone was raised. The seven anti-T8 mAb showed a spectrum of CTL blocking ability ranging from strong blocking with all five CTL clones tested to weak inhibition of only two out of five clones. mAb inhibition of CTL reactivity and cold target inhibition studies with one of the five CTL clones indicate a post-binding role of the T8 molecule. Functional epitope mapping based on CTL blocking with the anti-T8 mAb resulted in the definition of one nonfunctional epitope on the T8 molecule which is only expressed on mature T lymphocytes and a cluster of closely related functional epitopes expressed on both thymocytes and mature T lymphocytes. Not only allospecific cytotoxicity, but also nonspecific cytotoxicity induced anti-T3 mAb in these allospecific clones was inhibited by anti-T8 mAb in the absence of HLA class I expression on the target cell (Daudi cell line). The hierarchy of blocking with anti-T8 mAb and the classification of functional epitopes on T8 in anti-T3-induced nonspecific cytotoxicity were similar to those obtained in blocking of allospecific reactivity of the CTL clones. This analogy points to an identical function of the T8 antigen in both allospecific and anti-T3-induced nonspecific cytotoxicity. If HLA class I molecules are the counter structures of the T8 antigen, then these results argue against an adhesion-like function of the T8 structure. The combined results show that the T8 molecule has a regulatory role in CTL activation. It is postulated that the T8 antigen might serve as a receptor that transduces a negative feedback signal for T cell activation which prevents T cell triggering by nonspecific interaction.     

Selective monomorphic and polymorphic HLA class I antigenic determinant loss in surgically removed melanoma lesions

The analysis of human leukocyte antigen (HLA) class I allospecificity expression in malignant lesions has been hampered by the limited availability of HLA class I allospecificity-specific monoclonal antibodies (mAbs) which stain tissues in immunohistochemical (IHC) reactions. During the 12th International Histocompatibility Workshop, the HLA and cancer component made available a panel of mAbs capable of detecting monomorphic, locus- and allo-specific HLA class I antigenic determinants in surgically removed frozen tissue sections by IHC staining. In the present study, we have utilized this panel of mAbs to analyze the expression of HLA class I allospecificities in 33 primary and in 11 metastatic lesions surgically removed from HLA-typed patients with malignant melanoma, as this information contributes to determine the extent of HLA class I antigen abnormalities in melanoma lesions. HLA class I antigens were downregulated in six (18.2%) of the primary lesions and in six (54.5%) of the metastatic lesions. Selective loss of HLA-A and HLA-B antigens was detected in two (6.1%) and in one (3.0%), respectively, of the primary lesions, but in none of the metastases. HLA-A and HLA-B antigens were downregulated in three (9.1%) and four (36.4%) of the primary and metastatic lesions, respectively. Selective loss of one or more HLA class I allospecificities was found in 10 (33.0%) and two (18.0%) of the 33 primary and 11 metastatic melanoma lesions analyzed, respectively. HLA class I antigen abnormalities were present in 16 (48.5%) of the 33 primary lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, two lesions demonstrating selective abnormal reactivity with HLA-B locus-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A and HLA-B locus-specific mAbs, and seven lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). Furthermore, HLA class I antigen abnormalities were present in nine (81.8%) of the 11 metastatic lesions analyzed (i.e. six lesions demonstrating abnormal reactivity with HLA class I monomorphic-specific mAb, one lesion demonstrating selective abnormal reactivity with HLA-A locus-specific mAb, and two lesions demonstrating selective abnormal reactivity with HLA class I allele-specific mAb). It cannot be ruled out that the frequency of HLA class I allospecificity abnormalities is higher, as the expression of several HLA class I allospecificities could not be investigated because of the lack of appropriate probes. The frequency of HLA class I antigen defects in primary lesions was significantly correlated with primary lesion thickness, an important prognostic marker in melanoma, arguing for a potential clinical significance of HLA class I antigen abnormalities in melanoma. In conclusion, the results of the present study (i) demonstrate that the frequency of HLA class I allospecificity abnormalities in primary melanoma lesions is markedly higher than that of total HLA class I antigen downregulation described in the literature; (ii) corroborate our previous findings that staining of melanoma lesions with mAb to monomorphic determinants of HLA class I antigens does not detect selective HLA class I allospecificity loss; and (iii) demonstrate for the first time selective loss of antigenic determinants expressed on HLA class I molecules in melanoma lesions. The latter finding indicates that at least two mAbs recognizing distinct antigenic determinants on the HLA molecule being investigated should be used for IHC staining of tissue sections in order to prove that lack of immunostaining reflects actual loss of the corresponding HLA molecule and not selective loss of antigenic determinants.     

In vitro mutagenesis of HLA-B27: single and multiple amino acid substitutions at consensus B27 sites identify distinct monoclonal antibody-defined epitopes.

The consensus HLA-B27 sequence includes a unique constellation of amino acid residues along the peptide-binding cleft. To investigate the potential role of this region in the antigenic structure of HLA-B27, a panel of transfected cell lines was produced expressing 24 mutant B27 molecules with single or multiple substitutions within this constellation of residues. The cells were analyzed by flow cytometry with a panel of four anti-B27 mAb: ME1, GSP5.3, GS145.2, and B27M2. Previous studies have suggested that position 67 exerts a conformational effect on the ME1, GSP5.3, and GS145.2 epitopes. This was further supported in these studies by the observation that additional substitutions at the flanking residues 63 and 70 could reverse the disruption of these mAb epitopes by large residues at 67. Substitutions at positions 69-71 disrupted the binding of ME1 and GSP5.3, apparently by a direct effect. Individual substitutions at either of the two positions bearing residues unique to B27, 70 and 97, had no significant influence on the binding of any of the four mAb. The region of amino acid positions 63-71 in HLA-B27 thus appears to participate in the formation of at least three distinct epitopes shared by B27 and B7, identified by ME1, GSP5.3, and GS145.2.     

Four cytotoxic human-human hybridoma antibodies that react with HLA-B27

PBMC were isolated from a multiparous woman with HLA-B27 specific Abs in her serum. The HLA type of the donor was A2,9:B7. The PBMC were EBV transformed, and four cell lines making cytotoxic Abs to HLA-B27+ cells prepared. Hybridomas were constructed by fusing the EBV lines with the human fusion partner KR4. All four mAbs were of IgM isotype. One mAb (TrBH12) reacted specifically with B27+, B37+ and Bw47+ lymphoblastoid cell lines and with all B27+ PBMC except for a rare variant so far found only in one Norwegian family. Another mAb (Tr3B6) was cytotoxic for all B27+ cells tested, including the TrBH12- variant; in addition, it showed weaker cross-reactions to Bw42, B49 and a cell line with the probable phenotype B7,38. Supernatant from the Tr3B6 hybridoma was tested in lymphocytotoxicity against a panel of 658 individuals, 141 of whom were B27+. With this panel, Tr3B6 showed perfect correlation with HLA-B27. The two last mAbs (TrCG10 and TrBF1) reacted with all B27+ cells tested, but in addition showed quite extensive cross-reactions.     

Regulation of immunity to extraembryonic antigens in human pregnancy

Pregnancy results in the immunologic challenge of the female to a wide variety of allogeneic antigens. Particular attention has been given to antibodies directed to allotypic trophoblast antigens (TLX), for trophoblast form the true allograft interface between mother and fetus. Studies found that antibodies to paternal TLX allotypes are produced in women suffering from secondary recurrent abortions. These TLX antibodies are not directed to classical HLA private epitopes. In this report, treatment of lymphocytes with papain to remove HLA Class I did not decrease TLX antigen densities. These results suggest TLX antibodies are not directed to Class I epitopes, public or private. The allotypic nature of TLX antigens requires that a pregnant female must be able to regulate TLX immune responses to avoid rejection of the conceptus. One mechanism to specifically and systemically regulate TLX immunity is the idiotype anti-idiotype network. We provide preliminary evidence in this report for the presence of TLX idiotype network in a normal primigravida. Initially, no antipaternal TLX antibodies were detected in the serum of the primigravida, suggesting no TLX immunization had occurred. However, separation of Ab1 from Ab2 by absorption of primigravida serum with 2 degrees aborter Ab1 resulted in seroconversion. The primigravida's Ab1 was cytotoxic for paternal and 3rd-party lymphocytes in a non-HLA-restricted pattern. Primigravida's Ab2 was recovered from the Ab1 matrix by competitive elution by using platelets as source of TLX antigen. The Ab2 was found to inhibit cytotoxicity by 2 degrees aborter Ab1 as well as primigravida Ab1. This is evidence that the Ab2 recognizes a cross-reactive idiotype (CRI) on TLX antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

A unique murine monoclonal antibody recognizing HLA-B53, B37, B51, B52, +/-B44.

Monoclonal antibodies have played an important role in studying the biochemistry of the HLA-Class I molecules. Some murine anti-HLA mAbs can identify configurations of HLA epitopes that have never been reported in human allosera. One of these configurations is identified by an IgM mAb designated as: BHA-1441. This antibody was produced using a lymphoblastoid cell line typed as: A*02, A*25; B*38, B*4402/4405; C*0501, C*07, BW4, as the immunogen. A lymphocytotoxicity test of this mAb over a panel of 109 frozen, 452 fresh and, later, 44 DNA typed T cells revealed its specificity as B53, 37, 51, 52, +/- 44. All of the antigens recognized by this mAb share the Bw4 motif at positions 81-83, except for the HLA-B37, which shares only 82L and 83R. Furthermore, while B37 and B44 cross-react due to the aspartic acid (D) substitution at position 156, the reactivity with B53, B5 (51,52), B37 and 60% of B44 cells, makes it unlikely that the target epitope could be due only to the primary amino-acid sequence. The antibody-binding site might involve changes in tertiary structure and peptides bound by the MHC. BHA-1441 is an interesting tool to study and type the HLA-B53 antigen and its cross-reactive epitopes.     

A limited number of HLA epitopes are recognized from HLA class I-specific antibodies detected in the serum of sensitized patients

The goal of this study was to evaluate the epitope specificity of HLA class I-specific antibodies detected in the serum of sensitized patients awaiting retransplantation. The study group consisted of 22 sensitized from previous graft patients, who produced stable IgG HLA class I-specific antibodies. A total of 60 serum samples were screened and analyzed by two techniques in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISA technique. All recipients and donors were typed for class I HLA antigens by a standard lymphocytotoxicity technique. The epitope identification was based on class I HLA antigens sequencing, where the multiple immunogenic epitopes are differentially shared among various HLA antigens. The unique epitope configuration on one HLA antigen represents the private epitope of the specific HLA antigen while epitopes shared by more than one HLA antigen represent public determinants. In some HLA antigens (HLA-A1), more than one private epitope has been defined, while in others (HLA-B35, -B51), the private epitopes are not yet known. In a total of 36 antibody reactivity patterns, the majority of the definable IgG HLA class I-specific antibodies corresponded to the A-locus (75%), and only 25% had specificities against the B-locus antigens, although the number of incompatibilities concerning both loci were almost identical (29 for the HLA antigens of the A-locus and 26 for those of B-locus). All patients produced HLA class I-specific antibodies with specificities against the private epitopes of the immunogenic mismatched HLA antigen(s). In 6/21 cases (28.6%), HLA class I alloreactivity spreading to nongraft HLA antigens was detected and 9 public (shared) immunogenic alloepitopes were recognized. In conclusion, appling the epitope analysis of HLA class I-specific antibodies produced by sensitized from previous graft patients, we were able to define the immunogenic alloepitopes. We consider that the immunogenic alloepitopes, during transplantation course, are mainly private epitopes of mismatched HLA antigens and, in certain cases, shared epitopes between the donor alloantigens and other HLA antigens. This knowledge may offer the potential of transplanting sensitized patients through improved donor selection.     

Tripeptidyl peptidase II is dispensable for the generation of both proteasome-dependent and proteasome-independent ligands of HLA-B27 and other class I molecules

A significant fraction of the HLA-B27-bound peptide repertoire is resistant to proteasome inhibitors. The possible implication of tripeptidyl peptidase II (TPPII) in generating this subset was analyzed by quantifying the surface re-expression of HLA-B*2705 after acid stripping in the presence of two TPPII inhibitors, butabindide and Ala-Ala-Phe-chloromethylketone. Neither decreased HLA-B27 re-expression under conditions in which TPPII activity was largely inhibited. This was in contrast to a significant effect of the proteasome inhibitor epoxomicin. The failure of TPPII inhibition to decrease surface re-expression was not limited to HLA-B27, since it was also observed in several HLA-B27-negative cell lines, including Mel JuSo. Actually, HLA class I re-expression in Mel JuSo cells increased as a function of butabindide concentration, which is consistent with an involvement of TPPII in destroying HLA class I ligands. Inhibition of TPPII with small interfering RNA also failed to decrease the surface expression of HLA class I molecules on 143B cells. Our results indicate that TPPII is dispensable for the generation of proteasome-dependent HLA class I ligands and, without excluding its role in producing some individual epitopes, this enzyme is not involved to any quantitatively significant extent, in generating the proteasome-independent HLA-B27-bound peptide repertoire.     

A ligand-epitope in vitro analysis of major histocompatibility determinants expressed on B and T lymphocytes.

Human histocompatibility leucocyte antigen (HLA)-specific monoclonal antibody probes were used to determine the affinity constant, and cell-surface density of HLA class I and class II determinants. The measurements were estimated for single-cell units of B-lymphoblastoid cell line (B-LCL) and cloned activated T cells in different functional states. Each HLA subset showed unimodal affinity constant values for the interaction with the corresponding HLA-specific antibodies. Such values ranged between 2.2 x 10(7) M-1 (class I) and 4.0 x 10(7) M-1 (class II) for different histocompatibility epitopes. In both B and T cells there was a rank order of epitope expression, class I being highly expressed (5 x 10(6) epitopes/cell) followed by DR, DQ and DP, (1.1-3.0 x 10(6) epitopes/cell). Suppressive clones carrying functionally defined stimulating determinants previously designated 'DY' carried similar numbers of DR, DQ and DP binding sites to DY- non-suppressive clones, but showed selective increases of class II determinants reactive with broad class II-specific antibodies. The results are discussed in the context of the functional consequences of different patterns of HLA epitope expression in immune responses.     

Mapping of Bet v I epitopes by using murine monoclonal antibodies.

The different determinants of birch pollen extracts, as shown by SDS-PAGE analysis, range from 10 to 94 kDa. These determinants were then electrotransferred on nitrocellulose strips and allowed to react with human IgE Ab from sensitive patients in order to identify the allergenic determinants. Several minor (43, 35, 28 and 21 kDa) and the major (17 kDa) allergenic determinants were identified. Murine monoclonal antibodies (mAb) were then produced against the major allergenic determinant (Bet v I) and their specificity confirmed by immunoblot. One of them, mAb 3F10, was used to affinity-purify the Bet v I. The purity of this material was confirmed by SDS-PAGE analysis and its reactivity on immunoblot against human IgE ensured its biological activity. These mAb were then gathered on four families based on their pattern of reactivity with Bet v I. Indeed four different epitopes on the molecule were identified. Binding inhibition studies using two of them (mAb 5F9 and 8F12) suggested that the epitopes of Bet v I recognized by these mAb are not overlapping. On another hand, the binding of 8H7 and 3F10 was partially inhibited by 5F9 and the binding of 3F10, by 8F12. These data suggest that those two latter epitopes are somewhat overlapping. Finally, the mAb 5F9 could inhibit the binding of human IgE on the affinity-purified Bet v I up to 40% and then shares a common idiotope with human specific IgE Ab of allergic patients.     

Association of four HLA class III region genomic markers with HLA haplotypes

Abstract: We have studied restriction fragment length polymorphism (RFLP) in the region 300 kb centromeric to the HLA-B locus. Four probes were used: one was genomic DNA derived from the tumor-necrosis factor (TNF)-β gene, one was a cDNA for the BAT3 gene, and two single-copy genomic probes, R5A and M20A. The order of these markers from HLA-B towards the centromere is M20A, R5A, TNF and BAT3. The BAT3 and TNF-β probes each detected two allelic bands with Taq I and Nco I digestion, respectively; the R5A and M20A probes each detected three polymorphic allelic bands with BstEII digestion. To determine if these restriction polymorphisms are preferentially associated with certain HLA-B and -DR haplotypes, a total of 153 HLA haplotypes was analyzed. The haplotypes Al, B8, DR3 and A3, B7, DR2 were each associated with a distinct combination of polymorphisms identified at these four sites, thereby demonstrating that the strong linkage disequilibrium characteristic of these haplotypes extends also to this segment of the class III region. In contrast, haplotypes that are not in positive linkage disequilibrium, such as A1,B8,DR4 and A2,B7,DR3, showed ho preferential association with any of these polymorphisms. The antigens HLA-B27 and B35 were also found to be in positive linkage disequilibrium with RFLP patterns at three of these sites, and HLA-B14,B35,B44,Bw57 and Bw62 were found preferentially associated with polymorphisms at one or two of these sites, independent of the DR antigen present. These data further demonstrate that genetic linkage disequilibrium in the HLA class III region is complex and variable among different HLA halpotypes.     

Anti-HLA-B7,B27,Bw42,Bw54,Bw55,Bw56, Bw67,Bw73 monoclonal antibodies: specificity, idiotypes, and application for a double determinant immunoassay

The monoclonal antibodies (MoAbs) KS3 and KS4 are secreted by hybridomas constructed with splenocytes from a BALB/c mouse sequentially immunized with the cultured lymphoid cells JKu and LG-2 which share only the HLA-B27 specificity. Serologic and immunochemical assays have shown that the two MoAbs recognize the same (or spatially close) determinant expressed by HLA-B7,B27,Bw42,Bw54,Bw55,Bw56,Bw67, and Bw73 alloantigens. This determinant is spatially close but distinct from those defined by the anti HLA-B27 monoclonal antibodies described in the literature. The syngeneic antiidiotypic MoAb T12-105 and T12-211 elicited with MoAb KS4 were shown to recognize idiotopes within the antigen combining site of MoAb KS3 and KS4. Neither idiotope was detected on the anti HLA class I and anti HLA class II monoclonal antibodies tested. The MoAb KS4 in combination with the anti human beta 2-microglobulin MoAb NAMB-1 was utilized to develop a double determinant immunoassay (DDIA). The latter represents a sensitive method to detect and quantitate HLA-B27 antigens in spent culture medium of lymphoid cell lines and in serum. Typing for HLA-B27 antigens with the DDIA of sera from HLA typed donors yielded results highly correlated with those of the conventional lymphocytotoxicity assay.     

MULTIPRED2: a computational system for large-scale identification of peptides predicted to bind to HLA supertypes and alleles

MULTIPRED2 is a computational system for facile prediction of peptide binding to multiple alleles belonging to human leukocyte antigen (HLA) class I and class II DR molecules. It enables prediction of peptide binding to products of individual HLA alleles, combination of alleles, or HLA supertypes. NetMHCpan and NetMHCIIpan are used as prediction engines. The 13 HLA Class I supertypes are A1, A2, A3, A24, B7, B8, B27, B44, B58, B62, C1, and C4. The 13 HLA Class II DR supertypes are DR1, DR3, DR4, DR6, DR7, DR8, DR9, DR11, DR12, DR13, DR14, DR15, and DR16. In total, MULTIPRED2 enables prediction of peptide binding to 1077 variants representing 26 HLA supertypes. MULTIPRED2 has visualization modules for mapping promiscuous T-cell epitopes as well as those regions of high target concentration - referred to as T-cell epitope hotspots. Novel graphic representations are employed to display the predicted binding peptides and immunological hotspots in an intuitive manner and also to provide a global view of results as heat maps. Another function of MULTIPRED2, which has direct relevance to vaccine design, is the calculation of population coverage. Currently it calculates population coverage in five major groups in North America. MULTIPRED2 is an important tool to complement wet-lab experimental methods for identification of T-cell epitopes. It is available at http://cvc.dfci.harvard.edu/multipred2/.