Mutations in the third, but not the first or second, hypervariable regions of DR(beta 1*0101) eliminate DR1-restricted recognition of a pertussis toxin peptide.

We have examined the role of 12 polymorphic residues of the beta-chain of the HLA-DR1 class II molecule in T cell recognition of an epitope of pertussis toxin. Murine L cell transfectants expressing wild-type or mutant DR1 molecules (containing single amino acid substitutions in DR(beta 1*0101)) were used as APC in proliferation assays involving nine DR1-restricted T cell clones specific for peptide 30-42 of pertussis toxin. Four different patterns of recognition of the mutants were found among the pertussis-specific clones. Residues in the third hypervariable region (HVR) of DR(beta 1*0101) are critically important for all the T cell clones; amino acid substitutions at positions 70 and 74 abrogated recognition by all of the T cell clones, and substitutions at positions 67 and 71 eliminated recognition by most of the clones. In contrast, most single amino acid substitutions in the first and second HVR, predicted to be located in the floor of the peptide binding groove, had little or no effect on the proliferative responses of these clones. However, the involvement of beta-chain first and second HVR residues was demonstrated by the inability of transfectants expressing wild-type DR(beta 1*0404) (DR4Dw14) or DR(beta 1*1402) (DR6Dw16) to present peptide to these clones. These beta-chains have completely different first and second HVR compared with DR(alpha,beta 1*0101) although the third HVR are identical. These results illustrate the functional importance of third HVR residues of DR(beta 1*0101) and allow definition of the molecular interactions of the DR1 molecule with the 30-42 peptide.     

Polymorphic HLA-DR7 beta 1 chain residues that are involved in T cell allorecognition

The contributions to allorecognition of polymorphic amino acids in the HLA-DR7 beta 1 chain were analyzed by using mutant DR7 beta 1 chains with single amino acid substitutions at position 4, 11, 13, 25, 30, 37, 57, 60, 67, 70, 71, 74, or 78. Transfectants expressing mutant DR7 molecules were used as stimulators for six DR7-alloreactive T cell clones. The majority of the substitutions had profound effects on the ability of the DR7 molecule to stimulate one or more T cell clones. Nine of the 13 substitutions completely abrogated recognition by at least one clone. The finding that each of the substitutions in the beta-strands in the floor of the peptide binding groove affected T cell allorecognition supports the model of allorecognition in which the complex of a self-peptide bound to a class II molecule is recognized by the TCR. Interestingly, the substitution at position 4, which is predicted to be located outside the peptide binding groove, decreased the ability of the DR7 molecule to stimulate some clones. Each of the DR7-alloreactive T cell clones had a unique reactivity pattern in response to the different mutant molecules, indicating that the TCR of each clone recognized the DR7 molecule differently. Surprisingly, many of the mutant DR7 molecules induced proliferation by one or more clones that was greater than 125% of the proliferation induced by the wild-type DR7 molecule. These data indicate that multiple polymorphic residues, predicted in the class II model to be located in both the beta-strands and alpha-helix of the DR7 beta 1 chain, contribute to allorecognition of the DR7 molecule.     

Nonrandom selection of T cell specificities in anti-HLA-DR responses. Sequence motifs of the responder HLA-DR allele influence T cell recruitment

The self-restriction of Ag-specific T cell responses is interpreted as the result of a positive selection of the individual's T cell specificities for their compatibility with self-MHC molecules. If the T cell receptor (TCR) specificities in any given individual have an affinity for syngeneic MHC molecules, it is unclear how they interact with allogeneic MHC structures. To approach this question, we analyzed 123 alloreactive HLA-DR4 Dw4 or Dw14 specific T cell clones that were generated from responder/stimulator combinations with defined disparities in the HLA-DR beta 1-chain. Sets of T cell clones were established from three different HLA-Dw4+ responders and compared for their fine specificities. The majority of HLA-DR4 Dw14 specific T cell clones co-recognized HLA-DR1 Dw1+ (33 to 36% of all T cell clones) or HLA-DRw14 Dw16+ (26 to 33%) stimulators, both of which share very similar sequences in the third hypervariable region of the HLA-DR beta 1-chain with the HLA-DR4 alleles Dw4 and Dw14. These data suggest that sequence and structural similarities in the alpha-helical portions of the HLA-DR molecule impose a strong bias on the recognition of allotargets. The second haplotype of the responder did not appear to affect the typical fingerprint of T cell recognition except for the deletion of self-reactive TCR specificities. Nonrandom usage of TCR specificities in anti-HLA-DR responses was also found for HLA-DRw11/DRw13+ and HLA-DRw11/DR7+ T cell donors who did not share any obvious polymorphic sequence stretches with the allostimulators HLA-DR4 Dw4 or Dw14. T cell clones from an HLA-DRw11/DRw13+ responder functionally resembled the TCR specificities derived from the HLA-DR4 Dw4+ donors. T cell clones derived from an HLA-DRw11/DR7+ individual were characterized by a distinct cross-reactivity pattern preferring HLA-DRw13 Dw19+ (50 to 60%) and HLA-DR3+ (43 to 57%) stimulator cells. These findings suggest that the responder HLA-DR alleles influence the structural constraints in the recognition of allo-HLA-DR molecules in closely related and in completely disparate responder/stimulator combinations.     

Effect of natural polymorphism at residue 86 of the HLA-DR beta chain on peptide binding

Class I and class II MHC glycoproteins are highly polymorphic molecules that bind antigenic peptides and present them on cell surfaces for recognition by T lymphocytes. Even though MHC polymorphism has long been known to affect both peptide binding and recognition by the TCR, the role of individual amino acids of MHC proteins in these interactions is poorly understood. To examine the effect of a small number of amino acid residues on T cell stimulation, B lymphoblastoid cell lines homozygous for the closely related DR1 subtypes, Dw1 and Dw20, and the DR4 subtypes, Dw4 and Dw14, were compared for their ability to present an immunogenic influenza hemagglutinin peptide (HA307-319) to an Ag-specific, DR1,4-restricted T cell clone. B cell lines expressing DR1 Dw20 and DR4 Dw14 presented HA307-319 much less efficiently than DR1 Dw1 and DR4 Dw4 and bound a biotinylated analogue of the same peptide less well. Analysis of DRB1 gene sequences suggested that polymorphism at residue 86 had a major effect on peptide binding. Differences in binding of a set of HA307-319 analogues biotinylated at each residue to cells expressing DR1 Dw1 and DR1 Dw20 suggested that the polymorphism affected the interactions of many peptide residues with the class II molecule. In inhibition assays, DR1 Dw1 and DR4 Dw4 were shown to differ from DR1 Dw20 and DR4 Dw14 in their length requirements for peptide binding. Using a larger panel of homozygous B cell lines expressing many class II haplotypes, a Ser-309 substituted HA307-319 analogue was shown to bind to most B cell lines expressing Val-86 containing alleles (including DR1 Dw20 and DR4 Dw14) but failed to bind most B cell lines expressing Gly-86 alleles (including DR1 Dw1 and DR4 Dw4). The results indicated that polymorphism at residue 86 influenced the specificity and affinity of peptide binding and affected the conformation of peptide-DR protein complexes without completely eliminating T cell recognition.     

Purified HLA class II peptide complexes can induce adherence and activation of peptide-specific human T cell clones.

An initial event in T cell activation is the specific adherence of T cells via their T cell receptor to the MHC peptide complex. We have studied this adherence by incubating T cells with preformed HLA DR4Dw4 peptide complexes attached to a solid support. Adherence of sodium 51Cr-labeled T cell clones specific for the influenza hemagglutinin peptide, HA 307-319, was maximal after 15 min and was specific for the HLA DR4Dw4-HA 307-319 complex. The binding was temperature dependent and could be blocked with azide or protein kinase C inhibitors, indicating that for adherence the T cells need to be metabolically active and have a functioning protein kinase C pathway. The adherence could be blocked with CD4- or CD3-reactive murine mAb, suggesting that the TCR and CD4 molecules work in concert to induce strong adherence to the HLA DR4Dw4-HA 307-319 complex. A subsequent event in T cell activation is proliferation, which is thought to need additional proteins such as IL-1 or other adhesion molecules. MHC peptide complexes coated on microtiter plates also induced proliferation in the human T cell clones. Removal of any monocytes by treatment of human T cell clones with anti-CD14 in conjunction with C, followed by purification over a nylon wool column, did not abrogate proliferation. After prolonged culture of the T cell clones in plates coated with peptide-pulsed HLA DR4Dw4 in the presence of IL-2, the T cell clones continued to proliferate in response to peptide. These results suggest that human T cell clones do not require a second signal from a monocyte or other APC to proliferate.     

Analysis of DR beta and DQ beta chain cDNA clones from a DR7 haplotype

A cDNA library was constructed from a DR7, DRw53, DQw2 homozygous cell line, cDNA clones corresponding to DR beta and DQ beta chains were isolated, and the nucleotide sequences of the polymorphic first domains of these chains were determined. A novel screening strategy allowed rapid and simple identification of cDNA clones corresponding to both DR beta chains (DR7 beta1 and DR7 beta2): DR7 beta2 clones have a recognition site for the enzyme BssHII, whereas DR7 beta1 clones do not. The DR7 beta 1 sequence differs significantly from all previously described DR beta chains. As predicted by the presence of the BssHII site in DR7 beta 2 clones, the DR7 beta 2 sequence differs from the DR7 beta 1 sequence. The sequence of the DRw53-associated DR7 beta 2 chain is identical to the reported sequence of the DRw53-associated DR4 beta 2 chain. In addition, the sequence of the DQ beta chain from the DR7, DQw2 cell line is identical to the reported sequence of a DQ beta chain from a DR3, DQw2 cell. These findings raise interesting questions about the evolution of the DR3, DR4, and DR7 haplotypes.     

Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules

By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.     

HLA-DR2a is the dominant restriction molecule for the cytotoxic T cell response to myelin basic protein in DR2Dw2 individuals.

The HLA-DR2 restriction of the T cell response to myelin basic protein (MBP) was studied using murine L cells transfected with DRalpha and either DR2a or DR2b beta-chain cDNA. DR2a and DR2b represent the two isotypic DRbeta chains expressed in DR2Dw2 haplotypes. Eleven MBP-specific cytolytic T cell lines derived from patients with multiple sclerosis were isolated. Two of these cell lines recognized MBP-pulsed DR2-expressing L cell transfectants and four of them could only recognize the L cells if the adhesion molecule ICAM-1 was expressed in addition to HLA-DR2. Five of the six lines were restricted by HLA-DR2a; one line recognized Ag in conjunction with DR2b, but only if ICAM-1 was coexpressed. The remaining five lines did not lyse MBP-pulsed L cells. The ability of the DR2b molecules on transfected cells to stimulate T cells was confirmed with DR2b-allospecific T cell clones. Although five MBP-specific lines were restricted by DR2a, they recognized different parts of the MBP molecule, as demonstrated by the presentation of shorter peptides. Thus, our results suggest that DR2a is a dominant restriction molecule in MBP-specific responses by DR2+ MS patients. The results also indicate that the reported heterogeneity in MBP epitopes recognized by DR2-restricted T cells, may not be due to the use of different restriction elements but rather to the binding of different MBP peptides to DR2a molecules.     

Molecular mapping of interactions between a Mycobacterium leprae-specific T cell epitope, the restricting HLA-DR2 molecule, and two specific T cell receptors.

A systematic series of 89 single residue substitution analogs of the Mycobacterium leprae 65-kDa protein-derived peptide LQAAPALDKL were tested for stimulation of two HLA-DR2 restricted 65 kDa-reactive T cell clones from a tuberculoid leprosy patient. Some analogs with substitutions outside a "core" region showed enhanced stimulation of the T cell clones. This core region of seven or eight residues was essential for recognition, whereas substitution of amino acids outside this region did not affect T cell recognition although these residues could not be omitted. Thus these core residues interact directly with the presenting HLA-DR2 molecule and/or the TCR. Except for analogs of position 419 for clone 2B6, the majority of the nonstimulatory substitution analogs did not inhibit the presentation of LQAAPALDKL and thus probably failed to bind to the HLA-DR2 molecule. Unless all of the core residues are physically involved in binding to DR2, substitution at a position not directly involved in binding appears to have an influence on other residues that do bind to the DR2 molecule. Active peptide analogs with two or more internal prolines suggest that not all analogs need be helical for activity with clone 2F10.     

Mapping of distinct serologic and T cell recognition epitopes on an HLA-DR beta-chain.

A new DR beta-chain allele is defined that is identical to the previously described DR6b molecule except for the first hyperpolymorphic region, where the new allele displays the same polymorphisms found on DR8 and DR12 genes. Two distinct epitopes have been mapped on this new allele. The polymorphism in common with DRw8 and DRw12 is recognized by mAb GS313-9D11. However, alloreactive T cell clones specific for DR6b cells (Dw9) recognize this allele, whereas Dw8-specific T cell clones do not. The mAb determinant maps to the first beta-sheet and probably involves a polymorphic residue lying outside the helix. The binding of mAb 9D11 to this region does not interfere with TCR binding. Alloreactive T cell recognition is associated with polymorphisms located predominantly on the alpha-helical portion of the molecule.     

T cell activation-inducing epitopes of the house dust mite allergen Der p I. Proliferation and lymphokine production patterns by Der p I-specific CD4+ T cell clones.

Cloned human CD4+ T cell lines specific for the house dust mite Dermatophagoides pteronyssinus were used to map minimal T cell activation-inducing epitopes on the Group I allergen in D. pteronyssinus extracts (Der p I) molecule. Most of these Der p I-specific T cell clones expressed different TCR V alpha and V beta gene products. Using recombinant deletion proteins, three T cell epitopes were identified on the Der p I molecule; p45-67 and p117-143 were recognized by HLA-DR7-restricted T cells, whereas p94-104 was recognized in the context of HLA-DR2, DRw11 (DR5), and -DR8 molecules. This degenerate class II MHC restriction appears to be due to shared Phe and Asp residues at positions 67 and 70, respectively, in the third variable domain of the HLA-DR beta chain. All three T cell epitopes induced Th2-like cytokine production profiles by the Der p I-specific T cell clones, which were characterized by the production of very high levels of IL-4 and IL-5, as compared with those secreted by tetanus toxin-specific T cell clones derived from the same patients, but no or low amounts of IL-2 and IFN-gamma. This Th2-like production profile was, however, not an intrinsic property of the Der p I-specific T cells, but was dependent upon their mode of activation. Stimulation with Con A also induced very low or no measurable levels of IL-2 and IFN-gamma, whereas activation with TPA and the calcium ionophore A23187 resulted in the production of high levels of IL-4, IL-5, IL-2, and IFN-gamma. These results indicate that Der p I-specific T cell clones are not defective in their capacity to produce high levels of Th1 cytokines.     

Utilization of soluble fusion proteins for induction of T cell proliferation

A peptide display library was evaluated as a means to identify peptide binding motifs for class II molecules. Peptides expressed as part of a soluble fusion protein with a maltose binding protein (malE) were produced by Escherichia coli. Constructs containing the high-affinity binding influenza hemagglutinin peptide 307W–319 (mal-HA) or the low-affinity binding tetanus toxoid peptide 830–843 (mal-TT) were used as controls. mal-HA, but not mal-TT, inhibited synthetic biotinylated-HA peptide from binding to purified DR4 Dw4 molecules in a dose-dependent manner. The fusion-peptide presentation system was also evaluated for its ability to induce antigen-specific T cell proliferation. DR4 Dw4⁺ B cells pulsed with mal-HA, but not mal-TT, induced dose-dependent proliferation of an HA-specific DR4 Dw4-restricted T cell line to the same extent as synthetic HA peptide. Using this type of peptide display library, it may be possible to determine the antigenic specificity of T cell clones isolated from patients with autoimmune diseases.     

cDNA cloning and sequencing reveals that the electrophoretically constant DR beta 2 molecules, as well as the variable DR beta 1 molecules, from HLA-DR2 subtypes have different amino acid sequences including a hypervariable region for a functionally important epitope

Two-dimensional gel electrophoresis (2D-PAGE) of DR molecules from three different Dw subtypes (Dw2, Dw12, and FJO) of the HLA-DR2 haplotype reveals that at least two DR beta genes are expressed. Protein mixing experiments demonstrate that one of the two expressed DR beta molecules is electrophoretically variable (referred to as DR beta 1), and the other (DR beta 2) migrates constantly among DR2 subtypes. We have constructed cDNA libraries from Dw12 and FJO homozygous typing cells (HTC DHO for Dw12 and HTC FJO for FJO) and isolated DR beta cDNA clones. Four of these clones (FJO-13, DHO-8, FJO-6, and DHO-7) were sequenced, and the deduced amino acid sequences were compared with each other and with two published amino acid sequences for the DR beta molecules derived from a DR2-Dw2HTC. Prediction of the migration patterns on 2D-PAGE from the amino acid sequences of these and other DR beta molecules allows the tentative designation of the two full-length cDNA (DHO-8 and FJO-13) as coding for DR beta 2 molecules and the other two cDNA (DHO-7 and FJO-6) for DR beta 1 molecules. Amino acid sequence comparisons also show that the constantly migrating DR beta 2 molecules, as well as the electrophoretically variable DR beta 1 molecules, from Dw2, Dw12, and FJO have different primary amino acid sequences, including a clustered difference in the third hypervariable region of the polymorphic first domain.     

Clonal analysis of B and T cell responses to Ia antigens. IV. Proliferative T cell clones recognizing E beta and/or E alpha allodeterminants

The allospecific T cell recognition of the I-Ek molecule was assessed by using eight A. TH anti-A. TL proliferative T cell clones, all of which expressed the Thy-1-2+, Lyt-1+, Lyt-2-, Ia-, and p94,180+ cell surface phenotype. The use of panels of stimulating cells from homozygous of F1 hybrid strains indicated each T cell clone exhibited specificity for distinct alloactivating determinants including: i) a private E beta k-controlled determinant expressed in cis- or trans-complementing E beta kE alpha strains; ii) an apparently nonpolymorphic E alpha determinant resembling the serologic specificity Ia.7, i.e., present in all strains carrying E alpha and E beta expressor alleles; and iii) a series of conformational I-E determinants, the expression of which required a precisely defined combinatorial association of E beta plus E alpha chains. Two clones were found to be reactivated by cis- but not trans-complementing E beta k E alpha k strains, and another recognized an allodeterminant shared by the I-Ab molecule. Various I-Ek-reactive monoclonal antibodies (mAb) directed to epitopes presumably expressed on either E alpha (epitope clusters I and II) or E beta (epitope cluster III) chains inhibited the proliferative responses of seven clones recognizing private E beta k or unique E beta E alpha conformational activating determinants. By contrast, the restimulation of the clone directed to a nonpolymorphic E alpha determinant was selectively blocked by anti-Ia.7 mAb defining epitopes on the E alpha chains but not by those directed to the E beta chain. On the basis of these data, it was concluded that the recognition sites of most anti-I-Ek proliferative T cells were expressed on the E beta chain or the E beta plus E alpha interaction products, and that a minority of such alloreactive T cells could be activated through recognition of the E alpha chain per se.     

Immunochemical analysis of the Ia polymorphisms among the family of DR7-associated HLA-D specificities

Among cells that bear the serologically defined Ia alloantigen DR7, four T cell-defined HLA-D specificities have been described: Dw7, Dw17, Dw11, and Dw7L. Ia molecules expressed by cells homozygous for these specificities have been compared by using immunofluorescence and two-dimensional gel electrophoresis in order to identify the DR and DQ polymorphisms among the family of DR7-associated HLA-D specificities. Cells homozygous for each of the four HLA-D specificities have in common one DR molecule that is indistinguishable by these methods. Two DR-specific monoclonal antibodies, IIIE3 and 109d6, detect a second distinct DR molecule on Dw7, Dw17, and Dw7L cells. This second DR molecule is also very similar from cells of the three specificities. In contrast, a second DR molecule was not detected on four Dw11 homozygous cells. Therefore, these data raise the possibility that all DR homozygous cells do not express the same number of DR molecules. The DQ molecules expressed by DQw2-positive Dw7, Dw17, and Dw7L cells are also very similar, whereas DQw3-positive Dw11 DQ molecules are structurally different. Therefore, no DR or DQ structural polymorphisms were detected to correlate with the Dw7, Dw17, and Dw7L T cell-defined Ia polymorphisms.     

Analysis of the permissive association of a malaria T cell epitope with DR molecules.

In this study we examined the association of a promiscuous malaria T cell epitope, CS.T3, to different HLA-DR alleles. A large series of singly substituted or truncated variants of CS.T3 was prepared and tested for the ability to be recognised in association with, or to bind to, three distinct HLA-DR alleles (DR1, DRw11, and DRw14(w6)) and three natural variants of HLA-DRw11. We found that although association with the different DR molecules mapped to identical or closely overlapping regions of the peptide, distinct substitutions could drastically influence the capacity of the peptide to interact with one but not another of the three DR molecules tested. Based on analysis of the distribution of residues recognized by T cell clones restricted to the different DR alleles, we suggest that the peptide CS.T3 is not bound, at least for the three DR examined, as an alpha-helix. In addition we tested three subtypes of DRw11 as APC for the CS.T3 analogues and observed that the peptide is most likely bound in the same conformation to the three natural variants of the DRw11 molecule.     

Molecular studies of a rare DR2/LD-5a/DQw3 HLA class II haplotype. Multiple genetic mechanisms in the generation of polymorphic HLA class II genes

A cDNA library was constructed from a homozygous B lymphoblastoid cell line (REM) obtained from an individual of a long isolated American Indian tribe, the Warao. The REM cell line expresses serologically defined determinants, DR2 and DQw3, and the T lymphocyte-defined (Dw/LD) specificity, LD-5a. T cells can recognize differences between FJO (a DR2/DQw1 cell line that expresses the Dw specificity MN2) and REM for both DR and DQ molecules. cNDA clones encoding the polymorphic DR beta 1-, DR beta 2-, DQ beta-, and DQ alpha-chains were sequenced and compared with other DR and DQ gene sequences. The DR beta 1-sequence of REM is identical to the DR beta 1-sequence of FJO; the DR beta 2-sequence is also identical to that of FJO except for one amino acid difference at position 67 in the polymorphic first domain (Leu in REM, Phe in FJO) due to a single point mutation. The DQ beta-sequence is identical to that of DR4/DQw3 haplotype; the DQ alpha is different from the DQ alpha of DR4/DQw3 haplotype and identical to the DQ alpha of both the DR3/DQw2 haplotype of a Raji cell line and the DR5/DQw3 haplotype in deduced amino acid sequence. Taken together, these findings suggest that: 1) a single amino acid difference (position 67) in the third hypervariable region of the first domain of the DR beta 2-chain in the DR2 haplotype is apparently sufficient for stimulating T cell responses; 2) the DQw3 serologic specificity may be defined mainly by the DQ beta-rather than DQ alpha-chain; and 3) multiple genetic events have probably occurred to generate the rarely found REM (DR2/LD-5a/DQw3) haplotype.     

Functional definition of HLA-DR and -DQ epitopes specific for DR2-short,DwFJO haplotype

T-lymphocyte clones specific for the influenza A/Texas virus were obtained by limiting dilution of activated T cells from an HLA A2/3, B7/39, Cw -/-, DR2-short/2 short, DQw1/w1, DwFJO/FJO donor. Among the proliferating clones studied, and irrespective of their antigenic specificities, most of them were restricted by epitope(s) on HLA-DR molecules present only on DR2-short/DwFJO cells but not on DR2-negative or DR2-long positive (Dw2, Dw12, Dw-) cells. Two clones were restricted by epitopes borne by DQ products. Here again, these epitopes were present on DR2-short/DwFJO but not on DR2-long, DQw1 (Dw2, Dw12) cells, indicating that the DQwl molecules of DR2-long and DR2-short haplotypes are different. Taken together, these results indicate that the DR2-short, DwFJO haplotype is characterized by both HLA-DR- and DQ-specific molecules. Finally, one clone was restricted by an epitope shared by DR products from DR2 short/DwFJO, DRw11, and DRw13 haplotypes. This latter functional determinant has never been described until now.Abbreviations used in this paper APC antigen-presenting cells - HAU hemagglutinin units of influenza virus - HLA human leukocyte antigens - HTC homozygous typing cells - IL-2 interleukin 2 - mAb monoclonal antibody - MHC major histocompatibility complex - MLR mixed lymphocyte reactions - PBM peripheral blood mononuclear cells - %RR relative response percent