Different regions of the N-terminal domains of HLA-DR1 influence recognition of individual peptide-DR1 complexes

The contributions of individual amino acids in the polymorphic β chain and the conserved chain of HLA-DR1 to influenza HA-specific DR1-restricted and anti-DR1 allospecific T-cell recognition were analyzed. The genes encoding HLA-DR1 were subjected to site-directed mutagenesis in order to introduce single amino acid substitutions at 12 positions in the β₁ domain and 11 positions in the ₁ domain. The β₁-domain substitutions were all at polymorphic positions and introduced residues that are found in DR4 alleles. The amino acids introduced into the DR₁ domain were based on the sequences of other human and mouse class II chains. The responses of 12 DR1-restricted T-cell clones specific for two peptides of HA and seven anti-DR1 allospecific clones were studied. Substitutions at positions that point up from and into the peptide-binding site in the third variable region of the β₁-domain -helix caused substantial reduction in the responses of all the clones. Substitutions at multiple positions in the β₁-domain floor and in the ₁ domain influenced the anti-DR1 responses of the alloreactive and of the HA100-115-specific T-cell clones. In contrast, very few changes outside of the β₁ domain third variable region affected the responses of the HA306-324-specific DR1-restricted T-cell clones. These results suggest that a surprisingly limited region of the HLA-DR1 molecule is critically involved in T-cell recognition of HA306-324 by DR1-restricted T cells. However, the susceptibility of the HA100-115-specific and the anti-DR1 allospecific T-cell clones to substitutions at multiple positions in both N-terminal domains shows that the response to DR1-HA306-324 is unusual and may reflect the promiscuity with which this peptide binds to HLA-DR molecules. Human Immunology 40, 311–322 (1994)     

A family with an apparent HLA-DR triplet: evidence for exchange of functional HLA-DR beta-genes between different haplotypes

Serological and protein analysis showed that HLA-DR1 and DR2 short (s) segregated together as one haplotype, resulting in a HLA-DR triplet as found in a family study. Both DR1 and DR2 molecules were coordinately expressed and were shown to function as restriction elements in antigen presentation assays. This unique HLA-DR1, 2s haplotype was further studied by Southern blot analysis. Based upon well-known restriction fragment length polymorphisms for the involved gene sets, i.e. DR1 and DR2 along with the DR type from the other haplotype, the genes as identified by restriction fragment length polymorphism of the triplet could be established. Pseudogenes, which are included in the previously described gene sets of HLA-DR1 and DR2 are apparently lacking in the triplet. We therefore postulate that during an unequal crossing-over event the DR beta-pseudogene of DR2 could be exchanged by a functional DR1 beta-gene.     

Molecular characterization of a recombinant HLA-DR1/DR2 haplotype

Serologic analysis of two families identified an HLA-DR haplotype in which DR1 and DR2 cosegregated. DNA-RFLP analysis of these families with an HLA-DRB probe revealed a pattern of hybridization suggestive of a recombination between DR1 and DR15. Following amplification, cloning, and nucleotide sequencing of HLA-DRB-gene second-exon DNA sequences, three DRB amplification products associated with the novel haplotype were identified: these corresponded to DRB1*0101, DR2 pseudogene, and DRB5*0101. Clones representing the DRB1*1501 and DR1 pseudogenes were not identified: oligonucleotide typing with DRB1*1501-specific probes confirmed the absence of this gene within the DR1/DR2 haplotype. We postulate that the DR1/DR2 haplotype represents a recombinant between those of DR1-Dw1 and DR15-Dw2, and that the crossing-over may have been between the DRB1*0101 gene and the DR2 pseudogene. This is further supported by DNA-RFLP analysis with HLA-DQB and DQA CDNA probes, which revealed conserved linkage genes between the DQB1*0501, DQA1*0101, and DRB1*0101 genes.     

Functional difference between HLA-DR and HLA-DQ molecules in mixed lymphocyte reaction

In order to analyze the functional differences between HLA-DR and HLA-DQ molecules, we have established transfectants expressing HLA class II molecules. We investigated the contribution of these molecules in mixed lymphocyte reaction (MLR) using these transfectants. 1) The genomic clones encoding for DR alpha, DR beta, DQ alpha, and DQ beta from HLA-Dw 15 haplotype were isolated. These genes were introduced into murine L cells and two kinds of stable transfectants expressing either of HLA-DR4 and HLA-DQw4 were established. Expression of HLA class II molecules on transfectants was confirmed by FACS analysis using monoclonal antibodies specific for HLA class II molecules. 2) Primary MLR against class II transfectants and blocking experiments showed that DR molecules function as dominant stimulator molecules in allo MLR, whereas DQ molecules as well as DR molecules stimulate equally auto MLR. 3) We also determined the clone size of MLR reactive CD4+ T cells by the limiting dilution analysis. Frequencies of allo DR, auto DQ, and allo DQ reactive CD4+ T cells was estimated to be almost equal, but frequency of auto DR reactive CD4+ T cells was estimated to be far low. These results suggest the relatively high occurrence of auto DQ reactive clones which contribute significantly to auto MLR. These auto DQ reactive clones may not be eliminated as efficiently as DR reactive clones, because of lower expression of DQ molecules than DR molecules on bone marrow derived cells.     

Both HLA-DR and HLA-DQ determinants contribute to HLA-Dw typing

The respective contribution of HLA-DR and HLA-DQ gene products in the induction of allogeneic proliferative responses in primary mixed lymphocyte reaction and, therefore, in HLA-Dw typing, is still unclear or controversial. This is in part due to a strong linkage disequilibrium between HLA-DR and -DQ genes. We used DR- or DQ-restricted influenza-specific T-cell clones to define DR and DQ products on a large panel of allogeneic antigen presenting cells. With this functional screening assay, we identified two haplotypes with unusual DR/DQ associations. Cells of these haplotypes were then used as responder cells in mixed lymphocyte culture and stimulated by homozygous typing cells displaying DR or DQ incompatibilities. Our results indicate that DR or DQ incompatibilities alone can give rise, in both cases, to strong T-cell proliferation in a mixed lymphocyte reaction. This was further verified by blocking experiments of secondary mixed lymphocyte reactions by HLA-specific monoclonal antibodies. Anti-DQ, but not anti-DR, antibodies inhibited DQ-incompatible responses. Conversely, anti-DR, but not anti-DQ, antibodies could block DR-incompatible mixed lymphocyte reactions. Together, the results suggest that both HLA-DR and DQ gene products can be involved in HLA-Dw typing. Finally, in dual DR- and DQ-incompatible mixed lymphocyte reaction combinations, HLA-DR molecules seem to have an immunodominant effect, because the response is mostly inhibited by anti-DR antibodies. Immunodominance of HLA-DR allodeterminants may, at least in part, explain some of the controversial conclusions reported by others concerning the role of HLA-DQ molecules in HLA-Dw typing.     

Clonal analysis of HLA-DR and -DQ associated determinants: Their contribution to Dw specificities

In order to investigate the distribution of epitopes recognized by T-cell clones directed against HLA class II products, bulk primed cell populations were generated using cells matched for class I determinants but disparate for class II determinants. Cells were cloned by single cell deposition (FACS IV) or limiting dilution (1 cell/3 wells), and assayed for proliferative and cytolytic function with panels of well-characterized cells. All cytolytic clones generated from an anti-DR4/Dw4/DQw3 priming combination or an anti-DR2/Dw2/DQw1 priming combination lysed essentially all targets sharing the same Dw type as the sensitizing cell. In some cases, other targets were also lysed. For instance, some clones were lytic to targets bearing the same DR antigen but another Dw subtype including a few clones lytic to virtually all cells carrying that DR specificity. An occasional target cell expressing a different DR antigen from the sensitizing cell was also lysed by these clones, in some cases to the same extent of lysis seen on the specific target. Monoclonal antibody inhibition studies identified three groups of clones: the DQ directed clones and clones apparently directed at more than one DR product. However, the number of molecules detected for each haplotype remains to be investigated. Our data indicate that determinants detected on both DR and DQ products are associated with the Dw type of the sensitizing cell showing that there is polymorphism recognized by T cells on both DR and DQ that is subtypic to the serologically defined specificities. Thus, it appears that the bulk T-cell response is a composite of individual clones recognizing distinct determinants on these class II molecules. The implications of these findings for studies of HLA restricted recognition are discussed.     

The majority of HLA-DR3 alloreactive T cells is peptide specific, but does not recognize known DR3-bound sequences

Abstract: Rejection of transplants is frequently caused by activation of alloreactive T cells that recognize HLA/peptide differences between patient and graft. This T-cell response can be directed towards the HLA molecule, the HLA-bound peptide or towards a combination. More insight in the involvement of peptides in this process may help to find ways to avoid rejection using for example antagonist peptides. In recent years many naturally processed HLA-bound peptides have been identified. This raises the question of whether these, presumably abundant, peptides are involved in class ri-specific allorecognition. To investigate this, we first determined the proportion of peptide-specific alloreactive T cells in the alloresponse against HLA-DR3. For this purpose we have tested a panel of DR3-specific alloreactive T-cell clones against a DM-mutant (i.e. peptide loading deficient) cell line. We found that 59 out of 64 alloreactive T-cell clones were dependent upon the presence of DM for an optimal response. However, only 2 DM-dependent T-cell clones recognize known peptide sequences. Thus we conclude that most DR3-specific alloreactive T-cell clones are peptide specific and that the currently known DR3-bound peptides are not the main target for allorecognitioa Finally, we identified 4 T-cell clones that recognized the DM-mutant better than the wild-type cell line. The response against the wild-type cell line could not be restored with invariant chain derived peptides (CLIP). This provides additional evidence that DM can negatively select self-peptides other than CLIP, which can result in selection against peptides involved in allorecognition.     

A specific nucleotide sequence defines a functional T-cell recognition epitope shared by diverse HLA-DR specificities

An oligonucleotide probe that distinguishes the HLA-DR4, Dw14 allele from oother HLA-DR4-associated class II genes was used to identify a common nucleotide sequence shared between the DRβ1 locus encoding HLA-DR1 (Dw1), Dw16, and DRw10 haplotypes. The presence of this nucleotide sequence correlated with the ability of these haplotypes to stimulate an alloreactive proliferative T-cell clone (clone 14B) raised against a Dw14 + stimulator cell. Alleles of DRβ1 that differ by one specific nucleotide variation at codon 71 neither hybridize to the Dw14-specific oligonucleotide nor stimulate clone 14B. These data demonstrate the presence of a shared epitope present on serologically distinct DR alleles that is recognized as a specific allodeterminant by a single T-cell clone.     

Identification of cross-reactive T cell restriction epitopes located on the DR7 beta 1 and DR beta 4 molecules.

L cell fibroblasts transfected with HLA class II cDNA clones isolated from a cDNA library produced from a DR7 homozygous cell line were used as antigen-presenting cells (APC) for three HLA DR-restricted, diphtheria toxoid-specific T-cell clones in order to assess the antigen-presenting ability of the transfectants and to define the class II restriction of each clone. Class II-expressing transfectants are capable of presenting antigen to antigen-specific T-cell clones, although the transfectants are less efficient at antigen presentation than conventional APC. Paraformaldehyde fixation of transfectants prior to antigen pulsing abrogated antigen presentation, demonstrating that the transfectants require antigen processing. Antigen presentation by transfectants is completely inhibited by CD4-specific monoclonal antibodies (mAb) and one of four DR-specific mAb, whereas antigen presentation by conventional APC is only partially inhibited. Both the DR alpha:DR7 beta 1 and DR alpha:DR beta 4 (DR omega 53) molecules of the DR7 allotype serve as restriction elements for the diphtheria toxoid-specific T-cell clones. One clone is restricted by the DR7 beta 1 molecule, another clone by the DR beta 4(DR omega 53) molecule, and a third clone by a cross-reactive T cell epitope on DR7 beta 1 and DR beta 4(DR omega 53) molecules. The two DR beta 4(DR omega 53)-restricted clones react, however, differently with a panel of HLA-DR DR omega 53-positive human peripheral blood lymphocytes used as APC. Therefore the data presented here clearly document that the DR beta 4 (DR omega 53) chain may serve as restriction elements for DT-specific T-cell clones. They also provide the first evidence for functional cross-reactivity of the products of two different DR beta loci and in addition emphasize the high complexity of the supertypic HLA-DR omega 53 specificity.     

Mapping of allospecific T-cell recognition sites encoded by the HLA-DR4 beta 1-chain

Genes of the major histocompatibility complex (MHC) influence the immune system by their central role in the activation of T lymphocytes, which have to corecognize antigen in the association with MHC-encoded cell surface molecules. The location and number of sites on HLA class II molecules that interact with T-cell receptors remains unknown. Using a set of ten alloreactive human T-cell clones we have defined the molecular basis of T-cell interaction sites on the HLA-DR4 molecule. At least seven unique determinants are recognized that are confined to an immunodominant region encoded by the third hypervariable region (hvr) of the HLA-DR beta 1-chain. Substitutions at amino acid positions 71, 74, and 86 contribute similarly to determinants recognized by alloreactive T cells. A cluster of tightly overlapping sites stimulatory for distinct T cells is contained within the segment of amino acid residues 71 to 86, which is expressed within the HLA-DR4 as well as HLA-DR1 haplotype. Five of the ten T-cell clones are stimulated by HLA-DR1+ cells, suggesting that allospecific T-cell receptors directly interact with protein structures determined by the third hvr. These data provide evidence that the third hvr of the HLA-DR beta 1-chain encodes for a functional domain on the surface of the molecule that is recognized by a polyclonal T-cell response.     

Functional and biochemical subtypes of the haplotype HLA-DR3 in patients with celiac disease or idiopathic membranous nephropathy

HLA class II beta-chain polymorphism was investigated in the haplotype HLA-DR3 to determine if patients with HLA-DR3-associated diseases express normal or variant class II polymorphisms. Analysis was carried out by two-dimensional gel electrophoresis of immunoprecipitated HLA class II molecules, DNA hybridization with DR beta and DQ beta gene probes on Taq 1, Bam H1, or Rsa 1 digests, and mixed lymphocyte culture. Two subtypes of HLA-DR3 were identified in normal homozygous DR3 individuals on the basis of polymorphism in one of two DR beta chains detected, corresponding to differences in DR beta restriction fragment patterns. These polymorphisms exhibited significant linkage disequilibrium with the A1,B8,DR3 and B18,DR3 haplotypes, respectively. In proliferative experiments, cells with the B18,DR3-associated polymorphism strongly stimulated cells from donors with the B8,DR3-related polymorphism, suggesting that a T-cell epitope recognized by B8,DR3 cells lies on the B18,DR3-associated DR beta chain. In seven HLA-DR3 homozygous patients with celiac disease and three HLA-DR3-homozygous patients with idiopathic membranous nephropathy, only the normal patterns of HLA class II molecules were displayed, the B8,DR3 type occurring in all patients and the B18,DR3 type in one patient. These data suggest that celiac disease and idiopathic membranous nephropathy are not related to disease-specific HLA-DR beta or -DQ beta gene variants within the DR3 population that are revealed by these methods.     

Autoimmunization against the neutrophil-specific NA1 antigen is associated with HLA-DR2

A significant association of autoimmune neutropenia (AIN) in infants due to NA1-specific autoantibodies and HLA-DR2 is reported. Nineteen of 26 infants presenting with AIN and NA1 autoantibodies possessed the DR2 antigen, while only one out of six children with AIN, but non-NA1-specific autoantibodies, was DR2 positive. Furthermore, one adult patient with primary biliary cirrhosis and periods of neutropenia due to NA1 autoantibodies also carried the DR2 antigen. All DR2-negative patients were positive for DRw6. These findings indicate that a close relationship exists between autoimmunization against the NA1 antigen and HLA-DR2 and possibly also DRw6.     

Inhibitory effects on HLA-DR1-specific T-cell activation by influenza virus haemagglutinin-derived peptides

Collagen (CII) 263-272 peptide, an autoantigen in rheumatoid arthritis, is a specific human leukocyte antigen (HLA)-DR1/4-binding peptide recognized by T-cell receptors (TCR). The affinity of influenza virus haemagglutinin (HA) 306-318 peptide for the antigen-binding groove of HLA-DR1/4 molecules is higher than that of CII263-272. The HLA-DR1/4-binding residues of HA306-318 are located in the region 308-317. Altered HA308-317 peptides with substitutions of TCR-contact residues may inhibit HLA-DR1/4-specific T-cell activation by blocking the antigen-binding site of HLA-DR1/4 molecules. To evaluate the role of altered HA308-317 peptides in HLA-DR1-restricted T-cell activation, we synthesized three altered HA308-317 peptides. The specific binding of altered HA308-317 peptides to HLA-DR1 molecules was examined using flow cytometry. Effects of altered HA308-317 peptides on HLA-DR1-specific T-cell hybridoma were studied by measuring T-cell proliferation and surface expression of CD69 or CD25. The results showed that altered HA308-317 peptides were able to bind to HLA-DR1 molecules and competed with CII263-272 or wildtype HA308-317 peptide. Compared with wildtype CII263-272 or HA308-317, altered HA308-317 peptides did not stimulate significant T-cell proliferation and CD69 or CD25 expression. Furthermore, the altered HA308-317 peptides inhibited HLA-DR1-specific T-cell activation induced by CII263-272 or wildtype HA308-317 peptide, which may suggest an effective therapeutic strategy in inhibition of HLA-DR1-specific T-cell responses in autoimmunity.     

Molecular characterization of the HLA-DR2LUM haplotype

Abstract: Molecular studies of HLA-DRB, -DRA and -DQB1 genes in the variant DR2 haplotype, DR2LUM, were performed using the homozygous lymphoblastoid cell line, CTS. The results of HLA Class II gene RFLP and PCR analyses suggest that DR2LUM was created by a homologous recombination event between HLA-DR1 and HLA-DR15 haplotypes. Evidence for the presence of a recombinational "hotspot" in haplotypes possessing a DRB6 pseudogene is presented. The results of this study have important implications for detection of HLA-DR2 alleles in DRB gene oligotyping strategies, and suggest that the CTS cell line will be a useful addition to cell panels for characterizing HLA antisera.     

HLA-DR molecules enhance signal transduction through the CD3/Ti complex in activated T cells.

Crosslinking HLA-DR molecules by monoclonal antibodies (mAb) induces protein tyrosine phosphorylation and results in a secondary elevation of free cytoplasmic Ca2+ concentration ([Ca2+]i) in activated human T cells. Here we have studied the effect of DR on CD3-induced signal transduction in allospecific T-cell clones and T-leukemia (HUT78) cells. Co-crosslinking of DR with CD3 produced an enhanced [Ca2+]i response compared to that seen with CD3 alone. In contrast, CD2 responses were not enhanced by co-crosslinking with DR. Co-crosslinking CD45 in a tri-molecular complex of CD45, CD3, and DR completely abrogated the enhancing effects of DR on CD3-induced [Ca2+]i responses. In contrast, the enhancing effect of co-crosslinking CD4 on CD3 responses was not inhibited by co-crosslinking CD45. Thus, the DR-mediated accessory signals appear to be regulated differently from those provided by CD4 accessory molecules. The present data confirm, at the level of second messengers, recent findings suggesting that DR molecules have accessory functions in CD3/Ti-mediated T-cell responses.     

The HLA-DR2 haplotype is associated with an increased proliferative response to the immunodominant CD4(+) T-cell epitope in human interferon-beta

Human CD4(+) T-cell epitopes were identified in interferon-beta (IFN-beta)-1b. A prominent peptide epitope region was found that induced a proliferative response in 16% of all donors tested. Responses corresponded to the presence of the HLA-DR2 haplotype. Responsive donors expressing the HLA-DQ6 allele showed an increased level of proliferation to the epitope as compared to peptide-responsive HLA-DQ6 negative donors. A similar result was found for HLA-DR15-expressing donors. PBMC from donors expressing HLA-DR15 were more likely to proliferate in response to IFN-beta in a whole-protein in vitro assay than donors who did not carry this haplotype. It is striking that the common DQ6 allele HLA-DQB1(*)0602 is found in linkage disequilibrium with HLA-DRB1(*)1501, and this combination defines the HLA genotype associated with the development of multiple sclerosis. The HLA association between a response to IFN-beta and MS might explain the prevalence of neutralizing antibody development, and may underlie the etiology of the disease.     

Analysis of the T-Cell Receptors Utilized for Allogeneic HLA-DR Recognition: Comparison of Different Responder-Cell Donors Possessing an Identical HLA-DR Allele

We examined whether individuals with an identical HLA-DR type utilized the same T-cell receptors (TCRs) to recognize a given allogeneic HLA-DR molecule. CD4⁺ T cells from three responder-cell donors possessing the DRB1*0901 allele were stimulated with HLA-DRB1*0406 molecules, subjected to the primary mixed lymphocyte reaction (MLR) and the TCRs of the activated CD4⁺ T cells were analysed using single strand conformation polymorphism (SSCP) and random cDNA clone sequencing. The responder cells of each donor yielded many dominant SSCP bands in several TCRAV and TCRBV segments, but none of these dominant SSCP bands derived from two or three responders. Random cDNA sequence analysis demonstrated that the alloreactive TCRs were diverse, but each of the three responder-cell donors showed some dominant cDNA clones. However, no amino acid sequence identities or similarities among the dominant cDNAs of these donors were detected. These results indicate that certain T-cell clones from each individual's TCR repertoire pool expand preferentially as a result of allogeneic HLA-DR recognition but these clones are not necessarily common to different individuals, even when their responder cells possess identical DR alleles and are stimulated with the same alloantigen.     

Full-length DQβ cDNA sequences of HLA-DR2/DQw1 subtypes: Genetic interactions between two DQβ loci generate human class II HLA diversity

Two-dimensional gel electrophoresis of DQ molecules from three different Dw subtypes (Dw2, Dw12, and Dw21/FJO) of the HLA-DR2/DQw1 haplotype reveals that one β heterodimer of DQ molecule is expressed by each subtype and the DQB chain is electrophoretically variable among the three DR2/DQw1 subtypes. We have constructed cDNA libraries from the same homozygous typing cells used in the two-dimensional polyacrylamide gel electrophoresis analyses (HTC VYT for Dw2, HTC DHO for Dw12, and HTC FJO for Dw21/FJO) and isolated DQβ cDNA clones with full-length coding sequences for each subtype. The deduced amino acid sequences show that the DQβ chains of these three DR2/DQw1 subtypes are highly polymorphic and confirm their electrophoretic heterogeneity: for a mature protein of 229 amino acids, they differ with each other by 10–17 amino acids in the first domain and by 3–7 residues in the C-terminal sequence. Comparison among the available DQβ sequences representing the four major DQ specificities (DQw1, DQw2, DQw3, and DQw4) in the DQ subregion as defined by serologic method suggests that (1) DR2, Dw2, DQw1 and DR3, DQw2 haplotypes probably interact with each other to generate the DQw3 and DQw4 β alleles and (2) an evolutionary scheme may be proposed to relate the various β alleles of the four major DQ specificities.     

Association of HLA-DR1 with the allergic response to the major mugwort pollen allergen: molecular background

ABSTRACT: BACKGROUND: Mugwort pollen allergens represent the main cause of pollinosis in late summer. The major allergen, Art v 1, contains only one single immunodominant, solely HLA-DR-restricted T cell epitope (Art v 125-36). The frequency of HLA-DR1 is highly increased in mugwort-allergic individuals and HLA-DR1 serves as restriction element for Art v 125-36. However, Art v 125-36 also binds to HLA-DR4 with high affinity and DR1-restricted Art v 125-36 -specific T cell receptors can be activated by HLA-DR4 molecules. To understand the predominance of HLA-DR1 in mugwort allergy in spite of the degeneracy in HLA/peptide-binding and TCR-recognition, we investigated the molecular background of Art v 125-36 /MHC/TCR interactions in the context of HLA-DR1 compared to -DR4. RESULTS: The majority of Art v 125-36 -specific T cell lines and clones from HLA-DR1 carrying, mugwort pollen-allergic donors reacted to synthetic and naturally processed Art v 1-peptides when presented by HLA-DR1 or HLA-DR4 expressing antigen presenting cells. However, at limiting peptide concentrations DR1 was more effective in T cell stimulation. In addition, the minimal epitope for 50% of Art v 125-36 -specific T cells was shorter for DR1 than for DR4. In vitro binding assays of Art v 125-36 mutant peptides to isolated DR1- and DR4-molecules indicated similar binding capacities and use of the same register. In silico simulation of Art v 125-36 binding to HLA-DR1 and -DR4 suggested similar binding of the central part of the peptide to either molecule, but a higher flexibility of the N- and C-terminal amino acids and detachment at the C-terminus in HLA-DR1. CONCLUSIONS: The predominance of HLA-DR1 in the response to Art v 125-36 may be explained by subtle conformation changes of the peptide bound to DR1 compared to DR4. Computer simulation supported our experimental data by demonstrating differences in peptide mobility within the HLA-DR complex that may influence TCR-binding. We suggest that the minor differences observed in vitro may be more relevant in the microenvironment in vivo, so that only presentation by HLA-DR1, but not -DR4 permits successful T cell activation.     

HLA-DR molecules enhance signal transduction through the CD3/Ti complex in activated T cells

Abstract: Crosslinking HLA-DR molecules by monoclonal antibodies (mAb) induces protein tyrosine phosphorylation and results in a secondary elevation of free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in activated human T cells. Here we have studied the effect of DR on CD3-induced signal transduction in allospecific T-cell clones and T-leukemia (HUT78) cells. Co-crosslinking of DR with CD3 produced an enhanced [Ca²⁺]_i response compared to that seen with CD3 alone. In contrast, CD2 responses were not enhanced by co-crosslinking with DR. Co-crosslinking CD45 in a tri-molecular complex of CD45, CD3, and DR completely abrogated the enhancing effects of DR on CD3-induced [Ca²⁺]_i responses. In contrast, the enhancing effect of co-crosslinking CD4 on CD3 responses was not inhibited by co-crosslinking CD45. Thus, the DR-mediated accessory signals appear to be regulated differently from those provided by CD4 accessory molecules. The present data confirm, at the level of second messengers, recent findings suggesting that DR molecules have accessory functions in CD3/Ti-mediated T-cell responses.