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D D Eckels

Publications and source records attributed to D D Eckels.

At least 37 records · Page 2Linked to original sources
PubMed1993

Nonrandom T cell receptor usage in the allorecognition of HLA-DR1 microvariation.

Microvariation within the DR1 Ag family has created two DR molecules which differ only at beta-chain residues 85 (Val/Ala) and 86 (Gly/Val). TCR utilized by human alloproliferative T lymphocyte clones which can distinguish between these microvariants have been characterized by cDNA sequencing. The alpha- and beta-chain cDNA utilize a diverse set of variable (V) gene segments although the same V segment may be used by different individuals suggesting that V segment usage by the alloreactive T lymphocyte clones is nonrandom. There appears to be no difference in the repertoire of V segments utilized by T lymphocytes that preferentially recognize specific DR1 allelic products (DR(alpha,beta 1*0101) or DR(alpha,beta 1*0102)) and T lymphocytes that recognize both DR1 molecules. In contrast, the junctional regions of both alpha- and beta-chains are diverse in length and sequence although some common elements can be observed among TCR which share V gene segments. Two TCR which share V alpha and V beta gene segments differ in fine specificity for specific DR1 allelic products implicating the junctional regions of alpha- and beta-chains in the recognition of differentially bound peptides and/or in recognition of DR beta-chain residues 85 and 86. The stimulation of many diverse TCR by the limited allelic variation between DR(alpha,beta 1*0101) and DR(alpha,beta 1*0102) molecules suggests that the effect of DR microvariation on human immune responsiveness may be substantial.

Amino Acid Sequence↗
PubMed1992

Effects of localized HLA class II beta chain polymorphism on binding of antigenic peptide and stimulation of T cells.

The relationship between HLA-DR1 polymorphism and recognition of antigen by T cells was investigated. Two allelic variants of HLA-DR1, which differ by amino acid substitution at positions 85 and 86 of the beta chain, were characterized for the effect of substitution on recognition of foreign antigen by DR1-restricted T cells. Substitution of alanine and valine for valine and glycine residues at positions 85 and 86 of the DR1 beta chain resulted in deficient T-cell stimulation as demonstrated by the requirement for higher concentrations of antigen to induce maximal levels of T-cell proliferation, induction of lower levels of proliferation at optimal antigen concentrations, and slower kinetics of formation of stimulatory peptide-DR1 complexes. Direct binding studies employing both biotinylated and radioiodinated forms of antigenic peptide demonstrated quantitatively lower levels of peptide bound to substituted DR1 molecules and low levels of site-specific binding as assessed by competitive inhibition analyses. The effect of MHC class II polymorphism on peptide-binding affinity as opposed to induction of appropriate peptide conformation and the impact of polymorphism at DR1 beta chain positions 85 and 86 on allorecognition of HLA-DR1 are discussed.

Alleles↗
PubMed1992

The predictive value of HLA-DR oligotyping for MLC responses.

Comparison of HLA proteins between a patient and potential unrelated marrow donors is difficult because many similar, but not identical, HLA proteins are expressed in the human population. A reliable and practical method to detect these subtle differences is provided by oligotyping, a new technique that identifies polymorphic sequences in the genes encoding the HLA proteins. Oligotyping was used to compare polymorphic HLA-DR sequences in 286 pairs of samples from patients and potential unrelated donors who were serologically matched for HLA-DR specificities. Oligotyping detected HLA-DR differences in 53% of these pairs and all mismatched pairs were reactive in primary mixed lymphocyte cultures. Where HLA-DR disparity was not detected by oligotyping, 37% of the pairs were nonreactive in MLC. The remaining 63% often contained an allele associated with the HLA-DRw11 serological specificity. In the absence of HLA-DRB1*11, oligotyping was predictive of MLC reactivity for samples with HLA-DR2, -DR4, and DRw52. In clinical settings, the ability to predict MLC reactivity on the basis of precise HLA typing provides an alternative to MLC. Further, the relationship between specific polymorphic sequences and reactivity in MLC may lead to more fundamental insights into the mechanisms involved in alloreactive responses.

Amino Acid Sequence↗
PubMed1991

T cell receptor gene segment utilization by HLA-DR1-alloreactive T cell clones.

Transplantation of histoincompatible tissues leads to allograft rejection, which involves recognition of allogeneic MHC molecules by Ag-specific receptors expressed on T cells. The interaction of these molecules is highly specific yet poorly understood. We have investigated the relationship between TCR gene utilization and allo-MHC restriction patterns by using a one-way polymerase chain reaction to amplify the alpha- and beta-chain mRNA from a panel of 10 HLA-DR1-alloreactive T lymphocyte clones. Two previously unreported V alpha and five J alpha gene sequences were obtained. Although a few V alpha, V beta, and J alpha genes were utilized more than once, no correlation between TCR gene usage and DR1 alloreactivity was identified. At the sequence level, the presumed TCR alpha- and beta-chain CDR1 and CDR2 regions displayed limited diversity, whereas the CDR3 or junctional sequences were highly variable. Although most TCR probably interact with subtly different surface features of the DR1 alloantigen, we predict that TCR with similar CDR1 and CDR2 sequences would contact essentially identical regions of the DR1 molecule. The lack of sequence conservation in the junctional regions suggests that different endogenous peptides also may be recognized. Thus, alloreactive T cells may recognize not only allogeneic MHC molecules but perhaps also bound endogenous peptides.

Amino Acid Sequence↗
PubMed1990

Selective signal transduction through the CD3 or CD2 complex is required for class II MHC expression by human T cells.

Ag-dependent activation of human T cells results in high level expression of class II MHC molecules. As part of this process, Ag recognition by TCR generates a series of second signals including protein kinase C, tyrosine kinase, and Ca2+ mobilization. To investigate the role of these second messengers in class II MHC expression, purified T cells were first stimulated by PMA, ionomycin, OKT3 accompanied by IL-2, or the mitogenic anti-CD2 antibodies T112 and T113 and were then stained with FITC-conjugated anti-class I and -class II MHC antibodies for analysis by flow cytometry. OKT3 and IL-2 induced optimal expression of HLA-DR (DR) on 70% of T cells with high density. Despite their high mitogenicity, induction of class II MHC expression by PMA, even with co-stimulation by ionomycin, was reduced to less than 20% of T cells, with an intensity 50-fold lower than in OKT3/IL-2-stimulated T cells. Furthermore, PMA inhibited class II MHC expression by OKT3/IL-2-stimulated T cells in a dose-dependent manner and additional stimuli, such as IL-1, IL-4, IFN-gamma, TCR cross-linkers, or monocytes, did not restore class II MHC expression by PMA-activated T cells. DR beta mRNA analysis showed that the low induction of class II molecules by PMA extends to the transcriptional level. Interestingly, anti-T112 and anti-T113 induced not only proliferation of T cells but also DR expression on more than 90% of T cells. These results indicate that transduction of a specific signal, probably selective phosphorylation of the CD3 molecule, contributes to class II MHC induction in the process of T cell activation.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Selective expression of class II MHC isotypes by MLC-activated human T lymphocytes.

Although activated human T cells express class II MHC molecules, the biologic significance of this event is not understood. Using two-color flow cytometry, we have analyzed the expression of HLA-DR, -DQ, and -DP isotypes by T cells following activation by allogeneic lymphoblastoid B-cell lines. Within the CD3+ population, transient expression was observed at 1 day following initiation of culture, which preceded a dramatic and sustained increase around 6-7 days. DR expression was always highest, followed by DP and DQ with DP expression usually somewhat higher than DQ. At day 8, three populations were observed consisting of DR+DP+DQ+ (60%), DR+DP+ (69%), and DR+ (75%) T cells. Interestingly, DQ+ or DP+ but DR- T cells were not observed. These patterns of class II isotype expression were similar in CD2+, CD4+, and CD8+ subgroups and suggest that class II molecules are selectively expressed on T cells and may play a role in the regulation of T-cell responses to alloantigens.

Antibodies, Monoclonal↗
PubMed1990

Involvement of class II beta-chain amino acid residues 85 and 86 in T-cell allorecognition.

Alloreactive T-cell clones were derived by limiting dilution following priming to allogeneic cells bearing HLA-DR1 alloantigens. Clonal specificities were determined by extensive testing on a panel of allogeneic lymphoblastoid cell lines and by blocking studies with monoclonal antibodies specific for HLA-DR, -DQ, and -DP class II molecules. Out of nine DR1-positive cell lines, three failed to stimulate a subset of the T-cell clones in conventional proliferation assays. Proliferation by all of the clones was blocked by anti-DR antibodies, not by anti-DQ or anti-DP, which was consistent with the conclusion that the HLA-DR molecule was recognized. This DR1-associated polymorphism has been identified as Dw20 by the Tenth International Histocompatibility Workshop. The molecular basis for this altered recognition of the DR1 molecule was determined by allele-specific oligonucleotide hybridization and by DNA sequencing studies. The first, second, and third hypervariable regions of all nine DR1-positive cell lines were identical. Valine and glycine were found at positions 85 and 86 of the DR1 beta 1 chain in DR1 molecules from six of the nine lymphoblastoid cell lines, whereas alanine and valine were found in the three variant (Dw20) DR1-positive cells. By analogy with class I structure, residues 85 and 86 would be located at the extreme C-terminal end of the beta-chain alpha helix. Together or separately, these amino acid differences may define a T-cell recognition element on the DR1 molecule serving to contact allospecific T-cell receptors. Alternatively, if allorecognition involves recognition of a self peptide complexed with an allogeneic MHC molecule, then it is possible that the differences T cells recognize on DR1 class II proteins arise from peptide-specific interactions with residues 85 and 86.

Amino Acid Sequence↗
PubMed1990

Primary mixed lymphocyte responses to HLA-DP.

Combinations of peripheral blood lymphocytes, matched or mismatched for HLA-DP, were analyzed in primary one-way mixed lymphocyte culture experiments. Proliferative responses as correlated with tritiated thymidine uptake were assessed over a kinetic range of 5-15 days. A proliferative response was observed between DP-mismatched combinations, whereas combinations matched for DP and all other HLA alloantigens did not elicit significant proliferation. Optimal responses were observed 9 days after the combination of 1 x 10(5) responder and stimulator cells. Responses were blocked by anti-DP monoclonal antibodies. These studies demonstrate the complexity of the primary mixed lymphocyte culture system and suggest that DP alloantigens should be considered when anomalous responses are obtained.

Antibodies, Monoclonal↗
PubMed1989

T-cell identification of a private DQw5 subtype associated with DR1: contribution of endogenous peptide?

Human allospecific T-cell clones were generated against DR1 and DQw1 by limiting dilution. In proliferation experiments using a large panel of Epstein-Barr virus-transformed B-cell lines (LCL), eight T-cell clones (TLC) were found that responded only to the DR1+ LCLs* (9 of 9) and not the 94 other LCLs expressing DR specificities 2 through w9. TLCs* were analyzed further using monoclonal antibodies in blocking studies. As expected, most TLCs were blocked by anti-DR monoclonal antibodies (MoAbs)* and not by anti-DQ MoAbs. However, one clone, TLC 63.138, was not blocked by anti-DR MoAbs but was completely inhibited by anti-DQ MoAbs. This suggests that TLC 63.138 recognizes a private determinant on DQ molecules uniquely associated with DR1.

Antibodies, Monoclonal↗
PubMed1988

Structural model of HLA-DR1 restricted T cell antigen recognition.

Two human helper T cell determinants in influenza have been identified, one in the hemagglutinin and the other in the matrix protein (M1). Both were shown to be DR1 restricted by using transfected L cells to present antigen. Comparison of the sequences of the two peptides revealed a similar pattern that could account for their DR1 specificity if the peptides adopt a helical conformation. The model was supported by the demonstration that hybrid peptides, composed of the amino acids that interact with DR1 from one determinant and the residues that interact with the T cell receptor from the other, were recognized by each clone. The generality of the motif was confirmed by the finding that DR1 individuals respond to a ragweed peptide containing the defined pattern.

Allergens↗
PubMed1988

Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8. III. Family segregation analyses.

We have studied the complexity and fine specificity of the HLA-D region using a panel of T lymphocyte clones generated against alloantigens associated with HLA-DR1 through DRw8. After extensive testing in population studies, 89 clones were tested in proliferation assays with 14 families. Segregation patterns were analyzed for haplotype associations by calculating sequential lod scores to test the likelihood that genes encoding epitopes detected by TLCs were linked to HLA genes. Four general categories were identified: (1) clonal responses that segregated with the same HLA-D region haplotype in all informative pedigrees; (2) clonal responses that segregated with HLA in all pedigrees but not always with the same haplotype; (3) clonal responses that segregated with HLA in some families but failed to segregate in others or produced equivocal results; (4) clonal responses that did not segregate with HLA haplotypes.

Cell Line↗
PubMed1988

HLA DPw1 class II molecules on human T cells.

Human T cells express HLA class II antigens when activated by mitogens, alloantigens, or nominal antigens such as influenza virus. However, little is known about why they are expressed and the extent to which subsets of class II molecules (DR, DQ, and DP) are expressed. From studies with allocytotoxic antisera it is clear that DR and DQ molecules are expressed by T cells; cell surface expression of DP is more ambiguous because cellular typing methodologies are required. Alloreactive T-cell clones, specific for DPw1-associated antigens, were derived by limiting dilution in the presence of DPw1-positive stimulator PBLs and IL-2 and screened on panels of DPw1-positive and DPw1-negative PBL's in proliferation assays. Clones that recognized DPw1-associated determinants were then assayed for responses using as stimulators, irradiated T-cell clones derived from a DPw1-positive donor. Of seven DPw1-specific clones, one (TLC 56.26) was highly responsive to the alloantigens expressed on the stimulator T-cell panel. Six such clones gave a range of lower responses to T cells although capable of recognizing DP-associated determinants on PBL stimulators.

Antibodies, Monoclonal↗
PubMed1988

Presentation of influenza hemagglutinin peptide in the presence of limited allostimulation by HLA-DR1 transfected human fibroblasts.

A human fibroblast expressing HLA-DR1 antigen on its surface was generated by transfection with DR alpha and DR beta cDNAs. The ability of this transfected fibroblast line to process and to present antigens was analyzed by using human T-lymphocyte clones (TLCs) specific for HLA-DR1 alloantigen or restricted by DR1 in their recognition of influenza virus. TLC responses were measured in proliferative assays and were tested for blocking by monoclonal antibodies specific for MHC antigens. Two TLCs specific for a discrete segment (aa 306-320) of the influenza hemagglutinin molecule responded to the antigen added in peptide form but not as intact virion. The transfected fibroblast line thus appears unable to process antigen properly. A DR1-alloreactive TLC was able to respond to the transfected fibroblast. However, 24 other DR1-alloreactive TLCs and oligoclonal T-cell lines were unable to respond. These results suggest either that the conformation of the DR1 molecule on a transfected fibroblast allows peptide presentation but not allorecognition, or that self antigen processing and subsequent presentation by MHC antigens is necessary for allorecognition.

Antibodies, Monoclonal↗