Distinct T cell interactions with HLA class II tetramers characterize a spectrum of TCR affinities in the human antigen-specific T cell response.

The polyclonal nature of T cells expanding in an ongoing immune response results in a range of disparate affinities and activation potential. Recently developed human class II tetramers provide a means to analyze this diversity by direct characterization of the trimolecular TCR-peptide-MHC interaction in live cells. Two HSV-2 VP16(369-379)-specific, DQA1*0102/DQB1*0602 (DQ0602)-restricted T cell clones were compared by means of T cell proliferation assay and HLA-DQ0602 tetramer staining. These two clones were obtained from the same subject, but show different TCR gene usage. Clone 48 was 10-fold more sensitive to VP16(369-379) peptide stimulation than clone 5 as assayed by proliferation assays, correlating with differences in MHC tetramer binding. Clone 48 gave positive staining with the DQ0602/VP16(369-379) tetramer at either 23 or 37 degrees C. Weak staining was also observed at 4 degrees C. Clone 5 showed weaker staining compared with clone 48 at 37 degrees C, and no staining was observed at 23 degrees C or on ice. Receptor internalization was not required for positive staining. Competitive binding indicates that the cell surface TCR of clone 48 has higher affinity for the DQ0602/VP16(369-379) complex than clone 5. The higher binding affinity of clone 48 for the peptide-MHC complex also correlates with a slower dissociation rate compared with clone 5.

Gliadin antibodies in adult insulin-dependent diabetes--autoimmune and immunogenetic correlates.

Gliadin antibody (GA) tests used in screening for coeliac disease (CD) frequently yield positive GA results without accompanying CD in cases of diabetes mellitus type 1 (DM-1). To enlighten this phenomenon we screened 848 DM-1 patients for IgA- and IgG-GA. Subsequently, 16 out of 19 high titre GA patients (6 with CD) were compared with 37 low titre DM-1 patients matched for sex, age and disease duration, for autoimmune and immunogenetic markers. Chronic thyroiditis and thyroid peroxidase (TPO) antibody positivity were more frequent in the GA-positive than in the GA-negative sub-group (38 vs. 2.7%, p = 0.003, and 69 vs. 27%, p < 0.00, respectively). The tissue transglutaminase (tTg) IgA titres correlated with CD but not with GA. tTg IgG titres were lower in GA-positive individuals (p = 0.0012). GA-positivity correlated with a higher titre of factor XIII IgA antibodies (p < 0.001). GA-positive DM-I patients were characterised by a distinct immunogenetic profile; the risk of HLA DQB1*02 was lower among GA-positive patients than among GA-negatives (OR 0.4, preventive fraction 0.43). All CD patients were HLA DRB1*03-DQB1* 02-positive, but none of the five patients with normal biopsies. GA-positive patients instead had HLA DRB1*13 in 37.5% as compared to 8.6% in GA-negative (OR 6.4, etiologic fraction 0.32). Thus, the occurrence of positive GA in DM-1 is correlated to TPO antibody positivity, thyroiditis and factor XIII IgA antibodies, but inversely correlated to tTg IgG, and seems to be associated with another HLA haplotype than that previously found to be associated with CD.

MHC-peptide ligand interactions establish a functional threshold for antigen-specific T cell recognition.

Antigen-specific T cell recognition is dependent on the functional density of the TCR-ligand, which consists of specific MHC molecules and a specifically bound peptide. We have examined the influence of the affinity and concentration of exogenous peptide and the density of specific MHC molecules on the proliferation of a CD4+, DQA1*0501/DQB1*0201 (DQ2.1)-restricted, HSV-2-specific T cell clone. Using antigen peptide analogs with different mutations of known DQ2-anchor residues, T cell response was reduced in an peptide-affinity and - concentration specific manner. The decrease using weaker binding peptides was gradual as stimulation with a peptide with intermediate affinity yielded intermediate T cell proliferation and the poorest binding peptide induced an even weaker T cell response. MHC class II density on the APC was modified using DQ2 homo- and heterozygous B-LCLs as APCs, however this variation of MHC concentration had no effect on T cell proliferation. We interpret this as a reflection of a low threshold for activation of the T cell clone, in which peptide-MHC avidity is the over-riding determinant of the strength of ligand signal.

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

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

Microchimerism after liver transplantation: prevalence and methodological aspects of detection.

BACKGROUND: Microchimerism after liver transplantation is a readily observed phenomenon. The immunological implications, however, remain unclear. Moreover, methodological approaches and their detection limits in the study of allogeneic microchimerism have not been studied in detail. METHODS: Therefore, the aim of this study was to evaluate the single-step and nested formats of the polymerase chain reaction/sequence-specific priming (PCR-SSP) approach under standardized conditions. For that purpose, a panel of recombinant plasmid clones was generated by PCR cloning. The panel contained the allelic sequences of the second exon of DRB1 covering all DR specificities on a low-resolution level. Using this panel, limiting dilution assays for various DR sequences in the presence and absence of competitor DNA were carried out to determine the minimal number of copies required for detection by single-step and nested PCR-SSP. Subsequently, 22 liver transplant recipients were analyzed in a retrospective study for the presence of allogeneic microchimerism by nested PCR-SSP. RESULTS: Although at least 10 copies of template DNA could be detected by nested PCR-SSP overall, single-step PCR-SSP was on average 10(2) to 10(3) times less sensitive. Upon the addition of human competitor DNA, the detection limits decreased on average by a factor of 10. In addition, sequence-specific differences in amplification efficiency could be appreciated. Using nested PCR-SSP, peripheral blood allogeneic microchimerism could be observed in 17 of 22 HLA-DR-mismatched liver recipients. Recombinants representing recipient DRB1 specificities were used to exclude false-positive results by lack of cross-reactivities of the donor-specific primers and to evaluate negative results due to sample-related reduced amplification efficiencies in microchimerism-negative recipients. In donor/recipient combinations that differed by at least one DR specificity, allogeneic microchimerism was seen in 87.5% of the cases. In five chimerism-negative cases, sample-related problems were detected in two cases. CONCLUSION: The optimization and standardization of the detection of genomic HLA sequences at low copy number may be greatly facilitated using a clonal reference system. Furthermore, a clonal reference system may be used to conduct cross-priming experiments to exclude false-positive results and may allow the determination of sample-specific detection limits for donor-derived HLA-DR specificities in chimerism-negative patients. Our evaluation of the PCR-SSP approach for the study of allogeneic microchimerism indicated that nested PCR-SSP provides the most sensitive format when HLA sequences are targeted. Yet, the detection sensitivity may vary between individual alleles and specificities. Allogeneic microchimerism in liver recipients can be observed in the majority of patients. However, the detection may be subject to the degree of mismatching, the HLA-DR alleles involved, and sample-related impaired PCR amplification efficiency.

DQB1 promoter sequence variability and linkage in caucasoids.

Sequence variability in the upstream regulatory regions (URR) of HLA class II genes has been described as an additional mechanism of diversity in these polymorphic genes. For HLA-DQB1, 12 URR variants have been identified previously by sequence analysis of approx. 600 bp located immediately upstream of the first exon of the DQB1 gene. To investigate the distribution of these promoter alleles and their linkage with the structural portion of the DQB1 gene, a population-based study was carried out. Sequence information was utilized to develop 25 sequence-specific oligonucleotide probes to analyze enzymatically amplified locus-specific DNA. Supplemented with one sequence-specific primer pair to differentiate QBP1-6.2 from -6.3, all known 12 QBP1 alleles could be identified. Subsequently, 215 healthy, unrelated German controls were investigated for the distribution and linkage of DQB1 and QBP1 alleles. A total of 10 out of 12 known QBP1 alleles were observed. Since there was tight linkage between the promoter region and exon 2 of DQB1, the phenotype and genotype frequencies of the promoter alleles corresponded by and large to the frequencies observed for their linked DQB1 alleles. Exceptions were mainly seen for DQ5 and DQ6 haplotypes, as single DQB1 alleles could be linked to different, however, closely related QBP1 alleles and vice versa. Interestingly, for each DQB1 allele a single DQB1/QBP1 haplotype dominated (75.9 to 96.4%) the distribution. It is concluded that promoter and coding region variability are tightly linked by linkage disequilibrium. Exceptions are restricted to DQB1 DQ5 and DQ6 haplotypes. Since functional differences between different QBP1 alleles exist, the maintenance of haplotypic integrity may be of functional importance.