Sandra Reichstetter

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 VP16369–379-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 VP16369–379 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/VP16369–379 tetramer at either 23 or 37°C. Weak staining was also observed at 4°C. Clone 5 showed weaker staining compared with clone 48 at 37°C, and no staining was observed at 23°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/VP16369–379 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.

Mutational analysis of critical residues determining antigen presentation and activation of HLA-DQ0602 restricted T-cell clones

Three different HLA-DQ0602 restricted T-lymphocyte clones (clones 5, 44, and 48) specific for two different Herpes simplex virus type 2 (HSV-2) VP16 peptides were used in a series of proliferation assays with BLS-1 cell lines expressing mutated HLA-DQ0604 molecules as APC. Up to four residues in the peptide-binding region of DQ0604 were replaced by the respective DQ0602 residue. For all three clones, residue beta70 played a crucial role in TCR recognition; beta30 and beta57 were important, although beta86 was less significant. Clone 5 and 48, specific to the HSV-2 VP16 369--379 peptide, responded to the same mutated DQ0604 molecules. Both clones could be stimulated only when the antigen presenting DQ molecule contained the DQ0602-like Gly at position beta70. Stimulation of clone 44, which recognized a different HSV-2 VP16 epitope (VP16 40-50), was less restricted. Molecular homology modeling showed that the beta70Arg of DQ0604 partially covered the peptide around P5/P6. Interactions of beta70 with residues from the antigen-peptide and polymorphic residues at positions beta30 and beta57 can modulate this effect. Supported by molecular modeling data, we conclude that the distinct molecular topography of DQ0602 is not contributed by a single residue, but rather the interactions of various polymorphic DQ residues with particular antigenic peptides.

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.

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.

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<0.001, 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-1 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 DRBl*03-DQBl*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

Cytotoxic herpes simplex type 2-specific, DQ0602-restricted CD4+ T-cell clones show alloreactivity to DQ0601

Alloreactivity is one of the most serious problems in organ transplantation. It has been hypothesized that pre‐existing alloreactive T cells are actually cross‐reacting cells that have been primed by the autologous major histocompatibility complex (MHC) and a specific peptide. CD8+ cytotoxic T lymphocytes that are alloreactive and recognize a virus‐peptide that is presented by the autologous MHC have been reported. Here we demonstrate a cross‐reactivity that exists between DQ0602 restricted, herpes simplex type 2 VP16 40–50 specific CD4+ T‐cell clones, which can be alloreactive to DQ0601. Though most of the DQ0602 restricted T‐cell clones we isolated from two different donors were not alloreactive, weakly cross‐reacting T‐cell clones could be isolated from both donors. Two strongly cross‐reacting T‐cell clones with high affinity interaction of their T‐cell receptor (TCR) with both DQ0602/VP16 40–50 and DQ0601 could be isolated from one donor. DNA sequencing of the a fragment of the Vβ gene used in their TCR confirmed that these two T cells indeed are two independent clones. These clones are cytotoxic and produce cytokines of a T helper 2‐like pattern. Possible implications in a DR‐matched transplantation setting are discussed.