Structural analysis of TCRalpha and beta chains from human T-Cell clones specific for small nuclear ribonucleoprotein polypeptides Sm-D, Sm-B and U1-70 kDa: TCR complementarity determining region 3 usage appears highly conserved.

Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are systemic autoimmune diseases that are characterized by the presence of autoantibodies reactive with U small nuclear RNP (snRNP) autoantigens. Both B and T cells are important in the pathogenesis of the disease, and T- and B-cell immunity against snRNP polypeptides have been shown to be linked in vivo. Currently, several alternative hypotheses for the pathogenesis of these diseases have been proposed. These include loss of tolerance, modified self-antigens, molecular mimicry and nondirected immune activation. To help distinguish between the various models of disease pathogenesis, we have characterized the T-cell receptor (TCR) CDR3 from a large panel of well-characterized human T-cell clones and lines specific for individual snRNP polypeptides. The results presented here reveal highly restricted TCR usage across patients by the snRNP-reactive T cells based on the deduced amino acid sequence of the CDR3 loop. These data support the hypothesis that T-cell responses against self antigens in SLE and MCTD are antigen driven and that there are a limited number of T-cell epitopes present on the snRNP autoantigens.

T cell epitope mapping of the Smith antigen reveals that highly conserved Smith antigen motifs are the dominant target of T cell immunity in systemic lupus erythematosus.

B cell and T cell immunity to the Smith Ag (Sm) is a characteristic feature of systemic lupus erythematosus (SLE). We have shown that T cell immunity against Sm can be detected in SLE patients, and that T and B cell immunity against Sm are linked in vivo. TCR usage by Sm-reactive T cells is highly restricted and characteristic of an Ag-driven immune response. Sm is a well-characterized complex Ag consisting of proteins B1, B2, D1, D2, D3, E, F, and G. A unique feature of all Sm proteins is the presence of homologous motifs, Sm motif 1 and Sm motif 2. We used limiting dilution cloning and synthetic peptide Ags to characterize the human T cell immune response against Sm in seven SLE patients. We sought to determine the precise antigenic peptides recognized, the common features of antigenic structure recognized, and the evolution of the T cell response against Sm. We found there was a highly restricted set of Sm self-peptides recognized by T cells, with three epitopes on Sm-B and two epitopes on Sm-D. We found that T cell immunity against Sm-B and Sm-D was encoded within the highly conserved Sm motif 1 and Sm motif 2, and that immunity against these epitopes appeared stable. The present study supports the concept that T cell immunity to Sm is an Ag-driven immune response directed against a highly restricted set of self-peptides, encoded within Sm motif 1 and Sm motif 2, that is shared among all Sm proteins.

Analysis of T cell receptors specific for U1-70kD small nuclear ribonucleoprotein autoantigen: the alpha chain complementarity determining region three is highly conserved among connective tissue disease patients.

The U1-70kD autoantigen is a major target of B cell responses in patients with connective tissue diseases (CTD). T cell responses are important in the pathogenesis of CTD, however little is known about autoantigen-specific T cells in these diseases. We have recently proven that U1-70kD-reactive human T cells exist. To further characterize these autoreactive T cells, U1-70kD-reactive T cell clones have been generated from patients with CTD using either a recombinant fusion protein or synthetic peptides spanning the U1-70kD polypeptide. T cell receptors (TCR) isolated from the U1-70kD-reactive T cell clones were sequenced and the third complementarity-determining region (CDR3) compared to determine if a common motif was present. mAb blocking of antigen-induced proliferation was done to determine the HLA restriction element used in recognition of the U1-70kD autoantigen by T cells. The results presented here indicate that TCRAV CDR3 usage is highly restricted among U1-70kD autoantigen-specific human T cells clones derived from CTD patients with distinctive structural features. Furthermore, the recognition of the U1-70kD autoantigen occurs in the context of HLA-DR.

T cell receptor beta-chain third complementarity-determining region gene usage is highly restricted among Sm-B autoantigen-specific human T cell clones derived from patients with connective tissue disease.

OBJECTIVE: To determine the structure of T cell receptors (TCR) used by Sm-B-reactive human T cell clones, to map T cell epitopes on the Sm-B autoantigen, and to determine the HLA restriction element used in the recognition of Sm-B by T cells. METHODS: Sm-B-reactive T cell clones were generated from patients with connective tissue disease by using either a recombinant fusion protein or synthetic peptides. The TCR structure was defined with the use of polymerase chain reaction and DNA sequencing. Synthetic peptides were used to map T cell epitopes on Sm-B. HLA restriction element usage was defined by using monoclonal antibody blocking. RESULTS: Usage of the TCR third complementarity-determining region (CDR3) was highly restricted among Sm-B autoantigen-specific human T cell clones. Only amino acids 48-96 of the Sm-B2 autoantigen were recognized by T cells, and this occurred in the context of HLA-DR. CONCLUSION: TCR CDR3 gene usage is highly conserved by Sm-B autoantigen-specific T cell clones, and this appears to be related to the recognition of a limited number of T cell epitopes on the Sm-B autoantigen presented in the context of HLA-DR.

A polymorphic residue in the amino terminal alpha 1 hemi-domain of the mouse Ld class I molecule affects its assembly and surface expression.

In comparison to Dd and most other mouse major histocompatibility complex class I molecules, the Ld molecule is poorly expressed on the cell surface, has a lower affinity for beta 2-microglobulin and is trafficked more slowly to the cell surface. Previous studies using Ld-Dd exon-shuffled constructs and the chimeric Ddm1 molecules suggested that the Ld alpha 1 domain was responsible for this phenotype. Two constructs, one containing an Ld-Dd hemi-exon-shuffled alpha 1 exon and the other containing a Dd-Ld hemi-exon-shuffled alpha 1 exon, were inserted into either Ld or Dd to replace the intact alpha 1 exon. These constructs were transfected into mouse L cells. Flow cytometric analyses of the resulting transfectants indicate that the Dd-Ld alpha 1/Ld molecules, similar to the Dd alpha 1/Dd alpha 2/Ld molecules, were expressed at a higher level on the cell surface than either the Ld-Dd alpha 1/Ld molecules or intact Ld molecules. Analyses of the molecules in lysates suggested that a higher proportion of the Dd-Ld alpha 1/Ld molecules, like the Dd alpha 1/Dd alpha 2/Ld molecules, as compared to the Ld-Dd alpha 1/Ld and intact Ld molecules were assembled as detected by alpha 2 domain-reactive monoclonal antibodies. Pulse-chase and lysate stability studies suggested that the lower steady state levels of assembled Ld-Dd alpha 1 molecules resulted from a slower assembly rate rather than instability. Collectively, these studies suggest that residues in the amino terminal half of the Ld alpha 1 domain are responsible for its inefficient assembly, probably leading to its low cell surface expression. To determine which polymorphic residues in the amino terminal alpha 1 hemi-domain might influence this phenotype, several Ld point mutants, in which a Dd amino terminal alpha 1 hemi-domain residue was substituted into the corresponding position of Ld, were analysed. These analyses suggested that, while the residue at position 9 has only a slight effect on beta 2-microglobulin association, it has a striking effect on assembly and cell surface expression.

Association of Ld-restricted peptides with the wild-derived mouse Lw16 MHC class I molecule.

Previous serological analysis of untreated splenocytes and L cell transfectants expressing the wild-derived mouse major histocompatibility complex (MHC) class I molecule, Lw16, demonstrated the presence of two forms of this molecule in the cell lysates, one reactive with both the alpha 2 domain-reactive monoclonal antibody (mAb) 30-5-7 and the alpha 3 domain-reactive mAb 28-14-8 (30-5-7+ 28-14-8+), and the other reactive with only the latter of the two (30-5-7- 28-14-8+). Furthermore, the analysis suggested the presence of both forms on the cell surface. Due to the similarity of Lw16 to the inbred mouse-derived Ld molecule, we tested a panel of Ld-restricted and control peptides for their ability to bind to Lw16 molecules. Here, we report that two Ld-restricted viral peptides, lymphocytic choriomeningitis virus nucleoprotein (LCMV NP) 118-126 and murine cytomegalovirus (MCMV) pp89 168-176, significantly increase the number of Lw16 molecules on the cell surface as measured by the mAb 28-14-8, and the proportion of those molecules that are recognized by the mAb 30-5-7. This was further supported by an increase in mAb 30-5-7-reactive molecules in L.Lw16 cell lysates following treatment with either of these peptides. Examination of the stability of the different forms on the cell surface suggested that the 30-5-7+ Lw16 molecules induced with these peptides were unstable and probably lost their Ld-restricted peptides to generate 30-5-7- 28-14-8+ molecules; these latter molecules were also unstable. In contrast, putative 30-5-7+ and 30-5-7- 28-14-8+ Lw16 molecules on untreated cells were stable. Together, these results suggest that two Ld-restricted, viral peptides can induce assembly of or stabilize 30-5-7+ 28-14-8+ Lw16 molecules, mimicking endogenous self peptides. However, the association of the Ld-restricted peptides with Lw16 is apparently not optimal, since it results in unstable Lw16 molecules.

Slow egress of a mouse MHC class I molecule to the cell surface despite its strong association with beta 2-microglobulin.

Two H-2D region class I genes from the wild-derived mouse strain B10.GAA37 provisionally encoding the Dw16 and Lw16 molecules, respectively, were transfected into mouse L cells, and the expressed gene products were analyzed serologically by flow cytometry. As expected from nucleotide sequence comparisons, these analyses revealed that several Ld-reactive monoclonal antibodies (mAbs) recognize Lw16 and not Dw16. As detected by flow cytometry of intact L.Lw16 cells and B10.GAA37 splenocytes, and by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of immunoprecipitates from splenocyte lysates, the alpha 2 domain-reactive mAb 30-5-7 detected less Lw16 than did the alpha 3 domain-reactive mAb 28-14-8, suggesting the existence of two populations of Lw16 molecules: 30-5-7+ 28-14-8+ and 30-5-7- 28-14-8+. Sequential immunoprecipitation studies provided further evidence for these two Lw16 subsets; furthermore, the 30-5-7- 28-14-8+ subset was found predominantly on the cell surface and in association with beta 2-microglobulin (beta 2-m). Pulse-chase studies of B10.GAA37 splenocytes revealed that Lw16, like Ld, is trafficked slowly to the cell surface, whereas Dw16 is trafficked quickly, like most other mouse K and D region class I molecules. Despite these similarities, Lw16 and Ld differ in their association with beta 2-m, in that the immunoprecipitates of Lw16 contained much higher levels of radiolabeled beta 2-m per heavy chain. Together, these studies indicate that the slower trafficking of Lw16 to the surface does not result from a weaker association with beta 2-m, suggesting that other factors, such as peptide ligand-induced assembly, and/or retention by ER-resident proteins play an important role in the trafficking of major histocompatibility (MHC) class I molecules to the cell surface.