DRα:Eβ heterodimers in DRA transgenic mice hinder expression of Eα:Eβ molecules and are more efficient in antigen presentation

Abstract

HLA-DRA transgenic (tg) mice on H-2d background were constructed to study assembly, expression and function of DR alpha: E beta class II heterodimers when an alternate E alpha chain is available. Cytofluorimetric analysis and immunoprecipitation studies demonstrate that the majority (90%) of E beta d molecules on class II-positive splenocytes from DRA-tg mice are associated with DR alpha rather than E alpha chains. To characterize the functional role of the interspecies as compared with the wild-type I-E molecules, MHC restriction and T cell epitope immunodominance of synthetic peptides spanning the entire sequence of 65 kDa heat shock protein (hsp) from Mycobacterium tuberculosis were determined in hsp-primed DRA-tg and DBA/2 mice. A similar pattern of responsiveness was observed in both strains, but hsp epitopes recalled a higher response in DRA-tg as compared with DBA/2 mice. A panel of T cell hybridomas specific for two hsp peptides or a hen egg white lysozyme peptide presented by both DR alpha: E beta d and E alpha d: E beta d was studied in detail. Surprisingly, DR alpha: E beta d dimers present these peptides more efficiently than E alpha d: E beta d, even when the TCR was selected in mice expressing only E alpha d: E beta d molecules. The higher efficiency of antigen presentation by DR alpha: E beta d dimers does not appear to depend on increased binding affinity for peptides, as demonstrated by competition for antigen presentation, nor on increased efficiency in the interaction with CD4 molecules. Rather, the higher efficiency of antigen presentation could be explained by a more effective ligand-TCR interaction. This is consistent with molecular modeling based on the class II structure, indicating that 16 out of 17 substitutions between the first domain of E alpha d and DR alpha chains ile outside the peptide binding groove and are potentially available for interaction with the TCR.

Mice lacking all conventional MHC class II genes

“…[12][13][14]. The study of murine MHC-II transgenes in these mice is also complicated, because interallelic dimers may be generated by pairing of the residual endogenously encoded chains with the transgene-encoded components (29).…”

“…Mutant mouse lines genetically deficient in both the E␣ and A␤ genes-and thus lacking MHC-II complexes-have been used to ensure that phenomena observed derive from the activity of the human class II transgenes themselves. However, the possible pairing between human MHC-II molecules and the remaining murine E␤ and A␣ chains (12)(13)(14) complicates the interpretation of such experiments. It would therefore be desirable to express human MHC-II molecules in the complete absence of their murine counterparts.…”

Resistance to Adjuvant Arthritis Is Due to Protective Antibodies Against Heat Shock Protein Surface Epitopes and the Induction of IL-10 Secretion

“…Recombinant mammalian HSP60 was purchased from StressGen Biotech (Victoria, British Columbia, Canada). Synthetic 16-mer peptides overlapping by 10 amino acids of HSP65 were a kind gift from Dr. L. Adorini (Roche Milano Ricerche, Milan, Italy) (26). Synthetic 20-mer peptides overlapping by five amino acids of the mammalian HSP60 were a kind gift from Dr.…”

IL-12 Administration Reveals Diabetogenic T Cells in Genetically Resistant I-Eα-Transgenic Nonobese Diabetic Mice: Resistance to Autoimmune Diabetes Is Associated with Binding of Eα-Derived Peptides to the I-Ag7 Molecule

“…Backcrossed mice were screened for the absence of T and B cells, and for the presence of E␣ molecules on CD11b ϩ cells in the peripheral blood. Homozygous HLA-DRA tg mice on the DBA/2 (H-2 d ) background (26) were backcrossed onto the NOD background for over 10 generations. Backcrossed mice were selected for the presence of I-A g7 (mAb 10.3.62), the presence of DR␣ (mAb L243), and the absence E␣ molecules (mAb 14.4.4S) by cytofluorometric analysis of PBLs.…”

“…Backcrossed mice were selected for the presence of I-A g7 (mAb 10.3.62), the presence of DR␣ (mAb L243), and the absence E␣ molecules (mAb 14.4.4S) by cytofluorometric analysis of PBLs. DR␣ tissue distribution in tg mice is superimposable to that of E␣ (26,27). Transgene hemizygous NOD-DR␣, NOD-E␣, NOD-E␣-SCID mice, and transgene-negative littermates were used.…”