The I-Abm12 mutation has been used extensively to study the relationship between structure and function of murine class II major histocompatibility molecules. I-Abm12 differs from I-Ab by three amino acid replacements in the A beta chain, and the proposed structural model of the I-Abm12 molecule places these three amino acid substitutions along one of the alpha-helices where they may affect both antigen and TCR binding. Two of the substitutions, Ile----Phe67 and Thr----Lys71, are thought to point into the binding site, whereas the third substitution, Arg----Gln70, is thought to point up and hence, be available for binding to the TCR. These predicted orientations are consistent with serologic analysis of the bm12 molecule, which demonstrates that residue 70 is uniquely accessible to mAbs distinguishing I-Ab from I-Abm12. In this study we have determined the influence of each of these amino acid substitutions on the ability of the resulting molecules to stimulate a panel of I-Abm12 (allo) reactive T cell hybridomas. Our experiments indicate that reversion of the amino acid at position 70 from Gln (I-Abm12) to Arg (I-Ab) interferes with allorecognition by 33 of 35 I-Abm12-reactive hybridomas. On the other hand, many hybrids can tolerate amino acid substitutions at positions 67 or 71. Single amino acid substitutions at position 67, 70, or 71 are recognized by only a minority of I-Abm12-specific hybrids and usually the reactivity is greatly diminished. These data are most consistent with the idea that the amino acid at position 70 directly interacts with the TCR during allorecognition. The additional effects of residues 67 and 71 are consistent with a contribution by bound peptide to the allorecognition process.
We have generated an mAb, RR3-15, that recognizes murine TCRs containing the V beta 11 domain. Using this antibody to stain peripheral T cells, we have demonstrated that V beta 11-bearing T cells are largely absent from strains of mice that express the class II MHC molecule, I-E. Studies with F1 mice demonstrate that this effect is dominant, consistent with tolerance. The clonal deletion of V beta 11-bearing T cells appears to occur intrathymically, as immature but not mature V beta 11+ T cells are present in the thymus of I-E-bearing mice. Examination of B6 x DBA/2 recombinant inbred strains demonstrates that the expression of I-E molecules is necessary for the clonal deletion of V beta 11-bearing T cells, but that other non-MHC genes control the clonal deletion process, as well. Paradoxically, only a small fraction of V beta 11+ T cell hybridomas are I-E reactive.
T cells that express the T cell receptor V β 5.2 domain react with the class II major histocompatibility complex (MHC) molecule I-E, and V β 5.2 + T cells are deleted in mouse strains that express I-E glycoproteins. By examination of genetically defined recombinant inbred (RI) mouse strains, it was found that the deletion was dependent on the expression of I-E and one of a limited number of non-MHC gene products (cotolerogens). The gene encoding one of these cotolerogens maps to chromosome 12 and is linked to the endogenous provirus Mtv-9. These observations suggest that the I-E-mediated and minor lymphocyte-stimulating antigen (Mls)-mediated deletions of αβ T cells from the repertoire are similar; both require the expression of a class II MHC glycoprotein and a second non-MHC gene product.
On normal cells, the peptide-binding grooves of class II MHC proteins contain a wide spectrum of peptides. For some purposes, however, it would be helpful to have cells bearing class II proteins engaged by only one peptide species. In an attempt to make such cells we constructed a gene for a MHC class II beta-chain, IA beta b, covalently linked to a peptide, E alpha 52-68, which is known to bind to the peptide-binding groove of IAb. This gene, together with the gene for IA alpha b, was transfected into B lymphoma cells and fibroblasts. The IAb-E alpha complex was expressed on the surfaces of these cells where it could be recognized by a mAb and T cells specific for IAb plus E alpha 52-68. Most of the peptide on fibroblasts remained covalently attached to the IAb beta-chain, but the covalent linker and/or peptide were degraded to some extent on B lymphoma cells. Nearly all of the IAb expressed by transfected fibroblasts was occupied by the E alpha peptide. Of 16 IAb-reactive T cell hybridomas, only 3 could respond to the IAb-E alpha complex on fibroblasts, confirming the idea that recognition of MHC may often involve recognition of the peptides bound to the MHC as well.
Two variants of the AKR thymoma BW5147 have been isolated which can no longer express functional TCR alpha- and beta-chains. By generating hybridomas with these variant fusion lines, TCR of any normal T lymphocyte, including TCR-gamma/delta, can be studied at a clonal level, without interference of the BW5147-derived receptor chains. In this study one of the variants has been useful in identifying the reactivity to allogeneic MHC Ag of BW5147 itself.
The TCR alpha/beta variable element V beta 17a is expressed in all strains of mice carrying the V beta a complex in which about half of the V beta elements have been lost due to a large deletion. The mAb KJ23a detects V beta 17a containing alpha/beta receptors in these mice. Mouse strains with the V beta b complex carry a full complement of V beta genes including a structural gene for V beta 17 (V beta 17b), but no T cells reactive with KJ23a are present in these mice. Among random peripheral T cell hybridomas prepared from V beta b mice, occasional V beta 17b transcripts are found. The sequence of one of these transcripts reveals a single base difference from V beta 17a which results in a termination codon within the coding region inactivating the gene.
