Atassi, M. Zouhair

Abstract Previously we have reported the precise determination of the entire antigenic structure of sperm-whale myoglobin. This protein which is made up on a single polypeptide chain (153 residues, m.w. 17,816) has five antigenic sites located in exposed conformationally sensitive continuous portions of its polypeptide chain. The five antigenic sites occupy the following regions: Site 1: sequence 15–22; Site 2: sequence 56–62; Site 3: sequence 94–99; Site 4: sequence 113–119; Site 5: sequence 145–151. We investigated the genetic control of T lymphocyte proliferative response to myoglobin and its synthetic antigenic sites in mice (PETLES Assay). Two parts of the myoglobin molecule (sequences 1–6 and 121–127) that were not within antigenic sites were also synthesized and employed as controls. Also used as control was a synthetic ‘nonsense’ heptapeptide that did not resemble any part of myoglobin. Haplotypes H-2d, H-2f and H-2s gave substantial T lymphocyte proliferative response to whole myoglobin whereas haplotypes H-2d, H-2k, H-2p, H-2q and H-2r were low responders. Studies with the recombinant strains suggested that expression of a high responder allele in the I-A subregion was enough to give a good proliferative response. On the other hand expression of I-Cd gave a good response while I-Cs gave a poor response. In preliminary studies, mice were primed with whole myoglobin and then challenged in vitro with the synthetic peptides containing the antigenic sites. Among the high responder strains, H-2d haplotype responded to Sites 1, 2, 3 and 5, whereas H-2s and H-2f haplotypes responded only to Sites 1, 2 and 3. We were surprised to find that the low responder strains, haplotypes H-2b, H-2k and H-2r responded to Site 4 but failed to respond to Sites 1, 2, 3 and 5. Mice did not respond to the control peptides. Further studies are currently underway in recombinant strains and in F1 crosses and will be reported at the Ir Gene Workshop. We are also investigating the ability of synthetic antigenic sites in the priming process.

Abstract The genetic control of T lymphocyte proliferative response to the five synthetic antigenic sites of myoglobin, two synthetic nonantigenic control peptides, and one “nonsense” peptide was determined in independent and recombinant strains of mice. In all the strains examined, the nonantigenic control peptides and the “nonsense” peptide did not invoke a response in myoglobin-primed mice. Further, when mice were not primed with whole myoglobin, no response was obtained with any of the antigenic sites. Haplotypes H-2d, H-2f, and H-2s are higher responders to sites 1 and 2, whereas haplotypes H-2d and H-2s are high responders to site 5. Response to site 3 may be controlled by a non-H-2-linked gene. Site 4 can stimulate H-2b and H-2k haplotypes that are nonresponders to the whole myoglobin. Studies with the recombinant strains suggested that Ir genes to sites 1 and 2 map in the I-A subregion and I-C subregion and were designated Ir-Mb-1,2(A) and Ir-Mb-1,2(C). Ir genes to sites 4 and 5 mapped only in the I-A subregion and were designated Ir-Mb-4(A) and Ir-Mb-5(A). These studies suggest that individual antigenic sites in a molecule are controlled by unique Ir genes.

Abstract Studies on the genetic control of immune response to sperm whale myoglobin were initiated. As demonstrated in this paper, the T lymphocyte proliferative response to whale myoglobin is under H-2-linked Ir gene control. Mice of H-2d, H-2f, and H-2s haplotypes were high responders to the myoglobin, whereas haplotypes H-2b, H-2k, H-2p, H-2q, and H-2r were low responders. The Ir gene(s) was localized between H-2K and H2D regions, since the recombinant strain A.TL (KsIkSkDd) was a low responder and A.TH (KsIsSsDd) was a high responder. Further studies with recombinant strains revealed that the expression of the high-responder I-Ad or I-As alleles was sufficient to give a good response, since strains D2.GD (d d b b b b b b) and B10.HTT (s s s s k k k d) were high responders. The expression of the I-Cd allele in strains B10.A (k k k k k d d d) and B10.A(5R) (b b b k k d d d) also gave high response, and thus suggested a second Ir gene, derived from the H-2d haplotype. The finding that expression of the I-Cs allele in B10.S(8R) (k k ? ? s s s s) did not result in high response suggests the lack of the second Ir gene in the high-responder H-2s haplotype.