John F. Mccormick

Serological and biochemical analysis of Ia molecules in the I-A mutant B6.C-H-2.

Strain B6.C-H-2bm12 has a mutation in the I-A subregion of the mouse H-2 gene complex, which causes skin graft rejection, mixed lymphocyte reaction (MLR), and alterations in the expression of la antigens. The mutation affects the expression of Ia.3, 8, 9, 15, and 20 on normal spleen cells. When the spleen cells were stimulated with lipopolysaccharide (LPS), the expression of all la specificities were found except Ia.8. la molecules when internally labeled with 3H-leucine can be precipitated with antisera directed against Ia.3, 9, 15, and 20, but not Ia.8. When F18 are made between the mutant bm12 and unrelated haplotypes, Ia.3, 9, 15 and 20 can be detected by microcytotoxic assay on normal spleen cells, but not Ia.8. These studies suggest: (1) The mutation affects either the amount of la molecules expressed on normal spleen cell surfaces or the molecule is anchored improperly in the cell surface such that it is not accessible for cytotoxicity and radioiodination. (2) Specificity Ia.8, which may be a combinatorial determinant, is absent in the mutant because of a structural alteration in one of the chains, probably the ft chain. (3) The mutation does not involve the Ae chain. The significance of this finding in relation to I region-mediated allorecognition and antigen presentation is discussed.

Gene complementations to generate Ia antigens (Ia.23) on hybrid molecules.

Ia specificity 23 is a “combinatorial” antigen generated on a hybrid I region molecule, formed by the noncovalent binding of a 26,000− to 28,000-dalton β polypeptide chain (Ae) coded by a gene in the I-A subregion with a 32,000− to 35,000-dalton a chain (Ea) coded by a gene in the I-E subregion of the mouse H-2 gene complex. For expression of Ia.23, the Ae chain must be derived from the H-2d haplotype (I-Ad), while the Ea can be provided by I-Ed, I-Ek, I-Ep, I-Er, I-Ev, andI-Ew3, but not I-Eb, I-Ef, I-Eq, I-Ea, and I-Eu. With the exception of H-2u haplotype, all Ia.7 (I-E)-positive haplotypes can provide the permissive Ea chain for generating Ia.23 by trans-complementation. In the H-2d haplotype, Ia.23 is generated by cis-complementation of Ad with Ed. Lymphocytes of F1 animals expressed two I-E subregion coded hybrid Ia specificities; one formed by cis-complementation and another by trans-complementation. It is postulated that such hybrid determinants are involved in the recognition and generation of immune response to antigens such as GL-Phe and cytochrome C where dual Ir gene control has been demonstrated. It is also suggested that there are two types of Ia specificities: (1) allotypic la specificities expressed on the a or β chains (these could aid in the binding between the α and β chains such as la.7); and (2) hybrid Ia specificities which are unique interaction determinants formed by the specific association of the α and β chains (e.g., Ia.22,23). These interaction gene products may be involved in antigen recognition and presentation.

Identification of I-E alpha genes in H-2 recombinant mouse strains by F1 complementation

Nine recombinant H-2 mouse strains with crossovers in the I region between I-E-negative haplotypes f,q and I-E-positive haplotypes p,k were examined for I-E expression by microcytotoxicity dye exclusion assay. These recombinants were found to be negative for I-E expression. There are two possible genotypes in these recombinant mouse strains that could result in lack of I-E expression. Recombinants with crossovers between the E alpha gene and the S region would have both nonexpressed I-E alpha and beta genes (E beta fE alpha f, E beta qE alpha q) and recombinants with crossovers between the E beta and E alpha genes would have a nonexpressed E beta gene (E beta f or E alpha q) and a functional E alpha gene (E alpha k or E alpha p). To distinguish between these possible genotypes these recombinants were crossed to B10.A(4R), which carries a functional E alpha k gene but is I-E-negative due to a nonexpressed E alpha b gene. F1 mice were examined for transcomplementing I-E molecules by immunoprecipitation of 3H-leucine-radiolabeled detergent lysates of spleen cells with a monoclonal I-E antibody (14-4-4). Detection of a transcomplementing I-E molecule was confirmed by immunoprecipitation with a monoclonal antibody (H9-14.8) specific for the I-Ek beta polypeptide chain derived from B10.A(4R) and by tryptic peptide map comparisons. Five recombinant mouse strains were able to complement with B10.A(4R) in F1 mice to generate a transcomplementing I-E molecule, and thus have an expressed I-E alpha gene (E alpha k or E alpha p). Four recombinants did not complement with B10.A(4R) in the F1 expression of I-E molecules, and thus have nonexpressed I-E alpha genes (E alpha f or E alpha q).

Hybrid (combinatorial) Ia specificities: Gene complementations to generate Ia.22

Ia specificity 22 is expressed on a hybrid I-E molecule formed by the association of a beta chain (Ae) coded for by the I-A subregion and an alpha chain (Eα) encoded by the I-E subregion. Ia.22 can be generated by the complementation of Ab, AkAs, Ar with Ed, Ek, Evp, Er, Ew3, Eu, Ev but not Eb, Ef, Eq, and Es. With the exception of H-2p which does not complement with As to generate Ia.22, all Ia.7-positive (I-E) haplotypes can provide the permissive Eα allele. It is postulated that Ia.22 is a combinatorial Ia determinant generated by the association of the alpha and beta chains. These determinants are probably involved in the immune recognition of antigens under dual Ir-gene control.

Serological confirmation in Ia mutant B6.C-H-2bm12 of gene conversion

Recently it has been suggested that a short segment of the DNA sequence of the Ia beta gene in the mutant B6. C-H-2bm12 (bm12) is derived from the Eb beta gene by a gene-conversion-like mechanism, and that the same segment is also seen in the Ek beta gene. To obtain serological evidence for this idea, we produced an antibody against the “new” determination on the I-Abm12 molecule by immunizing A.BY mice with bm 12 cells. After absorption with B6 lymphocytes to remove antibodies against background antigens, the antisera lysed bm12 cells. Typical Ia peaks were obtained by immunoprecipitation. The absorbed antiserum reacted with B10.A (Ekβ), B10.A(5R) (Ebβ) and B10.S(9R) (ESβ), but not B10. The unabsorbed antiserum is specific for Ia when tested on A background mice. The antiserum lysed spleen cells of Ik strains (A/Sn, A.AL, A.TBR1, and A.TFR1) but not Ak strain, A.TBR13 (KsAkEbSbDb), confirming the presence of antibodies aganist the I-Ek molecule. This antiserum also lysed the cells from (A.BYxA.TFR5)F1, which expresses the transcomplementing EbβEKα molecule and from the (A.SWxA. TFR5)F1 which expresses the transcomplementing EsβEkα molecule. These data are consistent with DNA sequence analyses, and show the existence of a determinant (Ia.51) generated in the bm 12 mutant by a gene-conversion-like event that is also present in the I-Ek, I-Eb, and I-Es beta polypeptide chains.