Suresh Savarirayan

Proximity of the Mep-1 Gene to H-2D on chromosome 17 in mice

The Mep-1 gene on chromosome 17 in mice controls the activity of meprin, a kidney brush border metalloendopeptidase. Most inbred mouse strains of the k haplotype (e.g., CBA, C3H, AKR) are markedly deficient in meprin activity; these mice carry the Mep-1ballele. Mouse strains in which meprin activity levels are normal are designated Mep-1a Studies using congenic and recombinant strains mapped the Mep-1 gene telomeric to H-2D near the Tla gene. To further study the relationship between the major histocompatibility complex and Mep-1, a linkage study was conducted. Mep-1a F1 hybrids [C3H.A (KkDd) × C3H.OH (KdDk)] were backcrossed with Mep-1b C3H.OH (KdDk) parents. The progeny were assayed for H-2D markers, Pgk-2 isozymes, and meprin activity. Recombination between H-2D and Mep-1 occurred in 6 out of 284 mice, a crossover frequency of 2.1%. Mep-1 is therefore 2.1 crossover units telomeric to H-2D and approximately 0.6 crossover units from Tla. The Mep-1 locus provides a new genetic marker for the future mapping of this important area of the mouse genome.

Human HLA-DQβ chain presents minor lymphocyte stimulating locus gene products and clonally deletes TCR Vβ6+, Vβ8.1+ T cells in single transgenic mice

Abstract Minor lymphocyte stimulating locus (Mls) gene products in association with mouse major histocompatibility complex (MHC) class II molecules are known to determine the repertoire of T-cell receptor (TCR) in mature T cells. In order to test whether human class II molecules can present mouse Mls, HLA-DQ β transgenic mice were generated. The expression and function of the DQ β transgene were studied in the progeny of one selected founder which was H-2 f and H-2E negative. In these mice, DQ β molecules pairing with mouse A α chain and invariant chain are expressed on the cell surface in a tissue-specific manner. When the DQ β gene was bred into the Mls-1 a strain DBA/1 (H-2 q ), T cells bearing V β 6 and V β 8.1 TCR were clonally deleted in the thymus of DQ β + transgenics but not in DQ β -negative full sibs. Thus, the data presented here clearly demonstrate that the human MHC DQ β chain can present Mls in the clonal deletion of T cells. Our results also suggest the requirement for an interaction between CD4 and class II molecules (α chain) for clonal deletion of T cells to occur.

HLA-DQβ chain can present mouse endogenous provirus MTV-9 product and clonally delete Tcr Vβ5+ and Vβ11+ T cells in transgenic mice

The elusive Mls gene(s) are mouse mammary tumor virus genes. The endogenous cotolerogen involved in the clonal deletion of Tcr Vβ5.1, 5.2, and 11 in H-2E+ mouse strains has been narrowed down to MTV-9. We demonstrate that similar to H-2Eα molecules, human DQw6β chain mediated clonal deletion of Tcr Vβ5.1, 5.2, and 11 also requires the MTV-9 gene product. This shows that human class II molecules can present mouse retroviral antigen. Further, backcross analysis involving [B10.M(DQb)×DBA/1] suggest a second cotolerogen in the B10.M background in the clonal deletion of Vβ5-bearing T cells.

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).

Expression and function of mutant Ia antigen in transgenic mice.

Cell surface expression of Ia antigens requires the assembly of alpha and beta heterodimers. We have produced a double transgenic mouse with a wild form Ak alpha gene and a mutant Ak beta (Ak beta MB) gene with d-allele substitution at positions 63 and 65-67. Initial studies indicated that the Ak alpha and Ak beta MB transgenes are not expressed on the surface of lymphoid cells of the transgenic mice. However, when spleen cells were stimulated with LPS prior to FACS analyses, Ak/Ak MB assembly and subsequent surface expression was induced. The tail skins from transgenic founder mice were rejected by the parental mice indicating a role for the mutant antigen on the allograft. In addition, the Ak transgenic mice on H-2q/q background can partially delete V beta 6+ T cells, suggesting the presence of the transgene product in the thymus.

DNA restriction fragment analysis of Eαgenes in E-negative H-2 recombinant mouse strains

In the mouse, there are two classes of immune response (Ia) molecules, A and E, each with a distinct ~ chain with a molecular mass of 34 000 and a/3 chain with a molecular mass of 28 000. All the strains of mice have been found to express A molecules, while some strains do not express an E molecule on the cell surface (Jones et al. 1981). Mice With the b and s haplotypes synthesize only E;~ polypeptide chains, which can be detected in the cytoplasm but not on the lymphocyte cell surface. These mice have a deletion in the E~ gene that includes the promoter and part of the first exon; this prevents expression of the gene (Mathis et al. 1983). Similar deletions of the same size and location have been found in E-negative wild mouse strains (Dembid et al. 1984). Mice with q and fhaplotypes do not express an E molecule on the cell surface, and both the polypeptide chains are absent (Jones et al. 1981). Molecular genetic studies by Mathis and co-workers (1983) have shown that thefhaplotype synthesizes a normal amount of E~ mRNA which is predominantly of an aberrant size, while the q haplotype expresses very low levels of E~ mRNA. Recombinant mouse strains with crossovers between two E-negative haplotypes do not express an E molecule, as expected. In such recombinant mouse strains, the origin of the E~ allele cannot be determined by serology. These E~ genes can be identified by DNA restriction fragment analysis. In this study, we analyzed five E-negative recombinant mouse strains by DNA restriction fragment analysis (Table 1). They were typed by microcytotoxicity analysis for H-2K, H-2D, A, and E antigens. Immunoprecipitation analysis with anti-E serum confirmed the absence of E molecules. To determine if the B10.RBF, B10.RBQ1, B10. RKQ1, and B10. RKQ2 recombinant mouse strains have functional E~ genes, they were crossed to the E-negative f k strain A.TFR5 (E~E~) which has a functional E~ gene and a nonfunctional E f gene. If the recombinant has a functional E¢ gene, then transcomplementation will occur in F 1 between this E¢ polypeptide chain and the E~ gene from A.TFR5. These recombinants had functional E¢ genes derived from k or b haplotypes (Fig. 1). The recombinants were also analyzed by Ouchterlony immunodiffusion for S region-encoded C4 complement components Ss and Slp. The typing of B10.RBF and A.TFR4 showed that in these recombinants, the C4 genes are derived from thefhaplotype, which places the crossover point between the Ee gene and the C4 genes. The aUelic origin of the C4 genes of B10.RBQ1, B10.RKQ1, and B10.RKQ2 could not be determined by serology, since Ss and Sip antigens are not distinguishable between the b and q haplotypes. Thus, these studies placed the crossover in these recombinants between the E¢ gene and the H-2D gene. The five recombinant mouse strains were examined by restriction fragment analysis with two restriction endonucleases which cut within the E~ gene (Dembid et al. 1984). Recombinants B10.RBQ1, B10.RKQ1, and B10.RKQ2 have either the E~ or E q alleles, B10.RBF either

Expression of I-Aαk and allelic restriction on Aα/Aβ pairing in transgenic mice

Cell surface expression of class II major histocompatibility complex encoded (la) molecules depends on association of the component α and β chain into a stable heterodimer. Studies on cell lines transfected with MHC class II genes have revealed important limitations on the assembly of haplotype‐mismatched Aα:Aβ complex. In order to study α: β chain pairing restrictions in vivo a number of lines of transgenic mice carrying the Aαk gene were generated. The transgene was studied in the context of H‐2b, H‐2s, H‐24, H‐2u, H‐2f and H‐2v haplotypes. Initial FACS analysis of spleen and peripheral blood cells showed no Aαk expression. Spleen cells stimulated with LPS and analysed by FACS showed Aαk expression in three different H‐2b strains as well as H‐2u, but not in others. These data indicate that Aαk can associate with Aβb and Aαk, but not with Aβs,Aβq, Aβf, Aβv.