William P. Lafuse

Partial suppression of Theiler's virus‐induced demyelination in vivo by administration of monoclonal antibodies to immune‐response gene products (Ia antigens)

In vivo administration of monoclonal antibody reactive with major histocompatibility complex-encoded Ia molecules (I-A8) partially suppressed inflammation and demyelination in the spinal cord of SJL/J (H-28) mice persistently infected with Theilers murine encephalomyelitis virus. Demyelination was decreased if antibody was given at the time of virus inoculation or after inflammation had been established in the spinal cord. The decrease in demyelination was independent of isolation of infectious virus from the CNS or of serum titers of immunoglobulin to purified viral antigen. Thus, Theilers virus-induced demyelination is mediated, in part, by immune cells that carry Ia class II molecules.

Recombination hot spots within the I region of the mouse H-2 complex map to the Eβ and Eα genes

Recombinant mouse strains with crossovers in the I region of the H-2 major histocompatibility complex were examined by restriction fragment analysis for the presence of polymorphic restriction sites within the Eβ and Eα genes. Nine recombinant mouse strains were shown to have crossed over within a 5 kb DNA segment that contains the large intron between the second and third exons of the Eβ gene. These results are in accord with previous studies mapping a recombination hot spot within this gene. Seven recombinant mouse strains between the p and k haplotypes were shown to have crossed over in a 6 kb segment within the Eα gene. These results show the existence of a recombination hot spot within the Eα gene. Comparison of the H-2 haplotypes involved in these two recombination hot spots suggests that a specific DNA sequence in b, s, f, and q haplotypes may act to promote recombination in the Eβ gene and a specific DNA sequence in the p haplotype may act to promote recombination in the Eα gene.

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.

Biochemical evidence for multiple I-E Ia molecules

Sequential immunoprecipitation and isoelectric focusing analyses with monoclonal I-E-specific antibodies presented in this paper indicate the existence of multiple I-E molecules. In sequential immunoprecipitations with 13-4 (anti-Ia.7) and 17-3-3 (anti-Ia.22) monoclonal antibodies, 17-3-3 only partially cleared I-E molecules immunoprecipitated by 13-4. Similarly, 13-4 monoclonal antibody only partially cleared I-E molecules precipitated by 17-3-3 monoclonal antibody. These results suggested a minimum of three I-E molecules. One I-E molecule expresses both I3-4 and I7-3-3 determinants, a second I-E molecule expresses only 17-3-3 determinants, and a third I-E molecule expresses only 13-4 determinants. Isoelectric focusing analyses of I-E molcules immuno-precipitated by 13-4 and 17-3-3 showed differences in both Ae beta polypeptide chains and Eα alpha polypeptide chains. The sequential immunoprecipitation and isoelectric focusing analyses presented in this paper can be explained by a model in which there are at least two separate Ae genes being encoded within the I-A subregion and two separate Eα genes being encoded within the I-E subregion. The 17-3-3 monoclonal antibody would recognize a determinant on only one of two Ae beta polypeptide chains and the 13-4 monoclonal antibody would recognize a determinant on only one of two Eα polypeptide chains.

The genetic control of liver camp levels in mice.

Steady-state level of liver 3′,5′-cyclic monophosphate, cAMP, has been shown to be under genetic control linked to the mouseH-2 complex. Liver cAMP levels are associated withH-2 haplotype in fully segregating crosses of strains C3H and C57BL/10. In crosses involving strain A, other loci have an effect that swamps that ofH-2. Results withH-2 recombinants indicate that liver cAMP levels are affected by more than oneH-2-linked locus.

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.

Expression of Ia antigens by mouse Peyer's patch cells☆

Abstract Peyers patch cells of the mouse were characterized with respect to cell types and Ia antigen expression. Nearly 50% of patch cells are B cells and approximately 40% are T cells. Strong reactions by cytotoxicity and immunoprecipitation assays were obtained for I-A and I-E subregion Ia molecules. A subset of Peyers patch T cells express Ia antigens.

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.

Description of the H-2K-, H-2I-, and H-2D-gene products of the H-2v haplotype.

The prototype strain for the H 2 ~ haplotype is SM/J and the congenic strain B10.SM carries the H 2 ~ haplotype on the C57BL/10 background. H-2.21, the private antigenic specificity for the H 2 ~ haplotype was originally defined by antiserum [BALB/c x BI0.C(41N)]F 1 anti-SM (Snell et al. 1971). Since the H 2 v haplotype was positive for H-2.30 (an H 2 D r e g i o n specificity), H-2.21 was defined as the K v product. A new recombinant strain was identified between the parental haplotypes H 2 a and H 2 ~ (J. H. Stimpfling, unpublished). This new line was established as a congenic strain on the C57BL/10 background and designated B 10.SM(22R) with the haplotype symbol H 2 azl (with permission from P. D6mant of the H 2 Nomenclature Committee). When the B10.SM(22R) was originally described, it was shown to have the K k and D ~ haplotypes. In this report, we describe the complete serological and biochemical analysis of the recombinant and parent strains to define specificities associated with the K, I and D regions of the H 2 ~ haplotype. The presence of the K k allele in B10.SM(22R) was reconfirmed by the strong reaction with (A.TL x BZ.H)F1 anti-A.AL (anti-H-2.23, Table 1). Private specificity H-2.4 (marker for D e) was absent in the recombinant. The two anti-H-2.4 (anti-D e) antisera did contain cross-reacting antibodies to the DV-gene product, and these antibodies were directed against the H-2.43 specificity. Antiserum against the H-2.30 specificity showed weak reactivity with both B10.SM and BI0.SM(22R). This data substantiates the previous finding that the K region in the recombinant was derived from the H 2 parental chromosome, while the D region was derived from H 2 ~. Reactions of B10.SM(22R) with restricted anti-Ia sera demonstrated that specificities Ia.1, 2, 3, 17, 18, 19 ( I A ) and Ia.22 ( I -E) were expressed. These specificities are all characteristic of the I k allele. Complete absorption of (C3H.Q x B10.D2)F 1 anti-B10.AQR sera (anti-I k) by the recombinant spleen cells