Genetic and immunological characterization of naturally occurring recombinant B3 rats

The B-stock population of rats was bred for homozygosity at the loci controlling coat color. In this process, theAg-B1 andAg-B3 haplotypes became fixed in Hardy-Weinberg equilibrium. Extensive immunization and absorption studies showed that the specificities in the B-stock rats homozygous for theAg-B1 haplotype were the same as those found in the inbred F344 strain (Ag-B1), and that the specificities in the rats homozygous for theAg-B3 haplotype were the same as those found in the inbred BN (Ag-B3) strain. A homozygous line derived from the rats carrying theAg-B3 haplotype (B3) has the mixed lymphocyte reactivity and antibody responsiveness to poly (Glu⁵²Lys³³Tyr¹⁵) characteristic of the inbred strains in theAg-B4 group. Thus, it represents a naturally occurring recombination between the loci controlling MLR and immune responsiveness, on the one hand, and those controlling the Ag-B antigens on the other. Antibody responsiveness segregated with theAg-B3 haplotype in crosses between the B3 homozygotes and the low responder BUF and M520 strains; hence, this recombination is a stable one. There was no linkage of antibody formation or haplotype to coat color. The finding of a strain with a naturally occurring recombination in the major histocompatibility complex between the loci controlling mixed lymphocyte reactivity and the Ag-B histocompatibility antigens provides evidence for the separateness of these loci. Since the portion of the genetically determined mechanism controlling antibody responsiveness which is linked to the MHC was that characteristic of the MLR type, it too must lie outside the region defined by the serological specificities of theAg-B haplotype.     

Polymorphism of the major histocompatibility locus in the wild Norway rat

Specific alloantisera against the eight Ag-B groups found in inbred strains of rats were capable of reacting with all wild Norway rats (Rattus norvegicus) tested. Absorption studies, antisera production, and progeny testing involving wild rats showed that the antigenic specificities detected in the wild rat population were similar, if not identical, to the Ag-B antigens present in inbred strains. Xenoantisera prepared in rabbits against rat erythrocyte antigens (Ag-C1 and/or C2) reacted with erythrocytes from each wild rat tested. Progeny testing involving these erythrocyte antigens was identical to that observed in inbred strains. The restricted genetic polymorphism of theAg-B alleles in the wild rat population suggests that the functional and evolutionary significance of the major histocompatibility complex in the rat may not depend upon a high degree of genetic variability.     

Genetic analyses of alloreactions between recently wild and classical inbred strains of syrian hamsters: Evidence in favor of a major histocompatibility complex

Cytotoxic alloantisera were raised between recently wild and classical inbred strains of Syrian hamsters. Antisera produced by immunizing the classical inbred strains with tissue from the partially inbred, recently wild hamsters detect several specificities shared between the classical and recently wild strains. Reciprocal mixed lymphocyte reactions between the two different groups of hamsters suggest that the new source of hamsters possesses several unique MLR phenotypes which may represent new Hm-1 haplotypes. Moreover, several recently wild strains express MLR phenotypes quite similar if not identical to the Hm-1 ^a haplotype of the inbred strain, MHA. Genetic analyses of alloreactions between domestic inbred and recently wild strains suggest that a single locus or chromosomal region encodes the allodeterminants that induce strong MLR reactivity. Six unique MLR phenotypes have been defined which most likely represent haplotypes of the hamster MHC equivalent, Hm-1. Genetic linkage studies indicate that some alloantisera detect determinants encoded by loci closely linked to the MLR locus, and therefore define Hm-1 determinants. Moreover, other alloantisera recognize determinants encoded by a locus that is unlinked to Hm-1. These studies suggest that Syrian hamsters express a polymorphic MHC equivalent, Hm-1, which encodes determinants that induce both cell-mediated and humoral alloreactivity.     

Restriction by the major histocompatibility complex of antigen-induced T lymphocyte proliferation in new inbred guinea pig strains.

Employing new inbred guinea pig strains, JY 1, JY 2 and JY 3, established in this Institute in addition to strains 2 and 13, the authors investigated histocompatibility restriction in macrophage-T lymphocyte interaction. These five strains are known to possess distinct major histocompatibility complex (MHC) gene profiles (1, 2). This fact was supported by our results concerning the mixed leukocyte reaction (MLR) and cytotoxicity test with alloantisera. Using various combinations of T lymphocytes and peritoneal exudated cells (PECs) from these strains, in vitro proliferative responses of T lymphocytes from BCC-immune animals to PPD-pulsed normal PEC were tested. Successful activation of T cell response was observed not only in syngeneic combinations but also in allogeneic combinations among strains JY 1, JY 3 and strain 13 which share common Ia antigens detected by strain 2 anti-strain 13 alloantiserum. Because JY 1 and JY 3 seem to share a common B antigen differing from strain 13, it was suggested that identification in the I region of MHC is sufficient for effective antigen-presentation by the macrophage. Although a part of Ia is shared, no T lymphocyte activation was observed in the combination between JY 2 and JY 1 or JY 3, whereas strong MLR occurred in these allogeneic combinations. At the present stage of the study, it can be said that disparity in the part(s) of Ia antigens which is responsible for strong MLR cannot lead to effective T cell-macrophage interaction. These results support the concept that functional activation of primed, proliferating T lymphocyte requires the participation of gene products of macrophages coded for by the I region in MHC. By employing JY 1, JY 2 and strain 2, which appear to possess distinct B and Ia antigens, it was shown that the T lymphocyte and macrophage interactions essential for mitogen-induced T lymphocyte proliferation are not restricted by histocompatibility.     

Ag-B-linked analogue of Ia antigens in the rat.

The reactions of Lewis rat lymphocyte membrane antigens with two alloantisera, BN anti-Lewis and BN anti-Fischer have been studied. Three lines of evidence indicated that these antisera reacted with cell surface antigens homologous to Ia antigens of the mouse. 1) After absorption with Lewis platelets, the antisera killed only 40 to 50% of Lewis spleen cells. The majority of such cells were shown to be Ig-positive B cells by the examination of reaction patterns on lymphocytes after separation on nylon wool into T cell- and B cell-enriched subpopulations. 2) SDS-PAGE analysis of solubilized Lewis spleen cell antigens precipitated with these antisera revealed that the platelet-absorbed antisera reacted with molecules comparable in size to mouse Ia antigens (mw approximately equals 35,000 and 28,000). The unabsorbed sera reacted with these molecules and with additional molecules corresponding in size to mouse K and D antigens (m.w. = 45,000). 3) Neither of these antisera killed significant numbers of spleen cells from the partially congenic strain F.BN (seventh backcross homozygotes), a Fischer rat to which the Ag-B.3 allele is being transferred by repetitive backcrossing, indicating that the genes coding for these Ia-like antigens in the rat are linked to the Ag-B locus.     

Sharing of Ia antigens between species. II. Molecular localization of shared Ia determinants implies the existence of more than one I sublocus of the rat MHC.

A mouse alloantiserum B10.D2 anti-B10.BR (H-2d anti-H-2k) cross-reacted with rat lymphocyte surface glycoproteins with characteristics of Ia antigens. Sequential precipitation analysis on solubilized radiolabeled LEW rat lymphocyte antigens with this cross-reactive mouse alloantiserum and the rat alloantiserum BN anti-LEW (Ag-B3 anti-Ag-B1) revealed that the Ia-like antigen detected by the mouse alloantiserum also reacted with the rat anti-Ia antibodies. It was further shown that the rat alloantiserum also detected another set of Ia-like antigens that did not cross-react with the mouse alloantibody. Precipitation analysis with congenic rat strains confirmed that all Ia-like antigens precipitated by the rat alloantibody were encoded by Ag-B linked genes. Thus the shared Ia-like antigen must also be the product of Ag-B-linked gene(s) or be physically associated with such products. In addition, molecules bearing shared antigenic determinants were separable from at least some of the Ia-like antigens detected by the rat alloantiserum, possibly suggesting the existence of more than one sublocus coding for Ia antigens within the rat MHC.     

Serological studies on the major histocompatibility complex of new inbred strains of the guinea pig.

New inbred strains of guinea pigs, JY 1, JY 2, JY 3, JY 6, JY 9 and JY 10 have been established in this Institute. Serologic studies of guinea pig leukocyte antigens (GPLA antigens) were carried out in order to examine their major histocompatibility complex (GPLA complex). Antisera specific for Ia antigens were raised by cross-immunization of NIH strain 2 (NIH 2) and NIH strain 13 (NIH 13) guinea pigs, well known inbred guinea pigs. The sera identified four distinct Ia specificities, which were designated as Ia.2a, Ia2b, Ia.13a and Ia.13b. Six antigenic specificities different from the above Ia specificities were identified by sera obtained by appropriate immunization of the inbred guinea pigs and were designated as P.1, P.2, P.3, P.4, P.5 and P.6. Antigenic specificities of GPLA antigens recognized in inbred guinea pigs were : NIH 2 (Ia.2a, Ia.2b, P.1, P.2, P.O, P.4), NIH 13 (Ia.13a, Ia.13b, P.1, P.2, P.3, P.4), JY 1 (Ia.13a, Ia.13b, P.5), JY 2 (Ia.2b, Ia.13b, P.3, P.4, P.6), JY 3 (Ia.13a, Ia.13b, P.2, P.4, P.5), JY 6 (Ia.2b, Ia.13b, P.3, P.6), JY 9 (Ia.13a, Ia.13b, P.4, P.5), JY 10 (Ia.13a, Ia.13b, P.2, P.3, P.4, P.6), JY 9 (Ia.13a, Ia.13b, P.4, P.5), JY 10 (Ia.13a, Ia.13b, P.2, P.3, P.4, P.6). The correspondence of these specificities to those already reported was discussed and the P.2 or P.4 was considered to be an additional specificity of GPLA antigens that have not been reported yet. As the new inbred strains of guinea pigs were thus found to possess characteristic GPLA complex, which differ from each other and from those of NIH 2 and NIH 13 strain, they should be useful for studies of roles of the major histocompatibility complex in the immune system.     

Transplantation in miniature swine. V. Characterization of Ia antigens.

The complexity of the I region analog associated with the MHC of miniature swine has been probed by sequential antibody precipitation studies of Ia antigens. Treatment of solubilized lymphocyte preparations from MSLA homozygotes of the DD haplotype with excess AA anti-DD alloantiserum led to precipitation of only a portion of the total Ia antigens, as determined by secondary precipitation of the remaining material with CC anti-DD serum. The presumed I region of miniature swine must therefore code for more than one Ia antigen-bearing polypeptide chain. In addition, certain mouse alloantisera that had previously been shown to react with rat Ia antigens were tested for reactivity with swine Ia antigens. Anti-Iak mouse alloantisera precipitated Ia molecules from every swine extract tested, regardless of MHC type, precluding genetic mapping studies. However, sequential precipitation studies demonstrated that the cross-reactive mouse alloantisera reacted only with a subclass of swine Ia antigens, again suggesting genetic complexity of the pig Ia locus.     

The LEW.BN(2R) strain: a recombinant in the rat MHC

Cell-mediated cytolytic (CMC) responses resulting from immunizations between rat strains considered to be RT1 (Ag-B) identical (LEW.B3:BN) are capable of detecting a membrane determinant(s) controlled by a locus linked to RT1, which has been designated Ag-L. The Ag-L gene region has been isolated in a recombinant line, tentatively designated as LEW.BN(2R), and has been assigned the RT1r5 haplotype. The data presented demonstrate that the genes responsible for MLR stimulation in the 2R strain are of LEW origin. In addition, LEW.B3 anti-BN CTL appear to recognize multiple specificities, only one of which is in the 2R strain. Some of the remaining specificities in BN may be the result of interactions between undetected genes that have been separated in the LEW.B3 and 2R strains.     

Isolation of twelve monoclonal antibodies against Ia and H-2 antigens. Serological characterization and reactivity with B and T lymphocytes

The cell hybridization technique was used for the production of 12 monoclonal antibodies against H-2K^k, H-2D^b, I-A^k and I-E^k antigens. The strain distribution pattern indicated that three antibodies reacted with new H-2 and Ia determinants, respectively, while the majority of determinants defined by the monoclonal antibodies showed good correlation with H-2 and Ia determinants described by conventional alloantisera.Monoclonal Ia antibodies showed strong reactivity with about 90% of surface IgM positive B cells, but not with T cells. In double fluorescence studies, both I-A and I-E determinants were always found to be coexpressed on the same B cells. When the high sensitivity of the fluorescence activated cell sorter was utilized, about 30 to 40% of purified lymph node T cells were found to carry both I-A and I-E antigens, although in a much lower density than B cells. In conclusion, monoclonal Ia antibodies appear to display the same serological and cellular reactivity pattern as do conventional antisera.     

Sharing of Ia antigens between species. IV. Interspecies cross-reactivity of monoclonal antibodies directed against polymorphic mouse Ia determinants

The specificity of interspecies Ia cross-reactions has been analyzed by testing a panel of monoclonal antibodies (mAb) to mouse I-E and I-A antigens for reactivity with pig Ia antigens. Our earlier studies showed that mouse anti-I-E alloantisera recognized common determinants on Ia antigens of other species, whereas anti-I-A alloantisera showed much more limited cross-reactivity. These results were confirmed using a panel of 17 anti-I-E mAb, 10 of which were cytotoxic to pig cells. 2D gel electrophoretic analyses of precipitates with these mAb of 35S-labeled, NP40 solubilized pig cells revealed a limited set of protein spots that appeared to be identical to the subset of pig Ia antigens precipitated by A.TH anti-A.TL alloantiserum. Because the cross-reactive mouse sera were produced in mouse strains that do not express an I-E molecule (H-2b and H-2s), it was anticipated that the cross-reacting antibodies would be reactive with the monomorphic determinant of the I-E molecule, Ia.7. However, comparison of the reactivity of these mAb with pig cells and mouse cells revealed that the cross-reactivity on pig cells correlated not with Ia.7 but rather with detection of epitope(s) of the I-E molecule associated with inter-strain polymorphism. Anti-I-A cross-reactions were also detected, but were weaker and more limited. These findings may have implications for the evolution of Ia antigens in mammalian species.     

Detection of hybrid (combinatorial) Ia antigens using parent anti-F1 sera

Ia specificities 22 and 23 were initially identified by using conventional alloantisera and were mapped to the I-E subregion of k and d haplotypes on the basis of their reactivity with selected recombinants. Recently we found that Ia 22 and 23 are hybrid determinants on the basis of their expression on selected F1 cells, but absence from both parental cells. Initial attempts to detect hybrid Ia antigens by immunizing parents with F1 cells were unsuccessful. By utilizing lipopolysaccharide (LPS)-stimulated F1 spleen lymphoblasts as immunogens, 1 of the parents as recipient and the other parent cells for absorption of antisera, specific anti-Ia.22 and 23 antibodies were produced. The specificity of these parent anti-F1 sera was confirmed by cytotoxic and immunoprecipitation analyses.     

Analysis of class I MHC antigens in the rat by monoclonal antibodies

Monoclonal antibodies (mAb) were made against class I MHC antigens of the i (mAb 42,70,39) and u (mAb 68-D) haplotypes in the rat by using specific strain combinations in order to obtain reagents for identifying the products of the RT1.An, RT1.Au, and RT1.Eu loci. These antibodies were hemagglutinating only; were IgG except for mAb 68-D3, which had a defective heavy chain; reacted identically with MHC-congenic strains and with their inbred donor strains; and precipitated class I MHC antigens. Strain distribution, sequential immunoprecipitation, and peptide mapping studies were used to define the specificities of the mAb, and the assignments were checked by comparing the specificities of the mAb with those of haplotype-specific alloantisera. The specificities were the following: mAb 42, An; mAb 68-D, Au; mAb 70, Eu; and mAb 39, an antigen encoded by a locus different from A and E. This new locus was designated RT1.F, and the allele detected by mAb 39, as Fa. The serologic data place RT1.F between RT1.A and RT1.D. The plasma membranes of DA.1I(BI) lymphocytes contain comparable amounts of An, Eu, and Fa antigens but express them on the cell surface in the order An much greater than Eu greater than Fa.     

The syngeneic mixed leukocyte reaction: the genetic requirements for the recognition of self resemble the requirements for the recognition of antigen in association with self

We have used cells from inbred strain 2 and strain 13 guinea pigs in order to define further the role of Ia antigens in the syngeneic mixed leukocyte reaction (MLR). The guinea pig syngeneic MLR resembled the autologous MLR in man in that it demonstrated both memory and specificity. The Ia antigens appeared to be the proliferative stimuli in that the primary stimulator cell was an Ia-positive adherent peritoneal exudate cell (PEC) and the reaction could be specifically inhibited by anti-Ia sera directed to the stimulator cell. We also demonstrated the existence of two (2 x 13)F1 T cell populations that were capable of reacting to one or the other parental PEC in the absence of any known exogenous antigen. These results suggest that the syngeneic MLR may represent T cell activation mediated through a receptor for self Ia.     

Evidence for more than one Ia antigenic specificity on molecules determined by the I-A subregion of the mouse major histocompatibility complex.

Ia antigenic specificities determined by the I-A subregion of the mouse major histocompatibility complex have been examined in strain B10.D2 (H-2d), C57BL/10 (H-2b), and in a (C57BL/6xDBA/2) hybrid (BDF1; H-2b/d). Detergent solubilized, 3H-leucine-labeled antigen preparations were mixed with appropriate alloantisera and precipitation was induced either by addition of goat anti-mouse gamma-globulin or by addition of protein A-bearing Staphylococci. Sequential precipitation analysis showed that in strain B10.D2, Ia specificities 8 and 11 were co-precipitable, and that in strain C57BL/10, Ia specificities 8 and 9 were co-precipitable. In contrast, precipitation of specificities 9 and 11 from a BDF1 antigen preparation showed that these two Ia specificities were on separate molecules. The genetic implications of these data are discussed.     

Detection of at least two distinct mouse I-E antigen molecules by the use of a monoclonal antibody

In an attempt to determine whether the expression of more than a single Ia antigen is determined by the I-E subregion of the mouse major histocompatibility complex (MHC), sequential immunoprecipitation analyses were performed by using a monoclonal antibody and alloantisera reactive with I-E subregion products. 3H-leucine-labeled glycoprotein preparations obtained from H-2d spleen cells were precleared with the monoclonal antibody 14-4-4S and then examined for residual Ia activity precipitable by an alloantiserum detected by SDS-polyacrylamide gel electrophoresis. Residual Ia activity was observed for all three strains of the H-2d haplotype tested. The residual Ia activity could be detected by sera absorbed with B10.A spleen cells, indicating that products of the I-E subregion rather than of the I-C subregion were responsible for this activity. No separable I-Ek molecules were detected in products of B10.A cells with the use of combinations of two monoclonal antibodies (including 14-4-4S) and several appropriate alloantisera. These findings indicate the presence of at least two similar but distinct I-E antigens encoded by the H-2d haplotype.     

Detection of HLA-DRw (Ia-like) antigens on human T lymphocytes grown in tissue culture.

Human T lymphocytes that proliferate in the presence of conditioned medium from PHA-stimulated allogeneic peripheral blood cells were shown to express IPA antigens after the 8th culture day. Ia antigens as detected by xenogeneic antisera were present on 80 to 90% of the cultured cells which were also strongly reactive with xenogeneic antisera defining a human T cell antigen and formed E rosettes. Control cultures with PHA or no conditioned medium expressed T cell but not Ia antigens. These cultured T cells also express the same HLA-DRw determinants as the B cells of the donor they were derived from. Absorption of xenogeneic Ia, and HLA-DRw alloantisera with cultured T cells completely removed the reactivity of these sera for enriched peripheral blood B lymphocytes from normal donors.     

Antigenic specificity of opsonophagocytic antibodies in rabbit anti-sera to group B streptococci.

An opsonophagocytic assay has been developed which requires human polymorphonuclear leukocytes, immune serum, and complement for optimal killing of Group B streptococci. Only with all three of these components was killing of greater than 1.0 log10 of the initial inoculum achieved, using rabbit antisera directed to homologous strains of each of the five known serotypes of Group B streptococci. Titers of specific antisera which opsonized the strains and resulted in greater than 1 log 10 reduction of colony-forming units, ranged from 1:100 (serotype Ib) to 1:3200 (serotype Ia). Cross-reactions between serotype-specific sera and heterologous strains were seen in certain instances. Type Ic strain and serotype Ic antiserum demonstrated cross-reactions with types Ia and Ib which were explainable by known shared antigens among these types. The only other cross-reaction which resulted in greater than 1 log 10 reduction in colony-forming units was when unabsorbed antiserum to strain Ia was used to opsonize a strain of serotype III. Opsonization of 10 serotype III strains was demonstrated with a single type III antiserum. Killing of nine of these strains required polymorphonuclear leukocytes, complement, and antiserum, but one strain, D136C, the reference strain, could be killed (greater than 1 log 10 reduction in colony-forming units) without either complement or specific antiserum. Inhibition studies were performed utilizing large m.w. polysaccharide antigens extracted from each serotype. These antigens inhibited opsonization of homologous strains by homologous antisera with 50% inhibition points ranging between 0.5 and 4 mug.     

T(Iat)- and B(Iab)-cell alloantigens determined by theH-2 linkedI region in mice

The specificity of an antiserum directed againstI region associated (Ia) antigens is described. The serum was raised in (DBA/1×B10.D2)F₁ mice against lymphocytes of AQR mice, differing from the responder for theI region only. The serum reacts with Ia antigens expressed on B cells (Iab) as well as with Ia antigens expressed on T cells (Iat). Absorption studies indicate that B cells possess at least two Ia antigens, and one of these is shared by T cells. However, this shared antigen is not present on the surface of lymphocytes of thymectomized mice. Analysis of the strain distribution of Iab and Iat antigens revealed that the Iab antigens are present on lymphocytes of mice carrying theIA ^k subregion and that the Iat antigens are present on lymphocytes of mice carryingI region genes of theH-2 ^k haplotype located between theIA andIB subregions. This conclusion is based on the analysis of the antiserum's reactivity with T and B cells of the strains B10.A(2R), B10.A(4R) and B10.HTT: the serum reacts with B and T cells of B10.A(2R) but only with B cells of B10.A(4R) mice and only weakly with T cells of B10.HTT mice.Abbreviations ALG antimouse lymphocyte globulin from rabbits - B cells bone marrow derived lymphocytes - B10 C57BL/10Sn mice - D1D2F₁ (DBA/1×B10.D2)F₁ hybrid mice - GVHR graft-vs-host reaction - Ia I region associated antigen - Iab on B cells - Iat on T cells - MLR mixed lymphocyte reaction - T cells thymus-derived lymphocytes - Thy-1 thymus antigen 1, formerly called theta - Tx-Lyc lymphocytes of thymectomized, ALG treated, lethally irradiated and anti-Thy-1 treated bone marrow reconstituted mice - 2R B10.A(2R)/SgSn mice - 4R B10.A(4R) mice