Morten Simonsen

Nomenclature for chicken major histocompatibility (B) complex.

1 Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115 z Houghton Poultry Research Station, Huntingdon, Cambs PE17 2DA, United Kingdom 3 The Wistar Institute, Thirty-Sixth Street at Spruce, Philadelphia, Pennsylvania 19104 4 Department of Microbiology, New York University School of Medicine, 550 First Avenue, New York, New York 10016 5 College de France, Laboratoire de M6dicine Exp6rimentale, 11, Place Marcelin-Berthelot, 75231 Paris, Cedex 05, France 6 Institute for General and Experimental Pathology, University of Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria 7 Institute for Experimental Immunology, University of Copenhagen, N0rre Alle 71, DK-2100 Copenhagen 0, Denmark s Department of Immunology and MRC Group of Immunoregulation, University of Alberta, Edmonton, Alberta, Canada 9 Department of Animal Science, Iowa State University, Ames, Iowa 50011 10 Basel Institute for Immunology, Grenzacherstrasse 487, CH-4005 Basel, Switzerland ~1 Department of Avian Medicine, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30605 a Department of Medical Microbiology, Turku University, Turku, Finland 20520

The MHC haplotypes of the chicken.

The major histocompatibility complex (MHC) of Gallus gallus is the B complex of which three classes of cell-membrane antigens have been clearly defined by serological, histogenetic, and biochemical methods. Two of these classes are homologous to classes I and II of mammals (B-F and B-L, respectively), while the third (B-G) is a differentiation antigen of the erythroid cell-line; the mammalian homologue of this class is still undefined. The B haplotypes comprise at least one gene of each class that displays linkage disequilibrium of a remarkable strength. The present work is the first systematic comparison by serological and histogenetic methods of the allelic products (allomorphs) of 15 haplotypes, including all of the 11 that were accepted as “standard” B haplotypes at the recent international Workshop on the chicken MHC in Innsbruck, Austria. The analysis has revealed many similarities, but only four pairs of probable identities: G2 and G12, F4 and F13, L4 and L13, L12 and L19. It appears therefore that the B-G locus is comparable in its degree of polymorphism to the class I (B-F) locus. The “standard” haplotypes are almost all of White Leghorn derivation, and preliminary typings of other breeds of chickens, and of wild chickens, indicate the existence of a much wider spectrum of allomorphs.

The Elusive T Cell Receptor

Less than five years ago, at the Cold Spring Harbor Symposium on Antibodies, there was no one to question the assumption that recognition of antigen by potentially reactive lymphocytes is effectuated by specific immunoglobulin receptors. Nothing but nodding approval from the audience could, for example, meet either Mitchison or Jeme when, each in their fashion, they made axiomatic this generally shared assumption. The thought of multipotential lymphocytes referred to by Jeme (1967) as heresy seems but mild revisionism compared to the suggestion that recognition structures may not be immunoglobulins after all. It is nevertheless towards the latter suggestion we are becoming inclined with regard to cell mediated immunity. The present paper wiU describe our own investigations of the receptor mechanism involved in the GVH reaction in chicken embryos and discuss the findings in relation to conflicting findings in the literature. The principal approach has been to try neutralizing the GVH reaction with specific heteroand isoimmune sera. As a concurrently pursued interest we have investigated the same antibodies for their ability to inhibit antigenbinding cells in their rosette formation with sheep red blood cells (SRBC). Many of the best experiments have combined these two reactions by employing for the GVH reaction donor animals which were pre-immunized with SRBC. Three principal groups of antisera have been used, namely a) rabbit anti chicken L-chain (anti-L), b) rabbit anti chicken thymus (ATS), or bursa (ABS), and c) isoimmune chicken sera raised by cross-immunizing homozygous B 1/1 and B 2/2 birds (anti-Bl and anti-B2). The latter group, of course, are antisera directed against alloantigens determined by the major histocompatibility and blood group locus of the chicken, the B locus.

The chicken erythrocyte-specific MHC antigen. Characterization and purification of the B-G antigen by monoclonal antibodies

Mouse monoclonal antibodies with B-G antigen (major histocompatibility complex class IV) specificity were obtained after immunization with erythrocytes or partially purified B-G antigen. The specificities of the hybridoma antibodies were determined by precipitation of B-G antigens from 125I-labeled chicken erythrocyte membranes (CEM) followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. The B-G antigen had an approximate molecular mass of 46–48 kd in reduced samples, depending on the haplotype, and in unreduced samples contained either dimers (85 kd), when labeled erythrocytes were the antigen source, or trimers (130 kd), when B-G was purified and precipitated from CEM. The B-G antigen was unglycosylated as studied by (1) in vitro synthesis in the presence or absence of tunicamycin, (2) binding experiments with lectin from Phaseolus limensis, and (3) treatment of purified B-G antigen with Endoglycosidase-F or trifluoromethanesulfonic acid. Two-way sequential immunoprecipitation studies of erythrocyte membrane extracts with anti-B-G alloantisera and monoclonal antibodies revealed only one population of B-G molecules. Pulse-chase experiments have shown B-G to be synthesized as a monomer, with dimerization taking place after 20–30 min. No change in the monomers molecular mass due to posttranslational modifications was revealed. The antigen was purified from detergent extract of CEM by affinity chromatography with a monoclonal antibody, and then reduced and alkylated and affinity-purified once more. Finally, reverse-phase chromatography resulted in a pure product. The B-G antigen was identified in the various fractions by rocket immunoelectrophoresis. The final product was more than 99% pure, as estimated by SDSPAGE analysis followed by silver stain of proteins. The yield from the affinity chromatography step was 3–4 μg B-G/ml blood, calculated from Coomassie-stained SDS-PAGE of B-G using ovalbumin standards. The monoclonal antibodies were also used to identify the B-G (class IV) precipitation arc in crossed immunoelectrophoresis. No common precipitate with the B-F (class I) antigen was observed.

On the acquisition of tolerance by adult cells.

The phenomenon of acquired tolerance historically is connected very firmly with the prenatal or neonatal stage of development of an organism. Of course, it is fully legitimate to speak theoretically of the acquisition of tolerance in adult organisms. The question is whether such can occur and, if so, how to demonstrate it,. The chief purposc of this paper is to discuss the ways by which acquired tolerance may be demonstrated, not so much in the integrated organism, as usual, but rather in a population of spleen cells. I t has been asked sometimes whether anyone ever has seen a tolerant cell. I think the answer is “No.” However, I think that I have seen a t least some isolated spleen cell populations that behaved in a tolerant way and in accordance with all classic requirements. Among such cell populations there have been apparently tolerant cells whose origin was in adult spleens. However, before coming to these questions, I shall discuss a few points of analytic technique. ‘The basic technique employed is the graft-versus-host assay (GVHA), described two years ago (Simonsen et al., 1958), which is based on the fact that spleen and liver enlargement are constant and conspicuous signs in a rather early stage of the “runt disease,” being especially pronounced about ten days after grafting (Simonsen and Jensen, 1959). There are several reasons why the GVHA often is performed with litters of F1 hybrids: (1) Hybrid vigor usually ensures litters of good size and vitality. (2) The litters may be used at least 10 days after birth instead of 1 day, which is approximately the end point for use of litters not genetically tolerant of the graft. ( 3 ) They may be used to discriminate between immunologically competent cells of different genetic derivation; this is because 4to 10-day-old hybrids are sufficiently mature to eliminate foreign cells by the host response to the graft and a t the same time are young enough to allow a typical graft-versushost reaction to develop when they are injected I.P. with adult cells from one of their parental strains (TABLE 1). A warning is necessary here to those interested in using this method: it may fail entirely if the foreign graft (C in TABLE 1) belongs to the same H-2 group as the parental graft. Thus, if AKK spleen is grafted to (C3H X DBA/2), it gives as much splenomegaly as if adult C3H spleen had been grafted instead (AKR and C3H carry both the H-2k allele, while DBA/2 is H-2‘I). Apart from being an undesirable restriction in the applicability of the method, this finding proves that complete genetic tolerance of the graft is not recpired for the development of runt disease. It fits very well with the dem-

Major Histocompatibility Genes in Egg‐Laying Hens*

ABSTRACT: A common base population of White Leghorn was “synthesized” for a joint project on the genetics of egglaying, undertaken by animal breeding geneticists in 4 Scandinavian countries. After 6 to 7 generations of line selection for various egg‐laying parameters, MHC typing was undertaken of both the selection lines in Denmark, Norway, and Sweden and the respective control lines representing the common base population. Ten MHC haplotypes were defined which jointly accounted for about 95% of the MHC gene pool of the base population. The 2 haplotypes which were predominant in the base population, B15 and B19, responded very differently to the selection pressures applied.

Graft-versus-host-reactions: the history that never was, and the way things happened to happen.

No other reactions occurring in the clinic can show more dramatically than GvH reactions the importance of being histocompatible, if mixing your tissues with those of others you must. The severe clinical conditions calling for treatment with bone marrow grafting, which is of course the form of allotransplantation that is greatly endangered by the complication of GvH reactions, may themselves complicate both diagnosis, treatment, and biological interpretation of the events set in motion by the attack of grafted T lymphocytes on their host. However, what happens in tnan in these conditions is basically the same immunopathological events which can also be precipitated in experimental animals, if the following 3 conditions are met: 1. That the allograft contains T cells. 2. That the host contains cell membrane antigens which are recognized as foreign by the grafted T cells. 3. That the host is incapable for one reason or other of getting rid of the grafted T cells by an immunological counter-attack, which by definition would be host-versus-graft (HvG). Historically, GvH reactions were first observed in the Rockefeller Institute in New York by grafting of adult chicken tissues containing T cells, such as spleen tissue, onto the chorioallantoic membrane of allogeneic chicken embryos (Murphy 1916). This was decennia before anybody spoke about T cells and GvH reactions, and the immunological nature of the discovery was completely missed by Murphy, and probably also by his contemporary readers of the Journal of Experimental Medicine. I intend to discuss these findings in more depth because I am fascinated by the interplay in science of ideas and intellectual climate with chance and circumstance.

Mixed Lymphocyte Reactions (MLR) in rainbow trout (Salmo gairdneri) sibling

Suitable conditions for 2 way MLR were determined in random combinations of commercially cultured trout and were then applied to full siblings in two families reared for experimental purposes. The primary aim of the investigation was to determine whether the immunogenetic predictions from mammalian and avian experiments would apply, i.e. (i) that all full sibs, short of rare intra-MHC recombinants, can be assigned to a maximum of four different MHC genotypes, and (ii) that individuals of the same genotype are mutually histocompatible as tested in MLR. If both predictions apply, five or more siblings tested in all pairwise combinations should invariably display compatible pairs, so that a maximum of four groups of mutually incompatible individuals should emerge. The findings did not fit this model, and two alternative immunogenetic models are discussed.

Isolation and characterization of chicken and turkey beta2-microglobulin

Abstract Chicken and turkey beta2-m were isolated from citrated plasma in sequential use of three Chromatographie steps: affinity chromatography, gel filtration chromatography and anion-exchange chromatography. The purified protein was identified as beta2-m by reaction with a beta2-m specific monoclonal antibody and by the ability to recombine with the chicken MHC class I heavy chain. The purity was estimated by SDS-PAGE and IEF. The pI was between 5.1 and 5.3 for chicken beta2-m and 4.7 and 4.8 for turkey beta2-m, which fact is reflected in their different electrophoretic mobilities in agarose gel (turkey migrates in the alpha and chicken migrates in the beta region). The mol. wt of both chicken and turkey beta2-m was 14,500 estimated by SDS-PAGE whereas calculations based on the amino acid compositions gave mol. wts of 11,000. E M 280 was 15.9 for chicken beta2-m and 16.4 for turkey beta2-m. The amino acid compositions and sequences of the two avian beta2-m molecules have been compared with earlier data from the literature. The sequence of the 23 N -terminal amino acids was found to be identical in our preparations from both chicken and turkey, namely DLTPKVQVYSRFPASAGTKNVLN, and is incompatible with a previously published sequence also thought to be from turkey beta2-m. Reasons for our opinion that the molecules isolated and sequenced in this paper are the correct ones are given.

Analysis of class II genes of the chicken MHC (B) by use of human DNA probes

Class II genes of the major histocompatibility complex (MHC) in the chicken have been investigated by Southern blot analysis using human cDNA probes for DQα, DQβ, DRα, and DRβ. Both β probes but not the α probes cross-hybridized well with chicken DNA. The results indicated that the β probes hybridized with at least two β genes in the chicken MHC and there was no clear indication of a DQ-DR subdivision of chicken class II β genes. The possibility of using human β probes for MHC typing in the chicken was tested by using two homozygous individuals for each of 20 different, serologically defined, MHC (B) haplotypes originating from the domestic breeds of White Leghorn and Rhode Island Red, or from Red Jungle Fowl (the wild ancestral form). Genomic DNA samples from these individuals were digested with any one of the Eco RI and Pvu II restriction enzymes and hybridized with the DRβ probe. Restriction fragment length polymorphism (RFLP) was obtained with Pvu II only, which resolved seven different RFLP types. There was an excellent correlation between these RFLP types and the serological B typing since the RFLP type was identical within each pair of homozygotes. In addition to this broad survey of many haplotypes, a more detailed comparison was carried out on β21-like haplotypes originating from different breeds. No differences in restriction fragment patterns among these haplotypes could be resolved using any of the restriction enzymes Bg 111, Eco RI, Hind III, Pst 1, Pvu II, and Taq I.