B. M. Longenecker

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

Lymphoma induced by herpesvirus: Resistance associated with a major histocompatibility gene

Studies with crosses of inbred chicken lines demonstrate that resistance to Mareks disease, a herpesvirus-induced lymphoma of chickens, is associated with an allele (B21) of the major histocompatibility locus (theB locus). TheB21 allele is thus the first genetic marker for resistance to herpesvirus-induced neoplastic disease, and our studies suggest the means whereby similar associations might be found in man.

The major histocompatibility complex of the chicken

SummaryB identifies a system of two or more loci which have major effects on hostversus-graft and graft-versus-host reactions. LikeH-2 andHL-A, B encodes lymphocyte antigens which react with alloantibodies and allogenic lymphocytes. Like the mammalian MHCs,B has many alleleic variants. LikeH-2 alleles, some of these are associated with specific differences in immune competence and susceptibility to viral oncogenesis. UnlikeH-2, there is no evidence thatB histocompatibility andB serology represent separable genetic systems. In addition,B is associated with functional differences unknown in mammals. These include a general difference in immune competence and differences in the frequency of herpesvirus lymphoma and in reproductive and general fitness. We conclude that the different kinds of information available forB and the mammalian MHCs reflect differences in circumstance, sampling, and technique, and should be regarded as less fundamental than the immunogenetic similarities. We suggest thatB is the phylogenetic homologue ofH-2 andHL-A.

Organ specific metastasis with special reference to avian systems.

SummaryMany malignant tumors demonstrate a definite propensity for metastasis to specific organs despite the fact that tumor cells with the potential for metastasis may circulate randomly throughout the body. Current concepts of organ specific metastasis (OSM) center around the generation of tumor cell variants with enhanced capacity for metastasis to specific organs. At present three hypotheses, mechanical, seed and soil, and specific tumor cell adherence (STCA), stand out as possible explanations for OSM. These possible mechanisms of OSM are by no means mutually exclusive. Recent efforts to understand OSM have included the selection of organ-specific metastasizing variants from tumor cell lines and an examination of their surface and metastatic properties. OSM-selected cell lines from many different tumor systems have been used to examine the relative contributions of the three mechanisms. While examples of each mechanism have been reported, the relative contribution of each for different tumor systems may differ substantially. Therefore, generalizations about the behavior of tumors based on studies with just a few tumor lines and systems may not be valid.There is substantial evidence that cell surface molecules are important in the process of OSM and homing of lymphocytes to specific lymph nodes. Monoclonal antibodies have been produced against putative cell surface receptors and initial biochemical characterization has begun. There is much evidence that cell surface glycoconjugates can serve as specific recognition structures on normal cells and in addition, may play important roles in OSM. The role of these carbohydrates is discussed.The chick embryo as a model system is discussed as it offers several advantages for the study of metastasis in general and OSM in particular. A variety of human and murine tumors, including some freshly isolated, have been shown to grow and metastasize in these embryos. Furthermore, cell lines which have been selected for OSM in adults show similar patterns of metastasis in chick embryos indicating that this system may be an especially attractive one for the analysis of OSM.

A strong, preferential response of mice to polymorphic antigenic determinants of the chicken mhc, analyzed with mouse hybridoma (monoclonal) antibodies.

CBA/J mice were immunized with B2/B2 chicken RBCs (theB locus is the major histocompatibility complex (MHC) of the chicken). Spleen cells from these mice gave a much higher plaque-forming cell response when tested with RBCs bearing B2 alloantigens, than with RBCs bearing any other MHC alloantigens. Similarly, immunization with chicken RBCs of other genotypes also produced responses that were highest when tested on the genotype used for immunization. Spleen cells from mice immunized 3 days previously with B2/B2 RBCs were fused with mouse myeloma cells and hybrid clones which secreted anti-B2 antibodies were selected. These monoclonal antibodies could be divided into three groups: (1) those which react with all genotypes of RBCs (panreactive); (2) those which react with only certain genotypes of RBCs and detect “public” MHC antigens; and (3) those which react with only B2/B2 RBCs and detect “private” MHC antigens. Monoclonal antibodies which detect a private B2 alloantigen were shown to be excellent typing reagents, as all birds of an outbred population which possessed the B2 allele were easily detected using a simple one-step direct agglutination assay. No “false positives” were seen. The high and preferential response of the mouse to chicken MHC alloantigens suggested that mice might possess preexisting immunity to these antigens. In agreement with this hypothesis, normal mouse serum was found to have high titres of “natural” antibody against chicken MHC antigens.

Role of the Major Histocompatibility Complex in Resistance to Marek’s Disease: Restriction of the Growth of JMV-MD Tumor Cells in Genetically Resistant Birds

B21 is associated with resistance to Mareks disease (MD). Forty populations of chickens from all over the world were examined for the presence of the B21 allele. B21 was found in twelve of these populations and its presence was confirmed by GVH testing in all ten populations which were tested. The populations in which B21 was detected represent the extreme production types of the species and include the progenitor of the species, the Red Jungle Fowl. Our studies suggest that B21 may have strong survival value for the species. An allogeneic transplantable lymphoma of MD, the JMV tumor cell line, grows more slowly in MD resistant (B21/B21) chicks than in MD susceptible (B2/B2) chicks. This is the first direct evidence that genetic resistance to MD may involve an active (immunological?) restriction of tumor cell growth. JMV cells were further characterized as a transplant of B1 carrying lymphoblastoid cells, an allele which may be associated with susceptibility to MD.

A T-cell antigen system of chickens: Ly-4 and Marek's disease

A search was made for lymphocyte antigens associated with resistance or susceptibility to the T-cell lymphoma induced by the herpes virus of Mareks disease (MD), the experimental model for Burkitts lymphoma of humans. Antisera were produced by reciprocal immunization with whole blood between an MD-resistant and susceptible line of chickens compatible at the major histocompatibility complex (MHC), and were tested against lymphocytes of both lines. The lymphocytes were not agglutinated, immobilized, or lysed, but their ability to evoke graft-versus-host (GVH) splenomegaly was reduced. This inhibitory activity was line-specific, and these sera had a maximum limiting effect on GVH splenomegaly at a dilution of 1/50 and a minimum at 1/800 dilution. A test based on the differential limitation of GVH splenomegaly by a pair of alloantisera was used to identify the antigens in F1 and F2 generations. The segregation results established a locus,Ly-4, with two codominant alleles,Ly- 4a andLy-4b.Ly-4 is distinct from theA, B, orC blood group loci and from theBu-1 locus determining B-cell antigens, but may be linked to theTh-1 locus determining T-cell antigens (recombination frequency of 32 percent). Tentative evidence was obtained from comparisons of homozygous F2 and F3 progeny for association of theLy-4 allele characteristic of the susceptible line with increased incidence of MD.

A monoclonal antibody against chicken MHC class I (B-F) antigens

Monoclonal antibodies against major histocompatibility complex (MHC) antigens have been very useful in characterizing MHC gene products in several mammalian species (M611er 1978). The chicken MHC resembles mammalian MHCs in coding for typical class I and class II products (B-F and B-L antigens, respectively) and also controls synthesis of a third class of MHC products, the B-G antigens, which are not homologous to known mammalian MHC proteins (Pink et al. 1977). Monoclonal antibodies to B-G and B-L antigens have been prepared (Longenecker et al. 1979, Longenecker and Mosmann 1981, Ewert et al. 1984), but with the exception of the recent work of Crone and co-workers (1985), no monoclonal anti-B-F reagents have been described. Here we report on a monoclonal antibody, 37C.18, which reacts with a polymorphic determinant on chicken B-F antigens. We have also used the antibody to study the distribution of B-F antigens in different lymphoid organs. Monoclonal antibodies were produced as previously described (Longenecker et al. 1979) from spleens of mice given two i. v. injections (17 and 3 days before fusion) of 2 x 107 viable AL2 tumor cells (Shearman and Longenecker 1980). Culture supernatants were screened by indirect hemagglutination (Longenecker et al. 1979) and indirect immunofluorescence on frozen sections of bursa and thymus (Pink and Rijnbeek 1983). Antibody 37C.18 reacted positively in both assays with cells from birds carrying the B21 MHC haplotype. It was typed as IgG1 by Ouchterlony analysis, and its reactivity with cells from other chicken strains was examined by indirect immunofluorescence on suspensions of viable peripheral blood lymphocytes, purified by slow-speed centrifugation (Pink and Rijnbeek 1983). 37C.18 reacted in this assay with > 99% of lymphocytes from birds carrying haplotypes B4, BI2, B13, and B21 ; it did not react with cells from birds of MHC type B14, B15, or

Homing Receptors as Functional Markers for Classification, Prognosis, and Therapy of Leukemias and Lymphomas

Conclusions In the present minireview we have discussed several types of model systems which have given valuable clues to the ways in which leukemia and lymphoma cells mediate organ specific metastasis. Most patent is that these types of malignancies probably utilize normal receptor-mediated mechanisms for organ specific recognition. The animal models which have been most extensively studied indicate that the following three recognition mechanisms can be employed by metastatic lymphoma variants: (1) Normal organ cells have receptors for determinants on metastatic variants 17 (2) metastatic variants have receptors for determinants expressed on endothelial cells 2 , 19 ; and (3) an undefined interaction occurs between normal organ cells and metastatic variants 16 . A great deal of evidence suggests that organ specific metastasis is at least partially mediated through carbohydrate determinants which are recognized by receptors. It is possible that these receptors may be part of a larger family of recognition molecules which mediate normal cell-cell interactions. Despite this, a role for a “microenvironmental” mechanism cannot be ruled out and, indeed, is not mutually exclusive of the receptor-mediated adherence mechanism. In fact, in our bone marrow model our preliminary results suggest the existence of growth factors which are secreted by adherent bone marrow cells and which strongly stimulate the growth of the leukemia cell lines. Nevertheless, the avid binding of leukemia cells to the adherent cells of the bone marrow appears to be an important mechanism whereby leukemia cells are “held” in the bone marrow. The continued development of MAbs which recognize organ specific receptors on tumor cells, the isolation of these receptors, and their molecular biology should reveal whether a multigene family for organ specific recognition exists. These receptors will serve as functional markers in contrast to conventional markers such as T- and B-cell determinants. While it is true that T- and B-cell markers may indicate the probable cell lineage of the transformant, they do not, for example, explain why ALL grows in the bone marrow and not in the spleen. We suggest that the identification of “homing” receptors using MAbs will, for the first time, enable us to gauge the status of a malignancy, accurately determine the prognosis, and even allow us to choose the appropriate MAb to be used for the therapy of individual leukemias.

Histocompatibility typing by cellular radioimmunoassay

A quantitative cellular radioimmunoassay (CRIA) for histocompatibility typing is described. Chicken red blood cells (RBC) were incubated in microtiter plates with specific anti-MHC (B) alloantisera and the alloantibody bound measured indirectly by a second binding step with125I-labeled rabbit anti-chicken IgG. The assay is objective, highly consistent, and three to four orders of magnitude more sensitive than conventional hemagglutination assays. The new CRIA was used to detect minor subpopulations of cells in artificial cell mixtures; as few as 1% of relevant cells were easily detected. Erythrocyte chimerism was induced following the injection of B2/B2 chicken embryos with B15/B21 embryonic stem cells. Five weeks after hatching, erythrocyte chimerism was precisely quantitated by comparing the reaction of RBC from the putative chimeras with artificial cell mixtures using specific anti-B15/B21 alloantisera. The percent varied from 13–40% in 13 chimeric animals. The new CRIA was also used for the sensitive detection of tumor-specific antigens on a T-cell lymphoma. An unexpected finding was that anti-B15 alloantibody bound almost as well to B15/B21 heterozygous RBC as to B15/B15 homozygous cells, suggesting that either the concentration or the steric arrangement of B15 alloantigen at the erythrocyte surface may not conform to conventional expectations.