Jeffrey A. Frelinger

Antigenic relationships of murine coronaviruses: Analysis using monoclonal antibodies to JHM (MHV-4) virus

n Abstractn n Monoclonal antibodies were produced to JHMV-DL, a neurotropic member of the mouse hepatitis virus (MHV) or murine coronavirus group. Of 23 antibodies isolated, 10 were specific for the major envelope glycoprotein, gp180/90, 10 for the nucleocapsid protein, pp60, and 3 for the minor envelope glycoprotein, gp25. Eleven different MHV isolates were used in antibody binding assays to study antigenic relationships among the viruses. Each MHV isolate tested had a unique pattern of antibody binding, indicating that each is a distinct strain. Conservation of JHMV-DL antigenic determinants varied among the three proteins, with pp60 showing intermediate conservation, gp180/90 little conservation, and gp25 marked conservation in the different MHV strains. Monoclonal antibodies to pp60 proved most useful in delineating antigenic relationships among MHV strains. These antigenic groups correlated with pathogenic types, indicating that pp60 may be one of the gene products which mediates the distinct disease patterns manifested by different murine coronaviruses.n n

Resistance to fatal central nervous system disease by mouse hepatitis virus, strain JHM - I. Genetic analysis

Resistance of SJL/J mice to intracranial inoculation with the JHM strain of mouse hepatitis, a coronavirus, is dependent upon the age of the animals at inoculation. Animals 12 weeks of age or older are resistant, whereas those 6 weeks or younger are uniformly susceptible to viral infection. Spleen cells or thioglycolate elicited peritoneal exudate cells can transfer resistance from 12-week-old to 6-week-old recipients. Removal of the adherent cells from either spleen or peritoneal cells ablated protection. Adherent cells from 12-week-old mice were protective even after depletion of Ia- and Thy-1-bearing cells. Antiviral antibody, thioglycolate injection into 6-week-old animals, and nylon wool-purified T cells were ineffective in mediating resistance. Adherent cells transferred 4 days before virus challenge, but not after challenge, were protective. Thus, there is an age-related change in SJL mice that protects from acute central nervous system disease, which may be due to maturation of a specialized adherent cell population.

A new alloantigen, ly-8, recognized by c3h anti-akr serum.

A new membrane alloantigen, designated Ly-8.2, is defined by a C3H anti-AKR serum. The locus,Ly-8, which controls this determinant is not linked toThy-1, Ly-4, Ly-6, H-2, albino (c), or brown (b). Ly-8.2 has a unique strain distribution, and appears to be present on both T and B lymphocytes.

Brain Ia antigens have a bone marrow origin.

Our results, using radiation-induced bone marrow chimeras, demonstrate that the la antigen found in the brains of such animals is produced by cells having precursors in the bone marrow. These cells are not immediately blood borne since no IgM is detected in these brains. This rules out the obvious possibility of B-lymphocyte contamination as the source of la in the brain cell preparations. It thus appears that the central nervous system, like many other nonlymphoid organs, has a source of Ia-positive cells that are derived from bone marrow precursors.

Monoclonal antibodies reactive with H-2 determinants.

As part of our ongoing intensive study of the mouse H 2 major histocompatibility complex (MHC), it has become necessary to produce a large panel of monoclonal antibodies reactive with a single haplotype. We have chosen to begin an intensive study of the H 2 p haplotype because H 2 p represents an unusual H-2 type carried by relatively few inbred strains. The prototype strain is P/J and its congenic partner, B 10.P. H 2 p is also carried by the NB strain and its congenic partner C3H.NB. Since B 10.P carries a complete H 2 haplotype, shown by its expression of both I-A and I-E region determinants, and was of rather distinct lineage from other common inbred strains, we thought it would be useful as an immunogen for the production of monoclonal antibodies. In addition, two recombinant haplotypes are available that are carried by strains B 10.F(13R) and B10.F(14R). The recombinants separate K p in 13R from D p in 14R. This report describes 31 monoclonal antibodies that react with H-2P-encoded gene products as well as nine others produced against other H-2 types. All hybridomas were obtained by fusion of immunized spleen cells with a horseserum-adapted derivative of Sp2/0-Agl4 designated M5. This variant was supplied by Dr. J. Davie of Washington University, St. Louis, Missouri. Spleen cell donors were immunized prior to fusion with a mixture of spleen, lymph-node and thymus cells, at an approximate ratio of one donor per ten recipients. A minimum of 3 weeks following primary immunization the animals were boosted by intravenous infusion of spleen cells. Cells were fused 3 days later as previously described, using 34% polyethylene glycol (Aldrich, 1500), and then plated in 24-well Linbro plates at approximately 2 x 105/ml (Harmon et al. 1982). Monoclonal antibodies were initially screened in a onestep cytotoxic assay using Terasaki plates as previously described (Frelinger et al. 1974). Mesenteric lymph-node cells were used as targets. Supernatants were initially screened against the immunizing cell only. Positive cells

H-2 effects on cell-cell interactions in the response to single non-h-2 alloantigens. Iii. Evidence for a second ir-gene system mapping in the h-2k and h-2d regions.

Immune response (Ir) genes mapping in theI region of the mouseH-2 complex appear to regulate specifically the presentation of a number of antigens by macrophages to proliferating T cells. We have investigated the possibility that similarIr genes mapping in theH-2K andH-2D regions specifically regulate the presentation of target antigens to cytotoxic effector T cells. We report that the susceptibility of targets expressing specific non-H-2 H alloantigens to lysis by H-2-compatible, H-antigen-specific cytotoxic effector T cells is controlled by polymorphicH-2K/D genes. This control of susceptibility to lysis is accomplished through what we have defined operationally as antigen-specific regulation of non-H-2 H antigen immunogenicity. High immunogenicity of the H-4.2 alloantigen is determined by a gene mapping in theH-2K region ofH-2b. However, high immunogenicity of H-7.1 is determined by a gene mapping in theH-2D region ofH-2b. High immunogenicity of the H-3.1 alloantigen is determined by genes mapping in both theH-2K andH-2D regions ofH-2b. Therefore, genes mapping in theH-2K andH-2D regions serve a function in presenting antigen to cytotoxic effector T cells. This function is analogous to that played byI-regionIr genes expressed in macrophages which present antigen to proliferating T cells. We present arguments for classification of theseH-2K/D genes as a second system ofIr genes and discuss the implications of twoH-2-linkedIr-gene systems, their possible functions, and their evolution.

Epidermal Ia molecules from theI-A andI-EC subregions of the mouseH-2 complex

Immune response region-associated (Ia) antigens encoded by genes in theI-A orI-EC subregions have been detected on murine epidermal cells by indirect immunoprecipitation, using antisera produced against murine lymphoid cells. The Ia antigens encoded by genes in these subregions are composed of two polypeptides with approximate molecular weights of 33,000 and 28,000. The Ia antigens are not derived from contaminating B- or T-cell populations. The Ia molecules from lymphocytes and epidermal cells appear to have identical subunit structures and very similar, if not identical, molecular weights. The possible biological role of the Ia antigens on epidermal cells is discussed.

A cautionary note regarding Ia and H-2 typing of murine lymphoid tumors

1 Genetics Curriculum, Department of Bacteriology andlmmunology, University of North Carolina, Chapel Hill, North Carolina 27514 2 Basel lnstituteJor Immunology, 456 Grenzacherstrasse, Basel, Switzerland 3 Fred Hutchinson Cancer Research Center, Seattle, Washington 4 Department of Genetics, Washington University, St. Louis, Missouri 5 Department of Microbiology, University of Southern California Medical School, Los Angeles, California 90033

Ia antigens on non-lymphoid tissues Their origins and functions

Ia molecules are encoded in the 1 region of the mouse major histocompatibility complex (MIX) and expressed as surface antigens on certain immunologically active cells (reviewed in Ref. 1). B cells, subsets of T helper and T suppressor cells and accessory, antigen-presenting cells are Ia-positive (Ia+) while T killer cells and natural killer cells are not. This article discusses the nature and function of Ia+ cells found in non-lymphoid tissues, some of which are immunologically involved while others are not obviously so.

Partial amino acid sequences of mouse transplantation antigens.

The partial N-terminal amino acid sequences of the K and D gene products from theH-2q andH-2s haplotypes are presented. These data in conjunction with data already published demonstrate striking homology relationships among the transplantation antigens of mouse and other species. Moreover, these new data support the presence of certain sequence patterns noted in earlier sequence studies (e. g. no Kness or Dness, species-associated residues, and complex allotypes). These patterns place interesting constraints on the genetic organization and evolutionary history of the genes encoding the transplantation antigens which are discussed in this report.