Geoffrey Haughton - US grants

We will study the genetics of susceptibility to antigen-driven lymphomagenesis in B10.H-2[unreadable]a[unreadable]H-4[unreadable]b[unreadable] p/Wts (2[unreadable]a[unreadable]4[unreadable]b[unreadable]) mice. The objective is to determine whether a gene(s) associated with H-2[unreadable]a[unreadable] and/or H-4[unreadable]b[unreadable] determines susceptibility and/or whether an unlinked mutant gene is involved. The methods to be used are those of conventional Mendelian genetics employing segregation and linkage analysis. We will attempt to drive lymphomagenesis in 2[unreadable]a[unreadable]4[unreadable]b[unreadable] mice with antigens other than sheep erythrocytes (SRBC) to determine whether B-cell lymphomas bearing surface immunoglobulin specific for such antigens can be obtained and to delineate the range of preneoplastic B-cell clones occurring in 2[unreadable]a[unreadable]4[unreadable]b[unreadable] mice. The methods will employ hyperimmunization followed by syngeneic spleen cell transfer, as has proven successful with SRBC. We will continue to analyze the differentiation phenotype, the immunoglobulin gene arrangements, and the immunoglobulin idiotypes of the CH series of B-cell lymphomas in an attempt to understand the mechanism of antigen-driven lymphomagenesis and the possibility of a specific regulatory defect in 2[unreadable]a[unreadable]4[unreadable]b[unreadable] mice. The methods to be employed include PAGE of DNA restriction enzyme digests, computer analysis of flow cytometry data, and amino acid sequence analysis. We will utilize our growing collection of CH series B-cell lymphomas bearing surface immunoglobulin of known antigen reactivity to study the influence of mitogens, T-cell subsets and factors, and specific ligands on induced differentiation of B cells. The principle method to be used in these studies is that of Cunningham for enumeration of plaque-forming cells. (AG)

The major goal of this research is to generate a library of closely related, transplantable lymphomas in a single inbred strain of mice. Specifically, we aim to accomplish the following: (1) to induce a large number (more than 50) of primary lymphoid tumors in B10.H-2aH-4bp/Wts(2a4b) mice by procedures involving intense antigenic stimulation. Many of these tumors will be B-cell lymphomas (vide infra) with a minority of T-cell and macrophage tumors. Most of these tumors will be transplantable in 2a4b mice and most will convert readily to an ascites form; (2) from the above primaries, to establish a series of transplantable tumors such that 50 independently derived B-cell lymphomas are included. Cell suspensions from primary and early transplant generations of all tumors will be stored under liquid nitrogen; (3) to characterize, with respect to a battery of cell surface differentiation markers, the cells of each tumor which becomes established in transplantation; (4) to prepare specific xenogeneic anti-idiotype sera against the surface immunoglobulin of each independently derived B-cell lymphoma; (5) to determine the extent of crossreactivity between the idiotypes of the surface immunoglobulins of the 50 independently derived B-cell lymphomas; and (6) to catalog, with respect to surface differentiation markers, those B-cell, T-cell, and macrophage tumors which become established in transplantation and to make such tumors, together with histocompatible mice for transplantation, available for study by other interested investigators. At the end of the second year of this project, tumor induction is complete; over 700 primary lymphomas are stored under liquid nitrogen. About 35 B-cell and 50 T-cell tumors are characterized. Extensive idiotype sharing exists among the B-cell lymphomas. Antigen binding specificity of the IgM of 14 B lymphomas has been identified. One of the tumors, CH12, whose surface Ig reacts with sheep erythrocytes, has been used to study the mechanisms by which B cells may be induced to differentiate. These studies have led to definition of the surface IgM and 1-E molecules as true receptors which transduce differentiative signals across the cell membrane and formulation of a minimal model of MHC-restricted, antigen-specific signaling of B cells. This project has been renewed. (Ml)

The objectives of this proposal are to study the CH series B cell lymphomas which arose in B10.H-2aH-4bp/Wts mice and the normal Ly-1+ B cells from which they are derived. The CH lymphomas will be employed as models to study B cell differentiation. We will use tissue-culture adapted subclones of lymphomas, whose cells bear membrane IgM reactive with known antigens. We will concentrate on a group of tumors reactive with membrane phospholipids such as phosphatidyl choline, recognizable by the tumor IgM on sheep erythrocytes and bromelain treated mouse erythrocytes. We will study the influence of specific antigen, mitogens, lymphokines and MHC restricted T helper cells on the differentiation of the B cells. Differentiation will be detected as secretion of IgM, by localized hemolysis, or as a change in the immunoglobulin isotype produced. The latter will be detected by flow cytometry and by isotype specific ELISA of the supernatant from cultures grown to high density. We will sort and clone variant cells producing the changed isotypes and will compare the specificity and idiotype of the Ig produced with that of the IgM produced by the parent tumor. We will determine whether the same VH and VL genes are used for the parental IgM and the switched isotype. We will use restriction digest analysis of DNA, sandwich hybridization of RNA and nucleotide sequencing to elucidate the genetic mechanisms responsible for iusotype switching. We will use tissue culture, flow cytometry and ELISA to investigate the nature of signals which control isotype switching. We will also compare the specificity and idiotype of immunoglobulins produced by hybridomas derived from normal Ly- 1+B cells with those expressed by 27 previously characterized CH lymphomas. The objectives is to determine whether the CH lymphomas are derived from a random selection of the normal Ly- 1+B cells of syngeneic mice and to determine whether the Ig produced by normal Ly-1+B cells is as limited in specificity and idiotype as is that of the CH lymphomas. We will undertake exploratory experiments to test the importance of idiotype/antiidotype regulation in controlling the normal population of Ly-1+B cells.

DESCRIPTION (Adapted from applicants abstract): The goal of this project is to determine whether Ly-1 B cells differ fundamentally from other B cells in being incapable of high-rate somatic mutation of their assembled Ig V genes. Mutation will be sought in Ly-1 B cell lymphomas, in expanded populations of Ly-1 B cells in very old normal mice and in younger congenitally autoimmune mice. The project rests on four cardinal observations. First, Ly-1 B cells of normal young adult mice produce antibodies reactive with a range of self antigens; antibodies reactive with these same antigens are elevated in congenitally autoimmune mice. Second, the V gene assemblies expressed by Ly-1 B cells of normal young adult mice reveal the features of an antigen-driven primary response, but no evidence of the somatic mutation associated with a secondary response. Third, expanded clones of Ly-1 B cells develop in normal mice only in old age, but in autoimmune mice they appear in young adults. Fourth and finally, autoantibodies produced by mice with active autoimmune disease are products of a typical secondary response; they are produced by dominant clones of B cells whose assembled Ig V genes have been modified by multiple somatic mutations. The investigators will attempt to reconcile these potentially anomalous results. They will treat mice bearing lethal transplants of Ly-1 B cell lymphomas that express fully sequenced Ig genes (IgM, IgG or IgA) with subcurative doses of monoclonal anti-Id directed against "private" idiotopes and analyze the expressed Ig genes of late-arising recurrent tumors for evidence of somatic mutation. Dr. Haughton and coworkers will monitor the response to phosphatidyl choline (PtC) as an index of the immunobiology of Ly-1 B cells, using fluorescein-loaded synthetic liposomes and flow cytometry to detect the B cells and solid phase binding to detect the antibody. They will make hybridomas from clones of PtC binding Ly-1 B cells in the peritoneums of old normal and young autoimmune mice and sequence the expressed immunoglobulin gene assemblies, seeking evidence of somatic mutation. The investigators will explore the use of allotype-chimeric mice, derived from congenitally autoimmune (1pr) or immunodeficient (xid) strains to analyze the involvement of Ly-1 B cells in the generation of pathogenic autoantibody. Results will be useful in understanding the immunobiology of normal Ly-1 B cells, the possible involvement of Ly-1 B cells in autoimmune disease and in designing a strategy for specific immunotherapy of B cell malignancy.

In mice and humans, a minor population of B cells, B1 cells, is notable for its distinct array of cell surface antigens, its early appearance, its tissue distribution and its repertoire of antibody specificities. The antibodies produced are frequently auto reactive but there is no evidence that they are involved in autoimmune disease. B1 cells give rise to virtually all examples of human B cell CLL, B cell lymphomas in MDS patients and many murine B cell lymphomas. Two distinct hypotheses exist to explain the acquisition of different phenotypes. One states that the phenotypes are inherent in separate lineages of B cells (B1 and B2); they are determined in distinct precursors prior to the rearrangement of Ig genes. The phenotypes are fixed; B2 cells cannot be changed into B1 cells, nor vice versa. The other hypothesis states that commitment to a phenotype occurs after encounter with antigen; newly formed B cells, termed B0 cells, can be induced to differentiate along either of two pathways, termed B1 and B2, depending on the nature of the antigen. Multivalent antigen, leading to cross linking of surface Ig without involvement of T helper cells, induces the B1 phenotype, whereas cognate interaction with antigen and T helper cell induces the B2 phenotype. The majority of B cells are viewed as being virgin B0 cells that retain the capacity to differentiate along either pathway. This project is designed to resolve the issue by determining whether the B1 phenotype is programmed before or after expression of surface Ig. The immune response of mice to pyrrolidone (Py) will be characterized following immunization with the thymus independent (Ti) polymer, polyvinyl pyrrolidone (PVP36O) or the same hapten coupled to protein as a thymus dependent (TD) antigen. The phenotype of responding cells will be determined by FACS and ELISA spot analysis, and their derivation from fetal derived B cells or adult bone marrow by analysis of allotype chimeras. Antibodies produced in response to Ti and TD forms of Py will be characterized with respect to V gene usage, CDR3 assembly, isotype and the presence of somatic mutations, by analysis of hybridomas. A heavy chain VDJ assembly from a 1 light chain expressing hybridoma will be introduced into k chain deficient mice to yield a transgenic strain that produces large numbers of B cells bearing surface Ig reactive with Py. Immunizations will be done in vivo in transgenic mice and after B cell transfer to compatible recipients, as well as in vitro. Immunization with TD antigen will be supplemented with carrier specific T cells. Results from the project should demonstrate whether the strong neoplastic propensity of B1 cells is an inherent feature of a discrete lineage of cells or results from chronic antigenic stimulation of B0 cells with Ti antigens.

DESCRIPTION (Adapted from applicants abstract): The goal of this project is to determine whether Ly-1 B cells differ fundamentally from other B cells in being incapable of high-rate somatic mutation of their assembled Ig V genes. Mutation will be sought in Ly-1 B cell lymphomas, in expanded populations of Ly-1 B cells in very old normal mice and in younger congenitally autoimmune mice. The project rests on four cardinal observations. First, Ly-1 B cells of normal young adult mice produce antibodies reactive with a range of self antigens; antibodies reactive with these same antigens are elevated in congenitally autoimmune mice. Second, the V gene assemblies expressed by Ly-1 B cells of normal young adult mice reveal the features of an antigen-driven primary response, but no evidence of the somatic mutation associated with a secondary response. Third, expanded clones of Ly-1 B cells develop in normal mice only in old age, but in autoimmune mice they appear in young adults. Fourth and finally, autoantibodies produced by mice with active autoimmune disease are products of a typical secondary response; they are produced by dominant clones of B cells whose assembled Ig V genes have been modified by multiple somatic mutations. The investigators will attempt to reconcile these potentially anomalous results. They will treat mice bearing lethal transplants of Ly-1 B cell lymphomas that express fully sequenced Ig genes (IgM, IgG or IgA) with subcurative doses of monoclonal anti-Id directed against "private" idiotopes and analyze the expressed Ig genes of late-arising recurrent tumors for evidence of somatic mutation. Dr. Haughton and coworkers will monitor the response to phosphatidyl choline (PtC) as an index of the immunobiology of Ly-1 B cells, using fluorescein-loaded synthetic liposomes and flow cytometry to detect the B cells and solid phase binding to detect the antibody. They will make hybridomas from clones of PtC binding Ly-1 B cells in the peritoneums of old normal and young autoimmune mice and sequence the expressed immunoglobulin gene assemblies, seeking evidence of somatic mutation. The investigators will explore the use of allotype-chimeric mice, derived from congenitally autoimmune (1pr) or immunodeficient (xid) strains to analyze the involvement of Ly-1 B cells in the generation of pathogenic autoantibody. Results will be useful in understanding the immunobiology of normal Ly-1 B cells, the possible involvement of Ly-1 B cells in autoimmune disease and in designing a strategy for specific immunotherapy of B cell malignancy.