Molecular analysis of skewed Tcra-V gene use in T-cell receptor beta-chain transgenic mice.

The influence of beta-chain diversity on the expressed T-cell receptor (TCR) alpha-chain repertoire was investigated using transgenic mice which exclusively express a single rearranged TCR beta-chain gene. Analysis of these mice using alpha-chain-specific recombinant cDNA libraries showed that expression of the transgene-encoded beta chain results in significant skewing in Tcra-V gene segment usage vs nontransgenic mice. Skewing was most pronounced towards alpha chains using TCRA-V segments. Sequence analysis of Tcra-V8-containing genes from transgenic T cells revealed predominant use of a single Tcra-J segment (Tcra-J24), which was not detected in Tcra-V8 containing genes isolated from nontransgenic T cells. Further analysis revealed that co-expression of Tcra-V8 with Tcra-J24 in beta-transgenic mice is exhibited almost exclusively by CD4+ T cells, and is associated with a limited number of closely related N-regions. Analysis of transgenic CD8+ T cells demonstrated predominant co-expression of Tcra-V8 with another Tcra-J (Tcra-J30), together with a different, limited N-region sequence. We conclude that the composition of expressed beta chains can profoundly influence the selection of companion alpha chains expressed in the periphery, and that alpha-chain N and J regions play a crucial role in discriminating between class I vs class II major histocompatibility complex (MHC)-restricted recognition. Further, these results are in agreement with recent data concerning the crystal structure of the TCR, and most consistent with a model for TCR structure in which the complementarity determining region (CDR)3alpha domain participates in direct contact with the MHC.

Cellular competition in the development of ocular tissues in allophenic mice.

C57BL/6 mice exhibit impaired lens development as evidenced by competition studies with allophenic (chimeric) mice. Strain-specific differences in electrophoretic forms of glucose phosphate isomerase were exploited to determine the strain composition of various ocular and nonocular tissues in allophenic mice constructed from C57BL/6 and DBA/2 strains. As expected, there was variation from one animal to the next in overall mosaic composition, as well as variation among most ocular and nonocular tissues within individual animals. In contrast, when individual lenses from the same animals were assayed there was a marked and consistent deficiency of the C57BL/6 component. Control experiments indicated that these results are not the consequence of peculiarities of marker enzyme expression in the lens. Since lens formation occurs early in embryogenesis any anomaly at this stage could influence the normal induction of later ocular structures. We believe that the impaired lens-forming capacity of C57BL/6 mice may be the underlying cause of the 5-10% frequency of overt ocular defects in this strain.

Erythroid cell fusion in the early phase of Friend virus leukemogenesis.

Infection of susceptible strain mice with the oncogenic Friend erythroleukemia virus initially results in fulminant erythroid hyperplasia. Several weeks later a frank erythroid leukemia develops. At the earliest stages of Friend disease there is extensive cell fusion involving erythroid cells but not platelets and granulocytes. Fusion was detected in experiments with allophenic (chimeric) mice whose component strains express electrophoretically distinct forms of the dimeric enzyme glucose phosphate isomerase (GPI). Infection of such animals with the polycythemic strain of Friend virus resulted in the rapid development of Friend disease. Concomitant with the appearance of early disease symptoms was the appearance in the red cells of the heterodimeric form of GPI, an unequivocal consequence of cell fusion. Platelet and granulocyte samples from the same infected animals failed to exhibit the hybrid GPI form. Furthermore, no hybrid dimer was evident in red cells from chimeric mice in which blood formation had been stimulated by phenylhydrazine treatment. These observations suggest that the occurrence of cell fusion early after infection by Friend virus is a significant aspect in the rapid development of neoplastic disease.

Cross-reactive idiotypes and common antigen binding specificities expressed by a series of murine B-cell lymphomas: etiological implications.

A series of 27 B-cell lymphomas (designated the CH series), induced in B10.H-2aH-4b p/Wts mice by intense adoptive immunization with sheep erythrocytes, was found to represent a subset of the total B-cell repertoire. This subset was characterized by expression of a limited number of Ig heavy chain variable regions, as evidenced by the presence of cross-reactive idiotypes and common antigen binding specificities. Twenty-one of the 27 CH lymphomas studied were classified into five groups, defined by a particular cross-reactive idiotype; four of these groups were linked in a single network. Seven of 16 idiotypes defined by absorption analysis were present on lymphomas bearing either kappa or lambda light chains and so were localized to the heavy chain variable region. The surface Ig on 14 CH lymphomas was found to be specific for epitopes on certain erythrocytes (bromelain-treated autologous erythrocytes, sheep, and chicken erythrocytes) or E. coli. We propose that the CH lymphomas represent the malignant counterparts of a subset of idiotypically related, normal B cells in B10.H-2aH-4b p/Wts mice. Perturbation of this idiotype network, by hyperimmunization with an antigen for which some of the members are specific (sheep erythrocytes), increases the risk for neoplasia. Possible mechanisms for this are discussed.

Two separate functions of class II (Ia) molecules: T-cell stimulation and B-cell excitation.

We have evaluated the role of major histocompatibility complex-encoded class II (Ia) molecules as transmembrane signaling receptors in the T helper cell-dependent activation of B lymphocytes. For these studies, we utilized the murine B-cell lymphoma CH12, which expresses both I-A and I-E class II molecules. In addition, CH12 cells carry IgM of known antigen specificity and require both specific antigen and Ia-restricted T-cell help for the induction of antibody secretion. In this respect, they resemble normal resting B cells. We have studied the ability of antigen-specific or alloreactive T helper cells reactive with either the I-A or the I-E molecules on CH12 to be activated and their ability to stimulate antibody production by CH12. The results show that, although CH12 cells present antigen to T helper cells that interact with either the I-A or the I-E molecules, CH12 cells are stimulated to secrete antibody only by T helper cells reactive with their I-E molecules. Our data demonstrate that class II molecules are transducers of signals for B-cell excitation in addition to serving a restricting function for helper T-cell stimulation. Moreover, the data demonstrate that these two functions, T-cell stimulation and B-cell excitation, are discrete and need not be expressed by the same Ia molecule.

Three classes of signalling molecules on B-cell membranes.

The question of whether surface immunoglobulin and Ia molecules have a signalling function in helper T cell-dependent activation of B cells has been evaluated. Two sources of B cells have been used, one a purified population of hapten-binding B cells, the other a B-cell lymphoma, CH12, with known antigen specificity. Evidence is presented that both immunoglobulin and Ia molecules are receptors actively involved in the initial activation of resting B cells. Nevertheless, the requirements for ligand binding to either receptor can be bypassed under appropriate conditions, and the implications of this result for the function of these molecules is discussed. With respect to B-cell Ia, the authors present data that demonstrate two distinct functions of this molecule, one as a restricting element for T-cell activation, the second as a signalling receptor for B-cell excitation. On the CH12 surface, the I-A molecule fulfills the former function, but T-cell interactions with I-A fail to result in B-cell stimulation, suggesting that B-cell Ia may limit helper T cell-B cell interactions. We suggest that the binding of antigen surface immunoglobulin and binding of helper T-cell receptors to the appropriate Ia molecule(s) results in the activation of genes that encode for a third class of membrane B-cell receptors, those that bind B-cell stimulating factors.

Role of cell surface immunoglobulin in B-lymphocyte activation.

The role of cell surface immunoglobulin in helper T-cell-dependent B-cell activation was analyzed using a B-cell lymphoma, CH12, with known antigen specificity and activation properties similar to those of a resting B cell. Two sources of helper T cells were used, both selected such that they interact with H-2-encoded determinants on CH12 in the absence of the specific B-cell antigen, sheep erythrocytes. By this dissociation of the specificity of the T cells from that of the B cells, the requirement for antigen in the induction of CH12 to antibody secretion could be studied. The results show that both helper T-cell-B-cell interactions and surface immunoglobulin-antigen binding are involved in inducing B-cell differentiation, thus establishing a signalling function for the antigen receptor on B lymphocytes. Our data also show that the requirement for surface immunoglobulin-ligand interactions in B-cell activation can, under certain conditions, be circumvented, notably when high (nonphysiologic) multiplicities of T-cell help are used.

Induced differentiation of a B cell lymphoma with known antigen specificity.

We have previously described a murine B-cell lymphoma, CH12, the cells of which bear surface IgM reactive with sheep erythrocytes (SRbc) and which could differentiate to secrete hemolytic antibody. The question addressed in this paper was whether differentiation of CH12 cells could be influenced by interaction with regulatory T cells and antigen. If so, we wanted to know whether the conditions required differed from those known to govern similar interactions with normal B cells. We had two reasons for wanting to answer these questions. First, we wondered whether CH12 could be used as a clonal population of indicator cells to study the regulation of B cell differentiation and, second, we wanted to know the extent to which these neoplastic cells were still responsive to normal regulatory signals. The first addresses a major difficulty which must be faced in studies of normal B cell differentiation: to what extent is the interpretation limited by heterogeneity of the B cells used? The second relates to the nature of neoplasia and the possibility that neoplastic cells might be rendered harmless by inducing terminal differentiation. CH12 is one of a series of transplantable B cell lymphomas which arose in B10.H-2aH-4b p/Wts (2a4b) mice, following intense immunization with SRbc. It is a monoclonal tumor, all the cells of which bear membrane IgM(kappa) of a single idiotype, reactive with sheep and chicken Rbc and with bromelain-treated autologous mouse Rbc. The cells express KkAkEk and Dd antigens appropriate to the H-2a haplotype. During the latter stages of growth in vivo or in vitro, a small proportion (less than 3%) of the cells differentiate to secrete hemolytic antibody as measured by the Cunningham assay for plaque forming cells (PFC). We cultured CH12 cells for 3 or 4 days, together with antigen and spleen cells from primed animals, and assayed for PFC induction. Differentiation was induced by spleen cells from SRbc primed 2a4b mice in the presence of SRbc or ChRbc but not rabbit or human erythrocytes. Activity was depleted by treatment of the spleen cells with anti-Thy-1 or anti-Lyt-1 but not anti-Lyt-2 plus complement. Helper cells could also be induced by priming 2a4b mice with ChRbc but not rabbit or human Rbc. Neither of these last two would induce differentiation of CH12, even when both homologous antigen and SRbc were present in the cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigen-induced lymphomagenesis: identification of a murine B cell lymphoma with known antigen specificity.

CH12, a murine B cell lymphoma derived in B10 H-2aH-4bp/Wts mice after transfer of SRBC hyperimmunized spleen cells into an adult-thymectomized, sublethally irradiated, syngeneic recipient, is demonstrated to bear surface IgM specific for a determinant found on SRBC and ChRBC. The Ig specificity has been demonstrated by rosetting assays and complement-dependent hemolysis. The removal of CH12 surface IgM by capping with anti-mu or with anti-CH12 idiotype, but not with anti-gamma or with irrelevant anti-idiotype, eliminated the formation of rosettes between CH12 and SRBC or ChRBC. The absorption of CH12 Ig produced in vitro, with either SRBC or ChRBC but not with HRBC, removed all hemolysin activity against SRBC, demonstrating that only one CH12 product was responsible for the reactivity with both SRBC and ChRBC. CH12 has a surface phenotype of a relatively mature B cell expressing surface Ig (IgM-mu,kappa) and la antigens, but lacking Thy-1 or detectable Fc or C3 receptors. CH12 also expresses the antigen Lyt-1. Growth of CH12 in vivo or in vitro results in the generation of up to 3% direct PFC and serum hemolysin, which shows that CH12 is not irretrievably "frozen". The generation of PFC and serum hemolysin is associated with increased population density, and the rate of PFC and serum hemolysin accumulation cannot be explained by simple cell division. A continuously secreting hybridoma derived from CH12 was used to purify the CH12 IgM to facilitate studies of protein sequence and idiotype.