Ig lambda-producing B cells do not show feedback inhibition of gene rearrangement

In order to study the regulation of expression of Ig lambda genes we have analyzed lambda-producing hybridomas derived from transgenic mice which harbor a functionally rearranged kappa transgene. We also analyzed lambda-producing hybridomas from nontransgenic mice. Surprisingly, all but one of the transgenic lambda-hybridomas co-produce kappa L chains. Also, in contrast to transgenic kappa-hybridomas, most lambda-hybridomas have rearranged endogenous kappa genes despite the presence of transgenic kappa-chains and endogenous H chains. Analysis of spleen cells and hybridomas from nontransgenic mice shows that about 20% of lambda-producing B cells in the spleen co-produce kappa, and a similar proportion of lambda-hybridomas from normal spleens produce both kappa- and lambda-chains. The data argue strongly against the strictly sequential expression of kappa and lambda genes. We present a new model for the regulation of kappa and lambda gene expression, whose key feature is the distinction between a kappa cell lineage in which Ig gene rearrangement is susceptible to feedback by a complete antibody molecule at the pre-B cell stage, and a kappa lambda B cell lineage which does not show feedback inhibition during B cell development.     

Restricted Ig variable region gene expression among Ly-1+ B cell lymphomas

The majority of the characterized Ly-1+ B cell lymphomas of B10.H-2aH-4bp/Wts origin (the CH series) bear surface Ig related by Ag specificity or idiotype or both. To determine the genetic basis for these structural similarities, we have sequenced the VH and VL region genes expressed by 10 CH lymphomas, and have compared their VH and V kappa gene rearrangements by Southern blot analysis to one another and to those of four other CH lymphomas. Sequence analysis identified only five different VH, and seven different VL genes, and indicated that these V genes are essentially unmutated. CH lymphomas which express the identical VH gene share at least one idiotope. Thus, the basis for shared idiotype and specificity is due in most cases to the use of the same V gene. This restriction in V gene expression is not due to the preferential use of V genes of any particular VH family or VL group, as the expressed V genes belong to four different VH families and four V kappa groups, and include V lambda 1 and V lambda 2. We hypothesize that Ag selection accounts for the restriction in V gene usage among CH lymphomas.     

Ly-1 inducer and Ly-1,2 acceptor T cells in the feedback suppression circuit bear an I-J-subregion controlled determinant

An I-J-subregion controlled determinant is expressed on Ly-1 inducer and Ly-1,2 acceptor T cells in the feedback suppression circuit. Ly-1 T cells absorb the I-J antibody reactive with the Ly-1,2 acceptor T cell, suggesting that both inducer and acceptor T cells have the same I-J determinant. Since less than 10 percent of Ly-1 or Ly-1,2 T cells are killed by anti-I-J plus complement treatment, the I-J determinant demarcates functionally distinct subsets of both the Ly-1 and Ly-1,2 T-cell sets. This I-J determinant is not expressed on a detectable number of Ly-1 helper T cells which induce B lymphocytes to produce anti-sheep red cell antibody in tissue culture.     

Ly-1 inducer and Ly-1,2 acceptor t cells in the feedback suppression circuit bear an I-J-subregion controlled determinant

An I-J-subregion controlled determinant is expressed on Ly-1 inducer and Ly-1,2 acceptor T cells in the feedback suppression circuit. Ly-1 T cells absorb the I-J antibody reactive with the Ly-1,2 acceptor T cell, suggesting that both inducer and acceptor T cells have the same 1-J determinant. Since less than 10 percent of Ly-1 or Ly-1,2 T cells are killed by anti-I-J plus complement treatment, the I-J determinant demarcates functionally distinct subsets of both the Ly-1 and Ly-1,2 T-cell sets. This I-J determinant is not expressed on a detectable number of Ly-1 helper T cells which induce B lymphocytes to produce anti-sheep red cell antibody in tissue culture.Abbreviations used in this paper NMS normal mouse serum - BSS balanced salt solution - PFC plaque forming cells - Ig immunoglobulin - SRBC sheep red blood cells     

Immature B cells can pass through a VHDJH/germ line state in the Ig H chain gene rearrangements

SPL2-1-2, an Ig- murine immature B cell line, was established by the infection of immature B cells with tsOS-59, a temperature-sensitive mutant of Abelson murine leukemia virus. Southern blot and DNA cloning and sequencing analysis showed that SPL2-1-2 had a nonproductive VHQ52.DSP2.JH3 and a germ line (G) allele (VHDJH-/G), and that D to JH joinings followed by VH to DJH joinings progressed on the G allele during culture. These results indicated the existence of novel pathway of IgH gene rearrangements: G/G----DJH/G----VHDJH/G----VHDJH/DJH----VHDJH /VHDJH. This also implied that whether DJH/G state progressed to DJH/DJH or VHDJH/G state was stochastically determined, but not strictly controlled in an ordered fashion, although DJH/G state progressed more preferentially to DJH/DJH state than to VHDJH/G state.     

Induction of phorbol ester responsiveness in conventional B cells after activation via surface Ig.

The signals required to induce S phase entry in murine splenic B cells were found to be altered by prolonged treatment with low doses of anti-Ig antibody. Whereas fresh splenic B cells are stimulated by the combination of a phorbol ester protein kinase C agonist plus a calcium ionophore, anti-Ig-treated splenic B cells were stimulated by phorbol ester alone, in the absence of a comitogen. The majority of these phorbol ester responsive B cells expressed CD5. The phorbol ester responses of anti-Ig-treated splenic B cells paralleled those previously reported for untreated peritoneal CD5+ B cells in a number of respects: responses were not idiosyncratic to phorbol esters but occurred with nonphorbol protein kinase C agonists; phorbol ester responses were enhanced by IL-4; and, phorbol ester responses occurred rapidly and were greater at 24 than at 48 h. However, the effect of agents that act to raise intracellular levels of cAMP distinguished between anti-Ig-treated splenic B cells and untreated peritoneal B cells in that the phorbol ester responses of the former were enhanced whereas the responses of the latter were inhibited. The present results add a functional dimension to the phenotypic similarity between splenic B cells treated with anti-Ig and resident peritoneal B cells that constitutively express CD5; however, some differences in behavior were noted.     

Mechanism of Staphylococcus aureus exotoxin A inhibition of Ig production by human B cells

Staphylococcus enterotoxins and toxic shock syndrome toxin 1 are members of a family of exoproteins that are produced by staphylococci and bind specifically to MHC class II molecules. Upon binding to MHC class II molecules, these exoproteins are potent stimulators of T cell proliferation via interaction with specific TCR V-beta segments of both CD4+ and CD8+ T cells. These exoproteins also directly stimulate monocytes to secrete IL-1 and TNF-alpha. Furthermore, these exoproteins have a profound inhibitory effect on Ig production by PBMC. We examined the effects of Staphylococcus enterotoxin A (SEA) on proliferation and Ig production of highly purified human B cells. Our results demonstrated that the binding of SEA to MHC class II molecules on B cells does not alter their ability to proliferate in response to Staphylococcus aureus Cowan strain I (SAC) or to produce Ig in response to SAC plus rIL-2. In contrast, the anti-DR mAb L243 inhibited both B cell proliferation and Ig production. Unable to determine a direct effect of SEA on B cell function, we investigated whether the capacity of SEA to inhibit SAC-induced Ig production by PBMC was T cell-dependent. Our results demonstrated that in the presence of T cells, under appropriate conditions, SEA can either function as a nominal Ag for stimulation of B cell proliferation and Ig production or induce T cell-mediated suppression of Ig production. SEA-induced Ig production required T cell help, which was dependent on pretreatment of the T cells with irradiation or mitomycin C; Ig production was not induced by SEA in the absence of T cells or in the presence of untreated T cells. Furthermore, SEA inhibited Ig production in SAC-stimulated cultures of autologous B cells and untreated T cells; pretreatment of the T cells with irradiation or mitomycin C abrogated SEA-induced inhibition of Ig production. Thus, T cell suppression of SAC-induced Ig production was dependent on T cell proliferation. Similar results were observed with both SEA and toxic shock syndrome toxin 1.     

Ly-1 B helper cells in autoimmune "viable motheaten" mice

Previous work has demonstrated that Ly-1 B cells from normal C57BL/6J mice help the response of B cell subsets to the 4-hydroxy-3-nitrophenylacetyl hapten (NP). This regulatory cell population, called BH, preferentially helps the expression of plaque-forming B cells which express a predominant set of serologically related determinants collectively known as the NPb idiotype family. The specificity of BH cell activity in the NP system is a reflection of NPb idiotype-specific BH cell surface receptors. Thus, BH cells recognize autologous (i.e., idiotype) antigens. Given these observations and previous associations of increased Ly-1 B cell frequency in autoimmune mice, it was hypothesized that autoreactive Ly-1 BH cells may be present in high frequencies and in an activated state in autoimmune mice. To test this hypothesis the immunologic activity of BH cells in autoimmune viable motheaten (mev/mev) mice was studied. It was determined that splenic BH cells are approximately 10 times more frequent in viable motheaten than normal mice. The fact that BH cells from viable motheaten mice are activated was suggested by the presence of NPb idiotype-specific BH replacing helper activity in sera or B cell supernatants from these autoimmune mice. The soluble helper activities constitutively produced in mev/mev splenic B cell cultures and detected in mev/mev serum were resolved into two moieties, an NPb idiotype-specific immunoglobulin and a nonimmunoglobulin lymphokine(s) fraction. Purified mev/mev B cell-derived B cell maturation factor could substitute for the lymphokine moiety in the NPb idiotype helper cell assay. These results suggest that at least two signals, anti-idiotype immunoglobulin and a late-acting B cell maturation factor, are required for BH-dependent helper activity. The relationships of these results to current concepts of B cell activation mechanisms and the possible association of Ly-1 BH cells with autoimmunity are discussed.     

In vivo effects of hyperdiploid Ly-1+ B cells of NZB origin

Cells with increased chromosome number and DNA content have been found in the spleens of old NZB mice. These hyperdiploid cells are of clonal origin and demonstrate discrete IgH chain gene rearrangements by Southern blot analysis. In this report, hyperdiploid cells were analyzed by three-color flow cytometric techniques and found to be Ly-1+ B cells which were dull for Ly-1 and bright for surface IgM. These cells, unlike typical diploid Ly-1+ B cells, were negative for B220/6B2 and surface IgD. Hyperdiploid Ly-1+ B cells were found to be the predominant splenic subpopulation in animals receiving a spleen cell transfer from donors which possessed hyperdiploid Ly-1+ B cells. (NZB x DBA/2)F1 recipients of NZB spleen cells demonstrated a 10- to 1000-fold increase in Ly-1+ B cells in the spleen but showed no increased levels of Ly-1+ B cells in the peritoneum. Nearly all the splenic Ly-1+ B cells were hyperdiploid with the phenotype of the NZB parent. Cytogenetic analysis revealed that all the hyperdiploid cells were NZB donor cells. These findings suggest that the increase in splenic Ly-1+ B cells in the F1 recipients was due to expansion of injected splenic hyperdiploid Ly-1+ B cells of NZB origin. All of the F1 recipients of NZB hyperdiploid Ly-1+ B cells demonstrated a significant decrease in endogenous B cells as well as decreased serum IgM and anti-ssDNA autoantibodies. These studies suggest that hyperdiploid Ly-1+ B cells are different from typical peritoneal Ly-1+ B cells both in the lymphoid organs to which they home and in their proliferative capacity. NZB hyperdiploid Ly-1+ B cells, which may arise as a natural consequence of hyperactive Ly-1+ B cells, may play an immunoregulatory role in the spleen.     

Membranes from both Th1 and Th2 T cell clones stimulate B cell proliferation and prepare B cells for lymphokine-induced differentiation to secrete Ig.

Plasma membranes from the mitogen-activated mouse Th2 cell clone D10.G4.1 have recently been shown to provide the cell contact-dependent signals necessary for the induction of small B cell proliferation. Together with the Th2-derived lymphokines IL-4 and IL-5, these membranes stimulate production of Ig isotypes identical to those produced when B cells were stimulated by intact Th2 cells. In contrast, Th1 clones are poor inducers of Ig production in vitro. This could be solely due to differences in the lymphokines released by Th1 and Th2 cells or to differences in the cell-cell contact signals delivered by activated Th1 and Th2 cells. We report that membranes from three different activated Th1 clones induced strong Ag-independent proliferation of small dense B cells. The level of B cell proliferation was enhanced approximately fourfold by the addition of lymphokine-containing supernatant from Con A-activated Th2 cells and was unaffected by any of the lymphokine-containing supernatants from Con A-activated Th1 clones. As with D10.G4.1 membranes, Th1 membranes alone induced B cell proliferation but not secretion of Ig. However, addition of supernatant from Con A-activated D10.G41 cells, but not any supernatants from Con A-activated Th1 cells, induced Ig secretion of all isotypes. These effects were shown to not simply result from increased B cell numbers after stimulation with Th2 lymphokines. Thus, Th1 cell clones seem to poorly induce antibody responses entirely because of their lymphokine repertoire and not because of differences or deficiencies in the ability of these cells to deliver cell contact-dependent signals to B cells.     

Surface Ig receptor-induced nuclear AP-1-dependent gene expression in B lymphocytes.

The relationship between signals generated via the sIgR complex of B lymphocytes and subsequent changes in gene expression is poorly understood at the molecular level. To illuminate mechanisms that may couple these events, we examined the expression and function of tetradecanoyl phorbol acetate-response element (TRE)-binding proteins (i.e., activator protein 1, (AP-1)) in the murine B lymphoma cell line BAL-17.7.1 (BAL-17), which models primary B lymphocyte responses in a number of respects. Cross-linking of sIgR led to substantial induction of nuclear AP-1, in BAL-17 B cells, that bound the TRE, as detected by electrophoretic mobility shift assay. The sIgR-induced TRE-binding activity consisted of both Jun and Fos proteins, on the basis of immunoreactivity of nucleoprotein complexes with specific antisera. In addition, immunoprecipitation with specific antisera showed that de novo synthesis of Jun-B and c-Jun proteins, accompanied by c-Fos, was stimulated after cross-linking of sIgR on BAL-17 B cells. Transient transfection of BAL-17 B cells with reporter gene constructs showed that B cell AP-1 failed to trans-activate the TRE-containing human collagenase gene promoter, for which activity is dependent upon functional expression of cellular c-Jun. In contrast, sIg-induced AP-1 trans-activated a HSV-tk promoter that contained three TRE; this pattern of gene expression is consistent with the presence of functional Jun-B-containing AP-1 in B lymphocytes. These results are the first to attribute a functional role to sIgR-mediated AP-1 in B lymphoid cells and suggest that AP-1 functions to couple the sIgR complex to changes in nuclear gene expression.     

Human mb-1 gene: complete cDNA sequence and its expression in B cells bearing membrane Ig of various isotypes.

The transmembrane protein, IgM-alpha, a product of mb-1 gene, has been shown to be specifically associated with membrane-bound IgM on the plasma membrane of B lymphocytes. Recent studies have suggested that IgM-alpha may play a role in transducing signals from the Ag receptors during the activation of B cells. A large amount of information has been obtained in the mouse system regarding IgM-alpha and other components of the newly conceived B cell Ag receptor complex. Here we report the cloning and the nucleotide sequencing of cDNA clones of human mb-1, covering the entire length of the mRNA. At the amino acid sequence level, human and murine mb-1 share a high homology in their transmembrane and intracytoplasmic segments, suggesting an important biologic function for these regions of mb-1. A major difference, mainly in the 3' untranslated part, exists between our cDNA sequence and the published partial human mb-1 cDNA sequence. It has also been observed that human mb-1 is expressed not only by B cell lines expressing membrane-bound Ig of mu and delta isotypes but also those expressing membrane-bound Ig of alpha and gamma isotypes.     

Expression of Ly-6 on activated T and B cells: Possible identity with Ala-1

The antigens Ala-1 and Ly-6 were first thought to be different on the grounds that Ala-1 was present only on activated T and B lymphocytes while Ly-6 was present only on post-thymic T lymphocytes. In this paper, it is shown that Ly-6 is expressed on activated B cells, including PFC and LPS blasts, and that after typing of several recombinant inbred lines,Ala-1 andLy-6 remain genetically inseparable. Based on available data, it is most likely that Ly-6 is in fact Ala-1, although further testing is required to confirm the absence of Ly-6 from nonactivated lymphocytes.Abbreviations used in this paper Con A concavalin A - LPS lipopolysaccharide - SRBC sheep red blood cells - PFC plaque-forming cells - RI recombinant inbred strains - B6 C57BL/6 mice     

Distribution of the alloantigen Ly-4.2 on murine B cells

The Ly-4.2 alloantigenic specificity has been described as a possible marker for B cells in the mouse, as, on testing by the lymphocytotoxicity method, there was a restricted distribution of this specificity on lymphocytes. Using the highly sensitive method of immunoelectron microscopy with a hybrid antibody, it was found that only 5.5% of cells in the thymus carried the Ly-4.2 specificity, compared with 55.7% in the spleen and 20.9% in lymph nodes. This distribution suggests that Ly-4.2 is present on B cells, and confirms other functional and population studies. However, by immunoelectron microscopy Ly-4.2 was also detected on a few macrophages in the thymus, spleen, and lymph nodes. As with other alloantigens, the location of Ly-4.2 was found in restricted areas of various sizes on the cells bearing this specificity.