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A rat monoclonal antibody (NIM-R3) was found to be reactive with activated mouse B cells but neither activated T cells nor small resting lymphocytes, T or B. The antibody stains antibody-forming cell precursors within 48 hr of primary or secondary immunization in vivo, but not at longer times (>14 days) immunization. When added to cultures of spleen cells responding in vitro to dinitrophenylated bovine, IgG, the number of antibody-forming cells was increased. NIM-R3 also maintained the proliferation of purified B blasts in the absence of lipopolysaccharide, but did not activate small resting B cells in the presence of anti-Ig. NIM-R3 could replace B-cell growth factor (SCGF II) in cultures of the murine B-cell lymphoma BCL1 inducing proliferation but not differentiation. Finally, competition was demonstrated for binding sites on BCL1 cells between BCGF II and NIM-R3, but not between NIM-R.3 and T-cell replacing factor (B15-TRF). We suggest that NIM-R3 may represent a novel specificity and may be directed against the cell surface receptor for BCGF II, and in turn this receptor may be independent of the B 1 5-TRE and BSF1 receptors. [ABSTRACT FROM AUTHOR]

A rat monoclonal antibody against mouse Ia antigens was used to investigate the induction of Ia expression by activated T cells and renal epithelial cells during immune responses. The induction of low levels of surface Ia on activated helper and cytotoxic/suppressor T-cell phenotypes was directly demonstrated by differential fluorescence staining. Renal epithelial cells were observed to become strongly Ia-positive after primary immunization with alum-precipitated protein antigen, or after secondary challenge with the soluble antigen used for priming. [ABSTRACT FROM AUTHOR]

Experiments have been carried out to find out if the administration of an anti-IgD serum to mice interferes in anyway with their immune response to sheep red blood cells. This was done to test a hypothesis that a biological role for IgD might be as a critical cellular receptor for antigen. Our results show that the injection of anti-IgD two days before antigen results in suppression of primary responses and priming (the antigen dependent generation of memory cells) but has no suppressive effect on a secondary response. On this indirect evidence we conclude that it is likely that IgD is present on antigen-sensitive precursor cells but not on memory cells. [ABSTRACT FROM AUTHOR]

Surface IgM on murine splenic lymphocytes was capped using a rhodamine-coupled anti-μ chain system. The cells were then reacted with a polyspecific, fluorescein-coupled anti-mouse immunoglobulin (Ig) under non-capping conditions. A large number (31–41 per cent of total Ig-bearing cells) which gave red caps with anti-μ subsequently stained green peripherally with anti-Fab under the non-capping conditions. The remaining stained cells were divided between those showing only red caps (17–29 per cent of Ig-bearing cells) and those showing only green rings (37–45 per cent of Ig-bearing cells). These data are interpreted by assuming that most of the staining with the second reagent (fluorescein- coupled anti-mouse Ig) is due to the presence of surface associated IgD. [ABSTRACT FROM AUTHOR]

Purified IgM isolated from the serum of mice bearing the transplantable plasmacytoma MOPC 104E was reduced and alkylated and then analysed by sucrose density gradient centrifugation and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. On partial reduction a mixture of IgM subunits was obtained in the absence of covalently linked 19S IgM. When examined under dissociating conditions this mixture was found to consist of disulphide-linked 7S subunits (IgMs), small amounts of HL subunits and oligomeric IgM ora size intermediate between monomeric and pentameric IgM. In the absence of a dissociating agent and on sucrose density gradient, however, the mixture resolved into a 19S and a 7S peak. The 19S peak consisted primarily of oligomeric IgM and IgMs with small amounts of HL subunits. Thus alkylated IgMs and HL subunits of IgM can associate through non-covalent forces to form a molecule sedimenting at 19S, providing oligorneric forms are present. In the absence of oligomeric forms, IgMs HL subunits and heavy and light chains sediment at about 7S. The products of partial reduction which sediment at 7S and 19S could also be isolated by preparative polyacrylamide gel electrophoresis. When this was done J chain was absent in the former and present in the latter, raising the possibility that J chain does not disulphide bond to each of the five IgMs subunits, constituting an IgM molecule. Thus, within cells secreting IgM, J chain would be expected to mediate the formation of an oligorneric form of IgM. Once the oligomeric structure has been assembled, then non-covalent forces between this and IgMs subunits will cause the formation of a 19S structure, thereby facilitating the final assembly through disulphide bonds. [ABSTRACT FROM AUTHOR]

Membrane immunoglobulins are associated with a transmembrane disulphide-linked heterodimer composed of an α-chain (mb-1) and a β-chain (B-29). The relative surface expression of all of the polypeptide chains comprising the Ig-αβ complex has been investigated using surface labeling coprecipitation analysis and two-color flow cytometric analysis. The main conclusions are that mb-1 and B-29 are B-cell surface markers on immature and mature B cells, and that all components of the surface Ig-αβ complex are expressed in stoichiometrically equivalent amounts. Thus the complex was quantitatively precipitated from digitonin lysates of 125I-surface-labeled cells with anti-B-29, anti-mb-1 or anti-Ig. Secondly, by two-color FACS analysis there was a proportionality between the relative amounts of cell surface mb-1 or B-29 and surface IgM or IgD, but not other B-cell markers (class II, 8220, FcRγ, FcR∊). Finally there was an insignificant number of B cells expressing membrane Ig without α- and β-chains, and vice versa. Thus there appears to be a closely controlled relative synthesis and surface expression of all components of the B-cell receptor complex. [ABSTRACT FROM AUTHOR]

Three cell-surface molecules associated with surface Ig on murine B cells have been identified. Surface-iodinated resting B cells were lysed in digitonin or Nonidet P-40. The lysates were then examined by immunocoprecipitation with anti-Ig reagents and SDS-PAGE. By this procedure, a 73,000 MW heterodimer, composed of 35,000 and 38,000 M W disulphide-linked subunits, was found to be associated with both mIgM and mIgD. This association was detectable in the detergent digitonin, but not Nonidet P-40. As an alternative approach, resting B cell mIg was capped and internalized by anti-Ig induced mIg cross-linking. The resulting ‘modulated’ cells were then screened for surface reactivity with a panel of monoclonal antibodies. Most antibodies tested (anti-class I, -class II, -LFA, -FcRγ,, -FcRε) reacted equally well with control and modulated B cells. With two, however, there was a significant reduction in surface representation on modulated B cells. One determinant corresponds to a previously undescribed 90,000 MW marine surface marker, and the other was a polydisperse glycoprotein recognized by the mAb .J11D. [ABSTRACT FROM AUTHOR]

A rat monoclonal antibody (mAb) (NIM-R8), insolubilized by binding to plastic plates, induced a rapid and extensive formation of dendrite processes (`spreading') in 13 lymphocytes activated by anti-IgM and interleukin-4 (1L4) or anti-CD38 and IL4. In contrast, resting B cells were unable to spread similarly on the NIM-R8-coated plates. The NIM-R8 antibody recognized a 90000 MW surface glycoprotein (gp90) present on both B and T lymphocytes. The expression of this molecule was greatly increased after polyclonal (lipopolysaccarhide, anti4gM plus IL-4 or concanavalin A) activation. The NIM-R8 mAb with or without IL-2 or IL-4 was unable to induce proliferation of splenic lymphocytes. Following the demonstration that the NIM-R8 mAb recognizes the murine equivalent of human CD44, the induction of spreading of activated B lymphocytes was studied using a panel of mAb recognizing different epitopes of murine CD44. All of these different mAb induced similar spreading of activated B cells. The ligand-inducible spreading of activated B lymphocytes may be an important mechanism for providing an increased cell-surface area for cell-cell or cell-matrix interactions, and thus may be an important factor controlling the response of activated lymphocytes. [ABSTRACT FROM AUTHOR]

The CD4-specific monoclonal antibody (mAb) CC26, when tested on a total of 143 cattle, failed to react with T cells from 16% of animals and gave reduced intensity staining in a further 35% of animals. The results of family studies with groups of half- and full-siblings indicated that CC26 recognizes an allele of CD4 which is co-dominantly expressed in heterozygous animals. This was confirmed by sequential immunoprecipitation and by selecting transfectants expressing the CC26+ and CC26- allelic forms of CD4 following transfection of genomic DNA from a heterozygous animal. Biochemical studies also revealed an allelic difference in the relative molecular weight (Mr) of the CD4 molecule, one allele giving 49 000/52 000 MW bands and the other 52 000/57 000 MW bands in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of CD4+ cell lines with tunicamycin resulted in the appearance of a 47 000 MW band for both allelic forms indicating that the difference in Mr is due to glycosylation. All of the CC26+ alleles examined were of the low molecular weight form (Mlowr) whereas both Mlowr and Mhighr alleles were represented in CC26- animals. Thus, on the basis of Mr and reactivity with mAb CC26, three allelic forms of bovine CD4 can be identified, namely CC26+ Mlowr, CC26- Mlowr and CC26- Mhighr; it is proposed that these alleles are designated CD4.1, CD4.2 and CD4.3, respectively. The allelic difference detected by CC26 was present in both Bos taurus and B. endocyst cattle indicating that it had arisen prior to divergence of these subspecies. The Mhighr allele (CD4.3) was detected only in B. endocyst animals. [ABSTRACT FROM AUTHOR]

CD38 has been implicated in the regulation of both proliferation and rescue from apoptosis of B cells. The signalling events associated with CD38-mediated activation of murine B cells are, as yet, not well defined but it is clear that ligation of CD38 by a mitogenic antibody, NIMR-5, induces a calcium influx in resting B cells. Interestingly, however, cross-linking of CD38 does not mobilize intracellular stores of calcium. We now provide a rationale for these findings by demonstrating that CD38 is not coupled to the generation of inositol phosphates in resting B cells. We do, however, show that CD38 ligation stimulates one, or more, protein tyrosine kinase activities which may play a central role in the transduction of CD38-mediated signals leading to B-cell activation. [ABSTRACT FROM AUTHOR]

This study compares the effects of soluble intact (lgG) and F(ab')2 rabbit anti-μ and anti-δ antibodies on mouse B cells. The results show that while the F(ab')2 antibodies to both isotypes induce polyclonal B cell proliferation, the lgG antibodies are not mitogenic, but rather inhibit DNA synthesis induced by the homologous F(ab')2 fragments. Furthermore, intact anti-μ antibodies inhibit mitogenesis induced by F(ab')2 anti-δ, and vice versa. However, the intact antibodies to both isotypes promote early changes characteristic of B-cell activation, namely, increased la antigen expression, and priming for a facilitated proliferative response to F(ab')2 anti-Ig. In addition, F(ab')2 anti-μ and anti-δ both induce the breakdown of phosphatidylinositol phospholipids in B cells, an early consequence of Ig receptor cross-linking which may be involved in the induction of cell growth. These results therefore indicate that stimulating mature B cells via 1gM or lgD receptors produces indistinguishable early effects, and that cross-linking Fc and surface lg receptors by intact anti-Ig generates a dominant inhibitory signal, regardless of which isotype is involved. [ABSTRACT FROM AUTHOR]

Murine splenic B cells were stained with antibodies against mIg, Ia or FcR and then separated on the fluorescence-activated cell sorter on the basis of quantitative differences in marker expression; that is, they were fractionated into subpopulations bearing high or low densities of the marker. The separated cells were then tested for their relative capacities for T-dependent primary and secondary antibody responses, and for lipopolysaccharide responsiveness. There was no association between the surface density of any of these markers and the ability of the cells to proliferate in response to lipopolysaccharide. How- ever, a high level of surface Ia was associated with good primary and secondary T-dependent responses. The density of mIg or of FcR showed no association with the capacity for primary responses, but a low density of these two markers, especially FcR, was correlated with good secondary responsiveness. Thus, subpopulations of B cells selected on the basis of quantitative levels of membrane markers can also be distinguished by their functional properties. [ABSTRACT FROM AUTHOR]