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Anti-idiotope antibodies produced by mice immunized with a human IgG paraprotein complexed with various mouse monoclonal antibodies (McAbs) have been measured. All animals receiving more than one injection of the paraprotein (free or complexed with a mouse McAb) produced antibodies to the idiotypes of the paraprotein. Complexing with a McAb, especially an anti-Fc-γ McAb, enhanced the response. Antibodies to the idiotopes of mouse McAbs were more difficult to produce and their production was very dependent on the mode and schedule of the immunization. The best antisera were produced by mice receiving a course of injections of pre-formed complexes of the IgG paraprotein and McAbs. Four of five mice produced antibodies to the idiotopes of an anti-light chain McAb (C4) after a course of immunization (one primary plus four boosts) of an IgG-C4 complex. Two of the six mice receiving a similar course of injections of the paraprotein complexed with an anti- gamma McAb (A55) produced high titres of antibodies to A55 idiotypes. Responses were enhanced when complexes were prepared with a pool of McAbs. It is probable that the formation of large multi- cross-linked complexes containing the McAb under study is important in generating the response. Once a response is initiated, very high titres may be achieved. [ABSTRACT FROM AUTHOR]

Complexing a human IgGλ paraprotein with a monoclonal antibody (McAb) to a lambda-chain determinant markedly depressed the anti-lambda response of immunized mice. No anti-lambda was found in the serum after two or four injections of the complex, whereas high titres of anti-lambda chain antibody were found in the sera of mice immunized with the IgG paraprotein complexed with any single anti-gamma chain McAb or with a pool of anti-gamma chain McAbs. Responses to the highly immunogenic Fcγ portion of the molecule were not suppressed by complexing with a single anti-gamma chain McAb and were only slightly suppressed after complexing with a pool of anti- gamma chain McAbs. Some anti-Fcγ antibody was produced by all animals receiving a single injection of the immunogen complexed to any McAb, but free immunogen did not generate a primary response. Complexing the IgG with an anti-Fcγ McAb enhanced the response to the poorly immunogenic Cγ1 domain but no anti-C-γ1 was produced by mice receiving the IgG complexed with an anti-C-γ1 McAb. In immunizations with ‘free’ (Bence-Jones) lambda chain, all the antibody produced was directed against ‘free’-specific determinants. Cornplexing the Bence-Jones protein with McAbs to ‘free’ or ‘general’ determinants on the lambda chain did not enhance or suppress the response. It is concluded that the response to weakly or moderately immunogenic, but not to strongly immunogenic, regions of the molecule may be suppressed by complexing the immunogen with a McAb of appropriate specificity. Possible reasons for this result are discussed. [ABSTRACT FROM AUTHOR]

Proteolysis of lymphocyte surface membrane immunoglobulin (SIg) by trypsin, papain and pronase was investigated. Cleavage in the hinge region resulting in removal of Fabδ was distinguished from removal of the whole SIgD molecule using antisera specific for Cδl and Fcδ Using the sensitive direct rosette assay SIgD determinants were detected after digestion under conditions previously thought to remove IgD from the membrane. By increasing digestion times it was shown that whilst papain and pronase remove the whole IgD molecule, Fcδ but not Fabδ determinants survive trypsin. The implications of the results for structural differences between membrane and serum IgD and for models of insertion of SIgD into the plasma membrane are discussed. [ABSTRACT FROM AUTHOR]

Production of IgM, IgG and IgA was induced from human blood B lymphocytes by culturing with a CD40 MoAb and IL-2 for 9 days. Replacement of IL-2 by IL-10 markedly enhanced production of all three isotypes. High levels of immunoglobulin production also occurred when activated irradiated autologous T cells replaced the CD40 MoAb, and when IL-10 replaced IL-2 in these cultures a spectacular increase in IgG production occurred. The effectiveness of the T cell stimulus depended on the mode of purification of the T cells and the nature of the stimulant used to activate them. Differences in the kinetics and level of expression of CD40L on the various T cell preparations were observed, but did not account for variations in immunoglobulin-inducing efficiency. Immunoglobulin production from sIgD+ and sIgD- B cells was investigated. IgG and IgA were found in sIgD+ cultures, indicating that some isotype switching had occurred, but the major part of the IgG and IgA secreted was from cells already committed to these isotypes. Anti- IgD or anti-IgM MoAbs enhanced the proliferation of B cells induced by anti-CD40 antibody, but immunoglobulin production was not enhanced. Factors affecting the balance of proliferation and differentiation are discussed. [ABSTRACT FROM AUTHOR]

The mechanisms controlling immunoglobulin production have been studied in two types of immunoglobulin-secreting cell generated from human B cells. The first type (I) was produced by activation and transformation of B cells by co-culture with cells of an Epstein--Barr-virus-positive (EBV+) lymphoblastoid cell line (EBV-B-LCL). The second type (II) consisted of human/mouse hybrid cells produced by fusing human tonsil B cells with cells of a mouse plasmacytoma line. Both these methods, singly and in combination, have been widely used for initiation of cell lines secreting human monoclonal antibodies (MoAbs). The two cell types were of quite different phenotype with respect to human B cell antigens. In type I cells MHC Class II and the pan B antigens CD19 and CD37 were expressed at levels typical of cells at the B cell stage. The antigens CD23 and CD39 were expressed at the high levels characteristic of EBV-transformed B cells. Type II cells expressed few B cell antigens. MHC Class II, pan B and the CD23 and CD39 antigens were very weakly expressed and by 119 days post-fusion only CD38 was detectable on cells of the three lines studied; (D9 was on two and CD19 on only one of the three lines. Thus the phenotype of type I cells was influenced by EBV transformation but was otherwise typical of activated B cells. Whereas the human B antigen expression of the hybrid (type II) cells was at the low level encountered on human plasma cells. It is suggested that fusion of a human B cell to a mouse cell which is at the plasmacytoid stage of differentiation results in a switching off of the expression of human peripheral B antigens by a differentiation-linked mechanism. These results are considered in relation to the practical aspects of the production of human MoAbs and the theoretical aspects of control of the passage of B cells to a secretory stage of differentiation. [ABSTRACT FROM AUTHOR]

The properties of five monoclonal antibodies (McAbs) which react with free κ chains but not with IgGκ. IgMκ or IgAκ have been studied. IgG preparations stored for long periods sometimes contained low and high molecular weight material reacting as free κ. This could be removed by passing the preparation through an affinity column to which two of the McAbs had been coupled, but reactive material reappeared when the purified IgG was heated at 65°C. Two of the McAbs from one fusion reacted with F(ab')2 fragments of IgG whereas two from another fusion did not. Pairs of McAbs (one from each fusion) were much more sensitive in the detection of free κ. particularly monomer, than a single McAb. This provided the basis for a very sensitive method of detection of free κ chains in human body fluids and culture supernales. [ABSTRACT FROM AUTHOR]

BALB/c mice were immunized with a human γ2k paraprotein and hybridomas prepared by the Kohler & Milstein technique. Ten hybridoma clones producing high levels of antibody reacting specifically with the immunogen were isolated. Six of the monoclonal antibodies competed strongly with each other for sites on the antigen. In reverse passive haemagglutination tests immunoglobulins reacting with the antibodies were not detectable in normal or pathological sera. Polyclonal antisera to two of the monoclonal antibodies were prepared in rabbits; they were rendered specific by adsorption with Sepharose-normal mouse IgG and tested against the other monoclonal antibodies. A weak cross-reaction of one of the mouse antibodies with the immunogen was observed with one antiserum but none with the other. Furthermore, no repetition of the idiotypes of the two immunogens was observed in polyclonal responses of BALB/c mice immunized with the same paraprotein antigen. These results with monoclonal antibodies confirm those obtained with polyclonal antisera in showing that the antibody response of one species to the private idiotypic determinants of an immunoglobulin of another species is highly heterogeneous. [ABSTRACT FROM AUTHOR]

Maternal lymphocyte function, as assessed by stimulation with cells from a human lymphoid line, is normal in pregnancy. Maternal serum, however, contains immunosuppressivc factor(s), demonstrable by 29 weeks of pregnancy, and having a greater effect at 36 weeks on mixed lymphocyte reactions. These immunosuppressive factor(s) block all mixed lymphocyte reactions to the same extent and are not specific towards those stimulated by paternal antigens. Anti-H cell (DR) alloantibodies, presumably directed against foetal antigens, are not uncommon in maternal serum even in early pregnancy. There is some suggestion that the presence of these antibodies in early pregnancy is associated with selective inhibition of maternal lymphocyte stimulation by paternal antigen. No correlation was found between the presence or absence of serum suppressive factors and the clinical course of the pregnancy. [ABSTRACT FROM AUTHOR]

A sensitive rosette test utilizing antibody-coated red cells has been applied to the study of the immunoglobulins on the surface of blood lymphocytes in chronic lymphocytic leukaemia (CLL). Contrary to other reports, IgG has been found to be a common surface membrane immunoglobulin (SmIg) on CLL cells. The reasons for this variation are discussed. Evidence is presented to show that (a) the antiIgG-coated erythrocytes really are detecting IgG and not a cross-reacting substance, (b) the IgG is intrinsic to the cell and not cytophilically bound and (c) Fc-binding and other artefacts have been excluded. Expression of Ig by individual cells was studied by using mixtures of fluorescein- and rhodarnine-labelled red cells coated with various anti-Ig. Fifty-one cases of untreated CLL were tested and the lymphocytes of thirty-eight of these cases bore Ig of a single light chain type. These cases could be classified on the basis of their lymphocyte SmIg as follows: eighteen expressing M and D and G, eleven expressing D and G but not M, six expressing M and D but not G, and three expressing G alone. [ABSTRACT FROM AUTHOR]

The expression of immunoglobulin on the membrane of peripheral blood lymphocytes of patients with contact dermatitis has been investigated using a sensitive rosette technique. The proportion of such cells in these patients is increased compared to normal values. However, the absolute values obtained are very much lower than has been reported from previous studies. The re-evaluation of immunoglobulin expression is discussed against a background of improved methodology and the established finding that individual lymphocytes may express more than one isotype. [ABSTRACT FROM AUTHOR]

Two new cell lines with the phenotype of terminally differentiated B cells have been derived from the presentation bone marrow of a patient with plasma cell leukaemia. They express the same Immunoglobulin (A1-κ) as the original bone marrow cells. JJN-1 is an hypodiploid, slow-growing line with a plasmacytic morphology, which grows in medium with 15-20% fetal calf serum. When JJN-1 was stimulated with a supernatant ('ESG') containing B cell stimulatory factor 2 (BSF-2/IL-6), a hypotetraploid sub-line, JJN-2, was selectively stimulated. JJN-2 is dependent on ESG for survival. The stimulatory effect of ESG can be completely abrogated by an anti-BSF-2 monoclonal antibody. However, purified BSF-2 atone only produces sub-maximal stimulation of the lines. Both lines show complex karyotypic abnormalities, including 14q- and del(6q). JJN-1 and JJN-2 may be useful for the study of late B cell differentiation and for use as immunogens for the generation of anti-plasma cell monoclonal antibodies. [ABSTRACT FROM AUTHOR]

A mouse monoclonal antibody named BU11 which detects an antigen strongly expressed on human plasma cells is described. The antibody stains plasma cells in tonsil sections, fresh and cultured plasmacytoid cells from the bone marrow of patients with multiple myeloma and cells of the plasmacytoid cell line RPMI 8226 used as the immunogen. In vitro studies of pokeweed mitogen (PWM) stimulated peripheral blood B cells and Epstein-Barr virus (EBV) stimulated tonsil B cells show that the antigen is present mainly on cells coexpressing the OKT10 antigen and containing cytoplasmic immunoglobulin (elg). The BU 11 antigen is expressed weakly on some normal B cells and is not present on T cells, monocytes or granulocytes. The antigen is of molecular weight 58kD under reducing conditions and is biochemically distinct from previously described plasma cell antigens. [ABSTRACT FROM AUTHOR]

A collection of new and established FDC-reactive MoAb has been used in an immunohistological study designed to throw light on (a) the nature of FDC (which are of unknown lineage) as judged by their sharing of antigens with other cell types and (b) the reason for the strong expression of some B cell antigens on FDC. The MoAb were tested on: (I) sections of tonsil, (2) sections of lymph nodes from four cases of non-Hodgkin's lymphoma. (3) peripheral blood cells and (4) cells of cultured haemopoietic cell lines. Only one of the ten new MoAb bound to FDC and no other component of the tissues screened. It resembled R4/23, a MoAb known to be specific for FDC. The other nine antibodies showed a range of cross-reactivity patterns involving one or more of the following: monocytes, macrophages, platelets, epithelium, endothelium and connective tissue fibres. Some of the MoAb reacted with B lymphocytes and cells of B lymphoblastive lines but none showed the restricted FDC-staining pattern associated with MoAb which detect the CD23, P45 antigen. The findings are discussed in terms of the intrinsic or extrinsic nature of the antigens detected. [ABSTRACT FROM AUTHOR]

Occasionally IgM monoclonal proteins (MP) from benign gammopathies bind neural antigens and have been associated with demyelinating peripheral neuropathy, A monoclonal antibody (MoAb) bas been raised against a cross-reactive idiotypic determinant expressed by three out of 12 MP witb specificity for neural antigens. The determinant recognised by tbis MoAb is only expressed when both H and L chain are properly associated. The idiotypic determinant was not expressed by 130 MP randomly selected from patients without neuropathy nor in polyclonal immunoglobulin preparations obtained from healthy individuals. [ABSTRACT FROM AUTHOR]

Cells from sIgG+ B lymphoblastoid cell lines, sIgG+ B cell neoplasms and from tonsils, adult and cord blood and fetal spleen were tested for IgG subclass expression using a panel of monoclonal antibodies. IgG1 was in all cases the commonest subclass except on blood lymphocytes, when approximately equal numbers of cells expressing IgG1 and IgG2 were found. No evidence for multiple subclass isotype expression was found. [ABSTRACT FROM AUTHOR]

A comparative study of human B lymphoma cells activation by monoclonal (murine hybridoma) antibodies to μ heavy chains (Ma-μ) as compared to polyclonal (rabbit) antibodies to μ heavy chains (Ra-μ) has been carried out. Early events related to calmodulin activation such as 86Rb influx and changes in cell volume at 4 h could be induced by Ma-μ. One antibody (AF6) approached Ra-μ with regard to the strength of response obtained-However. Ma-μs including AF6 were deficient in inducing DNA synthesis underconditions where this was achieved with Ra-μ, Studies in one lymphoma, where stimulation of re-expressed surface IgM could be studied, revealed that Ma-μ was deficient m stimulating re-expressed sIgM. These findings raise questions with regard to polyclonal antibody to surface Ig as a model for B cell triggering by antigen and suggest that antigen-induced B cell triggering may be more complex Mum indicated by previous studies with polyclonal antibody. [ABSTRACT FROM AUTHOR]

The Level of 31-1-labelled thymidine ([³H]TdR) incorporation of blood cells of patients with coeliac disease was shown in two separate studies to be significantly lower than that of a normal control group. In the first study the `background' DNA synthesis in unstimulated cultures containing a standard number of blood lymphocytes was measured on days 4, 5 and 6. In the second study a standard volume of freshly drawn whole blood was added to culture medium and the[³H]TdR incorporation measured over the first 24 hr and from 48 to 72 hr. In all cases the [³H]TdR incorporation of cells of coeliac patients on a normal or a gluten-free diet was lower than that of the control group. It is suggested that sequestration of DNA-synthesizing cells in the mucosa of the small bowel may partly explain these results. In whole-blood cultures from patients with inflammatory bowel disease in remission [³H]TdR incorporation over the first 24 hr was raised in Crohn's disease but normal in ulcerative colitis. [ABSTRACT FROM AUTHOR]

Following immunization with various lambda Bence-Jones proteins, over 100 hybridomas were established. A majority of the monoclonal antibodies (McAb) produced were demonstrated to recognize epitopes common to all lambda chains or unique to the immunogen. Twenty-nine McAb exhibited profiles of ‘restricted’ reactivity. Analysis with a panel of serologically defined antigens allowed McAb recognizing the λII subgroup to be identified. Other McAb recognized epitopes selectively expressed by λII and λIII subgroup proteins. A further twenty-one McAb exhibited individual specificity profiles, some of which detect minor lambda chain subpopulations. Several of these McAb have potential for analysis of the clonality of specific antibody responses and for detecting monoclonal B-cell proliferations. [ABSTRACT FROM AUTHOR]

Thirteen monoclonal antibodies (McAbs) showing subclass-specific and subclass-restricted reactivity to human IgG have been produced by the hybridoma technique. Their specificity has been defined by passive haemagglutination (HA) and haemagglutination-inhibition (HAI) assays using a large panel of purified IgG paraproteins of known subclass. Their use in assaying human IgG subclasses in radial immunodiffusion (RID) and in purifying human subclasses by both positive and negative immunoabsorption chromatography is illustrated. [ABSTRACT FROM AUTHOR]

Antibody-producing hybridoma clones have been isolated following immunization of mice with human IgG. Twenty-five monoclonal antibodies (nine anti-Cγ3, fourteen anti-Cγ2, one anti-κ and one anti-λ) were selected for study of their cross-reactivity with the IgO of fifteen mammalian species and chicken immunoglobulin. Each antibody exhibited a unique reaction profile suggesting that human IgO expresses a very large repertoire of immunogenic epitopes. Whilst some antibodies showed a very restricted cross-reactivity profile for others a very wide reactivity profile was observed-including two clones producing autoantibodies. Antibodies demonstrating cross-reactivity between human Fcy and 7S chicken immunoglobulin allow its definitive assignment as a homologue of human lgG. Four clones demonstrated specificity for bovine IgG subclass γ1 and γ2 and the degree of reactivity allows their application to qualitative and quantitative assay systems. These studies suggest new perspectives for the characterization of immunoglobulins and the standardization of anti-immunoglobulin reagents. [ABSTRACT FROM AUTHOR]

Hybridomas have been produced from mice immunized with human IgG. Culture supernates were assayed for the presence of antibody-producing cells by passive haemagglutination. Hybridomas producing antibodies to human kappa (κ) and lambda (λ) light chains have been cloned and grown as ascitic tumours in BALB/c mice. The antigen-binding characteristics of the monoclonal antibodies, contained in the ascitic fluid, were assessed by haemagglutination inhibition, ELISA and radioimmunoassay systems and by the binding of radiolabelled antigen in analytical flat-bed iso-electric focussing gels. One monoclonal anti-κ reacted better with free than with combined κ chains: for another the reverse was true. Antibody fractions separated by DEAE chromatography of ascitic fluids were coupled to ox red cells with chromic chloride and compared with polyclonal antibodies for the detection of cell-surface immunoglobulins. [ABSTRACT FROM AUTHOR]

Peripheral blood monocytes, but not other leucocytes, from healthy donors, were shown to enhance the DNA synthesis of three cell lines. The effect was very marked on an epithelial (Chang) and lymphoid (CLA-4) cell line derived from normal tissues, and less marked on a carcinoma-derived line (HT-29). The enhancement was demonstrable over a wide range of monocyte:cell line ratios, and some activity was present in supernates from monocytes cultured alone or with a cell line. Furthermore, monocytes from gastric carcinoma patients did not enhance Chang and CLA-4 cells to the same extent, relative to the healthy donors. [ABSTRACT FROM AUTHOR]

Human peripheral blood lymphocytes activated by contact with X-irradiated cells from lymphoblastoid cell lines (LCL cells) acquire cytotoxic capacity directed against LCL cells as demonstrated by release of 51Cr from the labelled target. In a large series of cross-over experiments it was possible to demonstrate an element of specificity in the cytotoxic phase in the sense that lymphocytes activated by irradiated cells from line 'A' tended to kill target cells of line 'A' more efficiently than those of an unrelated line. This 'line-directed' specificity was not absolute and in some experiments could not be demonstrated at all. Several factors could be identified which tend to obscure line-directed specificity Both specific and non-specific cytotoxicity as observed in this in vitro system are probably relevant to the immunological defence of the intact organism against proliferating aberrant lymphoid cells. [ABSTRACT FROM AUTHOR]

Two monoclonal antibodies to major histocompatibility complex (MHC) class II antigens, which in combination identify β chains encoded by the SB and DR loci, were used lo investigate which of these gene products were expressed at the cell surface of unstimulated T cells and at various stages of mitogen-induced T-cell maturation. In tests on blood lymphocytes from healthy donors 12% of T cells expressed class II antigens, but only SB antigens were expressed. During activation of T lymphocytes, SB-coded antigens were expressed before DR antigens, and the kinetics of SB expression correlated with the proliferative response of T cells. These results and consideration of recent reports from other laboratories lead us to suggest that SB-coded class II antigens play a role in T-cell proliferation. [ABSTRACT FROM AUTHOR]

The cytotoxic properties of human lymphocytes activated in mixed cell cultures with allogeneic lymphocytes or LCL cells have been investigated. Both activation and cytotoxic reactions could be obtained with almost pure populations of lymphocytes. No supernatant factors appeared to be involved. Human thymocytes and blood lymphocytes from pigs, guinea-pigs or rabbits were ineffective. Fibroblasts from human skin were not effective activators of human blood lymphocytes. There were marked differences in the susceptibility of various target cells to the cytotoxic reaction which could not be accounted for by any known antigenic classification. Various LCL cells were shown to lack cytotoxic potential under a variety of experimental conditions and, by this criterion, lacked immunological competence. It is suggested that the growth of abnormal lymphoid cells in vivo may be prevented by the cytotoxic mechanism described. [ABSTRACT FROM AUTHOR]

Two out of nine rabbits receiving a course of immunization with homologous leucocytes produced antisera which caused blast formation and stimulated DNA synthesis of donor lymphocytes in vitro. Of the seven rabbits which failed to produce active antisera three had received only a short immunization course. The active antisera ‘transformed’ lymphocytes from some rabbits but not those of others. This stimulatory effect was not related to the red cell types or the γ-globulin allotypes of the rabbits. Heterologous antisera raised to rabbit leucocytes in rats and guinea-pigs were cytotoxic to rabbit leucocytes in vitro and also caused leucoagglutination but never stimulated DNA synthesis and blast formation.

A sensitive indirect haemagglutination technique is described which depends upon attachment of the protein antigen to the erythrocyte with difluorodinitrobenzene. The attachment of human gamma globulin to human erythrocytes has been thoroughly studied using sera containing the Rose-Waaler factor as the agglutinator. It has also been shown that horse and rabbit serum proteins and egg white may be attached to cells by the reagent. The technique is simple and sensitive but an absorption step is necessary to remove a non-specific agglutinin to cells which have been treated with the reagent. [ABSTRACT FROM AUTHOR]

The article presents information on a comparison of the responses of lymphocytes from blood, spleen, thymus and appendix of rabbits of different ages to stimulation "in vitro" with staphylococcal filtrate. Staphylococcal filtrate-responsive cells are present in the thymus of rabbits at birth and their appearance in spleen and appendix coincides with the lymphoid ontogeny of these tissues. Thymus lymphocytes survive less well in vitro than lymphocytes from peripheral blood but the temporal relationship of their responses to staphylococcal filtrate is similar. Extensive equilibration of migratory cells between the lymphoid tissues is to be expected in adult animals and for this reason, the results using cells from very young animals are the most informative.

The article presents a discussion related to the in vitro transformation of thymocytes and lymphocytes from humans, rabbits and guinea pigs and from thymomas. The response of human, rabbit and guinea pig thymocytes in culture to blastogenic stimulants such as phytohaemagglutinin and staphylococcal filtrate was usually considerably less than that seen with equivalent numbers of peripheral blood and lymph node lymphocytes. The in vitro growth of thymocytes was improved by growing them at higher cell concentrations but they still responded less well than other lymphocytes.

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