Selective inhibition of lipopolysaccharide-induced polyclonal IgG response by monoclonal Ly-5 antibody

We have identified two important molecules involved in the regulation of B cell differentiation, namely Lyb-2 and Ly-5. To gain further insight into the function of these two molecules, we examined the effect of monoclonal Lyb-2 and Ly-5 antibodies on lipopolysaccharide (LPS)-induced B cell growth and maturation. We found that Lyb-2 antibody does not have any effect on LPS-induced proliferation and on polyclonal IgM or total IgG responses. On the other hand, although Ly-5 antibody did not affect proliferation and polyclonal IgM responses, it strongly inhibited polyclonal IgG responses, presumably by direct action on B cells. This inhibition was not caused by direct suppressive effect of Ly-5 antibody or Fc receptor-mediated negative signaling. To exert maximal inhibitory effect, Ly-5 antibody had to be added to the culture during the initial 48 hr. However, the presence of Ly-5 antibody during the first 2 days did not cause a significant inhibition. It is thus likely that Ly-5 plays a critical role in the regulation of LPS-induced B cell maturation into IgG-secreting cells at a phase starting within 48 hr after LPS stimulation and continuing thereafter.     

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.     

In vitro maturation of B cells in chronic lymphocytic leukemia. I. Synergistic action of phorbol ester and interleukin 2 in the induction of Tac antigen expression and interleukin 2 responsiveness in leukemic B cells

Recent evidence indicates that interleukin 2 (IL 2), formerly thought to serve as growth factor exclusively for activated T cells, is directly involved in human B cell differentiation. We have investigated the role of IL 2 and IL 2 receptors (as defined by monoclonal anti-Tac antibody) in the phorbol ester-induced in vitro maturation of leukemic B cells from patients with chronic lymphocytic leukemia (CLL). Peripheral blood lymphocytes from B cells from CLL patients with high (greater than 10(5)/microliters) white blood cell counts were depleted of residual T lymphocytes and low-density cells (primarily macrophages) by consecutive steps of E rosetting, complement-mediated lysis of OKT3+ and OKT4+ cells, and Percoll density gradient centrifugation. No OKT3+ T cells were detectable in these cell populations before or after culture. When incubated for 3 days with phorbol ester plus recombinant human IL 2 (rIL 2), 12 to 57% of highly purified B cells from four of five tested patients expressed Tac antigen. Both phorbol ester and rIL 2 were required for maximal Tac antigen expression. Functional studies revealed that phorbol ester-activated (but not resting) CLL B cells responded to rIL 2 with [3H]thymidine incorporation and with enhanced secretion of IgM. Tac+ B cells were isolated in two cases on a fluorescence-activated cell sorter. In one patient, stimulation of Tac+ B cells with rIL 2 resulted in enhanced [3H]thymidine incorporation but no change in IgM secretion, as compared with Tac- B cells; in the second patient, stimulation of Tac+ B cells with rIL 2 did not result in [3H]thymidine uptake, but did result in significant IgM secretion. These findings indicate that certain leukemic B lymphocytes can be induced to express IL 2 receptors and respond to IL 2. The use of resting clonal B cell populations arrested at distinct stages of differentiation may help to better define the stage(s) at which IL 2 acts directly on B cells to induce proliferation and/or terminal differentiation.     

The inhibition of T cell proliferative responses by crosslinking CD7 and IgM-Fc receptors.

Previous work from our laboratory described a human T cell soluble ligand that inhibited T cell proliferative responses to mitogen and alloantigen by interacting with CD7 and/or the receptor for the IgM-Fc portion (FcR mu) on T cells. In this report, we used mouse anti-human CD7 monoclonal antibodies (mAb) and purified human IgM (HIgM) to substitute for the human ligand and examined the possible involvement of these receptors in the inhibition of T cell proliferation. Preincubation of human T cells with mouse anti-CD7 mAb, HIgM, mouse anti-human IgM (MAH IgM) alone, or any of these combinations as a primary antibody did not inhibit mitogen- or alloantigen-induced T cell replication. Similar effects were seen with the pretreatment of T cells with an irrelevant negative control primary mAb or a secondary-step goat anti-mouse immunoglobulin (GAM Ig), goat anti-human IgM-Fc (GAH Fc mu), or both. In contrast, the pretreatment of T cells with anti-CD7 and/or HIgM followed by the appropriate secondary-step crosslinking antibody significantly reduced their proliferative responses to mitogen and alloantigen. Similarly, crosslinking of CD7 and FcR mu on human transformed T cell lines inhibited their spontaneous proliferation. The inhibitory effect of crosslinking CD7 and FcR mu was not due to cytotoxic effects of these antibodies and appears to be temperature sensitive. These findings suggest that crosslinking CD7 and/or FcR mu appears to have a novel role in down-regulating T cell proliferation.     

Ly1+ PRO-B lymphocyte clones. Phenotype, growth requirements and differentiation in vitro and in vivo.

Several clones obtained from the bone marrow of a BALB/c mouse were found to contain the heavy and light chain Ig genes in the germline configuration, to express Ly1 and to carry the B cell lineage markers B-220, Lyb8 and BP-1; these clones are Pgp-1+, LFA-1+, J11d+, Mac-1+ and Thy1-, Lyt2-, L3T4-, GM1.2- and Ia-. Three clones analyzed in detail (Lyd9, LyH7 and Lyb9) have receptors for interleukin (IL) 2 and IL3 as assessed with the 7D4 and CC11 monoclonal antibodies respectively. They grow in rIL3 but not in rIL2 or rIL1; both rIL4 and rIL5 also promote their proliferation, albeit to a much lesser extent than rIL3. None of the interleukins tested alone or in various combinations promoted the clones to differentiate in vitro along the B cell pathway. Treatment with 5-Azacytidine (5-Aza) induced cell surface Ia expression but not rearrangement or expression of Ig genes. However, 5-Aza-treated Lyd9, LyH7 and Lyb9 cells co-cultured with X-ray irradiated accessory cells and LPS gave rise to Ly1+, IgM+ B lymphocytes (range 14-51%) including mu + kappa + (78-93%), and mu + lambda + (9-25%) B lymphocytes. In vivo, the Lyd9, LyH7 and Lyb9 clones gave rise to IgM+ B lymphocytes (8.5-17%) including mu + kappa +, and mu + lambda +, but not to Lyt2+ or L3T4+ T lymphocytes after 4-6 weeks of transfer into Scid mice. Our results indicate that Ly1+ IgM+ cells comprise a subpopulation of B lymphocytes that is derived from IL3-responsive Ly1+ PRO-B lymphocytes.     

Antibodies to nonpolymorphic determinants of the Thy-1 molecule inhibit T cell proliferation

The effect of anti-Thy-1 monoclonal antibodies on murine mixed lymphocyte reactions and concanavalin A-induced mitogenesis were investigated. It is demonstrated that rat antibodies against nonpolymorphic determinants of the murine Thy-1 antigen inhibited cell proliferation in the absence of complement. In contrast, antibodies against polymorphic determinants of Thy-1 had no effect on T cell activation. Inhibition of T cell proliferation did not depend on the isotype of the blocking antibody, because both IgM and IgG antibodies against monomorphic determinants were inhibitory, whereas IgM or IgG antibodies against allotypic determinants were inactive. In addition, the blocking activity could not be attributed to the xenogeneic (rat) origin of the antibodies to nonpolymorphic Thy-1 determinants, because rat anti-Thy-1.2 antibodies had no effect on cell activation. Thus, the efficacy of anti-Thy-1 antibodies as T cell inhibitors was determined by the antibody specificity. The suppressive mechanism of anti-Thy-1 antibodies was effective throughout the entire course of mixed lymphocyte reactions. Addition of antibodies at any time point during the first 90 hr of a 120-hr mixed lymphocyte culture resulted in significant suppression of the proliferative response. However, in some cases an early enhancement preceded suppression of the response. The modulation of proliferative responses by anti-Thy-1 did not result from a nonspecific mitogenic effect of the antibodies on T lymphocytes, because no effects were observed when antibodies were added to responder cells alone. These results suggest that the Thy-1 molecule, or a molecule that is located on the cell membrane in close proximity to the Thy-1 antigen, is involved in the activation of T lymphocytes.     

Antigen-antibody complexes bound to B-lymphocyte Fc gamma receptors regulate B-lymphocyte differentiation

We studied the effect of soluble antigen-antibody complexes on the responses of polyclonally activated murine B lymphocytes. For this, normal B lymphocytes were stimulated with rabbit F(ab')2 anti-mu and lymphokines. IgG complexes, particularly in antigen excess, inhibited the plaque-forming cell response (55-70%), while proliferation was unaffected. Maximal inhibition was obtained with small amounts (0.2-1.0 microgram/ml) of complexes. Neither antigen or antibody alone was inhibitory. Inhibition was mediated via binding of the IgG complexes to Fc gamma receptors of B lymphocytes: (1) neither T lymphocytes or adherent accessory cells were required; (2) IgM complexes did not inhibit; and (3) inhibition was not seen when monoclonal anti-Fc gamma receptor antibodies prevented binding of the IgG complexes to these receptors. Kinetic experiments showed that B lymphocytes are susceptible to this inhibitory signal for only a short time after stimulation. We conclude that IgG complexes bound to the Fc gamma receptors of B lymphocytes regulate B-lymphocyte differentiation.     

Ligation of HLA-DR molecules on B cells induces enhanced expression of IgM heavy chain genes in association with Syk activation

Signals transmitted by class II major histocompatibility complex are important regarding cell function related to antigen presentation. We examined effects of DR-mediated signaling on Ig production from B cells. Cross-linking HLA-DR molecules on B cells by solid-phase anti-HLA-DR monoclonal antibodies, led to an increased production of IgM, without proliferation or apoptosis. This event was accompanied by an enhanced expression of both membrane- and secretory-type IgM heavy chain mRNA. When peptide-pulsed B cells were co-incubated with an HLA-DR-restricted T cell clone treated by the protein synthesis inhibitor emetine, peptide-induced de novo expression of lymphokines and cell-surface molecules on T cells can be neglected. CD40-CD154 interaction was not involved in IgM enhancement, in such a system. The protein-tyrosine kinase inhibitors and the Syk inhibitor piceatannol, but not the Src inhibitor PP2 had a marked inhibitory effect on IgM secretion. Furthermore, ligation of HLA-DR on B cells using the F(ab')2 fragment of anti-DR monoclonal antibody, enhanced Syk activity. Our data suggest that HLA-DR on B cells not only present antigenic peptides to T cells, but also up-regulate IgM production, in association with Syk activation and without the involvement of Src kinases, hence the possible physiological relevance of Src-independent Syk activation.     

Class I-specific antibodies inhibit proliferation in primary but not secondary mouse T cell responses.

Murine T and B splenocytes were incubated with antibodies that recognize CD3 or surface IgM. These antibodies induced proliferation of their respective target cells. Once stimulated via their receptors, the proliferation of both CD4+ and CD8+ T but not B lymphocytes was inhibited by class I-specific antibodies or their monovalent Fab' fragments. The inhibition of proliferation was dependent on the site on class I molecules recognized by the antibodies used, with the alpha 1/alpha 2 domains of H-2K molecules representing the major site for inhibition. Only soluble antibody-mediated proliferation could be inhibited by class I-directed antibodies; proliferation induced by CD3-specific antibody immobilized on plastic was not inhibited. Primary allogeneic MLR was also inhibited by class I-specific antibodies. In contrast, neither secondary allogeneic MLR, secondary Ag-specific responses, nor proliferation of CTL clones or tumor cell lines were inhibited by class I-specific antibodies. These results suggest a role for class I molecules in regulation of TCR/CD3- but not surface IgM-mediated cell signaling, which depends on the form of stimulation and the stage of differentiation of T cells.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Antibodies reactive with class II antigens encoded for by the major histocompatibility complex inhibit human B cell activation

Although class II antigens encoded by genes in the major histocompatibility complex (MHC) are important as recognition structures for immunoregulatory cell interactions, the precise functional role of these molecules in the biological responses of B lymphocytes is unknown. In the studies described here, we have examined the effects of six monoclonal antibodies reactive with human class II MHC antigens on B cell activation and proliferation. Peripheral blood IgM+ B cells purified by fluorescence-activated cell sorter (FACS) techniques were stimulated with anti-mu antibodies, protein A-bearing Staphylococcus aureus (SAC), or in T cell-dependent activation cultures. The B cell proliferative responses induced by these stimuli were inhibited 68 to 90% by low concentrations (1 to 5 micrograms/ml) of antibodies reactive with class II MHC antigens. Antibodies specific for DR and DQ antigens were both effective inhibitors of B cell proliferation. This inhibition was not due to the binding of antibody to B cell Fc-IgG receptors, because IgM and IgG anti-class II antibodies were equally potent as inhibitors. When responses of B cells fractionated on the basis of cell size by forward angle light scatter were analyzed, anti-DR and anti-DQ antibodies inhibited the proliferation of small, resting IgM+ cells induced by T-independent as well as T-dependent stimuli. Activation-dependent increases in B cell size and RNA synthesis were similarly inhibited. In contrast, the responses of large B cells (that had been preactivated in vivo) to T cell-derived B cell growth factors were not affected by anti-class II antibodies. These data suggest that class II MHC molecules do not serve merely as cellular interaction structures but also directly participate in early events of the B cell activation cascade that precede cell enlargement or increased RNA synthesis. After activation and expression of receptors for growth factors, however, B cell class II MHC antigens no longer mediate signals required for mitogenesis.     

Blockade of NK cell lysis is a property of monoclonal antibodies that bind to distinct regions of T-200

The previously described NK inhibitory monoclonal antibody 13.1 is shown to immunoprecipitate a series of high m.w. glycoproteins homologous with the murine T-200/Ly-5 molecules. Not all antibodies to the human T-200 molecule, however, have an inhibitory effect on NK cell function. A comparison is made between two noninhibitory anti-T-200 antibodies, 13.5 and 13.6, and two inhibitory anti-T-200 antibodies, 13.1 and 13.3. All antibodies are of the IgG1 subclass. Sequential immunoprecipitation experiments show that these antibodies react with the same set of molecules. The differences in NK-blocking activity could not be explained by the amount of antibody bound per cell in NK-enriched populations, nor by the avidity with which they bound. It is shown by competitive radiobinding assays that the 13.1 and 13.3 antibodies define a region, termed region A, distinct from that defined by the nonblocking antibodies 13.5 and 13.6, termed region B. Region B is shown to reside between the membrane and region A. These findings show that the inhibition of NK lysis by anti-T-200 antibodies is a function of the site on that molecule to which these antibodies bind. This may also explain the ability of antibodies to the A region of T-200 to block selectively the lysis of myeloid and erythroid tumor targets, with no effect on the lysis of T lymphoma targets.     

Binding of monoclonal antibody to the Epstein Barr virus (EBV)/CR2 receptor induces activation and differentiation of human B lymphocytes

A panel of B cell-specific monoclonal antibodies that identify the CR2/EBV receptor were examined for their ability to mimic the T-independent mitogenic agent, EBV, and thus activate human peripheral blood B lymphocytes. Two of four different anti-CR2/EBV monoclonal antibodies, OKB7 and AB-1, produced a 50-fold to 200-fold dose-dependent stimulation of DNA synthesis of peripheral blood mononuclear cells. One of the other monoclonal antibodies, anti-B2, had slight activity, and the other, HB-5, was completely inactive. One of the mitogenic antibodies, OKB7, which directly inhibits binding and infection of B cells by EBV in the absence of a second anti-immunoglobulin antibody, was examined in further detail. Both the intact antibody in soluble form and its pepsin-derived F(ab')2 fragment stimulated DNA synthesis of unseparated B and T lymphocytes. Peak stimulation of DNA synthesis in peripheral blood mononuclear cells occurred between 4 to 6 days. B cells were responsible for incorporation of [3H]thymidine. However, T cells were required for activation of peripheral blood mononuclear cells by OKB7. OKB7, as well as the other mitogenic monoclonal anti-EBV/CR2 receptor antibody, also induced B cells to differentiate after 6 to 10 days of culture as indicated by polyclonal Ig secretion. IgM was the predominate immunoglobulin secreted. These studies thus indicate that certain epitopes on the EBV/CR2 receptor trigger B cells to divide and differentiate. This pathway of B cell activation, in contrast to that produced by EBV, is T cell dependent.     

Role of the LFA-1 molecule in cellular interactions required for antibody production in humans

The lymphocyte function-associated antigen 1 (LFA-1) has been shown to play a role in various T cell functions in mice and humans including cytotoxicity, and proliferation to allogeneic cells and foreign antigens. These functions have been defined with specific monoclonal antibodies and were additionally confirmed by the investigation of patients with inherited deficiency in membrane LFA-1 expression. In this paper, we report our studies on the potential role of the LFA-1 molecule in T lymphocyte-dependent antibody responses. In a patient with a complete lack of membrane expression of LFA-1, there was no in vivo antibody response to vaccinal antigens such as tetanus, diphtheria toxoids, and polio virus, and no in vivo or in vitro antibody production to influenza virus, whereas serum immunoglobulin levels and antibodies to polysaccharides (isohemagglutinins, antibody to mannan, and a polysaccharide from Candida albicans) were detected in correlation with in vitro production of anti-mannan antibody. The defective antibody response to polypeptides was not secondary to poor antigen-specific T proliferation, because the latter was found to be present. Similarly, in vitro antibody production to influenza virus of normal cells was blocked by several anti LFA-1 monoclonal antibodies specific for the alpha subunit of the molecule, if they were added from the beginning of the culture. The antibody production blockade could be achieved with monoclonal antibody concentrations that partially preserved T cell proliferation. The helper effect of an influenza virus-specific helper T cell clone was also blocked. The targets of the blockade were shown by incubation experiments to be T cells and monocytes. In contrast, anti-LFA-1 monoclonal antibodies had no effect on pokeweed mitogen-induced B cell maturation into immunoglobulin-containing cells and on the anti-mannan antibody production. These combined data demonstrate that the LFA-1 molecule plays a role in T cell dependent antibody production to polypeptidic antigens but not in the antibody response to polysaccharides, although the antibody response to mannan is T cell dependent. It is proposed that the LFA-1 molecule is required to some extent for a antigen-presenting cells-T lymphocyte interaction and for the maintenance of a close association between antigen-specific helper T cells and small resting B lymphocytes. Polysaccharidic antigens that exhibit repetitive antigenic determinants might cross-link membrane immunoglobulins on B lymphocytes, thus allowing B cells to pass through a first step of activation requiring cognate T-B cell interaction.     

Role of the interleukin 2 receptor in differentiation of a clone of Ly-1+ B cells

CH12.LX, an in vitro subclone of a murine B cell lymphoma that makes IgM reactive with sheep erythrocytes (SRBC), has cell surface receptors for the lymphokine interleukin 2 (IL 2). The binding of recombinant murine IL 2 to these receptors did not stimulate CH12.LX cells to differentiate and secrete antibody. However, the binding of either of two monoclonal antibodies (Mab) specific for the IL 2 receptor increased the proportion of CH12.LX cells that secrete hemolytic IgM. The effect did not require the presence of antigen. One of the Mab, 3C7, is known to block the binding of IL2 to its receptor on T cells, whereas the other, 7D4, which also reacts with the IL 2 receptor, does not block the binding of IL 2. The differentiation of CH12.LX induced by 3C7, but not that induced by 7D4, was inhibited by recombinant IL 2. Neither IL 2 (up to 200 U/ml) nor 3C7 (up to 10 micrograms/ml) had any significant influence on incorporation of [3H]thymidine; 7D4 at 10 micrograms/ml decreased thymidine incorporation by about 60%. Mitomycin C and hydroxyurea, which both inhibit the incorporation of [3H]thymidine into CH12.LX cells, also both induce antibody secretion. In both cases, the concentration necessary to cause differentiation is substantially lower than that needed to cause detectable inhibition of thymidine uptake. We conclude that the IL 2 receptor on CH12.LX cells is a functional signal transducing molecule, and we discuss the possible inverse relationship between proliferation and differentiation.     

细胞因子对脐血B细胞免疫球蛋白类别转换的调节

为了解细胞因子对新生儿Ｂ细胞免疫球蛋白类别转换的调节作用,在体外细胞培养的基础上,采用反向酶联免疫斑点法观察了脐血单个核细胞及添加重组细胞因子后脐血Ｂ细胞免疫球蛋白释放细胞数量（ＩｇＳＣｓ）。结果：正常脐血单个核细胞仅产生少量的ＩｇＳＣｓ。用抗ＣＤ３单抗刺激丝裂霉素Ｃ处理后的脐血Ｔ细胞,并补充ｒＩＬ－２、ｒＩＬ－４、ｒＩＬ－１０及其组合,可诱导脐血Ｂ细胞释放ＩｇＡ、ＩｇＧ和ＩｇＭ。这些结果提示：细胞因子的补充可促进体外新生儿Ｂ细胞免疫球蛋白的类别转换     

Activation of human B cells and inhibition of their terminal differentiation by monoclonal anti-mu antibodies

Anti-mu antibody preparations have been found to exert both positive and negative effects on B cell activation and differentiation. To explore these paradoxical influences of IgM cross-linkage on human B cells, three gamma 1 kappa murine monoclonal antibodies specific for human mu-chains (DA4.4, AB6.4, 145.8) were examined for their comparative effects on activation of B cells and inhibition of terminal plasma cell differentiation. All three antibodies appeared equally efficient in immunoprecipitation of surface IgM molecules; however, fluorescence-activated cell sorter analysis revealed that the DA4.4 and AB6.4 antibodies saturated the B cell surface IgM at slightly lower concentrations than did the 145.8 antibody. When the affinity-purified antibodies were added in varying concentrations to cultures of small resting B cells, all three antibodies induced B cell enlargement and DNA synthesis, but with varying degrees of efficiency (DA4.4 greater than AB6.4 much greater than 145.8). In striking contrast, large B cells isolated either by FACS or density gradient separation were unresponsive. The anti-mu-induced proliferative response of small B cells required relatively high B cell densities, but not T cells or the Fc portion of the antibody molecules. The maximal proliferative response was obtained during the third day of culture, and the response curve suggested that anti-mu induced only one round of B cell replication. All three antibodies were capable of completely inhibiting T cell factor-induced differentiation of large B cells into IgM plasma cells; both F(ab')2 fragments and intact anti-mu antibodies were effective in final concentrations as low as 1 microgram/ml. Significant suppression of IgG and IgA plasma cell differentiation was also achieved, but required higher concentrations of the anti-mu antibodies. For each antibody, there was a close correlation between the efficiency of inducing small B cell proliferation and of inhibiting large B cell differentiation into plasma cells. The results show that the B cell response to cross-linkage of cell surface IgM varies according to the differentiation stage. We postulate that the mature resting B cell represents the only stage in the life history of the B cell during which surface Ig cross-linkage leads to a positive signal, negative signals being the rule at other stages in B cell replication and differentiation.     

Monoclonal anti-GD3 antibodies selectively inhibit the proliferation of human malignant glioma cells in vitro

The frequently occurring alteration of ganglioside expression in tumor cells has been implicated to play a role in the uncontrolled growth of these cells; antibodies to such gangliosides might affect tumor cell growth. We have studied the effect of IgM monoclonal antibodies to two glioma-associated gangliosides, GD3 and GM2, on cell proliferation of four human glioma cell lines and one renal tumor cell line. Of the two anti-ganglioside antibodies tested, only the anti-GD3 antibody resulted in a significant (p<0.005) inhibition of cell proliferation as measured by thymidine incorporation and Brd-U labeling, after 24[emsp4 ]h incubation. The effect was not dependent on any serum factor and no increased cell death was observed. All cell lines contained higher or similar amounts of GM2 than GD3, and both antigens were shown to be expressed on the cell surface and accessible to antibodies. The selective effect of anti-GD3 antibodies as contrasted to the inactivity of anti-GM2 antibodies suggests a possible role for ganglioside GD3 in tumor cell proliferation.     

Inhibition of anti-IgM and growth factor-induced proliferation of guinea pig B cells by one of two distinct Fc gamma receptors on the cells

Guinea pig B cells were found to proliferate when co-stimulated with F(ab')2 of rabbit anti-guinea pig IgM and human 12-kDa B cell growth factor (BCGF), though the proliferation did not occur with the replacement of the F(ab')2 by its parent IgG antibody. In addition, the intact antibody inhibited the proliferation induced by F(ab')2 of anti-IgM and BCGF. Because both two distinct types of FcR for IgG on the B cells, one specific for IgG2 (Fc gamma 2R) and the other for both IgG2 and IgG1 (Fc gamma 1/gamma 2R), can bind rabbit IgG, we determined whether they participate in the inhibition of the B cell proliferation by intact anti-guinea pig IgM antibody. Blocking Fc gamma 1/gamma 2R by F(ab')2 of anti-Fc gamma 1/gamma 2R mAb significantly reversed the inhibitory effect of intact anti-IgM antibody. F(ab')2 of anti-Fc gamma 2R mAb, however, was not effective. Furthermore, guinea pig IgG1 and IgG2 anti-rabbit IgG antibodies suppressed similarly the B cell proliferation induced by F(ab')2 of rabbit anti-IgM and BCGF. These results show that between these two types of Fc gamma R on B cells, Fc gamma 1/gamma 2R alone is involved in the regulation of anti-IgM and BCGF-induced B cell proliferation, and inhibits the response when cross-linked to the surface IgM.     

Preparation and characterization of polyclonal and monoclonal antibodies against human interleukin 1 alpha (IL 1 alpha)

Polyclonal and monoclonal anti-human IL 1 alpha antibodies (Ab) have been established. These Ab neutralized human recombinant IL 1 alpha (rIL 1 alpha) activity effectively, but did not interfere with human rIL 1 beta, murine rIL 1 alpha, or human rIL 2 activity. Fifty percent of rIL 1 alpha activity (25 U/ml, or 2.5 ng/ml) was neutralized by less than 0.06 microgram/ml of rabbit anti-IL 1 alpha Ab (R-38.3G) and by less than 0.13 microgram/ml of monoclonal Ab (clone 28(3B1], respectively. In other experiments, 10 micrograms/ml of rabbit anti-IL 1 alpha Ab could effectively neutralize 50% of 2000 U of rIL 1 alpha activity, and the same amount of monoclonal Ab neutralized 50% of 500 U/ml of rIL 1 alpha activity. Not only IL 1 alpha activity in the thymocyte costimulator assay, but also IL 1-dependent IL 2 production by a human leukemic cell line, HSB.2 subclone, were blocked by these polyclonal or monoclonal Ab. In addition, pI 4.9 IL 1 activity produced by the myelomonocytic cell line THP-1 and by the Epstein-Barr virus-transformed B cell lines, were neutralized by these Ab, suggesting that these cell lines also produce IL 1 alpha. The specificity of these polyclonal and monoclonal Ab was further confirmed by immunochemical method (Western blotting), in which anti-IL 1 alpha Ab reacted with rIL 1 alpha in a specific manner. Furthermore, an enzyme-linked immunosorbent assay system has been developed that can detect low levels of IL 1 alpha activity (less than 0.3 ng/ml or less than 3 U/ml), which is still less sensitive than thymocyte comitogenic assay and considerably less sensitive than the D10 assay. Finally, anti-IL 1 alpha Ab-conjugated affinity columns were prepared, by which IL 1 alpha activity, but not IL 1 beta activity, was specifically adsorbed and eluted effectively.