Non-specific polyclonal antibody response induced by Mycoplasma pneumoniae

The ability of heat-killed Mycoplasma pneumoniae (MP) organisms to induce polyclonal antibody production in cultures of blood lymphocytes of healthy subjects was studied. MP induced both IgM and IgG production, with a predominance of IgM. Supernatants of MP-stimulated lymphocyte cultures were tested by an enzyme-linked immunosorbent assay for antibodies to measles, rubella, and herpes simplex virus. MP as well as pokeweed mitogen induced production of viral antibodies of IgG class in lymphocytes of donors who had serum antibodies to the corresponding viral antigens. The MP-induced non-specific antibody response was T-cell-dependent. Lymphocytes from four patients with MP pneumonia, collected nine to 13 days after onset of illness, were tested for in vitro Ig production in the absence of MP. These lymphocytes spontaneously produced increased amounts of IgM and/or IgG. Lymphocytes from three of these four patients spontaneously produced viral IgG antibodies to measles and/or varicella antigens, indicating that MP had induced non-specific activation of memory B cells in vivo. Spontaneous viral antibody production was not found in lymphocyte cultures of healthy donors. The non-specific activation of blood B cells in vitro is probably induced by non-specific helper factors from MP-activated T cells. It is possible that in vivo MP also may have a direct activating effect on B cells.     

Inhibition of T cell responses to alloantigens and polyclonal mitogens by Ly-5 antisera

The effects of Ly-5 alloantisera on the generation of cytotoxic T cells (CTL), on the effector phase of CTL killing, and on polyclonal mitogenesis were studied. Ly-5 antisera added at the beginning of mixed lymphocyte culture (MLC) suppressed the production of CTL in an allele-specific manner. Neither Ly-5.1 nor Ly-5.2 antisera inhibited the generation of cytotoxic effectors by Ly-5.1/Ly-5.2 heterozygous spleen cells; however, a combination of Ly-5.1 and Ly-5.2 antisera markedly suppressed the appearance of Ly-5 heterozygous CTL. Similarly, Ly-5 antisera inhibited the effector phase of CTL killing in an allele-specific manner. In addition, Ly-5 alloantisera specifically blocked concanavalin A and oxidative mitogenesis of splenocytes carrying the appropriate Ly-5 alloantigen. The results are discussed in light of a possible functional role of Ly-5 molecules in immune processes.     

Quantification of IgG monoclonal antibody clearance in tissues

Monoclonal antibodies are an important therapeutic entity, and knowledge of antibody pharmacokinetics has steadily increased over the years. Despite this effort, little is known about the extent of IgG antibody degradation in different tissues of the body. While studies have been published identifying sites of degradation with the use of residualizing and non-residualizing radiolabels, quantitative tissue clearances have not yet been derived. Here, we show that in physiologically-based pharmacokinetic (PBPK) models we can combine mouse data of Indium-111 and Iodine-125 labeled antibodies with prior physiologic knowledge to determine tissue-specific intrinsic clearances. Unspecific total tissue clearance (mL/day) in the mouse was estimated to be: liver = 4.75; brain = 0.02; gut = 0.40; heart = 0.07; kidney = 0.97; lung = 0.20; muscle = 3.02; skin = 3.89; spleen = 0.45; rest of body = 2.16. The highest catabolic activity (per g tissue) was in spleen for an FcRn wild-type antibody, but shifts to the liver for an antibody with reduced FcRn affinity. In the model developed, this shift can be explained by the liver having a greater FcRn-mediated protection capacity than the spleen. The quantification of tissue intrinsic clearances and FcRn salvage capacity increases our understanding of quantitative processes that drive the therapeutic responses of antibodies. This knowledge is critical, for instance to estimate the non-specific cellular uptake and degradation of antibodies used for targeted delivery of payloads.     

Melittin-specific monoclonal and polyclonal IgE and IgG1 antibodies from mice

Melittin, a bee venom peptide consisting of 26 amino acid residues, elicited high IgG and IgE antibody responses in mice of BALB/c and CAF1 strains, but not in mice of A/J, AKR, and C57BL/6 strains. Greater than 80% of the melittin-specific antibodies in sera of responder mice were found to bind the hydrophilic carboxyl-terminal heptapeptide of melittin. Three melittin-specific monoclonal antibodies were obtained from responder mice by the hybridoma technique. Two are of the IgG1 isotype and one is of the IgE isotype. One monoclonal antibody of the IgG1 isotype binds the carboxyl-terminal heptapeptide of melittin, while the other two monoclonal antibodies do not. However, competitive binding studies suggest that all three monoclonal Ig bind at the same, or adjacent, site of melittin. These findings, together with the known amphiphilic property of melittin, suggest that the immunogenicity of this peptide is a consequence of its binding to cell surface phospholipids.     

Selective deactivation of serum IgG: a general strategy for the enhancement of monoclonal antibody receptor interactions

Serum IgG is a potent inhibitor of monoclonal antibody (mAb) binding to the cell-surface Fcγ receptors (FcγRs), which mediate cytotoxic and phagocytic effector functions. Here, we show that this competition can be eliminated, selectively, by the introduction to serum of (i) an enzyme that displaces Fc from FcγRs and (ii) a modification present in the therapeutic mAb that renders it resistant to that enzyme. Specifically, we show that (i) EndoS (endoglycosidase S) cleaves only complex-type glycans of the type found on IgG but (ii) is inactive against an engineered IgG Fc with oligomannose-type glycans. EndoS thus reduces FcγR binding of serum IgG, but not that of engineered mAb. Introduction of both the engineered mAb and endoglycosidase in serum leads to a dramatic increase in FcγR binding compared to the introduction of mAb in serum alone. Antibody receptor refocusing is a general technique for boosting the effector signal of therapeutic antibodies.     

Immunoregulation of the anti-bovine serum albumin response by polyclonal and monoclonal antibodies

Monoclonal or polyclonal antibodies directed toward determinants on limited structures of bovine serum albumin (BSA) (P505-582) were shown to regulate the entire anti-bovine serum albumin (BSA) immune response when passively administered to mice 24 hr prior to immunization. Regulation was observed as suppression of the humoral IgG immune response toward all BSA determinants except those on fragment P505-582. By Day 21 suppression of humoral response was most pronounced toward determinants present on the carboxy terminal end of the molecule (N 307-582). These observations demonstrate that monoclonal antibodies directed against a single determinant on a protein molecule have the capacity to regulate the immune response to a multiplicity of determinants present on the same protein. The data lend support to concepts of antibody-induced regulation by induction of suppressor cells or idiotype recognition.     

Serum-mediated suppression of nonspecific B-cell activation. III. Selective inhibition of the polyclonal B-cell response by normal mouse serum

Normal, nonimmune adult serum is known to inhibit in vitro immune responses when present in sufficient amounts. The significance of inhibition of the immune response by serum, however, is not known. Previous work suggested that normal mouse plasma or serum (NMS) was selectively more inhibitory to nonantigen-specific (e.g., polyclonal) as compared to antigen-specific responses. This led to the hypothesis that constituents of serum (or plasma) may serve naturally to minimize the polyclonal type of antibody response, preserving immune specificity. The present study further examined the effect of NMS on polyclonal versus antigen-specific antibody responses. Under the in vitro assay conditions used, 0.5% NMS supported bacterial endotoxin (ET)-induced mitogenic and polyclonal B lymphocyte responses, antigen (SRBC, TNP-KLH)-specific antibody (IgM, IgG) responses, and antigen-induced or -specific T-lymphocyte proliferative responses, while 5% NMS inhibited all of these responses. However, antigen-specific T-lymphocyte responses could be restored by a 10-fold increase in the antigen concentration and antigen-specific antibody responses could be restored by the addition of ET (10 micrograms/ml) as adjuvant. On the other hand, the mitogenic response to ET remained suppressed regardless of ET concentration. Thus, despite significant reduction of the mitogenic and polyclonal properties of ET in 5% NMS (greater than 70% suppression), sufficient antigenic stimuli permitted optimal specific T- and B-cell responses. Many naturally occurring antigens, e.g., bacterial, fungal, and viral, have inherent B-cell mitogenic and polyclonal activity in addition to adjuvanticity and the presence of the serum inhibitory factor may serve to minimize their indiscriminate polyclonal stimulation of antibody.     

Selective inhibition of anti-nucleoside-specific antibody production by nucleoside-ricin A conjugate

The effect of the preincubation of peripheral blood lymphocytes, from SLE patients, with nucleoside-ricin A conjugates on spontaneous A,G,C,T antibody production was examined. Enhanced spontaneous anti-nucleoside-specific antibody (anti-A,G,C,T antibody) production by SLE B cells was selectively inhibited by pretreatment in vitro with nucleosides conjugated to the ricin A chain. The selective suppression was demonstrated by the lack of suppression of the anti-DNP response or of polyclonal IgG production by pretreatment that did suppress anti-A,G,C,T production by the lymphocytes of eight patients with SLE. Furthermore, pretreatment of B cells, but not of T cells, with nucleoside-ricin A conjugates inhibited the A,G,C,T antibody response by these B cells. Thus, (A,G,C,T)-BGG-ricin A conjugates bind directly to the nucleoside-specific B cells via their antigen receptors. This demonstration of the selective elimination of B cells might have therapeutic applications in SLE.     

Irreversible inhibition of a monoclonal antibody by a nitrophenyl ester

TEPC-15 is a phosphorylcholine-binding mouse myeloma protein which reacts with an ester-containing phosphorylcholine, thep-nitrophenyl ester of 6-(phosphorylcholine)hexanoic acid (PEPCH). The rate of nitrophenolate release mediated by the antibody ispH-dependent and increases with increasingpH. The antibody becomes inactive during the reaction with the ester. The inactive antibody is not reactivated even after treatment with hydroxylamine. Antibody activity is associated with the Fab' fragment. These observations together with thepH profile of the reaction suggest that the ester acylates a lysine side chain near the antibody-binding site.A preliminary discussion of this work was presented at the 78th Annual Meeting of the American Society of Biological Chemists, Philadelphia, Pennsylvania, 1987.     

Affinity purification of tetanus toxin using polyclonal and monoclonal antibody immunoadsorbents

Tetanus toxin has been immunopurified on immunoadsorbent columns derived from equine polyclonal antitoxin coupled to cyanogen bromide-activated Sepharose CL4B. Desorption of bound toxin in active form was achieved only when the immunoadsorbent was mixed with Sephadex G15 and this mixture overlaid on a further volume of Sephadex G15. With equine antibody, 64% of adsorbed toxin was recovered with a specific activity of 2400 limiting flocculation units (Lf)/mg protein N (1.2 X 10(8) minimum lethal doses (MLD)/mg protein N). Similarly prepared immunoadsorbent derived from murine monoclonal antitoxin of low affinity had improved desorption with less acidic desorbents, without the requirement for Sephadex G15; greater than 80% of adsorbed toxin was recovered with a specific activity of 3000 Lf/mg protein N (1.6 X 10(8) MLD/mg protein N).     

Target-cell recognition by cloned lines of influenza virus-specific cytotoxic T lymphocytes. Selective inhibition by a monoclonal H-2-specific antibody

A monoclonal H-2^d-specific antibody markedly inhibits target-cell lysis mediated by two influenza virus A/JAP/57-specific, H-2K ^d -restricted cloned CTL lines. Three other A/JAP/57-specific, H-2 ^d -restricted CTL clones (two of which are also restricted to H-2K ^d in target-cell recognition) are only minimally inhibited by this monoclonal antibody. The inhibitory effect of the antibody is not due to selective binding to certain cloned CTL lines but rather is due to blocking of a determinant on the target cell. The monoclonal antibody produces partial inhibition of lysis mediated by a heterogeneous population of A/JAP/57-specific, H-2 ^d -restricted CTL. Likewise the profound, selective inhibition of cytolysis produced by the H-2^d-specific monoclonal antibody could not be reproduced with a conventional H-2^d alloantiserum. These observations suggest that more than one site on a particular H-2K or H-2D molecule can serve as a determinant for H-2-restricted CTL recognition. They furthermore imply that there is more than one recognition structure (receptor) for self MHC products clonally distributed among a population of H-2-restricted CTL directed to a particular antigen.     

A monoclonal antibody detecting the Ly-9.2 (Lgp 100) cell-membrane alloantigen

A mouse monoclonal cell line (20-1.5) was produced by the cell fusion method and the antibody secreted by this line defined the Ly-9.2 specificity — the reciprocal specificity to that previously identified as the Lgp 100 or the T100 molecule. Although most concentrated on lymph-node cells, the antigen is also found on thymocytes, spleen and bone-marrow cells as well as liver and brain tissue. The monoclonal antibody precipitates a 100000 molecular weight moiety from thymocytes. The antigenic specificities appear to be highly immunogeneic and antibodies to these specificities contaminate many antisera. These sera are noncytotoxic as is the case with the monoclonal antibody even though it is of the IgG2a subclass. As with T100 or Lgp 100, theLy-9 locus appears to be linked to theH-25 locus.     

Role of Ly-6 in lymphocyte activation. I. Characterization of a monoclonal antibody to a nonpolymorphic Ly-6 specificity

In studies with alloantisera and monoclonal antibodies (mAb) a number of antigenic determinants have been defined that are the products of the Ly-6 locus on murine chromosome 2 and that are expressed primarily on B and T lymphoid cells. It remains controversial whether these antigenic determinants are encoded by a single gene or a multigene complex. We have characterized a new rat mAb, D7, which recognizes a cell surface antigen whose expression on nonactivated peripheral lymphocytes varies from strain to strain. The phenotype of the staining profile, i.e., high or low percentage of D7-positive cells, mapped to the Ly-6 locus as assayed by strain distribution studies, RI lines, and Ly-6 congenic strains. The binding of D7 to Ly-6.1-positive strains could be inhibited by mAb directed to the Ly-6E.1 specificity, whereas D7 could inhibit the binding of mAb specific for Ly-6A.2 to cells from Ly-6.2-positive strains. Coprecipitation studies followed by Western blot analysis confirmed that D7 reacts with both Ly-6E.1- and Ly-6A.2-bearing molecules. The most likely explanation for these findings is that Ly-6A.2 and Ly-6E.1 represent allelic specificities. Further dissection of the complexity of the Ly-6 antigen system and determination of its possible functional importance in lymphocyte activation should be greatly facilitated by the availability of xenogeneic mAb that recognize framework determinants on multiple Ly-6 products.     

Selective in vitro inhibition of an antibody response to purified acetylcholine receptor by using antigen-ricin A chain immunotoxin

Purified acetylcholine receptor (AChR) covalently coupled to the catalytically toxic A chain of ricin has been used to selectively eliminate rat lymph node cells involved in in vitro anti-AChR antibody responses. The resulting inhibition was specific in view of the lack of such inhibition of anti-Keyhole limpet hemocyanin antibody responses. Furthermore, when fractionated B cell or T cell populations were treated with AChR-A chain, both populations were found to be sensitive to the specific cytotoxicity. However, T cell cytotoxicity required higher concentrations of the immunotoxin. Furthermore, when AChR-immune lymphocytes were treated with AChR-A chain in vitro, they became unable to mediate secondary adoptive transfer responses in vivo. The abrogation of the anti-AChR adoptive response correlated with the lack of muscle weakness characteristic of experimental autoimmune myasthenia gravis. Thus, it is possible, in principle, to eliminate clones of antigen-reactive lymphocytes with antigen-ricin A chain immunotoxins. This lets open the possibility of using such agents in immunotherapeutic approaches to autoimmune disease.     

Effect of acetylation (O-factor 5) on the polyclonal antibody response to Salmonella typhimurium O-antigen

Antibodies directed against lipopolysaccharide (LPS) O-antigen are often critical in the immune response to Gram-negative pathogens. Mice were orally immunized with isogenic strains of Salmonella typhimurium that differ only in a minor modification of O-antigen, namely acetylation, mediated by the oafA locus. To specifically examine the effect of acetylation on the antibody response to O-antigen, antibody titers were determined against both acetylated and unacetylated LPS by ELISA. In mice immunized with an oafA+ strain, the median titer against acetylated LPS was 32-fold higher than the titer against unacetylated LPS. Mice immunized with the oafA- strain had an 8-fold higher titer against unacetylated LPS. Thus, acetylation of O-antigen alters recognition by the vast majority of individual antibodies. This differential antibody recognition of O-antigen had a statistically significant correlation with protection against subsequent challenge with virulent S. typhimurium.