Activation of clones producing self-reactive antibodies by foreign antigen and antiidiotype antibody carrying the internal image of the antigen.

Because we found in previous work that a high fraction of antibodies exhibiting various specificities bound to glutamic acid 50-tyrosine50 homopolymer (GT) and expressed pGAT cross-reactive idiotype (IdX), we studied the activation of clones producing multireactive antibodies in 1-mo-old MRL/lpr and C3H/HeJ mice bearing VHJ haplotype. The activation of such clones was studied after mice were immunized with GT in CFA, HP20 (an anti-Id MAb carrying the internal image of GT in the D region), and a synthetic peptide corresponding to the D segment of HP20. Our results indicate that immunized mice produced both GT- and self-reactive antibodies. Study of the immunochemical properties of MAb showed that they exhibit multispecific properties and bind with similar-affinity constants to GT or self-antigens such as DNA, Smith antigen (Sm), and IgG2a. An important fraction of antibodies obtained from MRL/lpr mice immunized with HP20 expressed pGAT IdX and some of these antibodies share IdX expressed on anti-DNA, Sm, and rheumatoid factor (RFs) antibodies. The hybridomas producing multispecific autoantibodies use heavy-chain- (VH) and light-chain-variable region (VK) genes from various V gene families, suggesting that they do not derive from the pool of GAT precursors. Sequencing of VH and VK genes of two antibodies show that they can use closely related VHJ558, unmutated VK1, or different VK genes than those used by anti-GT antibodies. Our data demonstrate that clones producing antibodies binding to GT and self-antigens with similar-affinity constants can be activated by foreign or anti-Id antibodies carrying the internal image of the antigen or even by a synthetic peptide corresponding to the D segment of anti-Id antibodies.     

Antieosinophil serum and the kinetics of eosinophilia in Schistosomiasis mansoni

Mice were exposed to different intensities of infection with Schistosoma mansoni (10, 50, or 200 cercariae) and the kinetics of peripheral and bone marrow eosinophilia was followed for as long as 20 wk. When the schistosomula (immature worms) were migrating from the lungs to the liver there was a mild, transient eosinophilia, but soon after the onset of egg laying by the schistosomes, a major and prolonged increase in eosinophils occurred. This was terminated in the heavier infections by the death of the animals, but showed a spontaneous decline beginning at 18 wk in the lightly infected mice. The effect of S. mansoni eggs on eosinophilia in the blood, bone marrow, and granulomatous lesions was then examined by injecting schistosome eggs into mice intraperitoneally, subcutaneously, and intravenously. While the host response was dependent on the route by which eggs were administered, primary peripheral and bone marrow responses were seen on intravenous injection, and secondary responses occurred on intravenous and subcutaneous injection. In unsensitized and egg-sensitized mice, eosinophils were first seen around eggs injected into the pulmonary microvasculature at 96 and 24 h respectively. When the granulomas were maximal in size eosinophils made up at least 50% of the lesions. Administration of antieosinophil serum profoundly suppressed eosinophils in the peripheral blood, eliminated mature eosinophils and markedly increased eosinophil precursors in the bone marrow, and ablated eosinophils from the tissue lesions, considerably reducing their area.     

Immune response to the carcinoembryonic antigen in patients treated with an anti-idiotype antibody vaccine.

We have generated an IgG1 murine monoclonal anti-idiotype antibody (Ab2) designated 3H1, which mimics a specific epitope on the carcinoembryonic antigen (CEA). Patients with CEA positive tumors are immunologically "tolerant" to CEA. We used 3H1 as a surrogate for CEA for vaccine therapy of 12 patients with advanced colorectal cancer. Each of the patients received a minimum of four intracutaneous injections of aluminum hydroxide precipitated 3H1 at either 1, 2, or 4 mg dosage per injection. 9 of 12 patients demonstrated anti-anti-idiotypic (Ab3) response to 3H1. All nine patients generated specific anti-CEA antibody demonstrated by reactivity with radiolabeled purified CEA; some cases were confirmed by immunoprecipitation of purified CEA. We also demonstrated Ab3 stained both autologous and allogeneic colonic tumors. 7 of 12 patients demonstrated idiotype specific T cell proliferative responses and four also showed T cell proliferation to CEA. Toxicity was limited to local reaction with mild fever and chills. All 12 patients eventually progressed after finishing 4-13 dosages. This is the first report demonstrating that a vaccine therapy is capable of breaking "immune tolerance" to CEA in patients with CEA positive tumors. Future studies will focus on treating patients with minimal residual disease.     

Chemically humanized murine monoclonal antibody against a cell nuclear antigen: usefulness in autoimmune diagnostics.

The cellular nuclear antigen SS-B/La is known to be a major antigenic target to an autoantibody in patients with Sjogren's syndrome and systemic lupus erythematosus. It is useful to detect an anti-SS-B/La antibody from patients' sera in a clinical point of view. We purified SS-B/La from rabbit thymus acetone powder by affinity chromatography with a murine anti-SS-B/La monoclonal antibody (1C3-H7). An enzyme-linked immunosorbent assay method, in which SS-B/La was used to coat a plate, was also successfully established. It is difficult to obtain a large volume of patient's serum with high antibody titer and high specificity as a positive control. We investigated whether or not a positive control from human could be replaced by a murine monoclonal antibody to SS-B/La. The 1C3-H7 was conjugated with a human IgG Fc' fragment using N-gamma-maleimidobutyryloxysuccinimide as a cross-linker. The chemically humanized murine monoclonal antibody (1C3-Fc') was recognized by antiserum specific for human IgG Fc fragment. 1C3-Fc' reacted to SS-B/La but not to other antigens. Furthermore, the titration curve of this conjugate ran parallel with those of patients' sera specific for SS-B/La. It is concluded that a chemically humanized murine monoclonal antibody is useful as a positive control in place of a human patient's serum.     

Therapy of murine leukemia with monoclonal antibody against a normal differentiation antigen

The ability of monoclonal antibodies against the Thy-1.1 differentiation antigen to inhibit the growth of transplanted syngeneic AKR/J SL2 leukemic cells has been previously demonstrated. In the present study we further examined therapy with monoclonal antibody of the IgG2a isotype, which was the most effective isotype studied. Intravenous infusion of ascites fluid containing the anti-Thy-1.1 monoclonal antibody 19-E12 1-2 h after tumor implantation led to inhibition of the growth of 3 X 10(5) but not 3 X 10(6) syngeneic SL2 leukemic cells. The achievement of the maximal therapeutic effect required the infusion of a dose containing 3.2 mg of antibody, which inhibited the growth of a subcutaneous inoculum of 3 X 10(5) SL2 leukemic cells in 83% of treated mice. Multiple doses of antibody were no more effective than a single dose given shortly after tumor implantation. The infusion of this relatively large 3.2-mg dose of antibody was required to infiltrate the subcutaneous space and saturate surface Thy-1.1 sites on leukemic cells in a subcutaneous tumor nodule. The failure of antibody to inhibit larger numbers of tumor cells was investigated. Growth of a subcutaneous tumor nodule in mice challenged with more than 3 X 10(5) cells resulted from the growth of Thy-1.1-bearing cells in spite of the presence of the infused anti-Thy-1.1 antibody on their surfaces. In contrast, metastatic growth was due to the emergence of variant leukemic cells lacking the Thy-1.1 antigen. Thus, treatment of transplanted T leukemic cells with an IgG2a anti-Thy-1.1 monoclonal antibody was effective in eliminating 3 X 10(5) antigen-bearing leukemic cells from the subcutaneous space and was very effective in preventing metastasis of leukemic cells expressing the target Thy-1.1 antigen. Therapy was limited by the failure of host mechanisms to eliminate larger numbers of subcutaneous leukemic cells coated with the infused antibody and by the emergence of variant leukemic cells lacking the target antigen.     

Adherent cell function in murine T-lymphocyte antigen recognition. IV. Enhancement of murine T-cell antigen recognition by human leukocytic pyrogen

A macrophage-dependent, antigen-specific murine T-cell proliferation assay was utilized to examine the role of soluble products of murine and human adherent cells in the activation of T lymphocytes. Highly purified human leukocytic pyrogen, and supernates from both murine and human mononuclear phagocytes-macrophages stimulated the immune T-cell proliferative response to the multideterminant antigens dinitrophenyl-ovalbumin and keyhole limpet hemocyanin. The implications of these studies and the relationship of leukocytic pyrogen to human lymphocyte-activating factor are discussed.     

Use of a murine monoclonal antibody for detection of circulating plasma DF3 antigen levels in breast cancer patients.

The murine monoclonal antibody (MAb), designated DF3, reacts with a 300,000-mol wt mammary epithelial antigen. A sequential double-determinant radioimmunoassay (RIA) has been developed to monitor circulating DF3 antigen. Using this assay, we have demonstrated that 33 of 36 normal women had plasma RIA antigen levels less than 150 U/ml. In contrast, 33 of 43 patients (76%) with metastatic breast cancer had RIA DF3 antigen levels greater than or equal to 150 U/ml. The difference between these two groups was statistically significant (P less than 0.001). Similar results have been obtained with a double-determinant enzyme-linked immunoassay (EIA). Only 6 of 111 age-matched normal subjects had EIA DF3 antigens levels greater than or equal to 30 U/ml, while 42 of 58 patients (72%) with breast cancer had levels equal to or above this value. Thus, similar patterns of specificity are obtained with the EIA or RIA. The elevation of circulating DF3 antigen levels in breast cancer patients has been confirmed by transfer blot assays. MAb DF3 reactivity occurred predominantly with circulating antigens of three different molecular weights ranging from 300,000 to approximately 400,000 mol wt. We also demonstrate that patients with both primary and metastatic breast cancer who were free of detectable disease at the time of sampling have DF3 antigen levels that are similar to those obtained from normal subjects. While patients with hepatoma (27%) and ovarian carcinoma (47%) also had elevated circulating DF3 antigen levels, the results suggest that DF3 antigen levels may be useful in distinguishing breast cancer patients from those with esophageal, gastric, colorectal, pancreatic, and lung carcinomas. Furthermore, the results of the RIA, EIA, and transblot analyses demonstrate that the measurement of circulating DF3 antigen levels provides a new and potentially useful marker to follow the clinical course of patients with metastatic breast cancer.     

Immunochemical characterization of the antigen recognized by the murine monoclonal antibody A7 against human colorectal cancer

The nature of the antigen recognized by the murine monoclonal antibody A7 (Mab A7) against human colorectal carcinoma was investigated using immunochemical and biochemical techniques. Binding activity of 125I-labeled Mab A7 was examined using various human cancer cell lines. Mab A7 gave the highly specific binding to colon cancer cell lines, SW1116 and WiDr, and gave only a very weak or no reactivity to gastric cancer cell lines, pancreas cell lines or lung cancer cell lines. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of the extractable antigen from SW1116 showed a single band at approximately 45,000 dalton formed by 125I-labeled Mab A7. Treatment of SW1116 with sodium periodate, pronase and ficin resulted in the loss of antigenic activity. These data strongly suggest that the antigen recognized by Mab A7 is composed of glycoprotein. Competitive binding analysis to the surface of the colon cancer cell line using polyclonal anti-CEA and Mab A7 as well as immunoblotting analysis using monoclonal anti-CEA and Mab A7 suggested that the antigen recognized by Mab A7 was different from CEA. Moreover, this antigen was also found in surgical specimens of colorectal cancer patients and its molecular property was identical to the antigen extracted from SW1116.     

Two-gene control of the expression of a murine Ia antigen.

Two dimensional polyacrylamide gel electrophoresis of Non-Idet P-40 extracts and of specific Ia immunoprecipitates from [35S]methionine-labeled mouse spleen lymphocytes has revealed that the cell surface expression of some Ia antigens appears to be controlled by two genes. One locus, which maps in the I-A subregion, is probably the structural gene for an Ia polypeptide chain. The second locus, which maps between the I-J and H-2D regions, controls whether this I-A encoded molecule (Ae) remains in the cytoplasm or is modified and expressed on the cell surface. Complementation between these two loci allowing surface expression of Ae can occur in the cis or trans chromosomal position. Both the I-A molecule and a polypeptide chain coded for by a locus in I-E are coprecipitated by anti-I-E antibodies, suggesting that these two chains are associated with each other as a multisubunit complex in the cell. Because the ability to complement I-A for Ae expression is a property only of those strains which synthesize an I-E-encoded protein, it is likely that the I-E product itself is regulating the expression of Ae. These observations suggest several mechanisms by which interaction between two I region loci can generate new cell surface molecules. As a result, they may have important implications for understanding the molecular basis of two gene control of immune responsiveness and immune suppression.     

Elevation of laminin and beta-subunit of prolyl 4-hydroxylase in the sera of human subjects with Schistosomiasis mansoni

Serum levels of several markers for liver fibrosis were measured utilizing three groups of human subjects related with schistosomiasis mansoni in northeast Brazil; (1) 20 Schistosoma mansoni egg-positives, who have never been administered with anti-schistosomal drugs, (2) 29 egg-negative inhabitants in the endemic area of schistosomiasis, and (3) 23 egg-negative Japanese immigrants in the non-endemic area. None of these sera were positive for antibody to the surface antigen of human hepatitis B (HBs) and circulating HBs antigen. There was no significant difference in the serum levels of N-terminal peptide of procollagen type-III between the egg-positive subjects and either of the egg-negative Brazilian or Japanese immigrants, whereas the mean value of serum laminin significantly increased in the egg-positive subjects. A significantly higher concentration of serum immunoreactive beta-subunit of prolyl 4-hydroxylase (IR beta PH) was also observed in the egg-positive subjects only in comparison with that of the egg-negative Brazilian. Serum laminin and IR beta PH concentrations of the egg-positive subjects did not correlate with the absorbance of enzyme-linked immunosorbent assay (ELISA) which utilized crude antigens isolated from schistosome adults or eggs. No significant difference in these two parameters was observed between two subgroups of the egg-negative Brazilian or Japanese immigrants divided according to the serological data by ELISA. These findings suggest that serum laminin and IR beta PH levels are worth further evaluation for their usefulness as the marker for liver fibrosis in schistosomiasis.     

Regulation of reaginic antibody production in mice. I. Suppression by antigen of IgE antibody production in vitro

Regulation of IgE production by antigen in a primed murine splenic lymphocyte culture system was described. Maximum IgE antibody production was found to occur when cells were cultured in the absence of exogenously added antigen. A cells and T lymphocytes did not affect the production of anti-DNP IgE antibody. By using a hapten-carrier antigen system (DNP-EA) for priming mice in vivo, it was found that the production of anti-DNP IgE by spleen cells in vitro was inhibited by hapten when coupled to homologous (EA) or heterologous (BGG) carrier, and was not enhanced or inhibited by homologous carrier. Anti-DNP IgE antibody production by cultures depleted of macrophages or T lymphocytes was found to be as sensitive to the suppressive effects of hapten as was the IgE production by whole spleen cell cultures. Both IgM and IgG secondary anti-DNP PFC responses in vitro were enhanced by the presence of the homologous hapten-carrier or carrier alone. DNP-BGG had no effect on the anti-DNP IgM or IgG PFC responses of the cultures. These data suggest that endogenous production of antibody (IgM or IgG) was not responsible for the observed suppression of the IgE response in vitrol The experimental results presented indicate that the regulation of the IgE production by antigen in the primed mouse splenic lymphocyte cultures was a consequence of the direct interaction of hapten with IgE B cells.     

Lung lymphocyte elimination by apoptosis in the murine response to intratracheal particulate antigen.

Pulmonary immune responses are suited to determine mechanisms of lymphocyte elimination, as lung inflammation must be regulated tightly to preserve gas exchange. The self-terminating response of primed C57BL/6 mice to intratracheal challenge with the T cell-dependent Ag sheep erythrocytes (SRBC) was used to test the importance of lung lymphocyte apoptosis in pulmonary immunoregulation. Apoptosis of alveolar and interstitial lymphocytes was demonstrated morphologically, by three independent methods to detect DNA fragmentation, and by surface expression of phosphatidylserine. Apoptotic lymphocytes were exclusively CD4-, CD8-, B220-, but many were CD3+ and Thy 1+. Inhibiting apoptosis by in vivo cyclosporine treatment prolonged lung lymphocyte accumulation following SRBC challenge. Experiments using mice homozygous for the lpr or gld mutations showed that pulmonary lymphocyte apoptosis depended on expression of Fas (CD95) and its ligand (Fas-L). Pulmonary inflammation increased on repeated intratracheal SRBC challenge of lpr/lpr mice, in contrast to the waning response in normal mice. These results confirm that in situ lymphocyte apoptosis contributes to termination of immune responses in nonlymphoid organs, probably because of activation-induced cell death, and may be important in inducing tolerance to repeated antigen exposure.     

Antiidiotypic antibodies to sperm in sera of fertile women that neutralize antisperm antibodies.

The presence of antiidiotypic antibodies (ab-2) to sperm was investigated in the sera of fertile, infertile, and virgin women using sperm-specific anti-FA-1 monoclonal antibody Fab'.ab-2 were detected in 71% (17/24) of sera from fertile women and in none (0/12) of the sera from virgin females by the enzyme-linked immunosorbent assay, Western blot procedure, and immunoprecipitation procedure. Sera from infertile women that had antisperm antibodies showed a minimal presence of ab-2, with only three sera (13%, 3/23) demonstrating the presence of low levels of ab-2. The ab-2 present in fertile women were capable of neutralizing the fertilization-inhibitory activity of anti-FA-1 antibody in a concentration-dependent manner in a human sperm penetration assay (SPA) of zona-free hamster oocytes. ab-2 were also capable of inhibiting the binding of antisperm antibodies to the sperm surface as determined by the immunobead binding technique. This is the first report demonstrating the presence of ab-2 in the sera of fertile women that are capable of neutralizing antisperm antibodies present in sera of infertile women. These findings suggest that the inability to detect antisperm antibody activity in the sera of fertile women may be due to higher levels of ab-2 present in these sera than levels found in sera of infertile women, although both groups may be producing antisperm antibody response after sexual exposure to sperm.     

Anti-Lsa. Case study of an antibody to a low-incidence antigen

An antibody to the low-incidence antigen Lsa (Lewis II) was discovered during routine pre-operative antibody screening of an 87-year-old white man. This example of anti-Lsa reacted only in the indirect antiglobulin test. The antibody did not react with Ls(a+) red cells treated with either ficin or papain. Treatment of the serum with 2-mercaptoethanol did not reduce the reactivity of the antibody. No antibody activity had been detected during previous hospital admissions.     

Computer modeling of the reaction between antigen and heterogeneous antibody in fixed antigen excess: a new method for measuring antibody affinity

Antigen-antibody binding experiments were performed by choosing antigen excess starting conditions and then diluting both the 125I-BSA antigen and anti-BSA proportionately so that the ratio between the reactants remained constant. The fraction antigen bound was measured at each dilution. Binding data were analyzed by computer using non-linear least squares regression to determine the affinity and affinity distribution of different antisera. Early anti-BSA was found to have a unimodal distribution with a binding constant in the range of 10(6)M-1. Intermediate anti-BSA had a bimodal distribution: 1/3 high affinity (1.0 X 10(9] and 2/3 low affinity (3.4 X 10(6)M-1). Late and hyperimmune rabbit BSA antibodies had unimodal affinity distributions with binding constants varying between 1.7 and 2.9 X 10(10)M-1. Antibody affinity can be readily determined by computer analysis of binding curves obtained in constant antigen excess conditions.     

Production of a murine monoclonal antibody with anti-HIV-1 neutralizing properties.

A mouse monoclonal hybridoma cell line producing IgG 1k to human immunodeficiency virus (HIV-1) gp120 envelope protein was cultured in several systems. A small-scale flask culture was essential for characterizing the culture variables of the hybridoma. A dialysis tubing culture appeared to be an excellent alternative to in vivo cultures of ascitic fluid, and gave high mouse monoclonal antibody (Mab) concentrations. Two continuous culture systems were both very effective in producing large amounts of Mabs. The hollow fiber system has the advantage of giving a concentrated product in the harvest. The ceramic core system, on the other hand, allows excellent monitoring of the cellular growth and production phases and gave the highest HIV antigen reactivity/micrograms of the produced IgG. Twelve grams of HIV-1 neutralizing Mabs were produced. The Mab was purified with a yield of 61%. The neutralizing capacity of the Mab was studied in vitro and shown to be excellent with 50% neutralizing titers using 5 ng Mab. The biological half-life of the Mab given intravenously to an HIV-infected individual was shown to be around 30 h.