Inhibitory effects of anti-interleukin 2 receptor and anti-L3T4 antibodies on delayed type hypersensitivity: the role of complement and epitope

Although it is often assumed that anti-T cell antibodies mediate immunosuppression by targeting T cells for destruction, other activities should be considered. To dissect the mechanisms by which anti-L3T4 and anti-interleukin 2 receptor (IL 2R) monoclonal antibodies (Mab) mediate immunosuppression, the effects of anti-L3T4 and two complement-fixing anti-IL 2R Mab of the same isotype, but defining functionally distinct epitopes, were probed in a delayed type hypersensitive (DTH) model using BALB/c as well as two C5-deficient mouse strains. Low doses of anti-L3T4 and the M7/20 anti-IL 2R Mab, which competitively blocks IL 2 binding, inhibit DTH in BALB/c mice whereas an anti-receptor antibody which does not block the IL 2 binding site did not effectively abrogate DTH. Interestingly, anti-L3T4, but not M7/20 anti-IL 2 Mab treatment blocked DTH in the C5-deficient strains. On the other hand, M7/20 does not cause immunosuppression solely by blocking the IL 2R from occupancy by IL 2 because binding to T blasts by M7/20 is equivalent in BALB/c and C5-deficient strains. Consequently, immunosuppression mediated by anti-IL 2R Mab is dependent on both IL 2 receptor site blockade and C5. Clearly, anti-L3T4 and M7/20 have disparate requirements for C5 in mediating immunosuppression. There can be no doubt that factors other than the cellular targeting patterns influence the immunosuppressive activities of Mab. Ideally, anti-T cell Mab should fix complement and inhibit T cell function.     

Establishment of a mouse hybrid-hybridoma secreting bispecific antibodies to bovine lactoferrin and horseradish peroxidase

Summary A mouse hybridoma HS@03A secreting anti-horseradish peroxidase (HRPO) monoclonal antibodies (IgG1) was established. A HAT sensitive clone of HS@03A was obtained by culturing the hybridoma cells in a 6-thioguanine supplemented medium. The resulting clone 03AR10-2 was fused with a 5-bromo-2-deoxyuridine resistant (HAT sensitive) clone of a mouse hybridoma HB8852 secreting anti-bovine lactoferrin (bLF) antibodies. Hybrid-hybridomas secreting bispecific antibodies were selected and a hybrid-hybridoma clone HH1-4-3 was established. The bispecific antibodies secreted by the hybrid-hybridoma HH1-4-3 were found to be useful for the analysis of bLF by competitive ELISA.     

Neutralizing monoclonal antibodies to human rotavirus and indications of antigenic drift among strains from neonates.

Cells producing neutralizing monoclonal antibodies to a serotype 3 human neonatal rotavirus strain RV-3 were derived by fusion of hyperimmunized mouse spleen cells with mouse myeloma cells. As ascites fluid, three rotavirus-neutralizing monoclonal antibodies were characterized by hemagglutination inhibition and reacted with 17 cultivable mammalian rotaviruses representing five virus serotypes, by fluorescent focus neutralization and enzyme immunoassay. Two antibodies, Mab RV-3:1 and Mab RV-3:2, reacted with the seven serotype 3 rotaviruses only. Mab RV-3:1 was shown to bind to the outer capsid glycoprotein gp34 of rotavirus when variants of SA 11 rotavirus were used, and it therefore appears to react with the major neutralization epitope of serotype 3 rotaviruses. The antibody Mab RV-3:3 was specific for an epitope of RV-3 rotavirus not present on any other rotavirus of any serotype tested, including another neonatal isolate of identical RNA electropherotype isolated from the same ward of the same hospital as RV-3 3 months earlier. These two viruses were also distinguishable by fluorescent focus neutralization, using antiserum to RV-3 virus. Western blot analysis showed binding of Mab RV-3:3 to the trypsin cleavage product of the outer capsid protein p86 of RV-3. This suggests that antigenic drift may have occurred among neonatal rotaviruses in Melbourne. These monoclonal antibodies will be useful in serotyping assays of rotaviruses directly in stool samples, and in further analysis of antigenic variation within the serotype.     

Role of the L3T4-antigen in T cell activation. II. Inhibition of T cell activation by monoclonal anti-L3T4 antibodies in the absence of accessory cells

We have used two monoclonal antibodies (Mab) to the L3T4 antigen to reexplore the role of this molecule in the process of T cell activation. Both Mab (Gk1.5 and 2B6) were capable of inhibiting Con A-induced IL 2 production by a number of antigen-specific T cell hybridomas in an assay system that was free of major histocompatibility complex (MHC) class II antigen-bearing cells. The inhibition produced by the anti-L3T4 Mab was specific, because other Mab to cell surface antigens expressed on the hybridomas were without inhibitory effects. These studies rule out the possibility that the mechanism of inhibition by anti-L3T4 in this model is mediated by blocking interaction of L3T4 with MHC class II products. Taken together, these results and those of other groups of investigators, are most compatible with a dual function for L3T4 in T cell activation. L3T4 might first interact with MHC class II molecules or other molecules on target or accessory cells; L3T4 would subsequently transmit a signal that would regulate the activation process. Mab to L3T4 might exert inhibitory effects at one or both of these steps.     

Accessory cell-T cell interactions involved in anti-CD3-induced T4 and T8 cell proliferation: analysis with monoclonal antibodies

The effect of monoclonal antibodies (Mab) directed at T cell and accessory cell (AC) surface molecules on OKT3-induced T4 and T8 cell proliferation was examined. Mab directed at nonpolymorphic class I (W6/32, MB40.5) and class II (L243) major histocompatibility complex (MHC)-encoded gene products, an epitope common to LFA-1, CR3, and the p150, 95 molecule (60.3), and a heterodimer present on monocytes (M phi) and activated T cells (4F2) inhibited M phi-supported OKT3-induced proliferation of both T4 and T8 cells. Moreover, an Mab directed at the CD4 molecule (66.1) inhibited OKT3-induced T4 but not T8 cell proliferation, whereas an Mab directed at the CD8 molecule (OKT8) inhibited T8 but not T4 cell responses. With the exception of 66.1, each inhibited OKT3-induced T cell proliferation when added as late as 15 hr after the initiation of culture. Inhibition could not be explained by competition for Fc receptors on the AC. A variety of other Mab including OKT11 and those directed at other HLA-DR and DQ determinants were not inhibitory. The inhibitory Mab were found to diminish T4 cell IL 2 production and IL 2 receptor expression. Consequently, IL 2 reversed some but not all of the Mab-mediated inhibition of T cell proliferation. In contrast to the effects noted with M phi-supported responses, 60.3 and 66.1 but neither L243 nor 4F2 inhibited OKT3-induced T4 cell proliferation supported by Ia- or IFN-gamma-treated Ia+ endothelial cells. None of the Mab tested inhibited T cell proliferation induced by the AC-independent stimuli OKT3 and phorbol myristate acetate (PMA) or calcium ionophore and PMA in the presence or absence of added AC. The data therefore suggest that the Mab inhibit OKT3-induced activation of T4 and T8 cells by preventing necessary interactions between AC and T cell surface proteins. Moreover, the results suggest that different arrays of interaction molecules are involved in OKT3-induced T cell proliferation depending on the nature of the AC and the responding T cell subset.     

Preparation of monoclonal antibodies to nuclear DNA of mammalian cells by immunization with group A streptococcal polysaccharide conjugated with synthetic polyelectrolytes

Monoclonal antibodies (MAb) were obtained by hybridization of spleen cells from BALB/c mice immunized with streptococcal group A polysaccharide (A-PS) conjugated with synthetic polyelectrolytes (PEL). These MAb reacted with nuclei from human and mouse cells. MAb reacting with nuclei were obtained after prolonged immunization with conjugates and were not formed by hybridization of spleen cells from non-immunized mice or by the immunization with PEL. The investigation of Mab (B1/2 and A5/2) reacting with nuclei has shown that these Mab are directed against DNA and do not react with other tissue substances. No cross-reactions of Mab with A-PS used for immunization have been revealed. Mab B1/2 and A5/2 belong to autoantibodies.     

Macrophage antigens associated with adhesion: identification by a monoclonal antibody specific for Lewis lung carcinoma cells

A monoclonal antibody specific for Lewis lung carcinoma (3LL) cells (Mab 5B5) was found to recognize antigens expressed on murine macrophages and on a macrophage hybridoma line upon cell adhesion on plastic surfaces. These antigens were also present on the surface of murine macrophage tumor M5076 cells which develop solid tumors and metastases. The M5076 tumor cells freshly isolated from the primary tumor and from hepatic metastases strongly bound Mab 5B5 but lost this capacity after adhesion. Freshly isolated thioglycolate-elicited peritoneal mouse macrophages were not labeled by Mab 5B5; however, after 1 h of adhesion, 50% of the adherent macrophages were directly incubated with Mab 5B5 prior to harvesting by scraping. Permeabilization of peritoneal macrophages by saponin showed that the antigens recognized by Mab 5B5 were present inside the cells before adhesion. Similar results were obtained with the 2C11-12 macrophage hybridoma cells. P388D1 cells (a weakly adherent macrophage tumor cell line), HL60 cells (a human promyelocytic cell line), and human monocytes were poorly labeled without permeabilization but were strongly labeled by Mab 5B5 upon permeabilization. The specificity of the monoclonal antibody in relation to the adherence capacity of these cells is discussed.     

Localization of atrial natriuretic peptide in pig granulosa cells isolated from ovarian follicles of various size

The aim of the current study was to present the spatial distribution of atrial natriuretic peptide (ANP) in short-term cultures of pig granulosa cells obtained from small, medium, and large ovarian follicles. The specific immunoreactivity was detected by three monoclonal antibodies recognizing different epitopes of the ANP molecule (Mab 6C3, Mab 6F11, Mab5D3). The specific ANP immunoreactivity detected by Mab 6C3 and Mab 6F11 showed dense staining of cytoplasm and was similar in granulosa cells from small and medium follicles. The strongest ANP immunostaining was observed in GC obtained from large follicles. The ANP immunostaining detected by Mab 5D3 had granular appearance moderately expressed in the submembrane region of granulosa cells of all types of follicles. Since ANP and ANP receptors are present in reproductive organs, the three anti-ANP antibodies may be an useful tool in further studies concerning the role of ANP in granulosa cell differentiation and function.     

Transfer of monoclonal antibodies into mammalian cells by electroporation

A simple rapid and reproducible procedure for transferring monoclonal antibodies into mammalian cells by electroporation is described. Two functionally different monoclonal antibodies (Mab 3F3 and Mab 2B4) specific for asparagine synthetase (EC 6.3.1.1) were used for electroporation into HeLa, HT-5, and L5178Y D10/R (L-asparaginase-resistant) cells. The conditions were optimized so that the viability of the electroporated cells was very high (80-90%), and 90% of the viable cells had antibody incorporated. Electropermeabilized cells were structurally intact, and the high voltage electric pulse had no inhibitory effect on overall cellular DNA and protein synthesis. Incorporated immunoglobulins showed unaltered structural integrity and were functionally active. L5178Y D10/R cells incorporated with an antibody (Mab 3F3) known to be a potent inhibitor of tumor asparagine synthetase showed increased dependence on an exogenous source of asparagine in the culture medium, while the growth of cells incorporated with a control (noninhibitory) antibody (Mab 2B4) remained unaffected. These studies demonstrate that electroporation can be employed successfully for large scale transfer of antibodies into cultured mammalian cells for the study of cellular metabolism.     

Induction of antibody responses to influenza virus in human lymphocyte cultures. I. Role of interleukin 2

The in vitro T cell-dependent antibody response of human lymphocytes to influenza virus X31 was used to study the role of T cell-derived lymphokines in antigen-specific responses. Supernatant from cultures of phytohaemagglutinin-stimulated, pooled human tonsil cells (PHA-MLR) was capable of replacing T cells and inducing T-depleted tonsil cells to secrete influenza-specific antibody. The T cell-replacing activity of PHA-MLR supernatant co-purified with interleukin 2 (IL 2) on Ultrogel AcA54 gel filtration and reversed phase-high performance liquid chromatography. PHA-MLR supernatant and IL 2 also enhanced B cell proliferation induced by anti-mu or Staphylococcal aureus strain Cowan I (SAC). A murine monoclonal antibody directed against the human IL 2 receptor (Mab 2A3) was used to completely block the enhancement of influenza-specific antibody production mediated by PHA-MLR supernatant, purified IL 2, and recombinant human IL 2. Mab 2A3 did not affect the T-independent B cell proliferation induced by anti-mu or SAC, but abrogated the enhancing effect of the PHA-MLR supernatant and IL 2 in this culture system. Immunofluorescence studies failed to demonstrate binding of Mab 2A3 to B cells activated by the X31 influenza virus and IL 2, or by SAC. By using Mab 2A3 to mask out IL 2 effects in the influenza-specific culture system, no other B cell differentiating activities were revealed in supernatants from lymphocytic cultures stimulated with a variety of mitogens. Thus, our results indicate that the production of influenza-specific antibodies by T-depleted human lymphocyte cultures is absolutely dependent on the presence of both antigen and IL 2.     

Characterization of NAD(P)H diaphorase from boar spermatozoa using specific monoclonal antibodies.

1. After immunization of BALB/c mouse, four monoclonal antibodies against soluble NADH diaphorase from ejaculated boar spermatozoa were produced and characterized. The monoclonal antibodies were designated as follows Mab 1F2, Mab 4E2, Mab 5B8, Mab 5D8. 2. These monoclonal antibodies react with other enzyme forms-sedimentary NADH and NADPH and soluble NADPH and inhibit (although not completely) their activity. It is supposed that different forms of the enzyme share some common epitopes. 3. Treatment of ejaculated boar semen with 2O-methylcholanthrene causes an increase of the activity of the soluble diaphorase form only. 4. These results lead to the assumption that the sperm diaphorase is a dynamic enzyme system consisting of four immunologically similar isoenzymes although their functions are different.     

CA215 and GnRH receptor as targets for cancer therapy

Two monoclonal antibodies (Mabs), RP215 and GHR106, were selected for the preclinical evaluations of anti-cancer drugs targeting various human cancers including those of the ovary, cervix, lung, and liver. Both Mabs were shown to react with pan cancer markers, which are over-expressed on the surface of almost all human cancers. RP215 Mab was shown to react with the carbohydrate-associated epitope(s) of cancer cell-expressed glycoproteins, mainly consisting of immunoglobulin superfamily (IgSF) proteins and mucins, generally known as CA215. GHR106 Mab was generated against the extracellular domain of human GnRH receptor, which is also highly expressed on the cancer cell surface. Preclinical studies were performed to evaluate the efficacy of these two Mabs as anti-cancer drugs for treating human cancers. High tumor specificity of RP215 Mab was demonstrated with immunohistochemical staining studies of various cancer cell lines, as well as normal and cancerous tissue sections. These two Mabs were shown to induce apoptosis as well as complement-dependent cytotoxicity upon treatment to many cultured cancer cells. Significant dose-dependent growth inhibition of tumor cells from several different tissue origins were demonstrated by nude mouse experiments. It was further demonstrated that GHR106 Mab can function as long-acting GnRH analogs in its biological actions. Efforts were made to generate human/mouse chimeric forms of the GHR106 Mab. Based on the results of these preclinical studies, we believe that these two Mabs, in chimeric or humanized forms, can be developed into suitable therapeutic agents for treatment of human cancers as anti-cancer drugs.     

Monoclonal anti-human tumor antibodies of six isotypes in cytotoxic reactions with human and murine effector cells

Eighty-seven murine monoclonal antibodies (MAb) produced against human tumors of various origins and representing six different immunoglobulin classes were tested for antitumor reactivity in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. Mouse splenocytes, thioglycolate-elicited mouse peritoneal macrophages, freshly obtained nonadherent human peripheral blood lymphocytes, and human monocytes were used as effector cells, and human or rabbit serum as the source of complement. Of all four effector cell types tested, mouse macrophages showed the highest cytotoxic activity, based on net cytotoxicity, minimum requirement for Mab concentration, and effector cell number. Different immunoglobulin classes were associated with characteristic patterns of reactivity with the various effector cells or complement, independent of the target cell type used. MAb able to mediate ADCC were found among all IgG subclasses, with IgG2a and IgG3 MAb inducing lysis with all effector cell types. IgM and IgA MAb were nonreactive in the various ADCC assays, but IgM MAb were highly cytotoxic with complement.     

Monoclonal antibodies against SSEA-1 antigen: binding properties and inhibition of human natural killer cell activity against target cells bearing SSEA-1 antigen

We describe the properties of three monoclonal antibodies (Mab) against stage-specific embryonic antigen-1 (SSEA-1) in terms of their binding activity to HL60, K562, OTF9, and SOTF9 tumor target cells and their functional activity in modulating human natural killer (NK) cytotoxicity assays in vitro against these target cells. Indirect binding, competition, and Western blot analyses indicate that the Mab AEC3A1-9 (3A1), ASSEA-1, and AECAB1-32 (AB1) recognize cell-defined SSEA-1 antigen with activity characteristic of the cell source (HL60 greater than OTF9 greater than K562 much greater than SOTF9). The addition of anti-SSEA-1 Mab to the NK cytotoxicity assay resulted in an inhibition of LU per 1 X 10(6) PBL that correlated closely with the expression of SSEA-1 antigen on the target cell. No significant inhibition was seen for seven other Mab. Inhibition of NK activity (greater than 30%) was observed in the presence of anti-SSEA-1 Mab for 18 of 21 and 6 of 7 human donors examined for HL60 and OTF9 target cells, respectively. The pretreatment of fixed competing cells with anti-SSEA-1 Mab reduced the efficacy of those cells to act as cold competitors in a standard NK cytotoxic assay. Taken together these data suggest that SSEA-1 determinants are important at some stage in the cytolysis produced by NK cells.     

Syngeneic anti-idiotypic monoclonal antibodies against an anti-human chorionic gonadotrophin antibody

Monoclonal antibody designated 1B10 (Mab 1B10) has been shown to be highly specific for the beta-chain of human chorionic gonadotrophin (HCG). We used this antibody to investigate its paratope using anti-idiotypic antibodies. Purified Mab 1B10 has been used to immunize syngeneic BALB/c mice to produce anti-idiotypic monoclonal antibodies. An enzyme immunoassay (ELISA) on Mab 1B10 coated plate was employed to screen the supernatants of growing hybridomas. The specificity of each antibody selected was assessed using an inhibition ELISA and immunoblotting. Monoclonal antibodies belonging to two categories were selected. (a) Those (designated Mab 4F8 and Mab 7G9) recognizing epitopes of the Ig molecule located in/or near the antigen-binding site of Mab 1B10. In ELISA these antibodies were shown to inhibit in a dose-dependent manner, the reaction of Mab 1B10 with its specific antigen; (b) those (Mab 2B8, Mab 3B8) reacting with epitopes located outside of the antigen binding site of the antiHCG antibody molecule and did not influence the reactions of Mab 1B10 and its antigen. Following immunization of syngeneic BALB/c mice monoclonal antibodies (Mab 4F8, Mab 7G9) were produced which recognized epitopes located on the variable region of Mab 1B10 since they did not react with other marine monoclonal antibodies of the same isotype. These antibodies inhibited the binding of Mab 1B10 to its corresponding epitope on the molecule of HCG and they can be defined as syngeneic anti-idiotypic antibodies.     

Inhibition of in vitro tumor cell growth by RP215 monoclonal antibody and antibodies raised against its anti-idiotype antibodies

RP215 monoclonal antibody (Mab) was shown to recognize carbohydrate-associated epitope(s) in the heavy chains of cancer cell-expressed immunoglobulins, designated in general as CA215 pan cancer marker. Growth inhibitions of tumor cells in vitro by RP215 Mab and antibodies against its anti-idiotype (anti-id) antibodies were investigated. Polyclonal rabbit anti-id antibodies and the corresponding rat anti-id Mabs were generated and characterized. Following immunizations in mice, antisera raised against anti-id antibodies were analyzed by typical immunoassays. It was observed that mouse anti-anti-id sera (Ab3) revealed binding affinity and specificity to CA215 that are comparable to those of RP215. Both RP215 and Ab3 were shown to induce apoptosis of cultured cancer cells in vitro by TUNEL and MTT assays. These experimental observations were consistent with that of in vivo tumor growth inhibition by RP215 in previous nude mouse experiments. Therefore, heterologous or homologous anti-id antibodies of RP215 that contain the internal image of its specific epitope in CA215 may serve as effective anti-cancer vaccines for therapeutic treatments of various cancers in humans. The relative stability of RP215-specific carbohydrate-associated epitope was compared to that of human IgG at extreme pH’s (≤2 or ≥12) or following NaIO₄ treatments. The major molecular forms of CA215 were further documented with various enzyme immunoassays and found to have similar secondary structures to those of normal human immunoglobulin G.     

Accessory cell independent proliferation of human T4 cells stimulated by immobilized monoclonal antibodies to CD3

The capacity of the monoclonal antibodies (Mab) 64.1 and OKT3 directed at CD3 molecules to induce T4 cell proliferation and interleukin 2 (IL 2) production was examined. Each was tested in soluble form or was immobilized by adhering it to the wells of plastic microtiter wells. Soluble anti-CD3 did not induce proliferation of accessory cell (AC)-depleted T4 cells. In contrast, immobilized anti-CD3 induced T4 cell IL 2 production and proliferation in the complete absence of AC. When T4 cells were stimulated with high density immobilized anti-CD3, responses did not require AC, IL 2, or Mab directed at the Tp44 molecule (9.3). In contrast, responses stimulated by lower densities of immobilized anti-CD3 were enhanced by IL 2, AC, and 9.3, and with even lower densities of immobilized anti-CD3 proliferation, required these additional signals. A variety of other immobilized Mab directed at T cell surface proteins including class I major histocompatibility complex encoded gene products, CD2, CD5, 4F2, and Tp44, did not induce proliferation even in the presence of IL 2. Anti-CD4 Mab (66.1) inhibited immobilized anti-CD3-stimulated T4 cell responses, with a greater degree of inhibition noted when lower densities of immobilized anti-CD3 were used to stimulate T4 cells. The data demonstrate that stimulation of T4 cells by anti-CD3 is completely AC independent when the antibody is immobilized onto a surface. Furthermore, the results indicate that maximal stimulation requires multiple interactions with anti-CD3 without internalization of the CD3 molecule. The observation that additional signals are required to support T4 cell proliferation when the density of immobilized anti-CD3 is diminished suggests that these are necessary only when insufficient interactions with the CD3 molecule have occurred to transmit a maximal activation signal to the cell. Finally, the results indicate that anti-CD4 provides a direct inhibitory signal to the T4 cell, the effect of which is inversely proportional to the intensity of the activation signal.     

Anti-interleukin 2 receptor antibody suppresses delayed-type hypersensitivity to foreign and syngeneic antigens

Delayed-type hypersensitivity (DTH) requires stimulation of antigen-specific helper T cells (Th). Because de novo expression of the interleukin 2 receptor (IL 2R) is a necessary step in T cell activation, we tested the capacity of anti-mouse IL 2R monoclonal antibody (Mab) and anti-Th Mab (anti-L3T4) to block DTH. We examined the effect of these Mab on two distinct DTH systems, i.e., to foreign hapten (trinitrobenzenesulfonic acid) and to this hapten present on syngeneic blasts. Both anti-IL 2R and anti-L3T4 Mab suppress DTH. Therapy is as effective treating with one injection just before challenge with the hapten as giving six daily injections. These data indicate that DTH is dependent on a discrete subset of activated IL 2R-positive T cells, because anti-IL 2R therapy, which targets few cells, is as effective as anti-L3T4 Mab treatment, which targets the entire Th subset.