Autoimmune effector cells. V.A monoclonal antibody specific for rat helper T lymphocytes inhibits adoptive transfer of autoimmune encephalomyelitis

The in vitro activation of spleen cells from donor rats immunized with myelin basic protein results in enhanced adoptive transfer of experimental allergic encephalomyelitis to syngeneic recipients. In this study, adoptive transfer was inhibited when monoclonal antibody W3/25, which defines an antigen on rat helper T lymphocytes, was included in the in vitro cultures. Partial inhibition was achieved with monoclonal antibody OX-3, which recognizes la antigen, whereas other anti-T cell monoclonal reagents were ineffective. These results support the conclusion that W3/25+T cells play an essential role in the induction of this autoimmune response.     

Interleukin 7 is a potent co-stimulator of myelin specific T cells that enhances the adoptive transfer of experimental autoimmune encephalomyelitis

Interleukin 7 (IL-7), originally described as a B cell growth factor, has recently been found to play a critical role in T and B lymphocyte development and function. This study evaluated the effects of IL-7 on myelin specific T cells. IL-7 strongly enhanced proliferation of proteolipid protein (PLP) 139-151 specific T cells in association with elevated secretion of the T cell growth factor IL-2. Co-stimulation with IL-7 preferentially increased the levels of pro-inflammatory cytokines secreted by PLP 139-151 specific T cells and adoptive transfer of these cells into naive recipients induced a profound enhancement of experimental autoimmune encephalomyelitis, an animal model for the human disease multiple sclerosis. These results suggest that IL-7 may be a critical co-stimulatory factor that enhances the extrathymic expansion of inflammatory T cells and may play an important role in the pathogenesis of a number of inflammatory autoimmune disorders.     

Autoimmune effector cells. III. Role of adjuvant and accessory cells in the in vitro induction of autoimmune encephalomyelitis

The present investigation shows that autoreactive effector cells that transfer experimental allergic encephalomyelitis (EAE) can be activated from spleens and lymph nodes of Lewis rats given a single injection of 25 micrograms myelin basic protein (BP) in incomplete Freund's adjuvant (IFA), despite the fact that the cell donors do not develop EAE. Rather, these donor rats are unresponsive to EAE when given an encephalitogenic emulsion of BP in complete Freund's adjuvant (CFA). Lymphoid cells from rats given a single injection of BP-IFA were almost as effective as cells from BP-CFA-treated rats with respect to transferring EAE after in vitro activation with BP or concanavalin A (Con A). Irrespective of whether donors received BP in IFA or CFA, BP-cultured spleen and lymph node cells (SpC and LNC, respectively) transferred EAE, whereas Con A-cultured SpC but not LNC exhibited effector cell activity. Con A-cultured LNC were able to transfer EAE if the cultures were reconstituted with irradiated adherent phagocytic cells (which could be obtained from normal Lewis rat spleens) or with conditioned medium from these adherent SpC. These findings indicate that accessory cells are required for in vitro induction of this T cell-mediated autoimmune response.     

Autoimmune effector cells. VI. Transfer of experimental allergic encephalomyelitis with spleen cells activated in mixed lymphocyte cultures

Although spleen cells (SpC) from Lewis rats that have been immunized with guinea pig myelin basic protein in complete Freund's adjuvant do not transfer experimental autoimmune encephalomyelitis (EAE) to syngeneic recipients, we report that effector cells of EAE can be activated in SpC suspensions during mixed lymphocyte reactions (MLR) to allogeneic SpC. Effector cell activation correlates with interleukin 2 (IL 2) production. The results are consistent with the hypothesis that autoreactive cells may be generated as a result of an immune response to exogenous antigens.     

Therapy of rat autoimmune disease by a monoclonal antibody specific for T lymphoblasts

The central role of T lymphocytes in the initiation, regulation and propagation of autoimmune diseases defines them as most suitable targets for selective immunotherapy. The recent advance in culturing human and animal T cell lines allows us to select monoclonal antibodies specific for differentiation antigens expressed by activated T lymphocytes. We selected a monoclonal antibody cytotoxic for a subpopulation of activated rat T cells. In vivo, this antibody effectively blocks immune responses to foreign antigens or autoantigen and prevents development of autoimmune diseases like experimental allergic encephalomyelitis and adjuvant arthritis. Even already established disease can be blocked by a single injection of antibody. Furthermore, this monoclonal antibody can be used to monitor the course of autoimmune disease progression from peripheral blood samples.     

Autoimmune effector cells. IV. Induction of experimental allergic encephalomyelitis in Lewis rats without adjuvant

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats without the aid of adjuvant. Radioresistant cells were activated from spleens of nonimmune donor rats by in vitro culture with myelin basic protein (BP). After adoptive transfer, these cells recruited EAE effector cells in normal syngeneic recipients, as demonstrated after transfer to secondary hosts. The induction of EAE without adjuvant may lead to a better understanding of the mechanisms of autoimmune disease induction.     

Equilibrium binding parameters of an autoimmune monoclonal antibody specific for double-stranded DNA

Two techniques have been developed to estimate binding parameters for Jel 241 under equilibrium conditions. Jel 241 is an autoimmune monoclonal antibody derived from an NZB/NZW mouse which binds to double-stranded DNA. Thermal denaturation profiles of poly[d(AT)] were measured in the presence of increasing concentrations of IgG Jel 241. From these data it was estimated that the IgG occludes 12 base-pairs on duplex DNA, and the binding to double-stranded DNA was at least four orders of magnitude greater than to single-stranded DNA. In addition, intrinsic association constants (K(O)) were measured by a gel filtration technique for the interaction of both Fab and IgG Jel 241 to native calf thymus DNA. K(O) for the IgG was only 60-fold greater than for the Fab fragment for which K(O) was 4.4 X 10(4) M-1 at an NaCl concentration of 150 mM. Also, K(O) for the Fab increased dramatically with decreasing ionic strength, suggesting that there are four phosphates involved in the interaction. These techniques should be applicable to most autoimmune antibodies which bind to nucleic acid polymers.     

Inhibition of human vitamin-K-dependent protein-S-cofactor activity by a monoclonal antibody specific for a Ca2+-dependent epitope

Protein S is an anticoagulant vitamin-K-dependent plasma protein functioning as a cofactor to activated protein C in the degradation of factors Va and VIIIa. A murine monoclonal antibody, HPS 7, specific for a calcium-stabilized epitope in human protein S, is described. The epitope was available in intact protein S, both in its free form and when protein S was bound to C4b-binding protein. It disappeared upon reduction of disulfide bridges and also after thrombin of chymotrypsin cleavage of protein S. Thrombin cleaves protein S close to the calcium-binding region containing gamma-carboxyglutamic acid (Gla). The cleaved protein still contains the Gla region, linked by a disulfide bridge, but it has a lower affinity for calcium and no protein C cofactor activity. The thrombin-mediated cleavage of protein S could be inhibited by HPS 7. The Ka for the interaction between protein S and the monoclonal was estimated to be approximately 0.7 X 10(8) M-1. Half-maximal binding between HPS 7 and protein S was observed at a calcium concentration of 0.50 mM, indicating that saturation of the Gla region with calcium was required for the interaction. The recently reported Gla-independent high-affinity calcium binding did not induce the epitope. The calcium-dependent binding of protein S to phospholipid vesicles as well as the protein C cofactor activity was inhibited by HPS 7. The data suggests that the epitope for HPS 7 is located in the Gla region of protein S or in the closely positioned thrombin-sensitive region.     

Importance of immunoglobulin isotype in therapy of experimental autoimmune encephalomyelitis with monoclonal anti-CD4 antibody

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by CD4+ T cells. Prior studies have established that monoclonal anti-CD4 antibodies can reverse EAE. To determine whether immunoglobulin isotype plays a role in the therapy of EAE with anti-CD4 antibody, an isotype switch variant family of the mouse IgG1 anti-rat CD4 antibody W3/25 was isolated with the fluorescence-activated cell sorter. The IgG1, IgG2b, and IgG2a W3/25 isotype variants all had identical binding capacities for rat CD4+ T cells. Although all three W3/25 isotypes showed some beneficial effects in the amelioration of EAE, the IgG1 and IgG2a W3/25 antibodies were superior to the IgG2b W3/25 in the treatment of EAE. Multiparameter fluorescence-activated cell sorter analysis of T cell subpopulations from treated rats showed that none of the antibodies of the W3/25 isotype switch variant family substantially depleted CD4+ target cells in vivo. These experiments demonstrate that immunoglobulin isotype is important in the monoclonal antibody therapy of autoimmune disease. They indicate that therapy of EAE may be successful without a major depletion of CD4+ lymphocytes. Immunotherapy may be optimized by selecting an appropriate isotype of a monoclonal antibody.     

A monoclonal antibody recognizing an epitope shared by receptors for growth hormone,prolactin, interleukin 2 and interleukin 6

Many membrane proteins are implicated in the regulation of cell functions by triggering specific signaling pathways. Porins are known potential modulators of cell proliferation and differentiation. We explored the possible involvement of this protein in signal transduction pathways in mouse gut macrophages. In the present work we have shown that porins can trigger signal transduction in mouse macrophages infected with S. typhimurium. Activation of macrophages by porins results in an increase in inositol trisphosphate and intracellular Ca2+ mobilization. There is a translocation of protein kinase C to the membrane which is accompanied by nitric oxide release within the macrophages. This effect is the outcome of the expression of nitric oxide synthase, which is dependent on Protein kinase C. Further, we observed that there is an increased binding of the porins on macrophages infected with S. typhimurium which results in activation of macrophages and triggering of specific signaling pathways. These results indicate that porins induce the production of nitric oxide via a protein kinase C dependent pathway. Nitric oxide plays a fundamental role in macrophage effector function where it has both communication and defensive function.     

Autoimmune effector cells. VIII. Cellular requirements for the induction of autoreactive T cells of experimental allergic encephalomyelitis in nonimmune rats

We utilized a system of sequential in vitro cell culture and adoptive transfer to investigate the sequence of events which lead to the activation of effector cells responsible for the induction of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. This procedure involves only naive (nonimmune) rats, and eliminates the requirement for adjuvants. Spleen cells (SpC) from naive donors were sensitized in vitro to myelin basic protein (BP), then transferred to intermediate (primary) hosts. Although these recipients did not develop EAE, they were primed for disease because they exhibited accelerated onset of active EAE when challenged with BP in adjuvant. Moreover, SpC from nonchallenged primary recipients transferred EAE to secondary recipients subsequent to in vitro exposure to antigen. The cells from the naive cultures which primed the intermediate recipients were radioresistant (1500 R); other studies have indicated that these are macrophages. In contrast, the cells which transferred EAE to the secondary recipients were radiation-sensitive T lymphoblasts. The finding that these cells also elicit disease in lethally irradiated (850 R) secondary recipients suggests that the transferred cells either are the actual effector cells of EAE or induce disease in collaboration only with radioresistant host cells.     

Production of a monoclonal antibody specific for Salmonella spp.

A monoclonal antibody (MAb) specific for heat-treated Salmonella spp. which shows no cross-reactivity with other enteric bacteria when tested by an ELISA system, has been developed. The MAb isotype is IgG2a and has been successfully purified using protein A. Preliminary work suggests that the MAb recognizes a protein present in the Boivin lipopolysaccharide extracts of Salmonella spp.     

In vitro sensitization and expansion with viable tumor cells and interleukin 2 in the generation of specific therapeutic effector cells

We have investigated the efficacy and immunologic characteristics of immune effector cells generated from cultures containing large numbers of viable tumor cells and interleukin 2 (IL 2) in the adoptive immunotherapy of experimentally induced pulmonary metastases from the newly developed, weakly immunogenic MCA 105 sarcoma in mice. The current culture conditions allowed increases of either normal or MCA 105 immune spleen cells up to 94-fold in 15 days. The in vitro expanded normal and MCA 105 immune cells displayed nonspecific in vitro cytotoxicity against several syngeneic tumor targets. However, therapeutically effective cells could only be obtained from cultures initiated with MCA 105 immune spleen cells. Immunotherapy with expanded immune effector cells could lead to the reduction of established 3 day pulmonary metastases, prolongation of survival, and cure of tumor in the majority of animals. The generation and proliferation of therapeutic effector cells in vitro depended on the presence in cultures of specific tumor stimulator cells as well as the presence of IL 2. Although immunotherapy with either fresh noncultured or secondarily in vitro-sensitized (IVS) MCA 105 immune spleen cells was immunologically specific, the efficacy of the adoptive cellular therapy with cultured but not fresh immune cells could be improved by the administration to tumor-bearing hosts of exogenous IL 2. In addition to numerical expansion, the IVS immune cells, on a per cell basis, afforded an eightfold to 10-fold increase in therapeutic efficacy when compared with fresh noncultured MCA 105 immune cells. Our results indicate that the current culture procedure induced in vitro antigenic stimulation and expansion of tumor-specific immune effector cells that was otherwise not possible by conventional mixed lymphocyte-tumor cultures.     

Successful adoptive immunotherapy of murine poorly immunogenic tumor with specific effector cells generated from gene-modified tumor-primed lymph node cells

We previously reported that cytokine gene transfer into weakly immunogenic tumor cells could enhance the generation of precursor cells of tumor-reactive T cells and subsequently augment antitumor efficacy of adoptive immunotherapy. We investigated whether such potent antitumor effector T cells could be generated from mice bearing poorly immunogenic tumors. In contrast to similarly modified weakly immunogenic tumors, MCA102 cells, which are chemically induced poorly immunogenic fibrosarcoma cells transfected with cDNA for IL-2, IL-4, IL-6, IFN-gamma, failed to augment the host immune reaction. Because priming of antitumor effector T cells in vivo requires two important signals provided by tumor-associated Ags and costimulatory molecules, these tumor cells were cotransfected with a B7-1 cDNA. Transfection of both IFN-gamma and B7-1 (MCA102/B7-1/IFN-gamma) resulted in regression of s.c. tumors, while tumor transfected with other combinations of cytokine and B7-1 showed progressive growth. Cotransfection of IFN-gamma and B7-1 into other poorly immunogenic tumor B16 and LLC cells also resulted in the regression of s.c. tumors. Cells derived from lymph nodes draining MCA102/B7-1/IFN-gamma tumors showed potent antitumor efficacy, eradicating established pulmonary metastases, but this effect was not seen with parental tumors. This mechanism of enhanced antitumor efficacy was further investigated, and T cells with down-regulated L-selectin expression, which constituted all the in vivo antitumor reactivity, were significantly increased in lymph nodes draining MCA102/B7-1/IFN-gamma tumors. These T cells developed into potent antitumor effector cells after in vitro activation with anti-CD3/IL-2. The strategy presented here may provide a basis for developing potent immunotherapy for human cancers.     

Adoptive transfer of experimental allergic encephalomyelitis: incubation of rat spleen cells with specific antigen.

Spleen cells from myelin basic protein (BP)-sensitized donor rats appear to be incapable of adoptively transferring experimental allergic encephalomyelitis (EAE) directly to normal recipients. It has been reported that in vitro incubation with concanavalin A (Con A) activates rat spleen cells so that they are capable of transferring EAE. We report here that incubation with specific antigen, BP, also permits transfer of disease with spleen cells. Data are presented in which activation of EAE spleen cells by Con A is compared with activation by BP. Cellular proliferation does not appear to be necessary for in vitro activation with specific antigen.     

Cytometrically detected specific binding of interleukin 2 to cells.

We have developed a new technique for detecting binding of interleukin 2 (IL-2) to cells. This technique involves incubating the cells with IL-2 and then analysing the cell surface with specific anti-IL-2 antibodies and flow cytometry. This binding was only detected on tumor cells that possessed the p55 subunit of the IL-2 receptor. The role of p55 was ascertained by inhibition of the binding with a monoclonal antibody to p55. Although p55 is necessary for cytometrically detected IL-2 binding, further studies demonstrated that p55 is not sufficient. Thus, cytometrically-detected binding is likely to involved the contribution of other IL-2 surface receptors. Interleukin-2 binding to peripheral blood T lymphocytes and to a non-transformed T-cell clone was also detected cytometrically and it was shown that this binding is regulated by the activation status of the cells. Whereas IL-2 binding to quiescent T cells could not be detected, upon activation abundant binding was seen. The functional consequences of this type of cellular binding were studied. Interleukin-2 binding to cells during a short pulse was shown to have significant long-term consequences both for T-cell proliferation and for the enhancement of major histocompatibility complex (MHC)-non-restricted cytotoxicity.     

CD4+ suppressor cells inhibit the function of effector cells of experimental autoimmune encephalomyelitis through a mechanism involving transforming growth factor-beta.

Nylon wool adherent, CD4+ T cells from the spleens of rats that have recovered from experimental autoimmune encephalomyelitis (EAE) inhibit the in vitro production of IFN-gamma, but not IL-2, by effector cells of EAE when cocultured in the presence of myelin basic protein Ag. When anti-transforming growth factor-beta (TGF-beta) antibodies are added to the co-cultures, IFN-gamma production is restored to normal levels. Irrelevant control antibodies have no effect. The same pattern of response was obtained with cells incubated in serum-free medium. In other experiments, purified TGF-beta was added to cultures of effector cells in the presence of antigen. TGF-beta inhibited the production of IFN-gamma by these cells in a dose-dependent manner, but had no apparent inhibitory effect on IL-2 production. Finally, supernatants from cultures containing effector cells and CD4+ suppressor cells plus Ag contained measurable amounts of TGF-beta, whereas supernatants from cultures of effector cells plus Ag contained no measurable amounts of TGF-beta. These results suggest that CD4+ Ts cells of EAE regulate effector cells of EAE through a mechanism that involves the secretion of TGF-beta and that the inhibitory function of this cytokine can be reversed with neutralizing antibodies directed against TGF-beta.     

Inhibition of iodothyronine transport into rat liver cells by a monoclonal antibody

The role of the rat liver plasma membrane in the regulation of uptake and subsequent deiodination of thyroxine (T4) or the biologically active thyroid hormone 3,3',5-triiodothyronine (T3) was investigated. Here we report on the production of monoclonal antibodies raised against rat hepatocytes. Two antibodies were selected. Antibody ER-22 did bind to a Mr 52,000 membrane protein and inhibited the 1- and 5-min uptake of both T4 and T3 by primary cultured rat hepatocytes in a dose-dependent fashion. As the uptake of T4 and T3 depends on the presence of a sodium gradient over the plasma membrane, the inhibitory potency of ER-22 on the Na+,K+-ATPase activity was investigated. No inhibition of the uptake of 86Rb+ could be determined, indicating that antibody ER-22 is not directed against the Na+,K+-ATPase but probably the carrier protein itself. Clearance of T3 from the medium and concomitant iodide production by cultured rat hepatocytes during a 20-h incubation in the presence of ER-22 were both inhibited by 50% with respect to a control incubation in the absence of monoclonal antibody, pointing to the importance of carrier-mediated transport in cellular uptake and metabolism of T3. A second monoclonal antibody did bind to two other plasma membrane proteins but did not inhibit transport of thyroid hormone.     

Administration of agonistic anti-4-1BB monoclonal antibody leads to the amelioration of experimental autoimmune encephalomyelitis.

4-1BB, a member of the TNFR superfamily, is a costimulatory receptor primarily expressed on activated T cells. It has been shown that the administration of agonistic anti-4-1BB Abs enhances tumor immunity and allogenic immune responses. Paradoxically, we found that the administration of an agonistic anti-4-1BB mAb (2A) dramatically reduced the incidence and severity of experimental autoimmune encephalomyelitis (EAE). Adoptive transfer of T cells from such treated mice failed to induce EAE, whereas anti-4-1BB treatment following adoptive transfer of encephalitogenic T cells did not prevent EAE pathogenesis. These results suggest that anti-4-1BB treatment during the induction phase inhibits autoreactive T cell immune responses rather than preventing T cell trafficking into the CNS. This was substantiated by the observations that draining lymph node cells from anti-4-1BB-treated mice failed to respond to Ag stimulation in vitro. In addition, we found that such treatment initially promotes the activation and proliferation of Ag-specific CD4+ T cells but subsequently increases their probability of undergoing activation-induced cell death, thereby inhibiting effector T cell responses. More importantly, 2A treatment also inhibits the relapse of EAE in a clinically relevant murine model of multiple sclerosis. This study indicates that the agonistic Ab against 4-1BB can potentially be used as a novel immunotherapeutic agent for treating autoimmune diseases.     

Protection from experimental autoimmune thyroiditis conferred by a monoclonal antibody to T cell receptor from a cytotoxic hybridoma specific for thyroglobulin.

A clonotypic mAb, AG7, has been prepared from splenocytes of CBA/J mice immunized with a cytotoxic T cell hybridoma, HTC2, specific for a pathogenic epitope of the thyroglobulin molecule in association with class I MHC Ag. AG7 binds to HTC2 cells but not to the other T cell hybridomas tested. Moreover, when used in functional studies, AG7 blocks the HTC2 capacity of specific target lysis. It also reacts with a determinant that comodulates with the CD3 Ag present on the surface of HTC2 cells. Immunoprecipitation of 125I-labeled solubilized HTC2 membranes demonstrated two bands located at 90 and 72 kDa under nonreducing conditions, which became a 46-kDa band under reducing conditions. Finally, when AG7 is injected into CBA/J mice, on day -1 before immunization with the pathogenic tryptic fragments of the thyroglobulin molecule, experimental autoimmune thyroiditis is abrogated. Thus, one of the multiple potential mechanisms of the protective immunity against EAT induced by HTC2 cells that we previously proposed, i.e., the generation of anti-clonotypic antibodies to HTC2 TCR, seems apparent.