Mesenchymal stem cells: stealth and suppression.

Human post-natal bone marrow contains mesenchymal stem cells (MSCs), which are capable of giving rise to multiple mesenchymal cell lineages. Large quantities of human MSCs can be readily obtained following a simple bone marrow aspiration procedure and subsequent expansion over a million-fold in culture. This extensive capacity for clinical scale expansion in vitro has facilitated the development of preclinical models as well as clinical studies designed to assess the safety, feasibility, and efficacy of transplanting allogeneic MSCs for a variety of indications. This review focuses on the rationale for performing clinical studies of MSC transplantation and will discuss the potential role that MSCs may have in the field of hematopoietic stem cell transplantation as well as for the repair or regeneration of bone, cartilage, and cardiac tissues.

Induction of apoptosis in activated T cell blasts by suppressive macrophages: a possible immunotherapeutic approach for treatment of autoimmune disease.

Large suppressive macrophages (LSM) were induced by restimulating spleen cells from rats with experimental autoimmune myasthenia gravis (EAMG) in vitro, with the autoantigen acetylcholine receptor (AChR) in the presence of cyclosporine A. LSM, purified from these cultures, are extremely potent suppressors of AChR-stimulated lymphoproliferative responses and antibody responses in vitro. In the present study, we have analyzed the factors that determine susceptibility of primed lymph node cells (pLNC) to suppression by LSM and examined the fate of these cells. We found three characteristics of pLNC that influenced their susceptibility to suppression. First, pLNC were required to be activated (by antigen in these experiments) in order for suppression to occur. Resting lymphocytes were not affected, even when they were present in cultures where antigen-activated lymphoblasts were being actively suppressed. Second, antigen specificity of the responder cells influenced their susceptibility to suppression by LSM. AChR-specific cells were relatively more susceptible to suppression by AChR-induced LSM than pLNC primed to an unrelated antigen, keyhole limpet hemocyanin. Third, T cell proliferation was suppressed by LSM to a far greater extent than antibody production by B cells. Using enriched T cell blasts generated from AChR-stimulated T cell lines, we found that LSM rapidly suppressed [3H]TdR uptake and induced DNA fragmentation assessed by the TUNEL assay (within 8 h of coculture) and induced morphological signs of apoptosis of T cells (within 24 h). Few, if any, blasts remained by 48 h of coculture. The ability to suppress an activated immune response permanently, without affecting nonactivated, bystander lymphocytes, holds promise that LSM, or their cellular products, could be used for immunotherapy of autoimmune diseases such as myasthenia gravis.

Factors that determine the severity of experimental myasthenia gravis.

Based on our current information, the robust differences in responses of B6 and bm12 mice after immunization with AChR are as follows: (1) The AChR-specific T cell repertoires are strikingly different. The epitope specificities, as well as the rearranged TCR alpha and beta chains and their CDR3 domains, are virtually nonoverlapping in the two strains of mice. (2) The AChR antibody responses are quantitatively different, both to Torpedo AChR and to the autoantigen--mouse AChR. (3) The isotype distribution of AChR antibodies favors IgG2b in B6 mice, but not in bm12 mice. (4) The clinical manifestations of EAMG are qualitatively and quantitatively different in the two strains. These considerations have led to the following scheme, illustrated diagrammatically in FIGURE 2, to explain the differences in EAMG in B6 and bm12 mice: (1) The MHC Class II of B6 mice binds the alpha 146-162 peptide of Torpedo AChR with high affinity, while the genetically altered MHC Class II of bm12 mice does not, as previously suggested (see FIGURE 2). (2) The alpha 146-162/MHC Class II complex occurs only in B6 mice and interacts with T cells having appropriate TCRs, resulting in their stimulation and expansion. Although T cells of appropriate specificity are also available in the bm12 strain, the relevant peptide/MHC Class II complex is not present. Therefore, very few T cells with specificity for alpha 146-162 are stimulated, and those that are stimulated have different TCRs. T cells with specificity for other AChR peptides are also present and expanded in both strains of mice, but they have less influence on the outcome of the immune response. (3) The alpha 146-162-specific T cells of B6 mice, in turn, interact strongly with AChR-specific B cells of B6 mice. These B cells present the same epitope/MHC Class II complex as the APCs and therefore interact well with the alpha 146-162-specific T cells (FIGURE 2). Thus, T cells of this specificity appear to provide more efficient help for AChR antibody production than T cells with specificity for other Torpedo AChR epitopes. This results in production of greater amounts of AChR antibodies, including a critical subset that cross-reacts with autologous mouse AChR. The higher autoantibody levels contribute to the greater susceptibility to EAMG and to the greater severity of manifestations in the B6 strain compared with the bm12 strain. (4) There is a bias in B6 mice toward the production of AChR antibodies of IgG2b isotype. We suggest that T cells specific for alpha 146-162 may contribute to this isotype bias. The IgG2b antibodies appear to have particularly potent "myasthenogenic" effects in rats and mice. (5) Finally, it should be emphasized that these differences in immunological and clinical aspects of EAMG in B6 and bm12 mice are relative rather than absolute. T cells that respond to AChR epitopes other than alpha 146-162 can also provide help for AChR antibody production, albeit less potent. In a sense, this model represents a special case of molecular mimicry. In this case, the source of the foreign antigenic molecule is injection rather than the more usual route of infection. The antigen (Torpedo AChR) is one that these mice would never naturally encounter, and the critical amino acid (lysine 155) of the key epitope (alpha 146-162) is present only in the AChR of electric organs of electric fish and not in the AChR of mice, chickens, cows, or humans. The important point is that a detail of the structure of the foreign antigen--that is, a particular peptide of Torpedo AChR--can determine the severity of an antibody-mediated autoimmune disease, depending on how it interacts with a detail of the structure of the MHC Class II molecule and, in turn, on how the peptide/MHC Class II complex interacts with the available T cell repertoire. (ABSTRACT TRUNCATED)

Immunotherapy of experimental autoimmune myasthenia gravis: selective effects of CTLA4Ig and synergistic combination with an IL2-diphtheria toxin fusion protein.

We examined the effects of CTLA4Ig treatment in an experimental model of myasthenia gravis (EAMG) induced by immunizing Lewis rats with purified Torpedo acetylcholine receptor (AChR). During a primary response, CTLA4Ig treatment inhibited AChR antibody production profoundly, and induced a shift of AChR antibody isotypes from the normally predominant IgG2 isotype pattern toward an IgG1 response. Challenge of rats previously treated with CTLA4Ig produced markedly lower AChR antibody responses compared to untreated controls, persistent inhibition of the IgG2b isotype, and no development of EAMG. Treatment of a secondary AChR response with CTLA4Ig or with DAB389IL2 (which kills lymphocytes expressing IL2 receptors) inhibited AChR antibody responses, and clinical EAMG moderately. In contrast, combined treatment with CTLA4Ig plus DAB389IL2 strikingly inhibited AChR antibody levels, and completely prevented EAMG. Our results suggest that the therapeutic benefit of CTLA4Ig may be due to overall inhibition of AChR antibody production as well as a shift in the antibody isotype repertoire.

How subtle differences in MHC class II affect the severity of experimental myasthenia gravis.

Myasthenia gravis is an autoimmune disorder characterized by muscle weakness, due to an antibody-mediated deficit of acetylcholine receptors (AChRs) at neuromuscular junctions. We analyzed the factors that determine the severity of experimental myasthenia gravis (EAMG) induced by immunization with Torpedo AChR, in two congenic strains of mice--B6 mice, which are highly susceptible to EAMG; and bm12 mice, which are relatively resistant, and differ only in a change of three amino acids in MHC Class II. We prepared large numbers of AChR-specific T cell hybridomas from each strain and characterized their epitope specificities and T cell receptor (TCR) gene usage: Half the B6 hybridomas responded to a single AChR peptide (alpha 146-162), and their TCR genes encoded restricted V alpha and V beta chains and CDR3 motifs. bm12 hybridomas had different epitope specificities and different, less restricted TCR genes. APCs were able to present AChR or AChR-derived peptides virtually exclusively to hybridomas of their own strain. Levels of antibodies to Torpedo and autoantibodies to mouse AChR were higher in B6 mice, and were biased toward the IgG2b isotype. We conclude that the "better fit" of MHC II, peptide, and TCR in the B6 mice enhanced cognate interactions of APCs with T cells, and T cells with B cells, resulting in a more abundant and pathogenic AChR antibody response, and thus more severe EAMG.

Oral tolerance in myasthenia gravis.

Because of the antibody-mediated pathogenesis of MG, it is of particular interest to understand the effects of oral administration of the autoantigen AChR on the disease process. It is now clear that feeding AChR prior to immunization can prevent clinical manifestation of EAMG. It initially primed, then inhibited, antibody responses to foreign (Torpedo) AChR and self (rat) AChR, with a delayed onset. Cellular responses to AChR, evaluated by lymphocyte proliferation and IL-2 production, were markedly inhibited. The effects were dependent on the dose and purity of the fed antigen. Tolerance to an orally administered unrelated antigen, OVA, was more prompt in development and more profound, illustrating the influence of the nature of the antigen on tolerance. The tolerance induced was antigen specific. Oral administration of AChR after immunization resulted in inhibition of the clinical manifestation of EAMG, concomitant with a paradoxical enhancement of the AChR-antibody responses. Both the clinical benefit and the antibody response appear to be dependent on the feeding protocol. These findings suggest that a molecule with less immunogenic potential than native AChR may be required for safe and effective oral treatment of ongoing disease.

Immunosuppression and induction of anergy by CTLA4Ig in vitro: effects on cellular and antibody responses of lymphocytes from rats with experimental autoimmune myasthenia gravis.

The pathogenic antibody response to acetylcholine receptor (AChR) in experimental autoimmune myasthenia gravis (EAMG) is T cell dependent. Therefore, it should be possible to design specific immunotherapeutic approaches to treat EAMG (and human MG) by interfering with AChR-specific helper T cells. Productive T cell activation by antigen requires at least two signals: one signal delivered through the T cell receptor by antigen and a second costimulatory signal delivered through the CD28 receptor via the B7 counterreceptor expressed on antigen-presenting cells. Here we show that interference with the B7 costimulatory signal, using a soluble CD28 analogue, CTLA4Ig, resulted in a profound decrease in IL2 production and significantly decreased lymphoproliferative responses and antibody responses by primed lymph node cells from rats with EAMG, when stimulated with AChR in vitro. Nonclonal AChR-specific T cell lines, when stimulated with AChR in the presence of CTLA4Ig, were also inhibited in their ability to proliferate and to produce the cytokines IL2 and IFN-gamma. They remained deficient in their ability to produce IL2 when restimulated with AChR plus fresh antigen-presenting cells and showed variable inhibition of proliferation. The induction of hyporesponsiveness was accompanied by the expression of functional IL2 receptors, as shown by vigorous proliferative responses to addition of exogenous IL2. These results indicate that specific antigen stimulation in the presence of CTLA4Ig can induce certain features typical of anergy. CTLA4Ig provides a promising approach for the immunomodulation of MG and other antibody-mediated autoimmune diseases.

Tolerance to acetylcholine receptor induced by AChR-coupled syngeneic cells.

Intravenous administration of acetylcholine receptor (AChR)-coupled syngeneic spleen cells induced AChR-specific tolerance in Lewis rats. Injection of 20 x 10(6) AChR-coupled spleen cells resulted in a significant reduction of the antibody response to AChR when the animals were subsequently immunized with AChR (up to 66% inhibition). Tolerance induced by AChR-coupled cells was antigen-specific: challenge with AChR plus an unrelated antigen, ovalbumin (OVA), resulted in significantly reduced serum antibody titers to AChR but normal or enhanced titers to OVA, compared to controls. Lymphocytes from these rats showed substantially reduced proliferative responses in vitro to AChR, and normal or enhanced responses to OVA. To investigate the role of Ia antigens in tolerance induction, AChR was coupled to Ia-positive or Ia-negative spleen cells. Injection of AChR-coupled Ia-positive cells induced antigen-specific tolerance to AChR; challenge with AChR plus OVA resulted in reduced serum antibody titers and lymphoproliferative responses to AChR, and normal or enhanced titers and cellular responses to OVA. Injection of AChR-coupled Ia-negative cells did not induce tolerance to AChR. These results demonstrate that AChR, which is known to be highly immunogenic, can be rendered tolerogenic without denaturation or linkage to toxic substances. The possible mechanisms of tolerance induced by AChR-coupled spleen cells are discussed.

Suppression of immune responses to acetylcholine receptor by interleukin 2-fusion toxin: in vivo and in vitro studies.

The pathogenesis of the autoimmune disease, myasthenia gravis (MG), involves an antibody-mediated attack against acetylcholine receptors (AChRs). Since the relevant antibody response is T cell dependent, a therapeutic strategy aimed at T lymphocytes actively participating in the immune reaction to AChR should result in relatively selective suppression of AChR antibody. During an active immune response, T cells express receptors for interleukin 2 (IL2). In this study, we have used a genetically engineered fusion protein comprised of the binding region of IL2 and the toxic portion of diphtheria toxin (DAB486-IL2), to attempt to treat an experimental animal model of MG in rodents. We examined the effects of treatment with DAB486-IL2 in vivo on primary, ongoing, and secondary antibody responses to purified Torpedo AChR. Treatment of mice with intraperitoneal injections of DAB486-IL2 beginning at the time of immunization inhibited the primary AChR antibody response by 50% during the treatment period. Ongoing and secondary antibody responses to AChR were not suppressed in vivo by treatment with DAB486-IL2. In comparison, DAB486-IL2 was far more potent in suppressing antibody responses and lymphoproliferation in cell culture. At a dose comparable to that given in vivo, cellular proliferation and antibody production were virtually eliminated in a secondary response in vitro. The suppressive effect of DAB486-IL2 was much more pronounced when it was given at the time of initial antigen stimulation, as compared with its effect when given during an already established antibody response. These findings suggest that the effect of the fusion toxin on AChR antibody production was due predominantly to inhibition of T cells rather than B cells.

T-cell vaccination in experimental myasthenia gravis: a double-edged sword.

Immunization with antigen-specific T cells has been used successfully in the treatment of several T cell-mediated experimental autoimmune diseases, including experimental allergic encephalomyelitis, thyroiditis, and adjuvant arthritis. The aim of this study was to determine whether T-cell vaccination could be used to down-regulate specifically the antibody response to AChR in experimental autoimmune myasthenia gravis (EAMG), an antibody-mediated disorder. We produced T cells specific for the acetylcholine receptor (AChR) by immunizing Lewis rats with torpedo AChR, harvesting the regional lymph node cells, and restimulating them in vitro with AChR. This cell population was expanded with IL2. The cells were then activated with concanavalin A (Con-A) and exposed to high hydrostatic pressure to augment their immunogenicity. We found that rats vaccinated with these cells did not manifest decreased antibody titers to AChR, when challenged. In fact, the antibody response to AChR was consistently potentiated by the vaccine treatment. This result could not be attributed to antigen carryover by the vaccinating cells or to induction of anti-idiotypic antibodies. Despite these results showing overall enhancement of the AChR antibody response, we found evidence of AChR-specific suppressor cells in the spleens of the vaccinated animals. Our observations indicate that T-cell vaccination can elicit both a positive immune response and a suppressive response in the same animal. If the T-cell vaccination strategy is to be useful for the treatment of MG, methods for amplifying the suppressive effect will need to be developed.

Antigen-specific suppressor macrophages induced by culture with cyclosporine A plus acetylcholine receptor.

Suppressor cells specific for the acetylcholine receptor (AChR) can readily be induced by culturing spleen cells from rats with experimental autoimmune myasthenia gravis in medium containing cyclosporine A plus AChR. We describe here the purification and characterization of novel antigen-specific suppressor cells, which appear to be macrophages, from these cultures. The cells were adherent to plastic, very large, and of low buoyant density. These properties were used to enrich the cells, first by adherence, followed by centrifugation on discontinuous Percoll density gradients. The enriched large suppressor cells (LSC) were shown to be potent suppressors of secondary immune responses to AChR, including both lymphoproliferation and antibody production. Furthermore, the LSC exhibited antigen specificity of suppression: LSC induced with AChR suppressed secondary antibody responses to AChR significantly more than to an unrelated antigen, keyhole limpet hemocyanin (KLH). In reciprocal experiments, LSC induced with KLH suppressed KLH responses significantly more than AChR responses. The LSC exhibited the morphologic, enzymatic, phenotypic, and radioresistance characteristics of macrophages. They were neither lymphocytes nor NK cells as defined by phenotypic and functional properties, respectively. Several possible mechanisms are postulated to explain how these remarkable cells may acquire antigen specificity.

The effect of thalidomide on experimental autoimmune myasthenia gravis.

Thalidomide is reported to have immunosuppressive and anti-inflammatory effects which have led to its use in the treatment of a number of immune-mediated disorders including leprosy, prurigo, discoid lupus, and Behcet's disease. In addition, thalidomide has recently been used to prevent immunological rejection phenomena following skin and bone-marrow grafts. The immune responses in these conditions are thought to be cell-mediated. However, little is known about the effectiveness of thalidomide in suppressing antibody-mediated immune responses. In the present study, we have examined the effect of thalidomide in a model antibody-mediated autoimmune disorder--experimental autoimmune myasthenia gravis (EAMG). To induce EAMG, Lewis rats were immunized with acetylcholine receptor (AChR) purified from the electric organ of Torpedo californicus. Groups of rats were treated daily, either with thalidomide in excess of doses reported to prevent graft-versus-host (GVH) disease in bone-marrow-transplanted rats, or with control treatments. Our results show that thalidomide failed to inhibit AChR antibody production despite good absorption and high blood levels of the drug. This suggests that thalidomide is not likely to be generally useful in the treatment of antibody-mediated autoimmune conditions. However the selective effect of thalidomide in suppressing certain presumably cellular immune responses, while sparing antibody production, is inherently interesting, and merits further study.

Treatment of experimental myasthenia gravis with total lymphoid irradiation.

Total lymphoid irradiation (TLI) has been reported to be effective in the immunosuppressive treatment of certain human and experimental autoimmune disorders. We have investigated the effects of TLI in Lewis rats with experimental autoimmune myasthenia gravis (EAMG) produced by immunization with purified torpedo acetylcholine receptor (AChR). The radiation is given in 17 divided fractions of 200 rad each, and nonlymphoid tissues are protected by lead shielding. This technique suppresses the immune system, while minimizing side effects, and permits the repopulation of the immune system by the patient's own bone marrow cells. Our results show that TLI treatment completely prevented the primary antibody response to immunization with torpedo AChR, it rapidly abolished the ongoing antibody response in established EAMG, and it suppressed the secondary (anamnestic) response to a boost of AChR. No EAMG animals died during TLI treatment, compared with six control animals that died of EAMG. TLI produces powerful and prompt immunosuppression and may eventually prove useful in the treatment of refractory human myasthenia gravis.

Induction of suppressor cells specific for AChR in experimental autoimmune myasthenia gravis.

Suppressor cells specific for acetylcholine receptor (AChR) were induced in a population of lymphocytes previously sensitized to AChR, obtained from rats with experimental autoimmune myasthenia gravis (EAMG). The lymphocytes were cultured with the immunosuppressive drug cyclosporin A plus purified AChR for 7 days. These cells, when mixed with lymphocytes from rats with EAMG in vitro, strongly suppressed the antibody response to AChR. They did not inhibit antibody responses to an unrelated antigen, an indication that suppression was specific for AChR. This approach should be a useful way to induce specific suppressor cells from sensitized populations of lymphocytes and may be applicable in the treatment of autoimmune diseases such as myasthenia gravis.