Mechanisms of tolerance induction by a gene-transferred peptide-IgG fusion protein expressed in B lineage cells

A gene therapy model has been designed to induce tolerance to multiple epitopes expressed in-frame on a soluble IgG fusion protein scaffold. Tolerance to the lambda repressor cI sequence p1-102 or its immunodominant epitopes (p12-26, p73-88) can be elicited when bone marrow (BM) or LPS blasts are transduced and injected into naive or even primed recipients. To explore the mechanism of tolerance, class II(-/-) (knockout, KO) BM cells were transduced with p1-102-IgG and transferred to irradiated recipients. These cells failed to induce tolerance to challenge with p1-102 epitopes, whereas transduced +/+ BM cells did. This supports the importance of class II MHC on the tolerogenic APC rather than secretion and representation in tolerogenesis. When BM cells from muMT KO mice were transfected with p12-26-IgG and injected into irradiated mice, these transduced BM cells also failed to induce tolerance to an immunodominant epitope. These results suggest the direct involvement of B cells in tolerance to p1-102 epitopes. IL-10 KO BM cells infected with a p12-26-IgG construct were still tolerogenic. Importantly, anti-CTLA-4 injections reversed tolerance in primed, but not in naive, recipients of transduced LPS blasts. These data emphasize the importance of MHC class II presentation, B cell involvement, and CTLA-4 engagement in induction and/or maintenance of tolerance.     

Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response.

Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.     

Gene transfer of Ig-fusion proteins into B cells prevents and treats autoimmune diseases

Based on the tolerogenic properties of IgG carriers and B cell Ag presentation, we developed a retrovirally mediated gene expression approach for treatment of autoimmune conditions. In this study, we show that the IgG-Ag retroviral constructs, expressing myelin basic protein (MBP) or glutamic acid decarboxylase in B cells, can be used for the treatment of murine models for multiple sclerosis and diabetes. Transduction of syngeneic B cells with MBP-IgG leads to the amelioration of ongoing experimental allergic encephalomyelitis induced by the transfer of primed cells from PLxSJL F(1) mice with ongoing disease and could be effective even after symptoms appeared. This effect is specific and does not involve bystander suppression because treatment with MBP-IgG does not affect disease induced after immunization with proteolipid protein immunodominant peptide plus MBP. Interestingly, if donor B cells are derived from gld mice (Fas ligand-negative), then tolerance is not induced with a model Ag although there was no evidence for Fas ligand-mediated deletion of target T cells. In spontaneous diabetes in nonobese diabetic mice, we were able to stop the ongoing autoimmune process by treatment at 7-10 wk with glutamic acid decarboxylase-IgG retrovirally transduced B cells, or attenuate it with B cells transduced with an insulin B chain (B9-23) epitope IgG fusion protein. Furthermore, IgG fusion protein gene therapy can also protect primed recipients from Ag-induced anaphylactic shock, and thus does not cause immune deviation. These results demonstrate proof of principle for future efforts to develop this approach in a clinical setting.     

Tolerance to host minor histocompatibility antigens after allogenic bone marrow transplantation. Specific donor-host unresponsiveness is maintained by peripheral tolerizing cells.

The development of graft-host tolerance after allogeneic bone marrow (BM) transplantation is more demanding than the acquisition of self-tolerance because both donor-derived mature T cells and immature thymocytes encounter host Ag. The mechanism involved in tolerization of mature T cells, contained in unmanipulated BM, remains undefined. In previous experiments, we showed in vivo unresponsiveness to host minor histocompatibility Ag (MiHA) in immunocompetent chimeras obtained after MiHA-incompatible BM transplantation. In this study, we wanted to determine: first, what was the specificity of this graft-host unresponsiveness, and second, whether peripheral cells were involved in tolerization? LP recipients were irradiated (9, 5 Gy), injected with 10(7) undepleted BM cells from B10 donors and studied 100 to 150 days later. (B10-->LP) chimeras were immunized in vivo and restimulated in vitro with cells displaying one or multiple incompatible MiHA. In bulk culture experiments, chimeras demonstrated specific CTL unresponsiveness to host MiHA but responded normally to third party MiHA. In limiting dilution analysis conditions, chimeras showed a profound deficit, but not a complete absence of anti-host CTL precursor. Studies with congenic stimulators/targets showed that graft-host tolerance was induced against both immunodominant (e.g., H-3.2) and nonimmunodominant (e.g., H-8.2) MiHA although at the CTL precursor level, it was more complete against the former. Furthermore, chimera spleen cells inhibited the generation of CTL activity against host- and donor-type MiHA but not against third party Ag. This specific suppressor activity was not T cell dependent, and was mediated by radiosensitive cells that are not found in freshly explanted organs from normal mice. Taken together, our results suggest that peripheral tolerization can be a remarkably efficient process to maintain tolerance to MiHA after BM transplantation. Thus, peripheral tolerizing mechanisms may contribute not only to the induction of tolerance to Mls superantigens or to the product of transgenes (if expressed at high levels) but also to a wide array of MiHA.     

Gene therapy for immunologic tolerance: using bone marrow-derived cells to treat autoimmunity and hemophilia

Bone marrow derived cells, especially B lymphocytes, have been shown to function as tolerogenic antigen-presenting cells (APC's) both in vivo and in vitro. In addition, it is well established that immunoglobulins can function as potent tolerogenic carriers for associated epitopes. We have taken advantage of these properties to develop a gene therapy approach to induce unresponsiveness in a number of animal models for clinical diseases. In our system, we engineered target peptide-IgG constructs into retroviral vectors and transduced hematopoietic cells to create tolerogenic antigen-presenting cells. In this review, we discuss the strategies and mechanism of our gene therapy approach mediated by B cells, as well as by bone marrow cells, for tolerance acquisition in various mouse models for autoimmune disease and hemophilia A. Our results show that MHC class II and co-stimulatory molecules must be expressed on the tolerogenic antigen presenting cells for efficacy. This therapy requires regulatory T cells for both the induction and maintenance of tolerance. The putative role of epitopes provided by the IgG carrier in this process. Studies in non-human primates and with human T cell clones in vitro are in progress to transition this approach to the clinic. The use of stem cells and B cell-delivered gene therapy in human clinical diseases may soon become a reality.     

Induction of graft versus leukemia effects by cell-mediated lymphokine-activated immunotherapy after syngeneic bone marrow transplantation in murine B cell leukemia

 The feasibility of inducing graft versus leukemia (GVL) effects with allogeneic T cells in recipients of autologous bone marrow transplantation (BMT) was studied in a murine model (BCL 1) of human B cell leukemia/lymphoma. Allogeneic cell therapy, induced by infusion with peripheral blood lymphocytes, a mixture of allogeneic spleen and lymph node cells and allogeneic activated cell therapy, induced by in vitro recombinant-interleukin-2(rIL-2)-activated allogeneic bone marrow cells in tumor-bearing mice, prevented disease development in adoptive BALB/c recipients. Concomitant in vivo activation of allogeneic lymphocytes with rIL-2 suppressed even more effectively the development of leukemia in secondary adoptive recipients of spleen cells obtained from treated mice. In contrast, in vivo administration of rIL-2 after syngeneic BMT, with or without equal numbers of syngeneic lymphocytes, led to disease development in secondary recipients. Our data suggest that effective cell therapy can be achieved after SBMT by allogeneic but not syngeneic lymphocytes and that anti-leukemic effects induced by allogeneic lymphocytes can be further enhanced by in vitro or in vivo activation of allogeneic effector cells with rIL-2. Therefore, cell therapy by allogeneic lymphocytes following autologous BMT could become an effective method for inducing GVL-like effects on minimal residual disease provided that graft versus host disease can be prevented or adequately controlled. Received: 14 May 1996 / Accepted: 6 August 1996     

Cellular aspects of tolerance. II. Unresponsiveness of B cells

The responsiveness of bone marrow cells from tolerant donors was examined by reconstitution of lethally irradiated tolerogen-free recipients. In these animals, stem cells from tolerant donors gave rise to immunologically competent antigen sensitive B cells. The antibody produced by these cells could be detected by a sensitive plaque assay in liquid and by antigen elimination. The antibody was not demonstrable by an assay which only detected plaque forming antibody which was highly avid or was formed in large quantity per cell. In lethally irradiated animals, partially purified B cells from a tolerant animal could not cooperate with T cells from normal donors to reconstitute immunological responsiveness to immunogenic doses of the tolerance inducing antigen. We concluded that antigen sensitive B cells in the bone marrow become unresponsive following administration of tolerogenic forms of antigen. Responsiveness of the reconstituted recipient animals was due to the differentiation of donor stem cells and subsequent antibody production by their descendants. Earlier contradictory findings could be unified in terms of these observations and conclusions.     

Introduction of the green fluorescent protein gene into hematopoietic stem cells results in prolonged discrepancy of in vivo transduction levels between bone marrow progenitors and peripheral blood cells in nonhuman primates

Background

The green fluorescent protein (GFP) has proven a useful marker in retroviral gene transfer studies targeting hematopoietic stem cells (HSCs) in mice. However, several investigators have reported very low in vivo peripheral blood marking levels in nonhuman primates after transplantation of HSCs transduced with the GFP gene. We retrovirally marked cynomolgus monkey HSCs with the GFP gene, and tracked in vivo marking levels within both bone marrow progenitor cells and mature peripheral blood cells following autologous transplantation after myeloablative conditioning.

Methods

Bone marrow cells were harvested from three cynomolgus macaques and enriched for the primitive fraction by CD34 selection. CD34⁺ cells were transduced with one of three retroviral vectors all expressing the GFP gene and were infused after myeloablative total body irradiation (500 cGy × 2). Following transplantation, proviral levels and fluorescence were monitored among clonogenic bone marrow progenitors and mature peripheral blood cells.

Results

Although 13–37% of transduced cells contained the GFP provirus and 11–13% fluoresced ex vivo, both provirus and fluorescence became almost undetectable in the peripheral blood within several months after transplantation regardless of the vectors used. However, on sampling of bone marrow at multiple time points, significant fractions (5–10%) of clonogenic progenitors contained the provirus and fluoresced ex vivo reflecting a significant discrepancy between GFP gene marking levels within bone marrow cells and their mature peripheral blood progeny. The discrepancy (at least one log) persisted for more than 1 year after transplantation. Since no cytotoxic T lymphocytes against GFP were detected in the animals, an immune response against GFP is an unlikely explanation for the low levels of transduced peripheral blood cells. Administration of granulocyte colony stimulating factor and stem cell factor resulted in mobilization of transduced bone marrow cells detectable as mature granulocyte progeny which expressed the GFP gene, suggesting that transduced progenitor cells in bone marrow could be mobilized into the peripheral blood and differentiated into granulocytes.

Conclusions

Low levels of GFP‐transduced mature cells in the peripheral blood of nonhuman primates may reflect a block to differentiation associated with GFP; this block can be overcome in part by nonphysiological cytokine treatment ex vivo and in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

Rapid deletional peripheral CD8 T cell tolerance induced by allogeneic bone marrow: role of donor class II MHC and B cells

Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy of total body irradiation and anti-CD154 mAb followed by allogeneic bone marrow (BM) transplantation. In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II(+) donor cells promoting this tolerance, we used donor BM lacking certain cell populations or used positively selected cell populations. Neither donor CD11c(+) dendritic cells, B cells, T cells, nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis tolerance in both strain combinations tested. In contrast, positively selected CD11b(+) monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR-transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific cell-mediated lympholysis tolerance, and deletion of donor-reactive CD8 T cells, whereas CD11b(+) cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.     

B cells induce tolerance by presenting endogenous peptide-IgG on MHC class II molecules via an IFN-gamma-inducible lysosomal thiol reductase-dependent pathway

We have previously demonstrated that splenic B cells, transduced with peptide-IgG fusion proteins, are efficient tolerogenic APCs in vivo. Specific hyporesponsiveness to epitopes encoded in the peptide-IgG fusion protein has been achieved to over one dozen Ags, and clinical efficacy has been established in animal models for several autoimmune diseases and hemophilia. Previous studies also demonstrated that tolerance in this system requires MHC class II expression by the transduced B cells. Yet, the mechanisms of this B cell tolerogenic processing pathway remain unclear. In this study, we show that MHC class II molecules on tolerogenic B cells present epitopes derived from endogenous, but not exogenous (secreted), peptide-IgG fusion protein. These class II epitopes from the IgG fusion protein are processed in lysosomes/endosomes in an IFN-gamma-inducible lysosomal thiol reductase-dependent manner. We suggest that the MHC class II presentation of endogenously produced fusion protein epitopes represents a novel mechanism for tolerance induced by peptide-IgG-transduced B cells. An understanding of this process might provide insights into central and peripheral tolerance induced by other professional and nonprofessional APCs.     

Infectious nickel tolerance: a reciprocal interplay of tolerogenic APCs and T suppressor cells that is driven by immunization

Previously, we reported that tolerance to nickel, induced by oral administration of Ni(2+) ions, can be adoptively transferred to naive mice with only 10(2) splenic T cells. Here we show that 10(2) T cell-depleted spleen cells (i.e., APCs) from orally tolerized donors can also transfer nickel tolerance. This cannot be explained by simple passive transfer of the tolerogen. The APCs from orally tolerized donors displayed a reduced allostimulatory capacity, a tolerogenic phenotype, and an increased expression of CD38 on B cells. In fact, it was B cells among the APCs that carried the thrust of tolerogenicity. Through serial adoptive transfers with Ly5.1(+) donors and two successive sets of Ly5.2(+) recipients, we demonstrated that nickel tolerance was infectiously spread from donor to host cells. After the transfer of either T cells or APCs from orally tolerized donors, the spread of tolerance to the opposite cell type of the recipients (i.e., APCs and T cells, respectively) required recipient immunization with NiCl(2)/H(2)O(2). For the spread of tolerance from a given donor cell type, T cell or APC, to the homologous host cell type, the respective opposite cell type in the host was required as intermediate. We conclude that T suppressor cells and tolerogenic APCs induced by oral administration of nickel are part of a positive feedback loop that can enhance and maintain tolerance when activated by Ag associated with a danger signal. Under these conditions, APCs and T suppressor effector cells infectiously spread the tolerance to naive T cells and APCs, respectively.     

Regulatory T cells induced by 1 alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance

1alpha,25-dihydroxyvitamin D3, the active form of vitamin D3, and mycophenolate mofetil, a selective inhibitor of T and B cell proliferation, modulate APC function and induce dendritic cells (DCs) with a tolerogenic phenotype. Here we show that a short treatment with these agents induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. Peritransplant macrophages and DCs from tolerant mice express down-regulated CD40, CD80, and CD86 costimulatory molecules. In addition, DCs from the graft area of tolerant mice secrete, upon stimulation with CD4+ cells, 10-fold lower levels of IL-12 compared with DCs from acutely rejecting mice, and induce a CD4+ T cell response characterized by selective abrogation of IFN-gamma production. CD4+ but not CD8+ or class II+ cells from tolerant mice, transferred into naive syngeneic recipients, prevent rejection of donor-type islet grafts. Graft acceptance is associated with impaired development of IFN-gamma-producing type 1 CD4+ and CD8+ cells and an increased percentage of CD4+CD25+ regulatory cells expressing CD152 in the spleen and in the transplant-draining lymph node. Transfer of CD4+CD25+ cells from tolerant but not naive mice protects 100% of the syngeneic recipients from islet allograft rejection. These results demonstrate that a short treatment with immunosuppressive agents, such as 1alpha,25-dihydroxyvitamin D3/mycophenolate mofetil, induces tolerance to islet allografts associated with an increased frequency of CD4+CD25+ regulatory cells that can adoptively transfer transplantation tolerance.     

Mechanisms of gene therapy for tolerance: B7 signaling is required for peptide-IgG gene-transferred tolerance induction

LPS-activated B cells, transduced with IgG fusion proteins, are highly tolerogenic APCs. To analyze the mechanisms for this B cell-delivered gene therapy, we first followed the fate of CFSE-labeled B cell blasts. These cells primarily localized to the spleen, where a small population persisted for at least 1 mo after injection. By day 7 after injection, approximately 95% of the transduced cells had divided at least once, presumably an effect of the in vitro LPS activation into the cycle, because resting cells did not divide. B cells from gld donors were not tolerogenic, initially suggesting a role for Fas ligand (FasL) in tolerance. Because transduced normal B cells expressed only low levels of FasL and did not kill Fas-expressing Jurkat or A20 B lymphoma cells in vitro, these data suggest that gld B cells are not tolerogenic due to unique characteristics of these B cells rather than the lack of functional FasL expression. The transduced B cell blasts displayed significant up-regulation of both B7 costimulatory molecules, and B7.2 up-regulation was maintained through day 7 in vivo. When B cells from B7 knockout donors were transduced to express Ig fusion proteins, they were not tolerogenic in two different mouse strains and Ag models. Moreover, anti-B7 Ab blocked tolerance induction in this model, a result consistent with a role for B7 in tolerance induction. We propose that tolerance may be induced in this model by B7-driven negative regulatory signaling, but tolerance is maintained by a lack of signal 2, because expression of B7 is eventually lost in vivo.     

In vivo activity of tumor necrosis factor (TNF) mutants. Secretory but not membrane-bound TNF mediates the regression of retrovirally transduced murine tumor.

We have previously demonstrated that murine tumor cells transduced with a retrovirus containing the cDNA encoding wild-type human TNF regress in vivo when injected into immunocompetent mice; this regression is T cell mediated. To determine whether membrane-associated or secreted TNF was responsible for tumor regression, we transduced a cloned murine fibrosarcoma 205 F4 with retroviruses encoding modified human TNF genes. The cloned tumor lines of one retroviral transduction expressed only membrane bound 26-kDa TNF. This TNF could not be cleaved or secreted, but was present on the cell surface. A second retrovirus caused the expression of only secretory 17-kDa TNF, as the transmembrane domain of the cDNA was deleted. The TNF produced by tumor cells transduced with either retroviral vector was functional in vitro as direct lysis of the TNF-sensitive target L929 by transduced tumor cells was demonstrated. The TNF present on 26-kDa expressing tumors was membrane bound as supernatants from cultured 17-kDa TNF expressing tumor cells but not 26-kDa TNF expressing tumors mediated the lysis of L929 cells. Both tumors were injected s.c. into syngeneic mice and tumor growth was measured serially. In repeated experiments, 26-kDa TNF expressing tumors grew progressively in all mice. In contrast, 17-kDa TNF expressing tumors grew for 10 days and then regressed with all animals free of tumor at 28 days. Tumor regression was abrogated by in vivo injection of an anti-TNF antibody. Similar results were obtained in a second tumor model, 203 E4. Thus regression of TNF transduced tumors in vivo requires secretion of TNF, as membrane-bound TNF is insufficient to elicit the host response.     

Prevention of GVHD by modulation of rat bone marrow with UV-B irradiation: II. Kinetics of migration of UV-B-irradiated bone marrow cells in naive and lethally irradiated rats

UV-B irradiation (700 J/m2) of bone marrow (BM) cells prior to transplantation into lethally gamma-irradiated (1050 rad) allogeneic rats prevents the development of GVHD and results in a stable mixed lymphohematopoietic chimerism. To better understand the underlying mechanisms of the development of stable radiation chimeras in this model, this study was designed to examine whether the dose (700 J/m2) of UV-B irradiation used for the modulation of the BM inoculum would affect the homing pattern of radiolabeled BM cells compared to that of thoracic duct lymphocytes (TDL) in the naive and lethally irradiated recipients. The results showed that intravenously administered, 111Indium-oxine-labeled, unmodified TDL home specifically to the spleen, lymph nodes, and BM compartments with a subsequent recirculation of a large number of cells from the spleen to the lymph nodes. In contrast, radiolabeled, unmodified BM cells migrate specifically to the spleen, liver, and BM with the lymph nodes, thymus, and nonlymphoid organs containing very little amounts of radioactivity. The stable concentrations of radioactivity in the lymphoid and nonlymphoid compartments between 3 and 72 hr after injection suggest that BM cells, unlike TDL, do not recirculate. The migration pattern of BM cells in the naive recipient was not significantly different from that seen in lethally irradiated animals except for the higher concentration of radioactivity in the spleen and BM of irradiated animals compared to that seen in naive recipients. The similarity of tissue localization of BM cells in naive or in irradiated syngeneic recipients to that of allogeneic recipients suggests that the homing of BM cells is not MHC restricted. Our findings of similarity between tissue localization of UV-B-irradiated labeled BM cells and unmodified BM cells in naive and lethally irradiated recipients suggest that a dose of 700 J/m2 of UV-B irradiation is not capable of impairing BM cell migration although it is sufficient to abolish the homing of TDL to the HEV-bearing organs. Thus, our results show that BM cells are less susceptible to cell damage by UV-B irradiation than lymphocytes thereby providing the rationale for ex vivo modulation (rather than elimination) of mature T-lymphocytes in the donor BM inoculum with UV-B irradiation. This relatively simple and effective approach to modulation of T-cells in donor BM inoculum may be potentially useful in preventing GVHD without endangering successful engraftment in larger animals and in man.     

Role of the thymus in control of autoreactivity or allotolerance in syngeneic and allogeneic bone marrow chimeras treated with bacterial adjuvants

Thy-1-bone marrow (BM) cells from C57BL/6 (B6) mice were transferred into thymectomized or non-thymectomized syngeneic B6----B6, allogeneic B6----C3H or semiallogeneic B6----(B6 X C3H)F1, irradiated mice, after which bacterial substances (bacillus Calmette Guérin [BCG] or Bordetella pertussis [Bp]) were administered within 3 days. The regulation of reactivity toward the host environment, i.e., autoresponsiveness in B6----B6 and allotolerance in B6---C3H, was investigated by monitoring a graft-vs-host (GvH)-like wasting syndrome, as well as the in vitro responsiveness of spleen cells from the reconstituted mice in a mixed leukocyte culture/cell-mediated lysis (MLC/CML) assay. The BCG-treated B6----B6 recipients developed a wasting syndrome and MLC/CML reactivity toward syngeneic target cells within 7 wk. This was never observed in BCG-treated but otherwise normal (i.e., nonreconstituted) mice, nor was it seen in any bone marrow chimeras that had been left without BCG treatment, irrespective of host/donor combination or thymectomy. The development of wasting syndrome as well as autoreactivity in BCG-treated B6----B6 mice could be prevented by thymectomizing the recipients before reconstitution or co-cultivating the donor BM cells with syngeneic spleen cells before reconstitution of nonthymectomized recipients. In the allogeneic or semiallogeneic combinations, the BCG treatment resulted in a wasting syndrome and CML/MLC reactivity toward C3H or (C3H X B6)F1 host-derived cells irrespective of thymic presence or absence. No breakdown of allotolerance, however, was retarded in the thymectomized mice, and it could be prevented by co-cultivation of donor BM cells with splenocytes of recipient genotype only if the cells were used to reconstitute thymectomized recipients. The breakdown of allotolerance in B6----C3H chimera was never accompanied by autoreactivity against B6 target cells. It is concluded that induction of autoreactivity and GvH in BCG-treated syngeneic BM chimeras, probably reflecting the breakdown of autotolerance, is strictly thymus dependent. In contrast, induction of anti-host reactivity in BCG-treated allogeneic chimeras may occur in the absence of a thymus and without concomitant autoreactivity, suggesting two independent levels of controls: one that is thymus dependent for the breakdown of auto- as well as allotolerance, and one that is thymus independent, unique for the breakdown of allotolerance.     

Infectious and noninfectious tolerance are blocked by a monoclonal antibody to T-suppressor factor

Two forms of hapten-specific unresponsiveness have been demonstrated following intravenous (iv) injection of hapten-conjugated syngeneic spleen cell based on the nature of the antigen-presenting cell (APC): I-J+, I-A- APC have been shown to induce T-suppressor cells (Ts cells) which are demonstrated upon adoptive transfer, while I-J-, I-A+ APC induce a nontransferable tolerance. In this paper we report that a monoclonal antibody specific for T-suppressor effector cells and factors (14-12) can block the Ts cells induced by I-J+, I-A- APCs and the tolerance induced by I-J-, I-A+ APCs. In addition, it sufficiently overcomes suppression such that injection of TNP-spl iv induces immunity rather than suppression. We show that the I-A+, I-J- TNP-spl, which induce nontransferable tolerance upon iv injection, are the cells which induce immunity in 14-12-treated recipients. These results demonstrate that injection of I-J-, I-A+ APC does not lead to clonal deletion and the tolerance induced by the iv injection of both I-J+, I-A- and I-J-, I-A+ APC operate via Ts cells.     

Cutting edge: in vitro-generated tolerogenic APC induce CD8+ T regulatory cells that can suppress ongoing experimental autoimmune encephalomyelitis

APC exposed to TGFbeta2 and Ag (tolerogenic APC) promote peripheral Ag-specific tolerance via the induction of CD8(+) T regulatory cells capable of suppressing Th1 and Th2 immunity. We postulated that tolerogenic APC might reinstate tolerance toward self-neuronal Ags and ameliorate ongoing experimental autoimmune encephalomyelitis (EAE). Seven days after immunization with myelin basic protein (MBP), mice received MBP-specific tolerogenic APC, and EAE was evaluated clinically. To test for the presence and the phenotype of T regulatory cells, CD4 and/or CD8 T cells from tolerogenic APC-treated mice were transferred to naive mice before their immunization with MBP. The MBP-specific tolerogenic APC decreased both the severity and incidence of ongoing EAE. Tolerance to self-neuronal Ags was induced in naive recipient mice via adoptive transfer of CD8(+), but not CD4(+) T cells. Rational use of in vitro-generated tolerogenic APC may lead to novel therapy for autoimmune disease.     

Neogenesis of cerebellar Purkinje neurons from gene-marked bone marrow cells in vivo.

The versatility of stem cells has only recently been fully recognized. There is evidence that upon adoptive bone marrow (BM) transplantation (BMT), donor-derived cells can give rise to neuronal phenotypes in the brains of recipient mice. Yet only few cells with the characteristic shape of neurons were detected 1-6 mo post-BMT using transgenic or newborn mutant mice. To evaluate the potential of BM to generate mature neurons in adult C57BL/6 mice, we transferred the enhanced green fluorescent protein (GFP) gene into BM cells using a murine stem cell virus-based retroviral vector. Stable and high level long-term GFP expression was observed in mice transplanted with the transduced BM. Engraftment of GFP-expressing cells in the brain was monitored by intravital microscopy. In a long-term follow up of 15 mo post-BMT, fully developed Purkinje neurons were found to express GFP in both cerebellar hemispheres and in all chimeric mice. GFP-positive Purkinje cells were also detected in BM chimeras from transgenic mice that ubiquitously express GFP. Based on morphologic criteria and the expression of glutamic acid decarboxylase, the newly generated Purkinje cells were functional.     

Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes. Developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice.

During the course of B lymphocyte development, newly emerging surface Ig+ B cells pass through a stage when Ag-Ag receptor interactions lead not to immune responsiveness but to a state of functional tolerance. We have explored the molecular basis of antigenic nonresponsiveness and tolerance susceptibility using tolerance-susceptible surface Ig+ splenic B lymphocytes from neonatal mice and anti-mu chain antibodies as a polyclonal ligand. In this population of cells, surface IgM is uncoupled from the inositol phospholipid (PI)-hydrolysis pathway at a point proximal to the receptor; anti-mu antibodies did not stimulate inositol phosphate generation despite the fact that PI-hydrolysis was observed after treatment with A1F4, implicating the existence of a functional G protein and phospholipase C. Further evidence for a difference early in the signal transduction pathway stems from the finding that anti-mu stimulation does not induce the expression of two immediate/early PKC-linked genes egr-1 and c-fos. This appears to be the primary signaling difference between the mature and immature B cells from the neonatal mouse splenic population, as these cells undergo a G0-G1 cell cycle phase transition when surface IgM is bypassed using phorbol diester and calcium ionophore. Interestingly, despite undetectable levels of PI-hydrolysis, we observed equivalent receptor-mediated changes in intracellular calcium when comparing the immature and mature populations. These results indicate incomplete coupling of surface IgM to the signal transduction machinery operative in mature, immunocompetent B cells and suggests a molecular mechanism accounting for the differential processing of surface IgM signals into activation vs tolerogenic responses observed in these two stages of B cell development.