Tolerance induction via a B-cell delivered gene therapy-based protocol: optimization and role of the Ig scaffold

Our previous studies indicated that antigen-specific tolerance could be achieved by the injection of LPS-activated B-cell blasts that were retrovirally gene-transferred with an IgG-antigen fusion construct. This system was shown to be effective for tolerance induction with a variety of inserted antigens ranging in size from a single peptide to a large multi-epitope protein in a variety of mouse strains. Moreover, it was shown to be effective in four animal models for human disease. To optimize the existing protocol, establish the role of the IgG H chain scaffold, and provide baseline for potential clinical applications, we examined the effects of different B-cell activators, including lipopolysaccharide (LPS), anti-CD40, CpG oligodeoxynucleotide (CpG-ODN), and anti-IgM plus IL-4, on B-cell proliferation, GFP transduction efficiency, and tolerance induction in vivo. The results show that all activators except CpG-ODN have similar effects on retroviral gene transfer and peptide-IgG-induced tolerance. Furthermore, dose-response analyses showed that T-cell tolerance could be induced with 10(5) peptide-IgG LPS B-cell blasts, but that 10(6) transduced B-cells were needed for humoral unresponsiveness. Transduced anti-IgM-induced blasts were tolerogenic at 10(6) cells, but no dose of transduced CpG blasts was tolerogenic. Finally, to examine the role of IgG scaffold, a retroviral construct encoding lambda repressor p1-102 and signal peptide of murine IgG heavy chain was engineered to allow secretion of the p1-102 domain in the same manner as that of p1-102-IgG fusion protein. The results demonstrate that not only is IgG scaffold important in tolerance induction and maintenance of the long-lasting immune hyporesponsiveness, but assembly of the IgG heterodimer may be required for the efficacy of this system.     

Genetically transferred central and peripheral immune tolerance via retroviral-mediated expression of immunogenic epitopes in hematopoietic progenitors or peripheral B lymphocytes.

BACKGROUND: Based on the hypothesis that IgGs are potent tolerogens and that immature lymphohematopoietic antigen-presenting cells (APC), and even mature peripheral B cells, may be effective APC for tolerance induction, we designed an immunoglobulin fusion protein retroviral expression vector to test the role of B cells in a novel gene therapy strategy for the transfer of immune tolerance. METHODS: An immunodominant epitope (residues 12-26 of the lambda repressor cI protein) was fused in frame to an IgG heavy chain in a retroviral vector, which was used to infect either bone marrow cells or activated peripheral B lymphocytes. These cells were transferred into syngeneic recipients, who were subsequently challenged with the 12-26 peptide in adjuvant. RESULTS: Bone marrow (BM) chimeras generated with retrovirally transduced bone marrow were shown to be profoundly unresponsive to the 12-26 peptide at both the humoral and cellular levels, but were competent to respond to an unrelated protein (lysozyme or PPD). Importantly, we also show that immunocompetent adult recipients infused with transduced mature, activated B lymphocytes, are rendered unresponsive by this treatment. Surprisingly, lymphoid-deficient BM progenitors from syngeneic SCID donors could also be transduced to produce tolerogenic APC. CONCLUSIONS: Our data suggest that activated B cells are sufficient to be effective tolerogenic APC in immunocompetent adult mice, but that nonlymphoid cells may also induce tolerance in reconstituted hosts. This approach for gene-transferred tolerogenesis has the potential to be maintained indefinitely, and it requires only knowledge of cDNA sequences of target antigens.     

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.     

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.     

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.     

Host dendritic cells alone are sufficient to initiate acute graft-versus-host disease

Alloantigen expression on host APCs is essential to initiate graft-vs-host disease (GVHD); however, critical APC subset remains to be elucidated. We compared the ability of dendritic cells (DCs) and B cells to initiate acute GVHD by an add-back study of MHC class II-expressing APCs (II(+/+)) into MHC class II-deficient (II(-/-)) mice that were resistant to CD4-dependent GVHD. Injection of host-derived, but not donor-derived, II(+/+) DCs or host-derived II(+/+) B cells, was sufficient to break GVHD resistance of II(-/-) mice and induced lethal acute GVHD. By contrast, host-derived II(+/+) B cells, both naive and LPS stimulated, failed to induce activation or tolerance of donor CD4(+) T cells. Similarly, in a model of CD8-dependent GVHD across MHC class I mismatch injection of allogeneic DCs, but not B cells, induced robust proliferation of donor CD8(+) T cells and broke GVHD resistance of chimeric recipients in which APCs were syngeneic to donors. These results demonstrate that host-derived DCs are critical in priming donor CD4(+) and CD8(+) T cells to cause GVHD, and selective targeting of host DCs may be a promising strategy to prevent GVHD.     

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.     

The use of haptenated immunoglobulins to induce B cell tolerance in vitro. The roles of hapten density and the Fc portion of the immunoglobulin carrier

The relationship between the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig), and their ability to induce tolerance was examined. It was found that adult B cells responding to a T-independent (TI) antigen were tolerized by TNP11 human gamma globulin (HGG), but not by TNP10F(ab')2 fragments of HGG. Increasing the hapten density on the F(ab')2 fragments overcame their inability to induce tolerance. Thus, a TNP17-F(ab')2 was an effective tolerogen. Murine myeloma proteins of different IgG subclasses were similarly tested. A TNP12-IgG2a and a TNP11-IgG1 induced tolerance, whereas two TNP11-12-IgG3 did not. However, a more heavily haptenated TNP18-IgG3 was tolerogenic. These results suggest that lightly haptenated immunoglobulins depend upon Fc receptor binding to induce tolerance in adult B cells. Non-Fc receptor-binding carriers are not tolerogenic unless they are more heavily haptenated. Finally, T cell and macrophage depletion experiments suggest that the tolerogens act directly on the B cells.     

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.     

The in vivo fate of APCs displaying minor H antigen and/or MHC differences is regulated by CTLs specific for immunodominant class I-associated epitopes

The goal of this work was to evaluate the fate of APCs following interactions with T cells in unprimed mice with a normal T cell repertoire. We elaborated a model in which male adherent peritoneal mononuclear cells were injected into the foreleg footpads of naive female recipients mismatched for either minor or major histocompatibility Ags. At various times after injection, APC numbers in the draining (axillary and brachial) lymph nodes were assessed using a Ube1y gene-specific PCR assay. Our experimental model was designed so that the number of APCs expressing the priming epitope was similar to what is observed under real life conditions. Thus, early after injection, the frequency of afferent lymph-derived APCs expressing the priming epitope was in the range of 101-102/106 lymph node cells. We found that APCs presenting some, but not all, nonself epitopes were killed rapidly after entrance into the lymph nodes. Rapid elimination of APCs occurred following interactions with MHC class I-restricted, but not class II-restricted, T cells and was observed when APCs presented an immunodominant (B6dom1/H7a), but not a nondominant (HY), epitope. Killing of APCs was mediated partly, but not exclusively, by perforin-dependent process. We propose that killing of APCs by CTLs specific for immunodominant MHC class I-restricted epitopes may be instrumental in regulating the intensity, duration, and diversity of T cell responses.     

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.     

Oral tolerance to nickel requires CD4+ invariant NKT cells for the infectious spread of tolerance and the induction of specific regulatory T cells

Previously, oral administration of nickel to C57BL/6 wild-type (WT) mice was shown to render both their splenic T cells and APCs (i.e., T cell-depleted spleen cells) capable of transferring nickel tolerance to naive syngeneic recipients. Moreover, sequential adoptive transfer experiments revealed that on transfer of tolerogenic APCs and immunization, the naive T cells of the recipients differentiated into regulatory T (Treg) cells. Here, we demonstrate that after oral nickel treatment Jalpha18(-/-) mice, which lack invariant NKT (iNKT) cells, were not tolerized and failed to generate Treg cells. However, transfer of APCs from those Jalpha18(-/-) mice did tolerize WT recipients. Hence, during oral nickel administration, tolerogenic APCs are generated that require iNKT cell help for the induction of Treg cells. To obtain this help, the tolerogenic APCs must address the iNKT cells in a CD1-restricted manner. When Jalpha18(-/-) mice were used as recipients of cells from orally tolerized WT donors, the WT Treg cells transferred the tolerance, whereas WT APCs failed to do so, although they proved tolerogenic on transfer to WT recipients. However, Jalpha18(-/-) recipients did become susceptible to the tolerogenicity of transferred WT APCs when they were reconstituted with IL-4- and IL-10-producing CD4(+) iNKT cells. We conclude that CD4(+) iNKT cells are required for the induction of oral nickel tolerance and, in particular, for the infectious spread of tolerance from APCs to T cells. Once induced, these Treg cells, however, can act independently of iNKT cells.     

T cell subsets and in vitro immune regulation in "infectious" transplantation tolerance.

CD4-targeted mAb therapy results in permanent acceptance of cardiac allografts in rat recipients, in conjunction with features of the infectious tolerance pathway. Although CD4(+) T cells play a central role, the actual cellular and molecular tolerogenic mechanisms remain elusive. This study was designed to analyze in vitro alloimmune responses of T lymphocytes from CD4 mAb-treated engrafted hosts. Spleen, but not lymph node, cells lost proliferative response against donor alloantigen in MLR and suppressed test allograft rejection in adoptive transfer studies, suggesting compartmentalization of tolerogenic T cells in transplant recipients. A high dose of exogenous IL-2 restored the allogeneic response of tolerogenic T cells, indicating anergy as a putative mechanism. Vigorous proliferation of the tolerogenic T cells in in vivo MLR supports the existence of alloreactive lymphocytes in tolerogenic T cell repertoire and implies an active operational suppression mechanism. The tolerogenic splenocytes suppressed proliferation of naive splenocytes in vitro, consistent with their in vivo property of dominant immune regulation. Finally, CD45RC(+) but not CD45RC(-) T cells from tolerant hosts were hyporesponsive to alloantigen and suppressed the proliferation of normal T cells in the coculture assay. Thus, nondeletional, anergy-like regulatory mechanisms may operate via CD4(+)CD45RC(+) T cells in the infectious tolerance pathway in transplant recipients.     

Retrovirally transduced mouse dendritic cells require CD4+ T cell help to elicit antitumor immunity: implications for the clinical use of dendritic cells

Presentation of MHC class I-restricted peptides by dendritic cells (DCs) can elicit vigorous antigen-specific CTL responses in vivo. It is well established, however, that T cell help can augment CTL function, raising the question of how best to present tumor-associated MHC class I epitopes to induce effective tumor immunity. To this end, we have examined the role of MHC class II peptide-complexes present on the immunizing DCs in a murine melanoma model. To present MHC class I- and II-restricted Ags reliably on the same cell, we retrovirally transduced bone marrow-derived DCs with the model Ag OVA encoding well-defined class I- and II-restricted epitopes. The importance of CD4+ T cells activated by the immunizing DCs in this model is demonstrated by the following findings: 1) transduced DCs presenting class I and class II epitopes are more efficient than class I peptide-pulsed DCs; 2) MHC class II-deficient DCs fail to induce tumor protection; 3) CD4+ T cell depletion abolishes induction of tumor protection; and 4) DCs presenting bovine serum Ags are more effective in establishing tumor immunity than DCs cultured in syngeneic serum. When MHC class II-deficient DCs were directly activated via their CD40 receptor, we indeed observed a moderate elevation of OVA-specific CTL activity. However, this increase in CTL activity was not sufficient to induce in vivo tumor rejection. Thus, our results demonstrate the potency of genetically modified DCs that express both MHC class I and II epitopes, but caution against the use of DCs presenting only the former.     

The Dermatophagoides pteronyssinus group 2 allergen contains a universally immunogenic T cell epitope

The use of T cell epitope-containing peptides for the induction of anergy in allergen sensitization is limited by genetic restriction that could be circumvented by using universally immunogenic epitopes. We attempted to identify such epitopes on Dermatophagoides pteronyssinus group 2 allergen (Der p 2), a major allergen of D. pteronyssinus T cells from BALB/c (H-2(d)), C57BL/6 (H-2(b)), C3H (H-2(k)), and SJL (H-2(s)) mice that were immunized with rDer p 2, recognized an immunodominant region encompassing residues 21-35. A synthetic 21-35 peptide (p21-35) induced strong dose-dependent in vitro T cell proliferation with cells of the four mouse strains and required processing for MHC class II presentation. Substitution of Ile(28) with Ala resulted in reduction of T cell proliferation in each strain. Ile(28) could represent an important MHC class II anchoring residue for T cell response to p21-35. An immunodominant T cell epitope of Der p 2 therefore behaves as a universal epitope and could be a suitable candidate for T cell anergy induction.     

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.     

Tolerance to antigen-presenting cell-depleted islet allografts is CD4 T cell dependent

Pretreatment of pancreatic islets in 95% oxygen culture depletes graft-associated APCs and leads to indefinite allograft acceptance in immunocompetent recipients. As such, the APC-depleted allograft represents a model of peripheral alloantigen presentation in the absence of donor-derived costimulation. Over time, a state of donor-specific tolerance develops in which recipients are resistant to donor APC-induced graft rejection. Thus, persistence of the graft is sufficient to induce tolerance independent of other immune interventions. Donor-specific tolerance could be adoptively transferred to immune-deficient SCID recipient mice transplanted with fresh immunogenic islet allografts, indicating that the original recipient was not simply "ignorant" of donor antigens. Interestingly, despite the fact that the original islet allograft presented only MHC class I alloantigens, CD8+ T cells obtained from tolerant animals readily collaborated with naive CD4+ T cells to reject donor-type islet grafts. Conversely, tolerant CD4+ T cells failed to collaborate effectively with naive CD8+ T cells for the rejection of donor-type grafts. In conclusion, the MHC class I+, II- islet allograft paradoxically leads to a change in the donor-reactive CD4 T cell subset and not in the CD8 subset. We hypothesize that the tolerant state is not due to direct class I alloantigen presentation to CD8 T cells but, rather, occurs via the indirect pathway of donor Ag presentation to CD4 T cells in the context of host MHC class II molecules.     

Inhibitory feedback loop between tolerogenic dendritic cells and regulatory T cells in transplant tolerance

An active role of T regulatory cells (Treg) and tolerogenic dendritic cells (Tol-DC) is believed important for the induction and maintenance of transplantation tolerance. However, interactions between these cells remain unclear. We induced donor-specific tolerance in a fully MHC-mismatched murine model of cardiac transplantation by simultaneously targeting T cell and DC function using anti-CD45RB mAb and LF 15-0195, a novel analog of the antirejection drug 15-deoxyspergualin, respectively. Increases in splenic Treg and Tol-DC were observed in tolerant recipients as assessed by an increase in CD4(+)CD25(+) T cells and DC with immature phenotype. Both these cell types exerted suppressive effects in MLR. Tol-DC purified from tolerant recipients incubated with naive T cells induced the generation/expansion of CD4(+)CD25(+) Treg. Furthermore, incubation of Treg isolated from tolerant recipients with DC progenitors resulted in the generation of DC with Tol-DC phenotype. Treg and Tol-DC generated in vitro were functional based on their suppressive activity in vitro. These results are consistent with the notion that tolerance induction is associated with a self-maintaining regulatory loop in which Tol-DC induce the generation of Treg from naive T cells and Treg programs the generation of Tol-DC from DC progenitors.     

Donor MHC class II antigen is essential for induction of transplantation tolerance by bone marrow cells

Posttransplant infusion of donor bone marrow cells (BMC) induces tolerance to allografts in adult mice, dogs, nonhuman primates, and probably humans. Here we used a mouse skin allograft model and an allogeneic radiation chimera model to examine the role of MHC Ags in tolerance induction. Infusion of MHC class II Ag-deficient (CIID) BMC failed to prolong C57BL/6 (B6) skin grafts in ALS- and rapamycin-treated B10.A mice, whereas wild-type B6 or MHC class I Ag-deficient BMC induced prolongation. Removal of class II Ag-bearing cells from donor BMC markedly reduced the tolerogenic effect compared with untreated BMC, although graft survival was significantly longer in mice given depleted BMC than that in control mice given no BMC. Infusion of CIID BMC into irradiated syngeneic B6 or allogeneic B10.A mice produced normal lymphoid cell reconstitution including CD4+ T cells except for the absence of class II Ag-positive cells. However, irradiated B10.A mice reconstituted with CIID BMC rejected all B6 and a majority of CIID skin grafts despite continued maintenance of high degree chimerism. B10.A mice reconstituted with B6 BMC maintained chimerism and accepted both B6 and CIID skin grafts. Thus, expression of MHC class II Ag on BMC is essential for allograft tolerance induction and peripheral chimerism with cells deficient in class II Ag does not guarantee allograft acceptance.     

Distinction between immunogenicity and tolerogenicity among HBcAg T cell determinants. Influence of peptide-MHC interaction

One purpose of this study was to examine the concept of T cell immunodominance employing a neonatal tolerance model. The extent to which a single T cell recognition site can represent the total T cell response to hepatitis B core Ag (HBcAg) was examined in the B10.S and B10 murine strains. It was shown that the entire B10.S T cell response to HBcAg was focused on a single immunodominant site represented by residues 120-131. This was demonstrated by exposing B10.S neonatal mice to p120-140 or p120-131, which resulted in a state of T cell tolerance to the entire HBcAg. In contrast, p120-140 contained an immunogenic T cell site for B10 mice, p129-140, but this site was nontolerogenic. Similarly, injection of p120-140 into (B10.S X B10)F1 neonatal mice resulted in tolerization of p120-131-specific, I-As-restricted T cells, but not of p129-140-specific, I-Ab-restricted T cells. The second purpose of this study was to attempt to explain the immunologic basis of an immunogenic yet nontolerogenic T cell determinant. It was shown that the p120-131 T cell site, which is immunogenic and tolerogenic in B10.S mice, could be converted into an immunogenic/nontolerogenic T cell site by a single amino acid substitution in either residue 127 or 129. Residues 127 and 129 were previously shown to be involved in interaction with MHC class II molecules (agretopic). These results demonstrated that the relative avidity of a peptide-MHC interaction can influence T cell tolerance induction. Furthermore, the results suggest that a higher threshold of peptide-MHC avidity may be required to induce T cell tolerance as compared to the threshold of peptide-MHC avidity required to immunize T cells.