Induction of central deletional T cell tolerance by gene therapy

Transgenic mice expressing an alloreactive TCR specific for the MHC class I Ag K(b) were used to examine the mechanism by which genetic engineering of bone marrow induces T cell tolerance. Reconstitution of lethally irradiated mice with bone marrow infected with retroviruses carrying the MHC class I gene H-2K(b) resulted in lifelong expression of K(b) on bone marrow-derived cells. While CD8 T cells expressing the transgenic TCR developed in control mice reconstituted with mock-transduced bone marrow, CD8 T cells expressing the transgenic TCR failed to develop in mice reconstituted with H-2K(b) transduced bone marrow. Analysis of transgene-expressing CD8 T cells in the thymus and periphery of reconstituted mice revealed that CD8 T cells expressing the transgenic TCR underwent negative selection in the thymus of mice reconstituted with K(b) transduced bone marrow. Negative selection induced by gene therapy resulted in tolerance to K(b). Thus, genetic engineering of bone marrow can be used to alter T cell education in the thymus by inducing negative selection.     

Induction of alloreactive CD4 T cell tolerance in molecular chimeras: a possible role for regulatory T cells

Induction of molecular chimerism following reconstitution of mice with autologous bone marrow cells expressing a retrovirally encoded allogeneic MHC class I Ag results in donor-specific tolerance. To investigate the mechanism by which CD4 T cells that recognize allogeneic MHC class I through the indirect pathway of Ag presentation are rendered tolerant in molecular chimeras, transgenic mice expressing a TCR on CD4 T cells specific for peptides derived from K(b) were used. CD4 T cells expressing the transgenic TCR were detected in mice reconstituted with bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b), albeit detection was at lower levels than in mice receiving mock-transduced bone marrow. Despite the presence of CD4 T cells expressing an alloreactive TCR, mice receiving H-2K(b)-transduced bone marrow permanently accepted K(b) disparate skin grafts. CD4+CD25+ T cells from mice reconstituted with H-2K(b)-transduced bone marrow prevented rejection of K(b) disparate skin grafts when adoptively transferred into immunodeficient mice along with effector T cells, suggesting that induction of molecular chimerism leads to the generation of donor specific regulatory T cells, which may be involved in preventing alloreactive CD4 T cell responses that lead to rejection.     

Defining the requirements for peptide recognition in gene therapy-induced T cell tolerance

Expression of a retrovirally transduced MHC class I Ag, H-2K(b) (K(b)), in bone marrow-derived cells leads to specific prolongation of K(b) disparate skin grafts. To examine the extent to which peptides derived from K(b) contribute to the induction of tolerance, retroviruses carrying mutant K(b) genes designed to enter separate pathways of Ag presentation were constructed. Thymectomized and CD8 T cell-depleted mice that had been irradiated and reconstituted with bone marrow cells expressing a secreted form of K(b) showed prolongation of K(b) disparate skin graft survival. Skin graft prolongation was not observed when similar experiments were performed using mice that were not CD8 T cell depleted. This suggests that hyporesponsiveness can be induced in CD4 T cells, but not CD8 T cells by Ags presented via the exogenous pathway of Ag processing. Modest prolongation of skin allografts was observed in mice reconstituted with bone marrow cells transduced with retroviruses carrying a gene encoding a mutant K(b) molecule expressed only in the cytoplasm. Prolongation was also observed in similar experiments in mice that were thymectomized and CD4 T cell depleted following complete reconstitution, but not in mice that were reconstituted and then thymectomized and CD8 T cell depleted. Thus, hyporesponsiveness can be induced in a subset of CD8 T cells by recognition of peptides derived from K(b) through both the direct and indirect pathways of Ag recognition, while CD4 T cell hyporesponsiveness to MHC class I disparate grafts occurs only through the indirect pathway of Ag recognition.     

Induction of central tolerance by mature T cells

Induction of immunological tolerance is highly desirable for the treatment and prevention of autoimmunity, allergy, and organ transplant rejection. Adoptive transfer of MHC class I disparate mature T cells at the time of reconstitution of mice with syngeneic bone marrow resulted in specific tolerance to allogeneic skin grafts that were matched to the T cell donor strain. Mature allogeneic T cells survived long-term in reconstituted hosts and were able to re-enter the thymus. Analysis of T cell development using transgenic mice expressing an alloantigen-reactive TCR revealed that expression of allogeneic MHC class I on adoptively transferred mature T cells mediated negative selection of developing alloreactive T cells in the thymus. Thus, mature allogeneic T cells are able to mediate central deletion of alloreactive cells and induce transplantation tolerance without the requirement for any other alloantigen-expressing cell type.     

Induction of robust diabetes resistance and prevention of recurrent type 1 diabetes following islet transplantation by gene therapy

We have previously shown that the development of type 1 diabetes (T1D) can be prevented in nonobese diabetic (NOD) mice by reconstitution with autologous hemopoietic stem cells retrovirally transduced with viruses encoding MHC class II I-A beta-chain molecules associated with protection from the disease. In this study we examined whether a blockade of the programmed death-1 (PD-1)-programmed death ligand-1 (PD-L1) pathway, a major pathway known to control diabetes occurrence, could precipitate T1D in young NOD mice following reconstitution with autologous bone marrow retrovirally transduced with viruses encoding protective MHC class II I-A beta-chain molecules. In addition, we examined whether the expression of protective MHC class II alleles in hemopoietic cells could be used to prevent the recurrence of diabetes in mice with pre-existing disease following islet transplantation. Protection from the occurrence of T1D diabetes in young NOD mice by the expression of protective MHC class II I-A beta-chain molecules in bone marrow-derived hemopoietic cells was resistant to induction by PD-1-PD-L1 blockade. Moreover, reconstitution of NOD mice with pre-existing T1D autologous hemopoietic stem cells transduced with viruses encoding protective MHC class II I-A beta-chains allowed for the successful transplantation of syngeneic islets, resulting in the long-term reversal of T1D. Reversal of diabetes was resistant to induction by PD-1-PDL-1 blockade and depletion of CD25(+) T cells. These data suggest that expression of protective MHC class II alleles in bone marrow-derived cells establishes robust self-tolerance to islet autoantigens and is sufficient to prevent the recurrence of autoimmune diabetes following islet transplantation.     

Mixed hematopoietic molecular chimerism results in permanent transgene expression from retrovirally transduced hepatocytes in mice

BACKGROUND: Cytotoxic immune elimination of transduced hepatocytes may limit gene therapy for inherited liver diseases. Using beta-galactosidase as a marker gene, we studied whether creation of mixed beta-galactosidase molecular hematopoietic chimerism could induce tolerance to beta-galactosidase-transduced hepatocytes. METHODS: Molecular hematopoietic chimerism was established in irradiated recipient mice by transplantation of either a mixture of wild-type and beta-galactosidase-transgenic bone marrow or autologous bone marrow stem cells that were transduced with beta-galactosidase lentiviral vectors. After transplantation, mice were hepatectomized and injected with beta-galactosidase recombinant retroviruses to transduce regenerating hepatocytes. We monitored the presence of beta-galactosidase-expressing hepatocytes as well as the appearance of anti-beta-galactosidase antibodies during the time. RESULTS: In control animals, anti-beta-galactosidase antibodies and cytotoxic T-lymphocyte (CTL) response developed as early as 3 weeks after gene transfer. Transduced hepatocytes disappeared concomitantly. In bone marrow transplanted mice, tolerance could be observed in a significant proportion of animals. Tolerance resulted in permanent liver transgene expression and was absent unless a chimerism above 1% was achieved, demonstrating a threshold effect. CONCLUSIONS: Creation of a molecular hematopoietic chimerism can result in transgene tolerance and evade immune rejection of retrovirally transduced hepatocytes. This strategy may be useful for hepatic inherited diseases in which the transgene product behaves as a non-self protein.     

Anti-bacterial immunity to Listeria monocytogenes in allogeneic bone marrow chimera in mice

Protection and delayed-type hypersensitivity (DTH) to the facultative intracellular bacterium Listeria monocytogenes (L.m.) were studied in allogeneic and syngeneic bone marrow chimeras. Lethally irradiated AKR (H-2k) mice were successfully reconstituted with marrow cells from C57BL/10 (B10) (H-2b), B10 H-2-recombinant strains or syngeneic mice. Irradiated AKR mice reconstituted with marrow cells from H-2-compatible B10.BR mice, [BR----AKR], as well as syngeneic marrow cells, [AKR----AKR], showed a normal level of responsiveness to the challenge stimulation with the listeria antigens when DTH was evaluated by footpad reactions. These mice also showed vigorous activities in acquired resistance to the L.m. By contrast, chimeric mice that had total or partial histoincompatibility at the H-2 determinants between donor and recipient, [B10----AKR], [B10.AQR----AKR], [B10.A(4R)----AKR], or [B10.A(5R)----AKR], were almost completely unresponsive in DTH and antibacterial immunity. However, when [B10----AKR] H-2-incompatible chimeras had been immunized with killed L.m. before challenge with live L.m., these mice manifested considerable DTH and resistance to L.m. These observations suggest that compatibility at the entire MHC between donor and recipient is required for bone marrow chimeras to be able to manifest DTH and protection against L.m. after a short-term immunization schedule. However, this requirement is overcome by a preceding or more prolonged period of immunization with L.m. antigens. These antigens, together with marrow-derived antigen-presenting cells, can then stimulate and expand cell populations that are restricted to the MHC (H-2) products of the donor type.     

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.     

Development of diabetogenic T cells from NOD/Lt marrow is blocked when an allo-H-2 haplotype is expressed on cells of hemopoietic origin, but not on thymic epithelium

Diabetes is a T cell-mediated process in NOD/Lt mice, with a major genetically recessive component of susceptibility linked to homozygous expression of the unique H-2g7 MHC haplotype. Heterozygous expression of the H-2nb1 haplotype derived from the NON/Lt strain confers diabetes resistance both in (NOD x NON)F1 hybrids and in NOD mice congenic for the H-2nb1 haplotype. However, diabetes resistance is abrogated in F1 hybrids by NOD/Lt bone marrow reconstitution. To establish whether the generation of beta cell autoreactive T cells from NOD/Lt bone marrow-derived precursors required at least heterozygous expression of the H-2g7 haplotype on thymic epithelium, adolescent thymectomized (NOD x NON)F1 mice were implanted with neonatal NON/Lt thymus grafts before lethal radiation and reconstitution with NOD/Lt bone marrow. Peripheral T cells maturing through this ectopic thymic implant exclusively expressed the NOD H-2g7 haplotype and were tolerant to H-2nb1 skin grafts. Nevertheless, diabetes developed in 32% of the NON/Lt thymus-grafted chimeras vs 38% of the sham-thymectomized NOD bone marrow chimeras. Thus, homozygous expression of the diabetes-resistant H-2nb1 haplotype on thymic epithelium failed to block development of a diabetogenic T cell repertoire. To examine if expression of H-2nb1 on hemopoietically derived APC could alter the diabetogenic potential of NOD/Lt marrow, diabetes-resistant NOD.NON-H-2nb1 congenic mice were mated with NOD/Lt mice to produce NOD-H-2g7/H-2nb1 heterozygous recipients. These were lethally irradiated and reconstituted with either NOD/Lt marrow alone, NOD.H-2nb1 homozygous congenic marrow alone, or a 1:1 mixture of the two marrow populations. By 25 wk of age, all of the MHC heterozygous recipients of NOD.NON-H-2nb1 marrow remained diabetes-free whereas 75% of the MHC heterozygous recipients of NOD/Lt marrow developed diabetes. A striking decrease in diabetes was observed when T cell precursors derived from NOD/Lt marrow interacted with H-2nb1 gene products on hemopoietically derived APC, inasmuch as only 7% of the MHC heterozygous recipients reconstituted with a 1:1 mixture of NOD/Lt and NOD.NON-H-2nb1 marrow developed diabetes. Peripheral leukocytes in all reconstitution classes expressed the MHC phenotype(s) of the marrow donor(s). Skin grafting confirmed that all reconstitution classes of MHC heterozygous recipients were tolerant to the H-2nb1 haplotype.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transgenic major histocompatibility complex class I antigen expressed in mouse trophoblast affects maternal immature B cells

We have produced transgenic mice using the mouse placental lactogen type II promoter to force and restrict the expression of the mouse major histocompatibility complex (MHC) class I molecule, H-2K(b), to the placenta. We show that the transgenic MHC antigen H-2K(b) is expressed exclusively in trophoblast giant cells from Day 10.5 until the end of gestation. This expression affects neither the fetal development nor the maternal tolerance to the fetus in histoincompatible mothers. We have used the 3.83 B cell receptor (BcR) transgenic mouse line to follow the fate of H-2K(b)-specific maternal B cells in mothers bearing H-2K(b)-positive placentas. Our results suggest that transgenic H-2K(b) molecules on trophoblast giant cells are recognized by 3.83 BcR-transgenic B cells in the bone marrow of pregnant females. This antigen recognition triggers the deletion of a bone marrow B cell subpopulation, including immature and transitional B cells. Their percentage decreases during the second half of gestation and is down to 8% on Day 17.5, compared to 22% in the (3.83 Tg female x Fvb) control group. This deletion might contribute to the process of maternal tolerance of the conceptus.     

Dissociation of hemopoietic chimerism and allograft tolerance after allogeneic bone marrow transplantation.

Creation of stable hemopoietic chimerism has been considered to be a prerequisite for allograft tolerance after bone marrow transplantation (BMT). In this study, we demonstrated that allogeneic BMT with bone marrow cells (BMC) prepared from either knockout mice deficient in both CD4 and CD8 T cells or CD3E-transgenic mice lacking both T cells and NK cells maintained a high degree of chimerism, but failed to induce tolerance to donor-specific wild-type skin grafts. Lymphocytes from mice reconstituted with T cell-deficient BMC proliferated when they were injected into irradiated donor strain mice, whereas lymphocytes from mice reconstituted with wild-type BMC were unresponsive to donor alloantigens. Donor-specific allograft tolerance was restored when donor-type T cells were adoptively transferred to recipient mice given T cell-deficient BMC. These results show that donor T cell engraftment is required for induction of allograft tolerance, but not for creation of continuous hemopoietic chimerism after allogeneic BMT, and that a high degree of chimerism is not necessarily associated with specific allograft tolerance.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. I. Preferential induction of tolerance to Mls antigens and resistance to allo-MHC antigens

Neonatal tolerance inducibility of self-major histocompatibility complex (MHC)-class II-associated antigens was compared with that of allo-class II antigens. BALB/c (H-2d, Mlsb) mice, less than 24 hr after birth, were intravenously injected with bone marrow cells of either (BALB/c X DBA/2)F1 (H-2d, Mlsb/a, semiallogeneic at the Mls locus) or (BALB/c X B10.BR)F1 (H-2d/k, Mlsb; semiallogeneic at the MHC), as antigens. The mice were tested for in vivo immune activity of class II-reactive T cells by means of the popliteal lymph node-swelling assay. They developed tolerance, irrespective of type of antigens, showing profoundly suppressed host-versus-graft reaction, and those tolerized to the allo-MHC antigens accepted skin grafts of the corresponding allogeneic mice. In the thymus and spleen of the Mls-tolerant mice, antigen-specific class II-reactive T-cell activity was completely abolished, without the apparent involvement of suppressor cells. In contrast, the activity in allo-MHC-tolerant mice was not reduced in either thymus or peripheral lymphoid organs, suggesting that systemic hyporesponsiveness is attributable to reversible suppression of immune competent cells. The resistance for cell-level tolerance induction to allo-class II antigens may not be ascribed to the active participation of allo-MHC antigens in prevention of or in escape from tolerance induction or both, since an injection of bone marrow cells of both Mls and H-2-semiallogeneic (DBA/2 X B10.BR)F1 (H-2d/k, Mlsa/b) mice could induce tolerance to Mlsa-H-2d antigens in newborn thymus cells.     

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.     

Bone marrow-derived macrophage expression of endogenous and transfected class II MHC genes during differentiation in vitro

C57BL/6 (H-2b) mice fail to express I-E molecules on the surface of their cells and thus are unable to respond to I-E-restricted antigens such as GL phi and cytochrome c. Previous experiments in our laboratory have involved developing a system for studying differentiation of bone marrow cells into mature macrophage to gain a better understanding of class II MHC gene expression and function. In this study, we have used this system to transfect the E alpha d gene (cosmid 17.2) into C57BL/6 bone marrow cells and subsequently observed I-E expression on bone marrow-derived macrophages (BMDM) after differentiation in vitro. By using a modified calcium phosphate protocol, we found that the optimal period for transfection of the bone marrow cells was after 2 days of culture in vitro. By using the anti-I-E monoclonal antibody (Ia.7) derived from hybridoma 14-4-4, we detected the I-E molecule on the surface of transfected macrophages by a radiobinding assay and immunoprecipitation. BMDM expressed the I-E product maximally at 5 days of differentiation, and expression then declined. Furthermore, we have found that the expression of the I-E molecule on transfected macrophage was dependent upon exposure to interferon-gamma. Expression of I-E molecules was also detected by the generation of an allogeneic response. Transfected BMDM were compared with (CB6)F1 BMDM for their ability to stimulate C57BL/6 T cells and they were found to be equally effective. By using these initial findings, we hope to further optimize the conditions for insertion and expression of class II MHC genes in bone marrow cells.     

Immunogenetic analysis of tolerance induction in anti-alloantigen delayed type hypersensitivity responses by portal venous pre-inoculation with allogeneic cells

The present study investigates some of the immunogenetic bases for tolerance of anti-allo-delayed type hypersensitivity (DTH) responses as induced by pre-inoculating allogeneic cells via portal venous (p.v.) route. BALB/c mice were injected with totally allogeneic C57BL/6 or H-2 incompatible BALB.B spleen cells via p.v. route. These mice not only failed to exhibit anti-H-2b DTH responses, but also abrogated the potential to generate H-2b-specific DTH responses as induced by the subsequent immunization with H-2b spleen cells via subcutaneous (s.c.) route. The p.v. presensitization with allogeneic spleen cells differing at either class I or class II of major histocompatibility complex (MHC) resulted in the tolerance induction of DTH responses to the respective allogeneic class I or class II MHC antigens. Moreover, the p.v. administration of the class I-positive allogeneic cell fraction depleted of class II-positive component into recipients differing at both class I and class II was capable of inducing anti-class I DTH tolerance. These results indicate that anti-allo-class I or class II DTH tolerance can be induced independently and that the existence of class II antigens on p.v.-presensitized cells is not necessarily required for the tolerance induction of anti-allo-class I DTH response.     

Mechanisms of Allografted Tumor Rejection: The Roles of T Cells in Allograft Rejection Mediated by a Type of Bone Marrow-Derived Macrophage

Allografted tumor rejection does not occur in the absence of T cells, but the main effector cells responsible for the rejection are allograft-induced macrophages (AIM). We examined the roles of T cells in the AIM-mediated rejection of Meth A (H-2) tumor cells from C57BL/6 (H-2^b) mice. Irradiation of C57BL/6 mice abrogated both the induction of AIM and the allograft rejection. Reconstitution of the irradiated mice with F1 (C57BL/6 X C3H/He: H-2^b/k) bone marrow cells led to the appearance of H-2^b/k haplo-type of AIM exclusively in the rejection site and to allograft rejection, indicating that radiosensitive cells prerequisite for both the induction of AIM and allograft rejection were bone marrow-derived cells, and that the progenitors of AIM existed in the bone marrow cells to be activated into AIM in the rejection site. To understand the role of T cells in the induction of AIM, we used adult-thymectomized, X-irradiated C57BL/6 mice reconstituted with F1 bone marrow (ATXBM). The ATXBM mice could neither induce AIM nor reject allogeneic Meth A cells, whereas adoptive transfer of F1 lymph node T cells to the ATXBM mice restored not only the induction of AIM but also rejection of the allograft. Among the lymph node T cells, CD4⁺, but not CD8⁺, cells were found to be essential for the activation of AIM progenitors to AIM; and CD8⁺ T cells were further required for rejection, at least in part, to enhance the number of AIM in the rejection site.     

Transfection and expression of syngeneic H-2 genes does not reduce malignancy of H-2 negative teratocarcinoma cells in the autologous host

Rejection of the MHC class I negative 402AX teratocarcinoma is accompanied by induction of tumor cell-encoded H-2K and H-2D antigens by the genetically resistant host. To determine whether MHC antigen expression is required for 402AX rejection, we have prepared H-2Db-transfected 402AX cells (402AX/Db). Transfectants express high levels of H-2Db, most of which is not associated with beta 2-microglobulin. MHC syngeneic and allogeneic mice susceptible to 402AX are resistant to 402AX/Db, suggesting that MHC class I antigen expression is required for tumor rejection. Autologous 129 hosts, however, are susceptible to 402AX/Db. 402AX cells transfected with the H-2Kb gene (402AX/Kb) are also lethal in the autologous 129/J host, but rejected by MHC syngeneic and allogeneic mice. Non-129 strain 402AX-susceptible mice pre-immunized with 402AX/Db or simultaneously challenged with 402AX/Db plus 402AX are immune to 402AX. Mice immunized with 402AX/Db produce MHC class I induction factor. 402AX/Db and 402AX cells are lysed equally by natural killer cells, indicating that in 402AX cells the expression of class I antigens is unrelated to NK susceptibility. These studies confirm the requirement for class I expression in 402AX immunity, but demonstrate that in the autologous host immunity requires additional factors beyond class I antigen expression.     

In vivo priming of mouse CTL precursors directed to product of a newly defined minor H-42 locus is under a novel control of class II MHC gene

In a previous study, we discovered a new mouse minor histocompatibility antigen encoded by a locus at 8.5 cM apart from the H-2 complex, and we have since named the locus H-42. One allele of H-42, which is named H-42a, had been elucidated, but the other alleles, which we tentatively named H-42b, have not been elucidated. In the present study, we explored MHC control on the anti-H-42a cytotoxic T lymphocyte (CTL) responsiveness in H-42b mice. In vivo immunization (i.v. injection) of H-42b mice with 5 to 30 X 10(6) spleen cells (SC) bearing allogeneic H-42a antigen but carrying H-2 complex (mouse MHC) matched with the H-42b mice failed to prime anti-H-42a CTL but induced stable and specific anti-H-42a CTL unresponsiveness, i.e., tolerance, in the H-42b recipient mice. In contrast, H-2 heterozygous H-42b F1 mice injected with SC bearing H-42a alloantigen on either of the parental H-2 haplotypes were effectively primed to generate anti-H-42a CTL. Exploration of the region or subregion in the H-2 complex of H-42a donor SC that should be compatible with H-42b recipient mice for the induction of their anti-H-42a CTL tolerance demonstrated that the compatibility at I region, most probably I-A subregion, but not at K, S, or D region, determined the induction of the tolerance. MHC class II compatible H-42a skin graft (SG) to H-42b mice, however, consistently primed the anti-H-42a CTL in the H-42b recipients. These results were discussed in several aspects, including uniqueness of MHC class II control on the CTL response to minor H-42a antigen, possibility of inactivation of responding anti-H-42a precursor CTL or helper T cells in H-42b mice by encountering the veto cells present in MHC class II-matched H-42a SC population, and significance of the present observations as a mechanism of CTL tolerance to self-components.     

Multiple splenic lymphoid cell subpopulations regulate H-2 antigen expression on teratocarcinoma cells in vivo

Undifferentiated murine 402AX teratocarcinoma cells do not express MHC antigens when passaged in vitro or in vivo in genetically susceptible host mice. When passaged in vivo in genetically resistant mice, however, the tumor cells become H-2b antigen positive regardless of the H-2 haplotype of the resistant host mouse. The present studies use monoclonal anti-H-2b antibodies to corroborate these earlier findings, which were performed with conventional antisera. Previous studies have established that host bone marrow plus lymphoid cells from resistant primed donors regulate tumor cell H-2b antigen expression. Using bone marrow and mature lymphoid cell reconstitution techniques, the present studies indicate that splenic Ig- cells from genetically resistant host mice are the most efficient lymphoid cell subpopulation in tumor cell H-2b antigen induction. Ig+ spleen cells also reconstitute the capacity to induce teratocarcinoma cell H-2 antigens but are less effective than Ig- spleen cells. Tumor cell H-2 antigen induction in C57BL/6 beige mice is impaired compared to C57BL/6 hosts, which suggests that host NK cells may also be involved in tumor cell H-2 antigen induction. Reconstitution of lethally irradiated resistant hosts for teratocarcinoma cell H-2 antigen expression requires bone marrow plus resistant primed lymphoid cell subpopulations; bone marrow alone is insufficient. These results indicate that multiple splenic lymphoid cell subpopulations requiring a radiosensitive host environment and/or factor for differentiation regulate teratocarcinoma 402AX H-2b antigen expression in vivo in genetically resistant mice.