ABSTRACT
Resistance to checkpoint-blockade treatments is a challenge in the clinic. We found that although treatment with combined anti-CTLA-4 and anti-PD-1 improved control of established tumors, this combination compromised anti-tumor immunity in the low tumor burden (LTB) state in pre-clinical models as well as in melanoma patients. Activated tumor-specific T cells expressed higher amounts of interferon-γ (IFN-γ) receptor and were more susceptible to apoptosis than naive T cells. Combination treatment induced deletion of tumor-specific T cells and altered the T cell repertoire landscape, skewing the distribution of T cells toward lower-frequency clonotypes. Additionally, combination therapy induced higher IFN-γ production in the LTB state than in the high tumor burden (HTB) state on a per-cell basis, reflecting a less exhausted immune status in the LTB state. Thus, elevated IFN-γ secretion in the LTB state contributes to the development of an immune-intrinsic mechanism of resistance to combination checkpoint blockade, highlighting the importance of achieving the optimal magnitude of immune stimulation for successful combination immunotherapy strategies.
Subject(s)
Antibodies, Monoclonal/pharmacology , CTLA-4 Antigen/antagonists & inhibitors , Drug Resistance, Neoplasm/drug effects , Interferon-gamma/pharmacology , Neoplasms, Experimental/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , T-Lymphocytes/drug effects , Animals , Antibodies, Monoclonal/immunology , CTLA-4 Antigen/immunology , CTLA-4 Antigen/metabolism , Cell Line, Tumor , Clonal Deletion/drug effects , Clonal Deletion/immunology , Drug Resistance, Neoplasm/immunology , Humans , Interferon-gamma/immunology , Interferon-gamma/metabolism , Male , Mice, Inbred C57BL , Mice, Knockout , Neoplasms, Experimental/immunology , Neoplasms, Experimental/metabolism , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Burden/drug effects , Tumor Burden/immunologyABSTRACT
The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αß(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVß2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Chimerism/drug effects , Clonal Deletion/immunology , Killer Cells, Natural/immunology , T-Lymphocytes, Regulatory/immunology , Adoptive Transfer , Animals , Bone Marrow Transplantation/immunology , CD4-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/drug effects , Clonal Deletion/drug effects , Cyclophosphamide/therapeutic use , Graft vs Host Disease/drug therapy , Graft vs Host Disease/immunology , Immunosuppressive Agents/therapeutic use , Killer Cells, Natural/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Perforin/immunology , Receptors, Antigen, T-Cell/immunology , Skin Transplantation/immunology , T-Lymphocytes, Regulatory/drug effectsABSTRACT
We propose clonal deletion--immunization followed by deletion--as a "new" way to achieve tolerance. Immunization of a donor results in specific stimulation of a clone of cells, which can then be killed by various agents, leaving the patient otherwise immunologically normal. The theory of clonal deletion is supported by experimental evidence as well as earlier experiences with kidney transplants and donor-specific transfusions. To date, 22 patients who underwent clonal deletion have been surviving for 1.5 to 2.5 years with only low-dose prednisone. In addition, those patients who required conventional immunosuppressive drugs were treated with the new Drugs Added When Needed (DAWN) protocol. With DAWN as a tactic ready for intervention, and by using antibodies to monitor the completeness of clonal deletion, we assure that patients are subjected to the minimal amount of drugs on a personalized basis. We suggest that risks involved in testing this new procedure are small and the benefits immeasurable.
Subject(s)
Clonal Deletion/drug effects , Graft Rejection/prevention & control , Graft Survival/drug effects , Immunization , Immunosuppression Therapy/methods , Immunosuppressive Agents/therapeutic use , Organ Transplantation/adverse effects , Transplantation Tolerance/drug effects , Animals , Graft Rejection/immunology , Humans , Treatment OutcomeABSTRACT
To kill antigen-specific target cells (TCs), cytotoxic T lymphocytes (CTLs) reorganise their microtubule cytoskeleton to deliver lytic granules to the TCs. We used two drugs that stabilise microtubules, paclitaxel and peloruside, to determine how the stabilising microtubule network affects CTL function in vitro and in vivo. In vitro, neither paclitaxel nor peloruside inhibited antigen-specific killing, lytic granule delivery to the cell surface, nor interferon-gamma release by murine CTLs. In contrast, in an in vivo murine model of antigen-induced killing, a single dose of paclitaxel had a significant inhibitory effect on killing by CTLs. Furthermore, the inhibitory effect of paclitaxel was not caused by specific deletion of the effector CTL population in drug-treated mice. The findings reveal that microtubule-stabilising drug treatment can lead to immediate impairment of CTL function without affecting lytic granule release. The results also suggest that patients undergoing taxane anti-cancer therapy may be impaired in their ability to fight infection before the anti-mitotic effects of paclitaxel are apparent.
Subject(s)
Clonal Deletion/drug effects , Mitosis/drug effects , Models, Immunological , Paclitaxel/pharmacology , T-Lymphocytes, Cytotoxic/immunology , Tubulin Modulators/pharmacology , Animals , Antigens/immunology , Clonal Deletion/immunology , Dose-Response Relationship, Drug , Interferon-gamma/immunology , Mice , Mice, Transgenic , Microtubules/immunology , Mitosis/immunology , Secretory Vesicles/immunologyABSTRACT
Autoimmune diseases are incurable. We have hypothesized that these diseases can be cured by the transplantation of bone marrow (BM) stem cells that have been genetically engineered to express self-Ag. Here we have tested this hypothesis in experimental autoimmune encephalomyelitis (EAE) induced by the self-Ag myelin oligodendrocyte glycoprotein (MOG). We show that, in mice, transplantation of BM genetically modified to express MOG prevented the induction and progression of EAE, and combined with antecedent corticosteroid treatment, induced long-term remission of established disease. Mice remained resistant to EAE development upon subsequent rechallenge with MOG. Transfer of BM from these mice rendered recipients resistant to EAE. Splenocytes from these mice failed to proliferate or produce IL-17, IFN-gamma, and GM-CSF in response to MOG(35-55) peptide stimulation and they failed to produce MOG autoantibody. Mechanistically, we demonstrated in vivo reduction in development of CD4(+) MOG(35-55)-specific thymocytes, indicative of clonal deletion with no evidence for selection of Ag-specific regulatory T cells. These findings validate our hypothesis that transplantation of genetically modified BM expressing disease-causative self-Ag provides a curative approach by clonal deletion of disease-causative self-reactive T cells.
Subject(s)
Autoantigens/immunology , Bone Marrow Transplantation , Clonal Deletion/immunology , Encephalomyelitis, Autoimmune, Experimental/immunology , Glycoproteins/immunology , Immune Tolerance/immunology , Peptide Fragments/immunology , T-Lymphocytes, Regulatory/immunology , Adrenal Cortex Hormones/pharmacology , Animals , Autoantibodies/genetics , Autoantibodies/immunology , Autoantigens/genetics , Clonal Deletion/drug effects , Clonal Deletion/genetics , Cytokines/genetics , Cytokines/immunology , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/therapy , Female , Gene Expression/genetics , Gene Expression/immunology , Glycoproteins/genetics , Immune Tolerance/drug effects , Immune Tolerance/genetics , Immunity, Innate/genetics , Immunity, Innate/immunology , Mice , Mice, Transgenic , Myelin-Oligodendrocyte Glycoprotein , Peptide Fragments/genetics , Thymus Gland/immunology , Transduction, GeneticABSTRACT
CD8+ cytotoxic T lymphocytes (CTLs) are important effector cells responsible for tissue destruction in several autoimmune and allograft-related diseases. To discover if pathogenic T cells could be selectively deleted, we investigated the ability of a toxin coupled to major histocompatibility complex (MHC) class I tetramers to kill antigen-specific CD8+ T cells. H2-D(b) tetramers were assembled using streptavidin conjugated to the ribosome-inactivating protein (RIP) saporin (SAP). These tetramers inhibited ribosome activity in vitro, retained the T-cell receptor (TCR)-binding specificity of their nontoxic counterparts, and were internalized by 100% of target cells, leading to cell death in 72 hours. Cytotoxicity was dependent on the tetramer dose and avidity for the T cell. A single injection of the SAP-coupled tetramer eliminated more than 75% of cognate, but not control, T cells. This work demonstrates the therapeutic potential of cytotoxic tetramers to selectively eradicate pathogenic clonotypes while leaving overall T-cell immunity intact.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Clonal Deletion/drug effects , Cytotoxins/pharmacology , Histocompatibility Antigens Class I/pharmacology , N-Glycosyl Hydrolases/pharmacology , Plant Proteins/pharmacology , Animals , Autoimmune Diseases/drug therapy , Autoimmune Diseases/immunology , Autoimmune Diseases/pathology , CD8-Positive T-Lymphocytes/pathology , Cell Death/drug effects , Cytotoxins/immunology , Histocompatibility Antigens Class I/immunology , Mice , Mice, Transgenic , Ribosome Inactivating Proteins, Type 1 , Saporins , Time Factors , Transplantation, HomologousABSTRACT
T cell depletion, sirolimus and "mega" dose donor specific bone marrow (DSBM) infusion promotes stable multilineage chimerism and indefinite survival of skin allografts in completely mismatched mice. The purpose of this study is to determine whether the addition of low dose busulfan can reduce the amount of DSBM required while preserving efficacy. C57BL/6 recipients of BALB/c skin allografts were treated with alphaCD4 and alphaCD8 monoclonal antibodies, DSBM, sirolimus and various doses of busulfan. The kinetics and phenotype of chimerism and the presence of clonal deletion of alloreactive T-cells were defined using flow cytometry. In vitro reactivity was determined using mixed lymphocyte culture. Second skin grafts confirmed the presence of tolerance. All doses of busulfan resulted in engraftment when combined with this regimen using a reduced dose of donor marrow. The level, kinetics and character of chimerism observed were dose related. Chimerism was associated with indefinite allograft acceptance (>200 days). Tolerance was documented both in vitro/in vivo and was associated with clonal deletion. Addition of a single low dose of busulfan to an established tolerance protocol reduced the required DSBM dose by over 80% while still promoting comparable levels of donor chimerism and donor-specific tolerance.
Subject(s)
Busulfan/pharmacology , Graft Survival/drug effects , Immune Tolerance/drug effects , Myeloablative Agonists/pharmacology , Skin Transplantation/immunology , Transplantation Chimera/immunology , Animals , Bone Marrow Transplantation/immunology , Clonal Deletion/drug effects , Clonal Deletion/immunology , Graft Survival/immunology , Immune Tolerance/immunology , Lymphocyte Depletion/methods , Mice , Mice, Inbred BALB C , Transplantation, HomologousABSTRACT
Costimulation blockade protocols are effective in prolonging allograft survival in animal models and are entering clinical trials, but how environmental perturbants affect graft survival remains largely unstudied. We used a costimulation blockade protocol consisting of a donor-specific transfusion and anti-CD154 mAb to address this question. We observed that lymphocytic choriomeningitis virus infection at the time of donor-specific transfusion and anti-CD154 mAb shortens allograft survival. Lymphocytic choriomeningitis virus 1) activates innate immunity, 2) induces allo-cross-reactive T cells, and 3) generates virus-specific responses, all of which may adversely affect allograft survival. To investigate the role of innate immunity, mice given costimulation blockade and skin allografts were coinjected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid), TLR4 (LPS), or TLR9 (CpG) agonists. Costimulation blockade prolonged skin allograft survival that was shortened after coinjection by TLR agonists. To investigate underlying mechanisms, we used "synchimeric" mice which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, coadministration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells and shortened skin allograft survival. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) up-regulated CD44, and 3) failed to undergo apoptosis. B6.TNFR2-/- and B6.IL-12R-/- mice treated with costimulation blockade plus LPS also exhibited short skin allograft survival whereas similarly treated B6.CD8alpha-/- and TLR4-/- mice exhibited prolonged allograft survival. We conclude that TLR signaling abrogates the effects of costimulation blockade by preventing alloreactive CD8+ T cell apoptosis through a mechanism not dependent on TNFR2 or IL-12R signaling.
Subject(s)
Graft Enhancement, Immunologic , Graft Rejection/prevention & control , Growth Inhibitors/administration & dosage , Skin Transplantation/immunology , Toll-Like Receptors/agonists , Animals , Antibodies, Monoclonal/administration & dosage , Apoptosis/immunology , CD40 Ligand/immunology , CD8-Positive T-Lymphocytes/drug effects , Cell Line, Tumor , Clonal Deletion/drug effects , Female , Graft Rejection/immunology , Lipopolysaccharides/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Mice, Transgenic , Radiation Chimera , Receptors, Interleukin/metabolism , Receptors, Interleukin-12 , Receptors, Tumor Necrosis Factor, Type II/metabolism , Toll-Like Receptor 4/metabolism , Transplantation, HomologousABSTRACT
The current study examines how responsiveness of T cells is affected by the avidity of the peptide/MHC engaged during positive selection of their thymocyte precursors. We used a thymus reaggregate culture system in which CD4(+)CD8(+) thymocytes from AND TCR transgenic mice were induced to undergo positive selection by pigeon cytochrome c (PCC) peptide or its analogs presented by I-E(k) class II MHC on a thymic epithelial cell line. When low-affinity peptide analogs drove positive selection, up to 100 microM was needed to produce >50% CD4(+) T cells, and these cells were highly responsive to PCC. In contrast, <0.2 microM high-affinity peptides was required to achieve similar selection efficiency, but the resultant cells failed to respond to PCC. However, these cells were not dead based on dye exclusion and capacity to respond to phorbal ester and to agonist if IL-2 was also present, supporting the view that non-responsiveness of cells selected on high-affinity peptides is a form of central T cell tolerance distinct from deletion. Cells selected on intermediate-affinity peptides showed variable responsiveness which was suppressed 5- to 10-fold by addition during reaggregate culture of antibody to the IL-7R. Similarly, supplementary IL-7 in the reaggregate culture produced CD4(+) T cells that were promiscuously responsive. Overall, this study demonstrates that the responsiveness of T cells is not rigidly controlled and that the presence of IL-7 during T cell development has the potential to negate central T cell tolerance and produce autoreactive T cells.
Subject(s)
CD4 Antigens/immunology , CD4-Positive T-Lymphocytes/immunology , CD8 Antigens/immunology , Clonal Deletion/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocyte Subsets/immunology , Animals , Carcinogens/pharmacology , Cells, Cultured , Clonal Deletion/drug effects , Columbidae , Cytochromes c/immunology , Interleukin-7/immunology , Mice , Mice, Transgenic , Phorbol Esters/pharmacology , Receptors, Antigen, T-Cell, alpha-beta/geneticsABSTRACT
Polycyclic aromatic hydrocarbons (PAH) are common environmental pollutants that suppress the immune system in part by inducing pro/pre-B cell apoptosis. The PAH-induced death signaling pathway resembles the signaling cascade activated during clonal deletion and modeled by B cell receptor cross-linking or by dexamethasone exposure of immature surface Ig(+) B cells in that apoptosis is mediated by NF-kappa B down-regulation. Because a PAH-induced, clonally nonrestricted deletion of B cells would have important implications for B cell repertoire development, the nature of the PAH-induced intracellular death signal was studied further. Particular emphasis was placed on the roles of growth arrest and c-Myc, p27(Kip1), and p21(WAF1) expression, because all of these elements contribute to clonal deletion. As in clonal deletion models, and as predicted by the down-regulation of NF-kappa B, PAH-induced death of pro/pre-B cells was at least partially dependent on c-Myc down-regulation. Furthermore, whereas dexamethasone induced a G(0)/G(1) cell cycle arrest, PAH had no effect on pro/pre-B cell growth, indicating that growth arrest and apoptosis occur by separable signaling pathways in this early phase of B cell development. Finally, in contrast to clonal deletion, PAH-induced pro/pre-B cell death was not dependent on p27(Kip1) or p21(WAF1) up-regulation but did coincide with p53 induction. These results distinguish the PAH-induced apoptosis pathway from that activated during clonal deletion and indicate that signaling cascades leading to growth arrest and/or apoptosis in pro/pre-B cells differ from those active at later B cell developmental stages.
Subject(s)
9,10-Dimethyl-1,2-benzanthracene/pharmacology , Apoptosis/drug effects , B-Lymphocyte Subsets/drug effects , Cell Cycle Proteins/physiology , Clonal Deletion/drug effects , Cyclins/physiology , Hematopoietic Stem Cells/drug effects , Proto-Oncogene Proteins c-myc/physiology , Tumor Suppressor Proteins/physiology , Animals , B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/metabolism , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , Cell Cycle Proteins/biosynthesis , Cell Line , Coculture Techniques , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Cyclins/biosynthesis , Dexamethasone/pharmacology , Down-Regulation/drug effects , Environmental Pollutants/pharmacology , Growth Inhibitors/pharmacology , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Mice , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Proto-Oncogene Proteins c-myc/biosynthesis , Signal Transduction/drug effects , Tumor Suppressor Proteins/biosynthesis , Up-Regulation/drug effectsABSTRACT
Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag "sequestration," or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by approximately 80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.
Subject(s)
Antigen Presentation , Antigens, Neoplasm/immunology , Apoptosis/immunology , CD8-Positive T-Lymphocytes/immunology , Clonal Anergy/immunology , Deoxycytidine/analogs & derivatives , Mesothelioma/immunology , Mesothelioma/pathology , Up-Regulation/immunology , Animals , Antigen Presentation/drug effects , Antigen-Presenting Cells/immunology , Antigen-Presenting Cells/metabolism , Antigens, Neoplasm/metabolism , Apoptosis/drug effects , CD8-Positive T-Lymphocytes/metabolism , Clonal Anergy/drug effects , Clonal Deletion/drug effects , Clonal Deletion/immunology , Cytotoxicity, Immunologic/drug effects , Deoxycytidine/administration & dosage , Dose-Response Relationship, Immunologic , Epitopes, T-Lymphocyte/immunology , Epitopes, T-Lymphocyte/metabolism , Growth Inhibitors/administration & dosage , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Immunization , Injections, Intraperitoneal , Mesothelioma/drug therapy , Mesothelioma/prevention & control , Mice , Mice, Inbred BALB C , Mice, Transgenic , Tumor Cells, Cultured , Up-Regulation/drug effects , GemcitabineABSTRACT
Negative selection refers to the selective deletion of autoreactive thymocytes. Its molecular mechanisms have not been well defined. Previous studies in our laboratory have demonstrated that retinoic acids, physiological ligands for the nuclear retinoid receptors, selectively inhibit TCR-mediated death under in vitro conditions, and the inhibition is mediated via the retinoic acid receptor (RAR) alpha. The present studies were undertaken to investigate whether ligation of RARalpha leads to inhibition of TCR-mediated death in vivo and to identify the molecular mechanisms involved. Three models of TCR-mediated death were studied: anti-CD3-mediated death of thymocytes in wild-type mice, and Ag- and bacterial superantigen-driven thymocyte death in TCR-transgenic mice expressing a receptor specific for a fragment of pigeon cytochrome c in the context of the E(k) (class II MHC) molecule. Our data demonstrate that the molecular program of both anti-CD3- and Ag-driven, but not that of superantigen-mediated apoptosis involves up-regulation of nur77, an orphan nuclear receptor, and bim, a BH3-only member of the proapoptotic bcl-2 protein family, proteins previously implicated to participate in the negative selection. Ligation of RARalpha by the synthetic agonist CD336 inhibited apoptosis, DNA binding of nur77, and synthesis of bim induced by anti-CD3 or the specific Ag, but had no effect on the superantigen-driven cell death. Our data imply that retinoids are able to inhibit negative selection in vivo as well, and they interfere with multiple steps of the T cell selection signal pathway.
Subject(s)
Carrier Proteins/antagonists & inhibitors , DNA-Binding Proteins/antagonists & inhibitors , Membrane Proteins , Proto-Oncogene Proteins , Receptors, Retinoic Acid/agonists , Receptors, Retinoic Acid/physiology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/immunology , Thymus Gland/cytology , Thymus Gland/immunology , Transcription Factors/antagonists & inhibitors , Animals , Antibodies, Monoclonal/administration & dosage , Apoptosis/drug effects , Apoptosis/immunology , Apoptosis Regulatory Proteins , Bcl-2-Like Protein 11 , Benzoates/administration & dosage , CD3 Complex/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , Carrier Proteins/biosynthesis , Clonal Deletion/drug effects , Clonal Deletion/immunology , Columbidae , Cytochrome c Group/administration & dosage , Cytochrome c Group/immunology , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Enterotoxins/administration & dosage , Injections, Intraperitoneal , Ligands , Male , Mice , Mice, Transgenic , Nuclear Receptor Subfamily 4, Group A, Member 1 , Protein Binding/drug effects , Protein Binding/immunology , Receptors, Cytoplasmic and Nuclear , Receptors, Retinoic Acid/metabolism , Receptors, Steroid , Retinoic Acid Receptor alpha , Retinoids/metabolism , Staphylococcus aureus/immunology , Superantigens/administration & dosage , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/metabolism , Tetrahydronaphthalenes/administration & dosage , Thymus Gland/drug effects , Thymus Gland/metabolism , Transcription Factors/biosynthesis , Transcription Factors/metabolismABSTRACT
Maturation arrest and interference with selection are two well-documented effects of cyclosporin-A (CsA) on the thymus. We recently hypothesized that these effects are related and owing to the reduced T-cell receptor (TCR)-CD3 complex-mediated signal transduction in thymocytes upon CsA treatment. In this hypothesis, the maturation arrest is the result of the additional depletion of thymocytes that normally survive by positive selection, whereas the impaired self-tolerance induction is caused by an increased survival of thymocytes that normally undergo negative selection. In this view, it is anticipated that CsA differentially affects thymocyte apoptosis during in vivo thymocyte maturation. Indeed, we report in this study a strong increase in apoptotic cells in the thymic cortex on in situ analysis. Simultaneously, the number of apoptotic cells had decreased at the cortico-medullary zone which is held to be the site for negative selection. Rapamycin (Rapa) also interferes with thymocyte maturation by inhibiting cytokine-driven proliferation. Hence, Rapa preferentially affects the early maturational stages of thymocyte development and is considered not to alter thymocyte selection and subsequent apoptotic events. Indeed, the number of apoptotic events appears not to be altered. However, possibly owing to the decrease in cortical macrophages, the apoptotic cells revealed an atypical enumeration around blood vessels. Taken together, our results favour the hypothesis that the dominant effect of CsA on the thymus is the reduction of the TCR-CD3 complex-mediated signal transduction in thymocytes upon interaction with stromal cells. Furthermore, the preferential localization of apoptotic cells next to blood vessels upon Rapa administration may indicate that endothelial cells are a back-up system for the removal of apoptotic cells.
Subject(s)
Apoptosis/drug effects , Cyclosporine/pharmacology , Immunosuppressive Agents/pharmacology , T-Lymphocytes/drug effects , Animals , Atrophy , Cell Differentiation/drug effects , Clonal Deletion/drug effects , Cyclosporine/antagonists & inhibitors , Endothelium, Vascular/physiology , Female , Immunosuppressive Agents/antagonists & inhibitors , Models, Immunological , Rats , Rats, Inbred Lew , Receptor-CD3 Complex, Antigen, T-Cell/drug effects , Receptor-CD3 Complex, Antigen, T-Cell/physiology , Self Tolerance/drug effects , Signal Transduction/drug effects , Sirolimus/pharmacology , Specific Pathogen-Free Organisms , T-Lymphocytes/cytology , Thymus Gland/drug effects , Thymus Gland/pathologyABSTRACT
The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a recognized mutagen and carcinogen in the colon and prostate of male rats and in the mammary gland of female rats. In the current study, we examined the mutagenicity of PhIP in the kidney of male and female lacI transgenic rats and its modulation by a dietary chemopreventive agent, conjugated linoleic acid (CLA). Sex-specific changes in mutation were observed following PhIP and CLA treatment. Exposure to 100 ppm PhIP through dietary supplementation for 47 days induced a lacI mutation frequency (MF) of 7.7 +/- 0.3 x 10(-5) and 4.7 +/- 1.0 x 10(-5) in the kidney of male and female rats, respectively. The PhIP-induced MFs in the kidney of male and female rats were significantly different from each other and were 300% (P < 0.001) and 60% (P < 0.05) higher than the corresponding controls, respectively. When rats were given CLA along with PhIP, CLA completely inhibited the formation of PhIP-induced mutations in the kidney of female rats, but not in male rats. Comparison of mutational spectra did not detect significant differences between male rats treated with PhIP and PhIP + CLA. However, unlike the -1 frameshifts induced by PhIP in the colon and prostate, which consist primarily of G:C deletions, -1 frameshifts in the kidney involved the loss of both G:C and A:T basepairs. Our data indicate that the kidney of the rats responds in a sex-dependent way to mutagenesis and antimutagenesis by PhIP and CLA. These differences may be related to hormonally regulated induction of P450 enzymes or cell proliferation.
Subject(s)
Anticarcinogenic Agents/pharmacology , Imidazoles/toxicity , Kidney/metabolism , Linoleic Acid/pharmacology , Mutagenesis , Mutagens/toxicity , Sex Characteristics , Analysis of Variance , Animals , Base Sequence , Clonal Deletion/drug effects , DNA Primers , Female , Gene Amplification , Isomerism , Kidney/drug effects , Male , Mutagenicity Tests , Polymerase Chain Reaction , Rats , Rats, Inbred F344ABSTRACT
Mixed hemopoietic chimerism has the potential to correct genetic hemological diseases (sickle cell anemia, thalassemia) and eliminate chronic immunosuppressive therapy following organ transplantation. To date, most strategies require either recipient conditioning (gamma-irradiation, depletion of the peripheral immune system) or administration of "mega" doses of bone marrow to facilitate reliable engraftment. Although encouraging, many issues remain that may restrict or prevent clinical application of such strategies. We describe an alternative, nonirradiation based strategy using a single dose of busulfan, costimulation blockade, and T cell-depleted donor bone marrow, which promotes titratable macrochimerism and a reshaping of the T cell repertoire. Chimeras exhibit robust donor-specific tolerance, evidenced by acceptance of fully allogeneic skin grafts and failure to generate donor-specific proliferative responses in an in vivo graft-versus-host disease model of alloreactivity. In this model, donor cell infusion and costimulation blockade without busulfan were insufficient for tolerance induction as donor-specific IFN-gamma-producing T cells re-emerged and skin grafts were rejected at approximately 100 days. When applied to a murine beta-thalassemia model, this approach allows for the normalization of hemologic parameters and replacement of the diseased red cell compartment. Such a protocol may allow for clinical application of mixed chimerism strategies in patients with end-stage organ disease or hemoglobinopathies.
Subject(s)
Antibodies, Blocking/administration & dosage , Bone Marrow Transplantation/immunology , Busulfan/administration & dosage , Hemoglobinopathies/genetics , Hemoglobinopathies/therapy , Immunosuppression Therapy , Lymphocyte Activation/immunology , Transplantation Tolerance/immunology , Animals , B7-1 Antigen/immunology , CD28 Antigens , CD4-Positive T-Lymphocytes , CD40 Antigens/immunology , CD40 Ligand/immunology , Cell Line , Clonal Deletion/drug effects , Clonal Deletion/genetics , Cytotoxicity, Immunologic/drug effects , Cytotoxicity, Immunologic/genetics , Hemoglobinopathies/immunology , Immunosuppression Therapy/adverse effects , Injections, Intraperitoneal , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, SCID , Radiation Chimera/immunology , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , Titrimetry , Transplantation Tolerance/drug effects , Transplantation Tolerance/geneticsABSTRACT
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.
Subject(s)
Apoptosis/drug effects , Membrane Glycoproteins/pharmacology , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , Thymus Gland/cytology , Thymus Gland/drug effects , Tumor Necrosis Factor-alpha/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP-Binding Cassette Transporters/genetics , Animals , Antibodies, Monoclonal , Apoptosis Regulatory Proteins , CD4 Antigens/analysis , CD8 Antigens/analysis , Cells, Cultured , Child, Preschool , Clonal Deletion/drug effects , Cytotoxicity, Immunologic , Flow Cytometry , Genes, RAG-1/genetics , Humans , Infant , Jurkat Cells , Lymphocyte Activation , Membrane Glycoproteins/metabolism , Mice , Mice, Knockout , Organ Culture Techniques , Receptors, TNF-Related Apoptosis-Inducing Ligand , Receptors, Tumor Necrosis Factor/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , TNF-Related Apoptosis-Inducing Ligand , Thymus Gland/immunology , Thymus Gland/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Methotrexate (MTX), a folate antagonist with multiple enzymatic targets, is used in the treatment of malignancies as well as in autoimmune and chronic inflammatory diseases, and ZD1694 (tomudex), a water-soluble quinazoline specific inhibitor of thymidylate synthase (TS), is used in the treatment of adenocarcinomas. In this study, we investigated the effects of these folate analogues on superantigen (SAg)-reactive peripheral T cells in vivo. In BALB/c mice, staphylococcal enterotoxin B (SEB)-induced cytokine secretion, IL-2R (CD25) expression and early deletion of a fraction of SEB-reactive V(beta)8(+) T cells were not impaired by either MTX (7 mg/kg/day) or tomudex (5 mg/kg/day). However, both MTX and tomudex prevented V(beta)8-selective T cell expansion and accelerated their peripheral elimination. Administration of thymidine (500 mg/kg/12 h) completely abrogated this effect, indicating that inhibition of TS but not that of other folate-dependent enzymes was the main mechanism involved. Furthermore, a marked increase of apoptotic cells restricted to the V(beta)8(+) T cell subset indicated that proliferation inhibition was associated with apoptosis. In contrast with peripheral V(beta)8(+) T cell deletion, MTX and tomudex did not prevent the increase of V(beta)8(+) thymocytes triggered by SEB. Experiments in C57BL/6-lpr/lpr mice further demonstrated that deletion of V(beta)8(+) T cells induced by folate analogues was independent of Fas-Fas ligand interaction. Our results provide evidence that folate analogues may selectively delete dividing peripheral T cells through TS inhibition, but do not interfere with other events triggered by SAg.
Subject(s)
Clonal Deletion/drug effects , Membrane Glycoproteins/physiology , Methotrexate/pharmacology , Nucleic Acid Synthesis Inhibitors/pharmacology , Superantigens/immunology , T-Lymphocyte Subsets/immunology , Thymidine/antagonists & inhibitors , fas Receptor/physiology , Animals , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Enterotoxins/administration & dosage , Fas Ligand Protein , Folic Acid Antagonists/pharmacology , Growth Inhibitors/administration & dosage , Growth Inhibitors/pharmacology , Injections, Intraperitoneal , Injections, Intravenous , Ligands , Lymphocyte Activation/drug effects , Lymphocyte Depletion , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Quinazolines/pharmacology , Staphylococcus aureus/immunology , T-Lymphocyte Subsets/enzymology , T-Lymphocyte Subsets/metabolism , Thiophenes/pharmacology , Thymidine/biosynthesis , Thymidylate Synthase/antagonists & inhibitorsABSTRACT
Hepatocellular carcinoma (HCC) often develops in patients with chronic liver diseases associated with hepatitis B (HBV) and hepatitis C (HCV) virus infections with high incidences. Particularly, post-therapeutic recurrence encountered after the curative treatment of the preceding HCC may limit the prognosis. Thus, prevention of HCC is of great significance. In the present review, immunopreventions with alpha-interferon and glycyrrhizin, as well as chemoprevention with acyclic retinoid, are discussed. alpha-Interferon prevents the development of HCC not only in patients with a long-term elimination of HCV (sustained virological responders), but in ones with normalized serum aminotransferases (sustained biochemical responders). Glycyrrhizin also suppresses serum aminotransferases and thereby prevents the tumor development, even though the compound does not have antiviral activity for HBV or HCV by itself. Therefore, suppression of hepatic necroinflammation by these drugs may serve to prevent hepatocarcinogenesis. In contrast, acyclic retinoid suppresses the post-therapeutic recurrence in cirrhotic patients who underwent curative treatment of preceding tumors. The retinoid induces the disappearance of serum lectin-reactive alpha-fetoprotein (AFP-L3), a tumor marker indicating the presence of unrecognizable tumors in the remnant liver, suggesting a deletion of such minute (pre)malignant clones (clonal deletion). As a molecular mechanism of the clonal deletion, a novel mechanism of apoptosis induction by the retinoid via tissue transglutaminase is implicated. In future, a combination of immunopreventive and chemopreventive therapies may give a clue to the further advances of cancer prevention, and thereby to the improvement of the prognosis of cirrhotic patients.
Subject(s)
Carcinoma, Hepatocellular/prevention & control , Liver Neoplasms/prevention & control , Tretinoin/analogs & derivatives , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Antineoplastic Agents/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Clonal Deletion/drug effects , Glycyrrhizic Acid/therapeutic use , Humans , Interferon-alpha/therapeutic use , Liver Neoplasms/drug therapy , Prognosis , Tretinoin/therapeutic use , alpha-Fetoproteins/drug effectsABSTRACT
A role for glucocorticoids in thymopoiesis has been suggested by studies using glucocorticoid receptor (GR) anti-sense transgenic mice, glucocorticoid synthesis inhibitors and GR antagonists. Unfortunately, no consensus has been reached on exactly how glucocorticoids influence T-cell development. The most recent approach, using GR knockout (GR(-/-)) mice, indicates that GR signaling is, in fact, dispensable in this entire process.
Subject(s)
Glucocorticoids/physiology , Stress, Physiological/immunology , T-Lymphocyte Subsets/immunology , Aminoglutethimide/pharmacology , Animals , Cell Differentiation/drug effects , Clonal Deletion/drug effects , DNA, Antisense/genetics , Glucocorticoids/antagonists & inhibitors , Glucocorticoids/deficiency , Glucocorticoids/genetics , Hematopoiesis/drug effects , Hormone Antagonists/pharmacology , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Lymphocyte Activation/drug effects , Metyrapone/pharmacology , Mice , Mice, Knockout , Mice, Transgenic , Mifepristone/pharmacology , Models, Biological , Receptors, Glucocorticoid/deficiency , Receptors, Glucocorticoid/drug effects , Receptors, Glucocorticoid/genetics , Receptors, Glucocorticoid/physiology , Stress, Physiological/physiopathology , T-Lymphocyte Subsets/cytology , T-Lymphocyte Subsets/drug effects , Thymus Gland/embryology , Thymus Gland/immunologyABSTRACT
We elucidated the possible role of chimerism in skin and heart allograft tolerance using cyclophosphamide (CP)-induced tolerance. When C3H (H-2k; Thy1.2, Mls-1b) mice were i.v. primed with 1x10(8) spleen cells (SC) from H-2 matched AKR (H-2k; Thy1.1, Mls-1a) mice and then treated i.p. with 200 mg/kg of CP, the survivals of both AKR skin grafts and heart grafts (HG) were permanently prolonged in a tolerogen-specific fashion. After this treatment, a minimal degree of mixed chimerism, the clonal destruction of Mls-1a-reactive CD4+Vbeta6+ T cells in the periphery, and the clonal deletion of Vbeta6+ thymocytes were all observed. When AKR SC and 100 mg/kg CP were used for conditioning, the AKR HG were permanently accepted, but the survival of the AKR skin grafts was only mildly prolonged. The clonal destruction of CD4+Vbeta6+ T cells in the periphery and the intrathymic clonal deletion of Vbeta6+ thymocytes were induced in both the SC and the 100 mg/kg CP-treated C3H mice. A minimal degree of mixed chimerism was detectable at 4 and 12 weeks after AKR SC and 100 mg/kg CP treatment, and still did not disappear at 40 weeks. The degree of mixed chimerism induced with SC and 100 mg/kg CP was significantly lower than that with SC and 200 mg/kg CP during the observation. No posttransplant cardiac allograft vasculopathy (CAV) was observed to develop, while both the Th1 type (interferon-gamma) and Th2 type (interleukin-4 and -10) cytokine expressions decreased in the AKR HG of the tolerant C3H mice treated with both AKR SC plus 200 mg/kg CP, and AKR SC plus 100 mg/kg CP. A second set of skin grafts from donor AKR mice survived for more than 100 days in a tolerogen-specific fashion in all C3H mice treated with AKR SC and 200 mg/kg CP and also accepted the AKR HG for over 200 days, while 80% of the C3H mice treated with AKR SC and 100 mg/kg CP and accepted the AKR HG for more than 200 days. These results strongly suggested the following conclusions: 1) the degree of chimerism can strongly influence the induction of skin and heart allograft tolerance, 2) posttransplant CAV does not develop in the donor HG maintained by chimerism-based CP-induced tolerance, 3) the mRNA expression of both Th1 and Th2 type cytokine decreased in the donor HG maintained by chimerism-based CP-induced tolerance, and 4) the induction of skin allograft tolerance is more difficult than the prevention of posttransplant CAV.