ABSTRACT
Langerhans cells (LCs) are epithelial APCs that sense danger signals and in turn trigger specific immune responses. In steady-state, they participate in the maintenance of peripheral tolerance to self-antigens whereas under inflammation LCs efficiently trigger immune responses in secondary lymphoid organs. It has been demonstrated in mice that LC-deprived epithelia are rapidly replenished by short half-life langerin-expressing monocyte-derived LCs (MDLCs). These surrogate LCs are thought to be progressively replaced by langerin(high) LCs arising from self-renewing epithelial precursors of hematopoietic origin. How LCs arise from blood monocytes is not fully understood. Hence, we sought to characterize key factors that induce differentiation of langerin(high)-expressing monocyte-derived Langerhans-like cells. We identified GM-CSF and TGF-ß1 as key cytokines to generate langerin(high)-expressing cells but only in serum-free conditions. These cells were shown to express the LC-specific TROP-2 and Axl surface markers and contained Birbeck granules. Surprisingly, E-cadherin was not spontaneously expressed by these cells but required a direct contact with keratinocytes to be stably induced. MDLCs induced stronger allogeneic T cell proliferations but released low amounts of inflammatory cytokines upon TLR stimulation compared with donor-paired monocyte-derived dendritic cells. Immature langerin(high) MDLCs were responsive to MIP-3ß/CCL20 and CTAC/CCL27 chemokine stimulations. Finally, we demonstrated that those cells behaved as bona fide LCs when inserted in a three-dimensional rebuilt epithelium by becoming activated upon TLR or UV light stimulations. Collectively, these results prompt us to propose these langerin(high) MDLCs as a relevant model to address LC biology-related questions.
Subject(s)
Blood Cells/physiology , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Keratinocytes/physiology , Langerhans Cells/immunology , Monocytes/physiology , T-Lymphocytes/immunology , Transforming Growth Factor beta1/metabolism , Antigens, Neoplasm/metabolism , Cell Adhesion Molecules/metabolism , Cell Differentiation , Cells, Cultured , Humans , Isoantigens/immunology , Lipopolysaccharide Receptors/metabolism , Lymphocyte Activation , Proto-Oncogene Proteins/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, IgG/metabolism , Self Tolerance , Ultraviolet Rays , Axl Receptor Tyrosine KinaseABSTRACT
Emerging knowledge regarding B cells in organ transplantation has demonstrated that these cells can no longer be taken as mere generators of deleterious Abs but can also act as beneficial players. We previously demonstrated in a rat model of cardiac allograft tolerance induced by short-term immunosuppression an accumulation in the blood of B cells overexpressing inhibitory molecules, a phenotype also observed in the blood of patients that spontaneously develop graft tolerance. In this study, we demonstrated the presence in the spleen of regulatory B cells enriched in the CD24(int)CD38(+)CD27(+)IgD(-)IgM(+/low) subpopulation, which are able to transfer donor-specific tolerance via IL-10 and TGF-ß1-dependent mechanisms and to suppress in vitro TNF-α secretion. Following anti-CD40 stimulation, IgD(-)IgM(+/low) B cells were blocked in their plasma cell differentiation pathway, maintained high expression of the inhibitory molecules CD23 and Bank1, and upregulated Granzyme B and Irf4, two molecules described as highly expressed by regulatory B cells. Interestingly, these B cells recognized specifically a dominant donor Ag, suggesting restricted specificity that could lead to a particular B cell response. Regulatory B cells were not required for induction of tolerance and appeared following Foxp3(+)CD4(+)CD25(+) regulatory T cells, suggesting cooperation with regulatory T cells for their expansion. Nevertheless, following transfer to new recipients, these B cells migrated to the allograft, kept their regulatory profile, and promoted local accumulation of Foxp3(+)CD4(+)CD25(+) regulatory T cells. Mechanisms of regulatory B cells and their cell therapy potential are important to decipher in experimental models to pave the way for future developments in the clinic.
Subject(s)
B-Lymphocytes, Regulatory/immunology , CD40 Antigens/immunology , Granzymes/immunology , Heart Transplantation , Plasma Cells/immunology , Signal Transduction/immunology , Transplantation Tolerance , Allografts , Animals , Antigens, CD/immunology , Cytokines/immunology , Isoantigens/immunology , Male , Rats , T-Lymphocytes, Regulatory/immunologyABSTRACT
Despite accumulating evidence for the importance of allospecific CD8(+) regulatory T cells (Tregs) in tolerant rodents and free immunosuppression transplant recipients, mechanisms underlying CD8(+) Treg-mediated tolerance remain unclear. By using a model of transplantation tolerance mediated by CD8(+) Tregs following CD40Ig treatment in rats, in this study, we show that the accumulation of tolerogenic CD8(+) Tregs and plasmacytoid dendritic cells (pDCs) in allograft and spleen but not lymph nodes was associated with tolerance induction in vascularized allograft recipients. pDCs preferentially induced tolerogenic CD8(+) Tregs to suppress CD4(+) effector cells responses to first-donor Ags in vitro. When tolerogenic CD8(+) Tregs were not in contact with CD4(+) effector cells, suppression was mediated by IDO. Contact with CD4(+) effector cells resulted in alternative suppressive mechanisms implicating IFN-gamma and fibroleukin-2. In vivo, both IDO and IFN-gamma were involved in tolerance induction, suggesting that contact with CD4(+) effector cells is crucial to modulate CD8(+) Tregs function in vivo. In conclusion, CD8(+) Tregs and pDCs interactions were necessary for suppression of CD4(+) T cells and involved different mechanisms modulated by the presence of cell contact between CD8(+) Tregs, pDCs, and CD4(+) effector cells.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Heart Transplantation/immunology , Immune Tolerance/immunology , T-Lymphocytes, Regulatory/immunology , Adenoviridae/genetics , Adoptive Transfer , Animals , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , Cell Proliferation , Dendritic Cells/cytology , Dendritic Cells/immunology , Flow Cytometry , Genetic Vectors/genetics , Heart Transplantation/methods , Male , Models, Animal , Rats , Rats, Inbred Lew , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Spleen/cytology , Spleen/immunology , T-Lymphocytes, Regulatory/cytology , Transduction, Genetic , Transplantation, HomologousABSTRACT
C-type lectin receptors have recently been described as playing crucial roles in immunity and homeostasis since these proteins are able to recognize pathogens as well as self-Ags. We identified the C-type lectin-like receptor-1, CLEC-1, as being overexpressed in a model of rat allograft tolerance. We previously described in this model the expression of numerous cytoprotective molecules by graft endothelial cells and their interplay with regulatory CD4(+)CD25(+) T cells. In this study, we demonstrate that CLEC-1 is expressed by myeloid cells and specifically by endothelial cells in tolerated allografts and that CLEC-1 expression can be induced in endothelial cells by alloantigen-specific regulatory CD4(+)CD25(+) T cells. Analysis of CLEC-1 expression in naive rats demonstrates that CLEC-1 is highly expressed by myeloid cells and at a lower level by endothelial cells, and that its expression is down-regulated by inflammatory stimuli but increased by the immunoregulators IL-10 or TGFbeta. Interestingly, we demonstrate in vitro that inhibition of CLEC-1 expression in rat dendritic cells increases the subsequent differentiation of allogeneic Th17 T cells and decreases the regulatory Foxp3(+) T cell pool. Additionally, in chronically rejected allograft, the decreased expression of CLEC-1 is associated with a higher production of IL-17. Taken together, our data suggest that CLEC-1, expressed by myeloid cells and endothelial cells, is enhanced by regulatory mediators and moderates Th17 differentiation. Therefore, CLEC-1 may represent a new therapeutic agent to modulate the immune response in transplantation, autoimmunity, or cancer settings.
Subject(s)
Endothelial Cells/immunology , Endothelial Cells/metabolism , Lectins, C-Type/biosynthesis , Lymphocyte Activation/immunology , Myeloid Cells/immunology , Myeloid Cells/metabolism , T-Lymphocyte Subsets/immunology , Up-Regulation/immunology , Amino Acid Sequence , Animals , Cell Line , Cells, Cultured , Endothelial Cells/pathology , Gene Expression Regulation/immunology , Graft Survival/genetics , Graft Survival/immunology , Heart Transplantation/immunology , Heart Transplantation/pathology , Immune Tolerance/genetics , Inflammation Mediators/physiology , Lectins, C-Type/antagonists & inhibitors , Lectins, C-Type/genetics , Lectins, C-Type/physiology , Lymphocyte Activation/genetics , Molecular Sequence Data , Rats , Rats, Inbred Lew , T-Lymphocyte Subsets/metabolismABSTRACT
Treatment with CD40Ig results in indefinite allograft survival in a complete MHC-mismatched heart allograft model in the rat. Here we show that serial second, third, and fourth adoptive transfers of total splenocytes from CD40Ig-treated recipients into secondary recipients led to indefinite donor-specific allograft acceptance. Purification of splenocyte subpopulations from CD40Ig-treated recipients demonstrated that only the adoptively transferred CD8(+)CD45RC(low) subset resulted in donor-specific long-term survival, whereas CD8(+)CD45RC(low) T cells from naive animals did not. Accepted grafts displayed increased indoleamine 2,3-dioxygenase (IDO) expression restricted in the graft to ECs. Coculture of donor ECs with CD8(+)CD45RC(low) T cells purified from CD40Ig-treated animals resulted in donor-specific IDO expression dependent on IFN-gamma. Neutralization of IFN-gamma or IDO triggered acute allograft rejection in both CD40Ig-treated and adoptively transferred recipients. This study demonstrates for what we believe to be the first time that interference in CD40-CD40 ligand (CD40-CD40L) interactions induces allospecific CD8(+) Tregs that maintain allograft survival. CD8(+)CD45RC(low) T cells act through IFN-gamma production, which in turn induces IDO expression by graft ECs. Thus, donor alloantigen-specific CD8(+) Tregs may promote local graft immune privilege through IDO expression.
Subject(s)
Graft Survival/physiology , Heart Transplantation/physiology , Indoleamine-Pyrrole 2,3,-Dioxygenase/physiology , Interferon-gamma/physiology , Recombinant Fusion Proteins/therapeutic use , T-Lymphocytes/immunology , Adoptive Transfer , Animals , Graft Survival/drug effects , Graft Survival/immunology , Heart Transplantation/immunology , Immune Tolerance/drug effects , Rats , Rats, Inbred Lew , Transplantation, Homologous/immunologyABSTRACT
Regulatory T cells (Treg) have been identified as playing a pivotal role in the control of tolerance and in the suppression of pathologic immune responses in autoimmune diseases, transplantation, and graft-versus-host disease. Treg expanded ex vivo by dendritic cells could be potential reagents to promote antigen-specific tolerance in vivo. However, in vivo studies have been carried out mostly in rodents and will need validation in primates before clinical application. We characterized macaque dendritic cell derived either from bone marrow with and without prior CD34+ cell selection (BMDC), or from CD14+ peripheral blood mononuclear cells (Mo-DC). We demonstrate that with a semi-mature phenotype, BMDC are superior to Mo-DC in their capacity to expand freshly isolated allogeneic macaque CD4+ CD25+ CD127- Foxp3+ Treg in vitro in the presence of interleukin-2. Moreover, the expanded Treg maintain their phenotype and suppressive activity. These data provide a step toward the use of macaque dendritic cell to expand Treg for future preclinical testing.
Subject(s)
Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Dendritic Cells/immunology , Lymphocyte Activation , Monocytes/cytology , Monocytes/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Antigens, CD34/immunology , Immunophenotyping , Interleukin-2 Receptor alpha Subunit/immunology , Lipopolysaccharide Receptors/immunology , Macaca fascicularis , Models, AnimalABSTRACT
Pseudomonas aeruginosa is a major threat for immune-compromised patients. Bacterial pneumonia can induce uncontrolled and massive neutrophil recruitment ultimately leading to acute respiratory distress syndrome and epithelium damage. Interleukin-22 plays a central role in the protection of the epithelium. In this study, we aimed to evaluate the role of interleukin-22 and its soluble receptor IL-22BP in an acute Pseudomonas aeruginosa pneumonia model in mice. In this model, we noted a transient increase of IL-22 during Pseudomonas aeruginosa challenge. Using an antibody-based approach, we demonstrated that IL-22 neutralisation led to increased susceptibility to infection and to lung damage correlated with an increase in neutrophil accumulation in the lungs. On the contrary, rIL-22 administration or IL-22BP neutralisation led to a decrease in mouse susceptibility and lung damage associated with a decrease in neutrophil accumulation. This study demonstrated that the IL-22/IL-22BP system plays a major role during Pseudomonas aeruginosa pneumonia by moderating neutrophil accumulation in the lungs that ultimately leads to epithelium protection.
Subject(s)
Interleukins/analysis , Lung/pathology , Neutrophil Infiltration , Pneumonia, Bacterial/pathology , Pseudomonas Infections/pathology , Receptors, Interleukin/analysis , Animals , Disease Models, Animal , Mice , Interleukin-22ABSTRACT
Receptor activating NF-κB ligand (RANKL) is a member of the TNF superfamily that plays a pivotal role in bone homeostasis as being the major osteoclastogenesis factor. RANKL also has pleiotropic effects in the immune system in which it is expressed by activated T and B cells and some innate lymphoid cells. RANKL-RANK interactions mediate lymph node organogenesis and immunoregulatory functions in autoimmune disease and carcinogenesis as well as cross talk between the immune system and bone. In this study, we show that basophils were the strongest RANKL mRNA-expressing cells amongst major leukocyte subsets in human blood. RANKL was preformed as an intracellular protein in resting basophils and was rapidly and strongly expressed on their surface upon stimulation with IL-3, but not other stimuli. This expression was stable for at least 6 days. Activated basophils could also release soluble RANKL in small quantities upon interaction with DCs or monocytes. In the blood, basophils were the sole cells to express membrane RANKL in response to IL-3. This study indicates that basophils should be considered as new players in the pleiotropic and complex RANKL-RANK interaction system and suggests a role for RANKL in the interaction between basophils and immune cells in inflammatory allergic tissues and secondary lymphoid organs.
Subject(s)
Basophils/immunology , Bone Resorption/immunology , Cell Membrane/metabolism , Dendritic Cells/immunology , Intracellular Space/metabolism , Osteoclasts/physiology , RANK Ligand/metabolism , Cell Communication , Cell Differentiation , Cells, Cultured , Humans , Interleukin-3/immunology , Protein Transport/immunology , RANK Ligand/genetics , Up-Regulation/immunologyABSTRACT
BACKGROUND: Dendritic cells secrete a population of "antigen-presenting vesicles," called exosomes, expressing functional class I and II major histocompatibility complex (MHC) and co-stimulatory molecules. The subcutaneous administration of syngeneic exosomes expressing tumor antigens has been shown to induce specific antitumor immune responses in vivo. The authors hypothesized that antigen presentation by exosomes, depending on the context of their administration, may induce tolerance rather than immunity. METHODS: The authors therefore tested the capacity of exosomes derived from donor bone marrow dendritic cells, given before transplantation, to modulate heart allograft rejection. RESULTS: The authors show here that donor type but not syngeneic exosomes induced a significant prolongation of allograft survival, with a few recipients having long-term graft survival. During the first week after transplantation, allografts from exosome-treated rats displayed a significant decrease in graft-infiltrating leukocytes and in the expression of interferon-gamma mRNA compared with allografts from untreated animals. Moreover, when tested in vitro, spleen CD4+ T cells from exosome-treated recipients displayed a significant decrease in anti-donor responses, suggesting a decrease in anti-donor T-cell responses. However, the authors also found that allogeneic donor-derived exosomes increased anti-donor MHC class II alloantibody production. CONCLUSIONS: The authors demonstrate an effect of allogeneic exosomes on the modulation of immune responses in vivo, suggesting that, like donor cells, exosomes can stimulate or regulate antigen-specific immune responses.
Subject(s)
Antibody Formation , Bone Marrow Cells/immunology , Dendritic Cells/immunology , Graft Rejection/immunology , Graft Survival/immunology , Heart Transplantation/immunology , Major Histocompatibility Complex , Tissue Donors , Animals , Endosomes/immunology , Endosomes/transplantation , Heart Transplantation/pathology , Isoantibodies/blood , Male , Rats , Rats, Inbred Lew , T-Lymphocytes/immunology , Time Factors , Transplantation, Homologous/immunologyABSTRACT
Human blood DCs encompass pDCs and two subsets of mDCs: CD1c(+) mDCs and CD141(+) mDCs. The rare CD141(+) DC population is thought to be the equivalent of mouse CD8α(+) cDCs that play a significant role in antigen cross-presentation. Here, we analyzed by Q-PCR TLR1-10 expression in blood DC subsets. Whereas CD1c(+) DCs express all TLR except TLR9, CD141(+) DCs present a more restricted pattern with high expression of TLR3 and -10, expression of TLR1,-2, -6, and -8, and lack of TLR4, -5, -7, and -9. The in vitro analysis of isolated mDC subset reponsiveness to an extensive panel of TLR ligands confirmed these results, with CD141(+) DCs responding only to TLR1/2, -3, and -7/8. The cytokine/chemokine production profile of isolated CD141(+) DCs was also more restricted, as they produced mainly proinflammatory cytokines but no IL-12 and to a lower level, in comparison with CD1c(+) DCs, except for CXCL10, CCL5, and IFN-ß. In contrast, with the use of a whole blood assay, we found that CD141(+) DCs produce IL-12 in response to TLR1/2, -3, and more surprisingly, -9. Finally, both mDC subsets are potent inducers of Th1 response, particularly after TLR3 triggering. Taken together, these data confirmed functional differences between blood mDC subsets. The major response of CD141(+) mDCs to TLR3 ligand and their cytokine production pattern suggest a role for these cells in antiviral immunity.
Subject(s)
Antigens, CD/immunology , Cytokines/immunology , Dendritic Cells/immunology , Toll-Like Receptors/immunology , Antigen Presentation , Antigens, CD/genetics , Antigens, CD1/genetics , Antigens, CD1/immunology , Cells, Cultured , Cytokines/metabolism , Dendritic Cells/classification , Dendritic Cells/cytology , Flow Cytometry , Glycoproteins/genetics , Glycoproteins/immunology , Humans , Immunomagnetic Separation , Immunophenotyping , Protein Isoforms/classification , Protein Isoforms/genetics , Protein Isoforms/immunology , Toll-Like Receptors/classification , Toll-Like Receptors/geneticsABSTRACT
Tregs represent an interesting therapeutic tool to modulate immune responses that could be deleterious in autoimmune diseases and in transplantation. However, phenotype and functions of Tregs do not seem to be stable, and recent data suggest that FoxP3-expressing Tregs can be driven to produce IL-17. In this study, we have analyzed the role of pDCs versus cDCs on Treg responses and underlined that pDCs have an intrinsic, unique capacity to induce IL-17 secretion from T cells. We showed in rats that FoxP3(+) Tregs were able to secrete IL-17 only when stimulated by allogeneic, mature pDCs but not cDCs. In addition, in rats and mice, mature pDCs but not cDCs inhibited in vitro Treg-suppressive functions and in the presence of Tregs, supported Th17 differentiation from naive T cells through secretion of high amounts of IL-6. These data suggest an important role for pDCs in modulating or switching Treg function and allowing Th17 differentiation.
Subject(s)
Dendritic Cells/immunology , Interleukin-17/metabolism , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes/immunology , Animals , Cell Differentiation , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Forkhead Transcription Factors/metabolism , Interleukin-17/immunology , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-6/metabolism , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Rats , Rats, Inbred Lew , T-Lymphocytes/metabolismABSTRACT
Members of the Slfn protein family have been implicated in the regulation of cell growth, hematopoietic cell differentiation, and T cell development/differentiation in the thymus. Ten members of this family have been described in the mouse, and they have been divided into three subgroups based on the overall sequence homology and the size of the encoded proteins. We have identified Slfn3, a member of Subgroup II, as an overexpressed gene in CD4(+) CD25(+) T cells in the periphery. Interestingly, we demonstrate that upon activation and proliferation, Slfn3 mRNA is down-regulated in CD4(+) CD25(+) Tregs and up-regulated in CD4(+) CD25(-) Teffs. Moreover, TGF-beta inhibits the expression of Slfn3 in anti-CD3/CD28-activated CD4+ T cells, and the same conditions induce FoxP3 mRNA. Our results suggest that Slfn3 could have a role in T cell differentiation and activation.
Subject(s)
Proteins/metabolism , T-Lymphocytes, Regulatory/metabolism , Animals , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression Profiling , Gene Expression Regulation/drug effects , Humans , Interleukin-2 Receptor alpha Subunit/metabolism , Lymphocyte Activation/drug effects , Mice , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/drug effects , Transforming Growth Factor beta/pharmacologyABSTRACT
Anergy and suppression are cardinal features of CD4(+)CD25(+)Foxp3(+) T cells (T regulatory cells (Treg)) which have been shown to be tightly controlled by the maturation state of dendritic cells (DC). However, whether lymphoid organ DC subsets exhibit different capacities to control Treg is unclear. In this study, we have analyzed, in the rat, the role of splenic CD4(+) and CD4(-) conventional DC and plasmacytoid DC (pDC) in allogeneic Treg proliferation and suppression in vitro. As expected, in the absence of exogenous IL-2, Treg did not expand in response to immature DC. Upon TLR-induced maturation, all DC became potent stimulators of CD4(+)CD25(-) T cells, whereas only TLR7- or TLR9-matured pDC induced strong proliferation of CD4(+)CD25(+)Foxp3(+) T cells in the absence of exogenous IL-2. This capacity of pDC to reverse Treg anergy required cell contact and was partially CD86 dependent and IL-2 independent. In suppression assays, Treg strongly suppressed proliferation and IL-2 and IFN-gamma production by CD4(+)CD25(-) T cells induced by mature CD4(+) and CD4(-) DC. In contrast, upon stimulation by mature pDC, proliferating Treg suppressed IL-2 production by CD25(-) cells but not their proliferation or IFN-gamma production. Taken together, these results suggest that anergy and the suppressive function of Treg are differentially controlled by DC subsets.
Subject(s)
Cell Proliferation , Dendritic Cells/immunology , Plasma Cells/immunology , Spleen/immunology , T-Lymphocytes, Regulatory/immunology , Animals , B7-2 Antigen/immunology , Clonal Anergy/immunology , Dendritic Cells/cytology , Interferon-gamma/immunology , Interleukin-2/immunology , Interleukin-2/pharmacology , Interleukin-2 Receptor alpha Subunit/immunology , Plasma Cells/cytology , Rats , Rats, Sprague-Dawley , Spleen/cytology , T-Lymphocytes, Regulatory/cytology , Toll-Like Receptor 7/immunology , Toll-Like Receptor 9/immunologyABSTRACT
The immune tolerance to rat kidney allografts induced by a perioperative treatment with anti-CD28 Abs is associated with a severe unresponsiveness of peripheral blood cells to donor Ags. In this model, we identified an accumulation in the blood of CD3(-)class II(-)CD11b(+)CD80/86(+) plastic-adherent cells that additionally expressed CD172a as well as other myeloid markers. These cells were able to inhibit proliferation, but not activation, of effector T cells and to induce apoptosis in a contact-dependent manner. Their suppressive action was found to be under the control of inducible NO synthase, an enzyme also up-regulated in tolerated allografts. Based on these features, these cells can be defined as myeloid-derived suppressor cells (MDSC). Interestingly, CD4(+)CD25(high)FoxP3(+) regulatory T cells were insensitive in vitro to MDSC-mediated suppression. Although the adoptive transfer of MDSC failed to induce kidney allograft tolerance in recently transplanted recipients, the maintenance of tolerance after administration of anti-CD28 Abs was found to be dependent on the action of inducible NO synthase. These results suggest that increased numbers of MDSC can inhibit alloreactive T cell proliferation in vivo and that these cells may participate in the NO-dependent maintenance phase of tolerance.
Subject(s)
Cell Differentiation/immunology , Cell Movement/immunology , Immune Tolerance , Kidney Transplantation/immunology , Myeloid Cells/cytology , Myeloid Cells/immunology , Animals , B7-1 Antigen/biosynthesis , B7-2 Antigen/biosynthesis , CD11b Antigen/biosynthesis , Cell Adhesion , Cells, Cultured , Cytotoxicity Tests, Immunologic , Immunophenotyping , Male , Models, Immunological , Myeloid Cells/metabolism , Nitric Oxide Synthase Type II/biosynthesis , Nitric Oxide Synthase Type II/physiology , Rats , Rats, Inbred Lew , Spleen/cytology , Spleen/immunology , Spleen/pathology , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunologyABSTRACT
Toll-like receptors (TLRs) play a crucial role in the initiation of innate responses following microbial infection and also in adaptive immune responses by orchestrating the activation of different cell populations. TLRs are expressed at high levels in antigen-presenting cells and recent studies have demonstrated the expression and biological role of TLRs in mouse and human CD4(+) T cells. In this study, we analyzed TLR mRNA expression in rat CD4(+) T cells using stringent quantitative reverse transcription-PCR conditions enabling a direct comparison of the levels of each TLR. We show that TLR3, 5, 6 and 9 mRNAs are the highest TLRs expressed in rat CD4(+) T cells and that TLR5 mRNA is highly expressed in regulatory CD4(+) CD25(+) T cells. In addition, we show that the TLR9 ligand (TLR9L), CpG oligodeoxynucleotide, synergizes with anti-CD3 to induce proliferation of both CD4(+) CD25(-) and regulatory CD4(+) CD25(+) T cells and that TLR9L partially abrogates the suppressive activity mediated by regulatory CD4(+) CD25(+) T cells. This loss of suppression is in part due to the direct effect of TLR9L on effector T cells that are rendered more resistant to the regulation exerted by regulatory T cells. To our knowledge, this is the first study describing the expression of TLR mRNA in rat CD4(+) T cells and the capacity of TLR9L to directly regulate rat T cell responses. Thus, TLR9L may rapidly increase the host's adaptive immunity by expanding effector cells and also by attenuating the suppressive activity mediated by regulatory T cells.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , T-Lymphocyte Subsets/immunology , Toll-Like Receptor 9/biosynthesis , Animals , CD4-Positive T-Lymphocytes/metabolism , Cell Proliferation , CpG Islands , Interleukin-2 Receptor alpha Subunit/metabolism , Ligands , Oligodeoxyribonucleotides , RNA, Messenger/analysis , Rats , Rats, Inbred Lew , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocyte Subsets/metabolismABSTRACT
Chronic rejection (CR) is a major cause of long-term graft loss that would be avoided by the induction of tolerance. We previously showed that renal transplant patients with CR have lower numbers of peripheral CD4(+)CD25(high) T cells than operationally tolerant patients, patients with stable graft function and healthy volunteers (HV). We explored here the profile of CD4(+)CD25(high) blood T cells in these patients focusing on their expression of the regulatory T cells (Treg) gene Forkhead Box P3 (FOXP3) and their suppressive function. We show that CR is associated with a decreased number of CD4(+)CD25(high)FOXP3(+)T cells with normal regulatory profile, whereas graft acceptance is associated with CD4(+)CD25(high)FOXP3(+)T cell numbers similar to HVs. These data suggest that Treg numbers, rather than their intrinsic suppressive capacity, may contribute to determining the long-term fate of renal transplants.
Subject(s)
CD4 Antigens/immunology , Forkhead Transcription Factors/immunology , Graft Rejection/immunology , Immunity, Cellular/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Kidney Transplantation/immunology , T-Lymphocytes/immunology , Adult , Aged , Cell Proliferation , Disease Progression , Female , Follow-Up Studies , Graft Rejection/blood , Graft Rejection/pathology , Humans , Immune Tolerance , Kidney Transplantation/pathology , Male , Middle Aged , Prognosis , Time FactorsABSTRACT
We have previously shown that human monocyte-derived dendritic cells (DC) express indoleamine 2,3-dioxygenase (IDO), as well as several other enzymes of the kynurenine pathway at the mRNA level upon maturation. The tolerogenic mechanisms of this pathway remain unclear. Here we show that LPS-treated DC metabolize tryptophan as far as quinolinate. We found that IDO contributes to LPS and TNF-alpha + poly(I:C)-induced DC maturation since IDO inhibition using two different inhibitors impairs DC maturation. IDO knock-down using short-hairpin RNA also led to diminished LPS-induced maturation. In line with these results, the tryptophan-derived catabolites 3-hydroxyanthranilic acid and 3-hydroxykynurenine increased maturation of LPS-treated DC. Concerning the molecular mechanisms of this effect, IDO acts as an intermediate pathway in LPS-induced production of reactive oxygen species and NF-kappaB activation, two processes that lead to DC maturation. Finally, we show that mature DC expand CD4(+)CD25(high) regulatory T cells in an IDO-dependent manner. In conclusion, we show that IDO constitutes an intermediate pathway in DC maturation leading to expansion of CD4(+)CD25(high) regulatory T cells.
Subject(s)
Dendritic Cells/enzymology , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Lymphocyte Activation/immunology , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Cell Differentiation/immunology , Cells, Cultured , Dendritic Cells/cytology , Dendritic Cells/immunology , Humans , Immunohistochemistry , Indoleamine-Pyrrole 2,3,-Dioxygenase/immunology , Lipopolysaccharides/immunology , Lymphocyte Culture Test, Mixed , NF-kappa B , Reactive Oxygen Species/metabolism , Tryptophan/metabolismABSTRACT
Dendritic cells (DC) are a heterogeneous population of APC endowed with specific functions. The nature of the DC subset involved in the course of an immune response to a specific pathogen might be important for inducing the appropriate effectors. In addition, each DC subset might also exhibit intrinsic functional plasticity. In the rat, spleen DC can be separated into three morphological and phenotypical distinct subsets, namely CD4+, CD4-, and plasmacytoid DC (pDC), whose frequencies are strain dependent. We correlated the expression of TLR and nucleotide-binding oligomerization domain 2 (NOD2) in these DC subsets to their in vitro responsiveness to specific ligands. CD4- DC expressed high levels of TLR1, 2, 3, and 10 mRNA, low TLR4, 5, 6, 7, and 9, and very low, if any, TLR8. pDC had a restricted repertoire characterized by high TLR7 and 9. CD4+ DC expressed all TLR and 10-fold higher levels of NOD2 mRNA than CD4- and pDC. Upon stimulation by TLR and NOD2 ligands, each DC subset responded in quite a stereotyped fashion. TLR2/6, 3, 4, 5, 9, and NOD2 triggering induced CD4- DC to mature and produce high IL-12p40, low IL-10, and TNF-alpha. TLR7/8 and 9 triggering induced pDC to mature and produce copious amounts of IL-6, IL-12p40, and TNF-alpha and low IFN-alpha. CD4+ DC were very poor producers of inflammatory cytokines. This study suggests that the nature of spleen DC responses to pathogens is dependent on subset specific-stimulation rather than intrinsic plasticity.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Spleen/immunology , Spleen/metabolism , Toll-Like Receptors/biosynthesis , Animals , Cell Differentiation/immunology , Cell Separation , Cell Survival/immunology , Cells, Cultured , Cytokines/biosynthesis , Dendritic Cells/classification , Dendritic Cells/cytology , Interleukin-10/biosynthesis , Interleukin-12/biosynthesis , Interleukin-12 Subunit p40 , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/physiology , Leukocyte Count , Ligands , Lymphocyte Culture Test, Mixed , Nod2 Signaling Adaptor Protein , Protein Subunits/biosynthesis , RNA, Messenger/biosynthesis , Rats , Rats, Inbred BN , Rats, Inbred Lew , Rats, Sprague-Dawley , Spleen/cytology , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolism , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
We previously reported the characterization of a MHC class II(low) CD4- CD103+ (CD4-) subset of dendritic cells (DC) in rat spleen that exhibit a Ca2+-, Fas ligand-, TRAIL- and TNF-alpha-independent cytotoxic activity against specific targets in vitro. In this study, we demonstrate that this DC subset was also found in lymph nodes. Freshly extracted and, therefore, immature CD4- DC exhibited a potent cytotoxic activity against a large panel of tumor cell lines as well as primary endothelial cells. The cytotoxic activity of immature CD4- DC required cell-to-cell contact and de novo protein expression. CD4- DC-mediated cell death resembled apoptosis, as evidenced by outer membrane phosphatidylserine exposure and nuclear fragmentation in target cells, but was caspase as well as Fas-associated death domain and receptor-interacting protein independent. Bcl-2 overexpression in target cells did not protect them against DC-mediated cell death. Immature CD4- DC phagocytosed efficiently apoptotic cells in vitro and, therefore, rapidly and specifically engulfed their victims following death induction. Maturation induced a dramatic down-regulation of the killing and phagocytic activities of CD4- DC. In contrast, CD4+ DC were both unable to kill target cells and to phagocytose apoptotic cells in vitro. Taken together, these data indicate that rat immature CD4- CD103+ DC mediate an unusual cytotoxic activity and can use this function to efficiently acquire Ag from live cells.
Subject(s)
Antigens, CD/biosynthesis , Apoptosis/immunology , CD4 Antigens/metabolism , Caspases/physiology , Cell Differentiation/immunology , Cytotoxicity Tests, Immunologic , Dendritic Cells/immunology , Integrin alpha Chains/biosynthesis , Phagocytosis/immunology , Adaptor Proteins, Signal Transducing/physiology , Animals , Cell Communication/immunology , Cell Death/immunology , Cell Line , Cell Line, Tumor , Dendritic Cells/metabolism , Fas-Associated Death Domain Protein , Histocompatibility Antigens Class II/metabolism , Humans , Jurkat Cells , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Protein Biosynthesis/immunology , Proteins/physiology , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Proto-Oncogene Proteins c-bcl-2/genetics , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Receptor-Interacting Protein Serine-Threonine Kinases , Spleen/cytology , Spleen/immunology , Spleen/metabolismABSTRACT
We identified a novel rat gene specifically overexpressed in tolerated heart allografts in a model of tolerance induced by donor-specific blood transfusion (DST). We named this gene TORID, for tolerance-related and induced transcript. We show that TORID expression can be attributed to non-T cells infiltrating tolerated grafts. Interestingly, TORID overexpression was also observed in long-term grafts from a different model of tolerance in which chronic rejection does not occur. Comparison of the predicted amino acid sequence of TORID and of its human counterpart LR8 showed an homology with the four-transmembrane CD20/FcepsilonRIbeta family proteins. We investigated TORID expression in naive rat immune cells and lymphoid tissues. TORID was found to be preferentially expressed in cells of the myeloid lineage such as macrophages and dendritic cells (DCs). Its expression dramatically decreased following activation/maturation. Similar results were obtained in human monocyte-derived DCs. Interestingly, TORID overexpression in bone marrow-derived DCs alters expression of MHC II and CD86 and production of IL12p40 following activation. These results suggest that TORID may be involved in the control of DC maturation and may, therefore, play a role in the induction or maintenance of allograft tolerance.