ABSTRACT
Immunological requirements for rejection and tolerance induction differ between various organs. While memory CD8+ T cells are considered a barrier to immunosuppression-mediated acceptance of most tissues and organs, tolerance induction after lung transplantation is critically dependent on central memory CD8+ T lymphocytes. Here we demonstrate that costimulation blockade-mediated tolerance after lung transplantation is dependent on programmed cell death 1 (PD-1) expression on CD8+ T cells. In the absence of PD-1 expression, CD8+ T cells form prolonged interactions with graft-infiltrating CD11c+ cells; their differentiation is skewed towards an effector memory phenotype and grafts are rejected acutely. These findings extend the notion that requirements for tolerance induction after lung transplantation differ from other organs. Thus, immunosuppressive strategies for lung transplant recipients need to be tailored based on the unique immunological properties of this organ.
Subject(s)
CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Differentiation , Graft Rejection/immunology , Graft Survival/immunology , Lung Transplantation , Programmed Cell Death 1 Receptor/metabolism , Allografts , Animals , Graft Rejection/pathology , Immunosuppression Therapy , Mice , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
Antibody-mediated rejection (AMR) is an increasingly recognized form of lung rejection. C4d deposition has been an inconsistent finding in previous reports and its role in the diagnosis has been controversial. We conducted a retrospective single-center study to characterize cases of C4d-negative probable AMR and to compare these to cases of definite (C4d-positive) AMR. We identified 73 cases of AMR: 28 (38%) were C4d-positive and 45 (62%) were C4d-negative. The two groups had a similar clinical presentation, and although more patients in the C4d-positive group had neutrophilic capillaritis (54% vs. 29%, P = .035), there was no significant difference in the presence of other histologic findings. Despite aggressive antibody-depleting therapy, 19 of 73 (26%) patients in the overall cohort died within 30 days, but there was no significant difference in freedom from chronic lung allograft dysfunction (CLAD) or survival between the two groups. We conclude that AMR may cause allograft failure, but that the diagnosis requires a multidisciplinary approach and a high index of suspicion. C4d deposition does not appear to be a necessary criterion for the diagnosis, and although some cases may respond initially to therapy, there is a high incidence of CLAD and poor survival after AMR.
Subject(s)
Complement C4b/metabolism , Graft Rejection/etiology , Graft Survival/immunology , HLA Antigens/immunology , Isoantibodies/adverse effects , Lung Transplantation/adverse effects , Postoperative Complications , Female , Follow-Up Studies , Graft Rejection/metabolism , Graft Rejection/pathology , Humans , Male , Middle Aged , Prognosis , Retrospective Studies , Risk Factors , Survival Rate , Tissue DonorsABSTRACT
Ischemia-reperfusion injury-mediated primary graft dysfunction substantially hampers short- and long-term outcomes after lung transplantation. This condition continues to be diagnosed based on oxygen exchange parameters as well as radiological appearance, and therapeutic strategies are mostly supportive in nature. Identifying patients who may benefit from targeted therapy would therefore be highly desirable. Here, we show that C-C chemokine receptor type 2 (CCR2) expression in murine lung transplant recipients promotes monocyte infiltration into pulmonary grafts and mediates graft dysfunction. We have developed new positron emission tomography imaging agents using a CCR2 binding peptide, ECLi1, that can be used to monitor inflammatory responses after organ transplantation. Both 64 Cu-radiolabeled ECL1i peptide radiotracer (64 Cu-DOTA-ECL1i) and ECL1i-conjugated gold nanoclusters doped with 64 Cu (64 CuAuNCs-ECL1i) showed specific detection of CCR2, which is upregulated during ischemia-reperfusion injury after lung transplantation. Due to its fast pharmacokinetics, 64 Cu-DOTA-ECL1i functioned efficiently for rapid and serial imaging of CCR2. The multivalent 64 CuAuNCs-ECL1i with extended pharmacokinetics is favored for long-term CCR2 detection and potential targeted theranostics. This imaging may be applicable for diagnostic and therapeutic purposes for many immune-mediated diseases.
Subject(s)
Lung Transplantation/methods , Molecular Imaging/methods , Receptors, CCR2/physiology , Reperfusion Injury/diagnostic imaging , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Monocytes/metabolism , Peptide Fragments/metabolism , Positron-Emission Tomography/methods , Reperfusion Injury/immunology , Reperfusion Injury/metabolism , Signal TransductionABSTRACT
De novo induction of organized lymphoid aggregates at nonlymphoid sites has been observed in many chronic inflammatory conditions where foreign antigens such as infectious agents, autoantigens or alloantigens, persist. The prevailing opinion in the field of transplantation is that lymphoid neogenesis within allografts is detrimental to the establishment of immune tolerance. These structures, commonly referred to as tertiary lymphoid organs (TLOs), are thought to contribute to graft rejection by generating and propagating local alloimmune responses. However, recent studies have shown that TLOs rich in regulatory Foxp3(+) cells are present in long-term accepting allografts. The notion that TLOs can contribute to the local downregulation of immune responses has been corroborated in other chronic inflammation models. These findings suggest that contrary to previous suggestions that the induction of TLOs in allografts is necessarily harmful, the induction of "tolerogenic" TLOs may prove advantageous. In this review, we discuss our current understanding of how TLOs are induced and how they regulate immune responses with a particular focus on alloimmunity.
Subject(s)
Graft Rejection/immunology , Immune Tolerance/immunology , Lymphoid Tissue/immunology , Organ Transplantation , Allografts , Animals , HumansABSTRACT
Immune responses against lung-associated self-antigens (self-Ags) are hypothesized to play a role in the development of chronic lung graft rejection. We determined whether immune responses to lung self-Ags, K-alpha-1-tubulin (Kα1T) and Collagen V (Col-V) in the absence of alloimmunity, could promote airway inflammation and fibrosis. Following syngeneic murine orthotopic lung transplantation (LTx) we administered antibodies (Abs) to either Kα1T or Col-V or in combination to both of these self-Ags. As compared to recipients of isotype control Abs, Kα1T Abs and/or Col-V Abs-treated recipients had marked lung graft cellular infiltration and bronchiolar fibrosis. This inflammation was also associated the accumulation of Kα1T and Col-V-specific interferon-γ+ and IL-17+ T cells. Notably, the administration of Abs to Kα1T led to cellular and humoral immune responses to Col-V prior to development of fibrosis, and vice versa, indicating that epitope spreading can occur rapidly in an alloantigen independent manner. Collectively, these data support a model of chronic LTx rejection where the progressive loss of self-tolerance through epitope spreading promotes airway fibrosis. Strategies that target autoreactive Abs may be useful to inhibit chronic rejection of lung grafts.
Subject(s)
Bronchitis/prevention & control , Lung Transplantation , Animals , Autoantibodies/immunology , Bronchitis/immunology , Male , Mice , Mice, Inbred C57BL , T-Lymphocytes/immunologyABSTRACT
Although T cells are required for acute lung rejection, other graft-infiltrating cells such as neutrophils accumulate in allografts and are also high glucose utilizers. Positron emission tomography (PET) with the glucose probe [(18)F]fluorodeoxyglucose ([(18)F]FDG) has been employed to image solid organ acute rejection, but the sources of glucose utilization remain undefined. Using a mouse model of orthotopic lung transplantation, we analyzed glucose probe uptake in the grafts of syngeneic and allogeneic recipients with or without immunosuppression treatment. Pulmonary microPET scans demonstrated significantly higher [(18)F]FDG uptake in rejecting allografts when compared to transplanted lungs of either immunosuppressed or syngeneic recipients. [(18)F]FDG uptake was also markedly attenuated following T cell depletion therapy in lung recipients with ongoing acute rejection. Flow cytometric analysis using the fluorescent deoxyglucose analog 2-NBDG revealed that T cells, and in particular CD8(+) T cells, were the largest glucose utilizers in acutely rejecting lung grafts followed by neutrophils and antigen-presenting cells. These data indicate that imaging modalities tailored toward assessing T cell metabolism may be useful in identifying acute rejection in lung recipients.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Fluorodeoxyglucose F18 , Graft Rejection/diagnostic imaging , Lung Transplantation , Positron-Emission Tomography , Radiopharmaceuticals , Animals , CD8-Positive T-Lymphocytes/drug effects , Cells, Cultured , Flow Cytometry , Graft Rejection/etiology , Graft Rejection/immunology , Image Processing, Computer-Assisted , Immunosuppression Therapy , Immunosuppressive Agents/pharmacology , Lymphocyte Depletion , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Transplantation, Homologous , Transplantation, IsogeneicABSTRACT
Early immune responses are important in shaping long-term outcomes of human lung transplants. To examine the role of early immune responses in lung rejection and acceptance, we developed a method to retransplant mouse lungs. Retransplantation into T-cell-deficient hosts showed that for lungs and hearts alloimmune responses occurring within 72 h of transplantation are reversible. In contrast to hearts, a 72-h period of immunosuppression with costimulation blockade in primary allogeneic recipients suffices to prevent rejection of lungs upon retransplantation into untreated allogeneic hosts. Long-term lung acceptance is associated with induction of bronchus-associated lymphoid tissue, where Foxp3(+) cells accumulate and recipient T cells interact with CD11c(+) dendritic cells. Acceptance of retransplanted lung allografts is abrogated by treatment of immunosuppressed primary recipients with anti-CD25 antibodies. Thus, events contributing to lung transplant acceptance are established early in the graft and induction of bronchus-associated lymphoid tissue can be associated with an immune quiescent state.
Subject(s)
Dendritic Cells/immunology , Lung Transplantation/immunology , Lymphoid Tissue/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes/immunology , Animals , Antibodies, Monoclonal/administration & dosage , CD11c Antigen/metabolism , Dendritic Cells/drug effects , Forkhead Transcription Factors/metabolism , Graft Survival/drug effects , Humans , Immune Tolerance/drug effects , Interleukin-2 Receptor alpha Subunit/immunology , Lymphocyte Depletion , Lymphoid Tissue/drug effects , Lymphoid Tissue/growth & development , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Models, Animal , Reoperation , T-Lymphocytes/drug effects , T-Lymphocytes, Regulatory/drug effectsABSTRACT
Graft rejection remains a formidable problem contributing to poor outcomes after lung transplantation. Blocking chemokine pathways have yielded promising results in some organ transplant systems. Previous clinical studies have demonstrated upregulation of CCR2 ligands following lung transplantation. Moreover, lung injury is attenuated in CCR2-deficient mice in several inflammatory models. In this study, we examined the role of CCR2 in monocyte recruitment and alloimmune responses in a mouse model of vascularized orthotopic lung transplantation. The CCR2 ligand MCP-1 is upregulated in serum and allografts following lung transplantation. CCR2 is critical for the mobilization of monocytes from the bone marrow into the bloodstream and for the accumulation of CD11c(+) cells within lung allografts. A portion of graft-infiltrating recipient CD11c(+) cells expresses both recipient and donor MHC molecules. Two-photon imaging demonstrates that recipient CD11c(+) cells are associated with recipient T cells within the graft. While recipient CCR2 deficiency does not prevent acute lung rejection and is associated with increased graft infiltration by T cells, it significantly reduces CD4(+) T(h)1 indirect and direct allorecognition. Thus, CCR2 may be a potential target to attenuate alloimmune responses after lung transplantation.
Subject(s)
Lung Transplantation/methods , Monocytes , Animals , Chemokines , Graft Rejection/immunology , Inflammation , Lymphocyte Count , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Monocytes/immunology , Monocytes/metabolism , Monocytes/physiology , Pneumonia/metabolism , T-Lymphocytes/metabolism , Transplantation, HomologousABSTRACT
Lung allografts are considered to be more immunogenic than other solid organs. Little is known about the effectiveness of immunosuppressive regimens after lung transplantation. Herein, we describe a novel model of murine vascularized orthotopic lung transplantation we used to study the effects of costimulatory blockade on lung rejection. Transplants were performed in the Balb --> B6 strain combination. Recipients were either not immunosuppressed or received perioperative CD40/CD40L and CD28/B7 costimulatory blockade. Nonimmunosupressed Balb/c --> B6 lung transplants had severe acute rejection 7 days after transplantation and CD8(+) T cells outnumbered CD4(+) T cells within the allografts. Alternatively, B6 recipients that received perioperative costimulatory blockade had minimal inflammation and there were nearly equal numbers of CD8(+) and CD4(+) T cells in these grafts. Approximately one third of graft-infiltrating CD4(+) T cells expressed Foxp3. CD4(+) T cells isolated from these grafts induced apoptosis of alloreactive CD8(+) T cells that were stimulated with donor splenocytes in vitro. In contrast with wild-type B6 recipient mice, we observed severe rejection of Balb/c lungs 7 days after transplantation into Bcl-2 transgenic B6 recipients that had received costimulatory blockade. CD8(+) T cells outnumbered CD4(+) T cells in these immunosuppressed Bcl-2 transgenic recipients and, compared with immunosuppressed wild-type B6 recipients, a lower percentage of graft-infiltrating CD4(+) T cells expressed Foxp3, and a higher percentage of graft-infiltrating CD8(+) T cells expressed intereferon-gamma. Thus, our results show that perioperative blockade of the CD40/CD40L and CD28/B7 costimulatory pathways markedly ameliorates acute rejection of lung allografts in wild type but not Bcl-2 transgenic recipients.
Subject(s)
Lung Transplantation/physiology , Proto-Oncogene Proteins c-bcl-2/genetics , Transplantation, Homologous/physiology , Abatacept , Animals , CD4-Positive T-Lymphocytes/immunology , Gene Expression Regulation , Graft Rejection/immunology , Immunoconjugates/immunology , Lung Transplantation/immunology , Lung Transplantation/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
In lung grafts, ischemia-reperfusion signals rapidly induce the recruitment and differentiation of host monocytes into macrophages and dendritic cells. The nature of ischemia-reperfusion signals are antigen independent, but have been hypothesized to initiate Toll-like receptor (TLR) and interleukin (IL)-1R-mediated signaling pathways that are thought to potentiate alloimmune responses. We wondered whether MyD88, an adaptor molecule critical for both TLR and IL-1R-mediated inflammatory responses, regulated monocyte differentiation in a mouse model of vascularized orthotopic lung transplantation. Orthotopic left lung transplants were performed in the following syngeneic combinations: CD45.1(+) B6 --> CD45.2(+) MyD88(-/-) and CD45.1(+) B6 --> CD45.2(+) B6. One day later, recipient-derived dendritic cells and macrophage numbers were assessed in the bronchiolar lavage by FACS analysis. Compared with the bronchiolar lavage of wildtype recipients, MyD88(-/-) recipients had lower numbers of dendritic cells in lung graft airways that were of recipient origin. Lower numbers of newly differentiated lung graft dendritic cells was coincident with the appearance of higher numbers of undifferentiated monocytes in the lung airways of MyD88(-/-) recipients as compared with wild-type recipients. Moreover, adoptive transfer experiments demonstrated that MyD88(-/-) monocytes were poorer at differentiating into lung dendritic cells as compared with wild-type monocytes. Taken together, these data show that MyD88 regulates graft-infiltrating monocyte differentiation and suggests a mechanism by which TLR/IL-1R-signaling pathways control adaptive responses in lung allografts through controlling monocyte fate.
Subject(s)
Lung Transplantation/physiology , Monocytes/physiology , Myeloid Differentiation Factor 88/physiology , Animals , Cell Differentiation , Dendritic Cells/immunology , Leukocyte Common Antigens/immunology , Lung Transplantation/immunology , Macrophages/cytology , Macrophages/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Monocytes/cytology , Signal Transduction/immunology , Transplantation, Homologous/immunology , Transplantation, Homologous/physiologyABSTRACT
Outcomes after lung transplantation are markedly inferior to those after other solid organ transplants. A better understanding of cellular and molecular mechanisms contributing to lung graft injury will be critical to improve outcomes. Advances in this field have been hampered by the lack of a mouse model of lung transplantation. Here, we report a mouse model of vascularized aerated single lung transplantation utilizing cuff techniques. We show that syngeneic grafts have normal histological appearance with minimal infiltration of T lymphocytes. Allogeneic grafts show acute cellular rejection with infiltration of T lymphocytes and recipient-type antigen presenting cells. Our data show that we have developed a physiological model of lung transplantation in the mouse, which provides ample opportunity for the study of nonimmune and immune mechanisms that contribute to lung allograft injury.
Subject(s)
Lung Transplantation/methods , Pulmonary Circulation/physiology , Animals , Lung Transplantation/pathology , Mice , Mice, Inbred C57BL , Models, Animal , Pulmonary Artery/pathology , Pulmonary Veins/pathology , Transplantation, Isogeneic/pathology , Transplantation, Isogeneic/physiologyABSTRACT
Ischemia reperfusion (I/R) injury following lung transplantation is exacerbated by the destruction of the endothelial cell barrier leading to pulmonary edema and dysregulated activated lymphocyte migration. Sphingosine 1-phosphate (S1P), a G-coupled protein receptor (GPCR) agonist, has been previously shown to promote endothelial cell tight junction formation and prevent monocyte chemotaxis. We asked if S1P treatment could improve pulmonary function and attenuate I/R injury following syngeneic rat lung transplantation. In comparison to vehicle-treated recipients, S1P administered before reperfusion significantly improved recipient oxygenation following transplantation. Improved graft function was associated with reduced inflammatory signaling pathway activation along with attenuated intragraft levels of MIP-2, TNF-alpha and IL-1beta. Moreover, S1P-treated recipients had significantly less apoptotic endothelial cells, pulmonary edema and graft accumulation of neutrophils than did vehicle-treated recipients. Thus our data show that S1P improves lung tissue homeostasis following reperfusion by enhancing endothelial barrier function and blunting monocytic graft infiltration and inflammation.
Subject(s)
Edema/prevention & control , Lung Transplantation/adverse effects , Lung Transplantation/physiology , Lysophospholipids/therapeutic use , Reperfusion Injury/prevention & control , Sphingosine/analogs & derivatives , Animals , Biomarkers/analysis , Bronchoalveolar Lavage Fluid/chemistry , Caspase 3/analysis , Chemokine CXCL2 , In Situ Nick-End Labeling , Inflammation , Interleukin-1beta/analysis , Models, Animal , Monokines/analysis , Peroxidase/metabolism , Rats , Rats, Inbred F344 , Sphingosine/therapeutic use , Tumor Necrosis Factor-alpha/analysisABSTRACT
Ischemic cardiomyopathy leading to congestive heart failure remains the leading source of morbidity and mortality in Western society and medical management of this condition offers only palliative treatment. While allogeneic heart transplantation can both extend and improve the quality of life for patients with end-stage heart failure, this therapeutic option is limited by donor organ shortage. Even after successful transplantation, chronic cardiac rejection in the form of cardiac allograft vasculopathy can severely limit the lifespan of the transplanted organ. Current experimental efforts focus on cellular cardiomyoplasty, myocardial tissue engineering, and myocardial regeneration as alternative approaches to whole organ transplantation. Such strategies may offer novel forms of therapy to patients with end-stage heart failure within the near future.
Subject(s)
Cardiomyoplasty/methods , Myocardium , Regeneration , Tissue Engineering/methods , Animals , Child , Fetal Heart/surgery , Heart Transplantation , Humans , Infant, Newborn , Models, Animal , Myoblasts, Skeletal/transplantation , Myocytes, Cardiac/transplantation , Stem Cell TransplantationABSTRACT
Since vascular endothelium is now recognized as an immunologically active tissue, a better understanding of the relationship between endothelial cells and T lymphocytes is critical to the field of solid organ transplantation. Investigations of endothelial cell-T cell interactions have been limited by methodology. We developed a flow cytometric method allowing for concurrent investigation of multiple cell populations within the same culture that can be applied to these complex interactions. Allogeneic CD8+ or CD4+ T cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) were added to a murine endothelial cell monolayer, in which endothelial proliferation was not inhibited by irradiation or addition of a cell cycle-blocking agent. At specific time points, the coculture was analyzed by flow cytometry. T-cell proliferation could be detected by gating on the T-cell subset and evaluating the CFSE fluorescence peaks. By directly analyzing cellular division, we minimized erroneous interpretation of the data encountered by previous studies, which utilized (3)H-thymidine incorporation as sole measure of proliferation. Further subgating on cells that divided facilitated the study of CD8+ lymphocyte activation, differentiation, and acquisition of effector function. By gating on the endothelial cell population, phenotypic changes such as upregulation of surface MHC molecules or immune-mediated apoptosis could be detected. In conclusion, we present a flow cytometric approach that could have important applications for clinical immunological monitoring in allogeneic or xenogeneic transplantation, and might provide the requisite information to better tailor immunotherapy to prevent chronic rejection.
Subject(s)
Cell Communication , Endothelium, Vascular/cytology , Flow Cytometry/methods , T-Lymphocyte Subsets/physiology , Animals , Apoptosis/physiology , Cell Line , Cell Size , Coculture Techniques , Endothelium, Vascular/immunology , Endothelium, Vascular/physiology , Fluoresceins/metabolism , Fluorescent Dyes/metabolism , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class II/metabolism , Humans , Male , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Perforin , Phenotype , Pore Forming Cytotoxic Proteins , Succinimides/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/physiology , Thymidine/metabolism , Transplantation, HomologousABSTRACT
A 50-year-old woman underwent single lung transplantation for advanced chronic obstructive pulmonary disease. Shortly after the procedure, it was discovered that the donor suffered from both a renal cell carcinoma and a spindle-cell sarcoma of the ascending aorta, which had metastasized to the spleen. The patient was emergently listed for a retransplantation and underwent bilateral lung transplantation after a new donor became available 4 days after the initial transplantation procedure. After 24 months, the patient is without evidence of malignancy. This case illustrates the role of immediate retransplantation for patients who have inadvertently received thoracic organs from donors harboring occult malignancies.
Subject(s)
Emergency Medical Services , Lung Transplantation , Tissue Donors , Adult , Aortic Diseases/pathology , Carcinoma, Renal Cell/pathology , Female , Humans , Kidney Neoplasms/pathology , Middle Aged , Neoplasms, Multiple Primary/pathology , Reoperation , Sarcoma/pathology , Sarcoma/secondary , Splenic Neoplasms/pathology , Splenic Neoplasms/secondaryABSTRACT
Vascular endothelium is an important site for a wide array of immunological processes such as inflammation, atherosclerosis and allograft rejection. Culture methods of mouse vascular endothelium would provide an important in vitro correlate to immunological murine in vivo models. We describe a simple method to culture mouse vascular endothelium from thoracic aorta. Our cultured cells express typical phenotypic (CD105, CD31, CD106), morphological and ultrastructural (intercellular junctions, Weibel-Palade bodies) markers of vascular endothelium. They also possess functional receptors for uptake and processing of acetylated low-density lipoproteins. The mouse vascular endothelium within our system expresses high levels of MHC class I and MHC class II after activation with IFN-gamma. In addition, these cells express the accessory molecules CD80 and CD54, while they lack constitutive expression of CD86 and CD40, providing them the means to function as antigen presenting cells. Alloreactive CD4(+) and CD8(+) T lymphocytes demonstrate evidence of DNA synthesis after co-culture with activated vascular endothelium indicating their commitment to proliferation. In conclusion, we describe a simple culture system to isolate and grow mouse vascular endothelium, which provides a powerful tool to study biological interactions in vitro.