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Zimmerer JM, Ringwald BA, Chaudhari SR, Han J, Peterson CM, Warren RT, Hart MM, Abdel-Rasoul M, Bumgardner GL. Invariant NKT Cells Promote the Development of Highly Cytotoxic Multipotent CXCR3 +CCR4 +CD8 + T Cells That Mediate Rapid Hepatocyte Allograft Rejection. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2021; 207:3107-3121. [PMID: 34810223 PMCID: PMC9124232 DOI: 10.4049/jimmunol.2100334] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/08/2021] [Accepted: 10/13/2021] [Indexed: 12/22/2022]
Abstract
Hepatocyte transplant represents a treatment for metabolic disorders but is limited by immunogenicity. Our prior work identified the critical role of CD8+ T cells, with or without CD4+ T cell help, in mediating hepatocyte rejection. In this study, we evaluated the influence of invariant NKT (iNKT) cells, uniquely abundant in the liver, upon CD8-mediated immune responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type) and iNKT-deficient Jα18 knockout mice (cohorts CD4 depleted) were transplanted with allogeneic hepatocytes. Recipients were evaluated for alloprimed CD8+ T cell subset composition, allocytotoxicity, and hepatocyte rejection. We found that CD8-mediated allocytotoxicity was significantly decreased in iNKT-deficient recipients and was restored by adoptive transfer of iNKT cells. In the absence of both iNKT cells and CD4+ T cells, CD8-mediated allocytotoxicity and hepatocyte rejection was abrogated. iNKT cells enhance the proportion of a novel subset of multipotent, alloprimed CXCR3+CCR4+CD8+ cytolytic T cells that develop after hepatocyte transplant and are abundant in the liver. Alloprimed CXCR3+CCR4+CD8+ T cells express cytotoxic effector molecules (perforin/granzyme and Fas ligand) and are distinguished from alloprimed CXCR3+CCR4-CD8+ T cells by a higher proportion of cells expressing TNF-α and IFN-γ. Furthermore, alloprimed CXCR3+CCR4+CD8+ T cells mediate higher allocytotoxicity and more rapid allograft rejection. Our data demonstrate the important role of iNKT cells in promoting the development of highly cytotoxic, multipotent CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of allogeneic hepatocytes engrafted in the liver. Targeting iNKT cells may be an efficacious therapy to prevent rejection of intrahepatic cellular transplants.
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Affiliation(s)
- Jason M Zimmerer
- Comprehensive Transplant Center, Department of Surgery, The Ohio State University College of Medicine, Columbus, OH
| | - Bryce A Ringwald
- Medical Student Research Program, The Ohio State University College of Medicine, Columbus, OH
| | - Sachi R Chaudhari
- Comprehensive Transplant Center, Department of Surgery, The Ohio State University College of Medicine, Columbus, OH
| | - Jing Han
- Biomedical Sciences Graduate Program, The Ohio State University College of Medicine, Columbus, OH; and
| | - Chelsea M Peterson
- Comprehensive Transplant Center, Department of Surgery, The Ohio State University College of Medicine, Columbus, OH
| | - Robert T Warren
- Comprehensive Transplant Center, Department of Surgery, The Ohio State University College of Medicine, Columbus, OH
| | - Madison M Hart
- Comprehensive Transplant Center, Department of Surgery, The Ohio State University College of Medicine, Columbus, OH
| | | | - Ginny L Bumgardner
- Comprehensive Transplant Center, Department of Surgery, The Ohio State University College of Medicine, Columbus, OH;
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Zimmerer JM, Ringwald BA, Elzein SM, Avila CL, Warren RT, Abdel-Rasoul M, Bumgardner GL. Antibody-suppressor CD8+ T Cells Require CXCR5. Transplantation 2019; 103:1809-1820. [PMID: 30830040 PMCID: PMC6713619 DOI: 10.1097/tp.0000000000002683] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
BACKGROUND We previously reported the novel activity of alloprimed CD8 T cells that suppress posttransplant alloantibody production. The purpose of the study is to investigate the expression and role of CXCR5 on antibody-suppressor CD8 T-cell function. METHODS C57BL/6 mice were transplanted with FVB/N hepatocytes. Alloprimed CD8 T cells were retrieved on day 7 from hepatocyte transplant recipients. Unsorted or flow-sorted (CXCR5CXCR3 and CXCR3CXCR5) alloprimed CD8 T-cell subsets were analyzed for in vitro cytotoxicity and capacity to inhibit in vivo alloantibody production following adoptive transfer into C57BL/6 or high alloantibody-producing CD8 knock out (KO) hepatocyte transplant recipients. Alloantibody titer was assessed in CD8 KO mice reconstituted with naive CD8 T cells retrieved from C57BL/6, CXCR5 KO, or CXCR3 KO mice. Antibody suppression by ovalbumin (OVA)-primed monoclonal OVA-specific t-cell receptor transgenic CD8+ T cells (OT-I) CXCR5 or CXCR3 CD8 T-cell subsets was also investigated. RESULTS Alloprimed CXCR5CXCR3CD8 T cells mediated in vitro cytotoxicity of alloprimed "self" B cells, while CXCR3CXCR5CD8 T cells did not. Only flow-sorted alloprimed CXCR5CXCR3CD8 T cells (not flow-sorted alloprimed CXCR3CXCR5CD8 T cells) suppressed alloantibody production and enhanced graft survival when transferred into transplant recipients. Unlike CD8 T cells from wild-type or CXCR3 KO mice, CD8 T cells from CXCR5 KO mice do not develop alloantibody-suppressor function. Similarly, only flow-sorted CXCR5CXCR3 (and not CXCR3CXCR5) OVA-primed OT-I CD8 T cells mediated in vivo suppression of anti-OVA antibody production. CONCLUSIONS These data support the conclusion that expression of CXCR5 by antigen-primed CD8 T cells is critical for the function of antibody-suppressor CD8 T cells.
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Affiliation(s)
- Jason M. Zimmerer
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
| | - Bryce A. Ringwald
- Medical Student Research Program, The Ohio State University College of Medicine, Columbus, OH
| | - Steven M. Elzein
- Medical Student Research Program, The Ohio State University College of Medicine, Columbus, OH
| | - Christina L. Avila
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
| | - Robert T. Warren
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
| | | | - Ginny L. Bumgardner
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
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Zimmerer JM, Liu XL, Blaszczak A, Avila CL, Pham TA, Warren RT, Bumgardner GL. Critical Role of Macrophage FcγR Signaling and Reactive Oxygen Species in Alloantibody-Mediated Hepatocyte Rejection. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2018; 201:3731-3740. [PMID: 30397035 PMCID: PMC6289737 DOI: 10.4049/jimmunol.1800333] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/06/2018] [Accepted: 10/03/2018] [Indexed: 12/24/2022]
Abstract
Humoral alloimmunity negatively impacts both short- and long-term cell and solid organ transplant survival. We previously reported that alloantibody-mediated rejection of transplanted hepatocytes is critically dependent on host macrophages. However, the effector mechanism(s) of macrophage-mediated injury to allogeneic liver parenchymal cells is not known. We hypothesized that macrophage-mediated destruction of allogeneic hepatocytes occurs by cell-cell interactions requiring FcγRs. To examine this, alloantibody-dependent hepatocyte rejection in CD8-depleted wild-type (WT) and Fcγ-chain knockout (KO; lacking all functional FcγR) transplant recipients was evaluated. Alloantibody-mediated hepatocellular allograft rejection was abrogated in recipients lacking FcγR compared with WT recipients. We also investigated anti-FcγRI mAb, anti-FcγRIII mAb, and inhibitors of intracellular signaling (to block phagocytosis, cytokines, and reactive oxygen species [ROS]) in an in vitro alloantibody-dependent, macrophage-mediated hepatocytoxicity assay. Results showed that in vitro alloantibody-dependent, macrophage-mediated hepatocytotoxicity was critically dependent on FcγRs and ROS. The adoptive transfer of WT macrophages into CD8-depleted FcγR-deficient recipients was sufficient to induce alloantibody-mediated rejection, whereas adoptive transfer of macrophages from Fcγ-chain KO mice or ROS-deficient (p47 KO) macrophages was not. These results provide the first evidence, to our knowledge, that alloantibody-dependent hepatocellular allograft rejection is mediated by host macrophages through FcγR signaling and ROS cytotoxic effector mechanisms. These results support the investigation of novel immunotherapeutic strategies targeting macrophages, FcγRs, and/or downstream molecules, including ROS, to inhibit humoral immune damage of transplanted hepatocytes and perhaps other cell and solid organ transplants.
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Affiliation(s)
- Jason M Zimmerer
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210; and
| | - Xin L Liu
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210; and
| | - Alecia Blaszczak
- Medical Scientist Training Program, College of Medicine, The Ohio State University Wexner Medical Center, Columbus, OH 43210
| | - Christina L Avila
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210; and
| | - Thomas A Pham
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210; and
| | - Robert T Warren
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210; and
| | - Ginny L Bumgardner
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Wexner Medical Center, Columbus, OH 43210; and
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DeTemple DE, Oldhafer F, Falk CS, Chen‐Wacker C, Figueiredo C, Kleine M, Ramackers W, Timrott K, Lehner F, Klempnauer J, Bock M, Vondran FWR. Hepatocyte-induced CD4 + T cell alloresponse is associated with major histocompatibility complex class II up-regulation on hepatocytes and suppressible by regulatory T cells. Liver Transpl 2018; 24:407-419. [PMID: 29365365 PMCID: PMC5887891 DOI: 10.1002/lt.25019] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/02/2017] [Revised: 11/07/2017] [Accepted: 12/16/2017] [Indexed: 12/21/2022]
Abstract
Hepatocyte transplantation is a promising therapeutic approach for various liver diseases. Despite the liver's tolerogenic potential, early immune-mediated loss of transplanted cells is observed, and longterm acceptance has not been achieved yet. Patients deemed tolerant after liver transplantation presented an increased frequency of regulatory T cells (Tregs), which therefore also might enable reduction of posttransplant cell loss and enhance longterm allograft acceptance. We hence characterized hepatocyte-induced immune reactions and evaluated the immunomodulatory potential of Tregs applying mixed lymphocyte cultures and mixed lymphocyte hepatocyte cultures. These were set up using peripheral blood mononuclear cells and primary human hepatocytes, respectively. Polyclonally expanded CD4+ CD25high CD127low Tregs were added to cocultures in single-/trans-well setups with/without supplementation of anti-interferon γ (IFNγ) antibodies. Hepatocyte-induced alloresponses were then analyzed by multicolor flow cytometry. Measurements indicated that T cell response upon stimulation was associated with IFNγ-induced major histocompatibility complex (MHC) class II up-regulation on hepatocytes and mediated by CD4+ T cells. An indirect route of antigen presentation could be ruled out by use of fragmented hepatocytes and culture supernatants of hepatocytes. Allospecific proliferation was accompanied by inflammatory cytokine secretion. CD8+ T cells showed early up-regulation of CD69 despite lack of cell proliferation in the course of coculture. Supplementation of Tregs effectively abrogated hepatocyte-induced alloresponses and was primarily cell contact dependent. In conclusion, human hepatocytes induce a CD4+ T cell alloresponse in vitro, which is associated with MHC class II up-regulation on hepatocytes and is susceptible to suppression by Tregs. Liver Transplantation 24 407-419 2018 AASLD.
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Affiliation(s)
- Daphne E. DeTemple
- Regenerative Medicine and Experimental Surgery, Department of General, Visceral and Transplant SurgeryHannover Medical SchoolHannoverGermany
| | - Felix Oldhafer
- Regenerative Medicine and Experimental Surgery, Department of General, Visceral and Transplant SurgeryHannover Medical SchoolHannoverGermany
| | - Christine S. Falk
- Institute of Transplant Immunology, Integrated Research and Treatment Centre TransplantationHannover Medical SchoolHannoverGermany,German Centre for Infection Researchpartner site Hannover‐BraunschweigHannoverGermany
| | - Chen Chen‐Wacker
- Institute for Transfusion MedicineHannover Medical SchoolHannoverGermany
| | | | - Moritz Kleine
- Regenerative Medicine and Experimental Surgery, Department of General, Visceral and Transplant SurgeryHannover Medical SchoolHannoverGermany
| | - Wolf Ramackers
- Regenerative Medicine and Experimental Surgery, Department of General, Visceral and Transplant SurgeryHannover Medical SchoolHannoverGermany
| | - Kai Timrott
- Regenerative Medicine and Experimental Surgery, Department of General, Visceral and Transplant SurgeryHannover Medical SchoolHannoverGermany
| | - Frank Lehner
- Regenerative Medicine and Experimental Surgery, Department of General, Visceral and Transplant SurgeryHannover Medical SchoolHannoverGermany
| | - Juergen Klempnauer
- Regenerative Medicine and Experimental Surgery, Department of General, Visceral and Transplant SurgeryHannover Medical SchoolHannoverGermany
| | - Michael Bock
- Department of Gastroenterology, Hepatology and EndocrinologyHannover Medical SchoolHannoverGermany,German Centre for Infection Researchpartner site Hannover‐BraunschweigHannoverGermany
| | - Florian W. R. Vondran
- Regenerative Medicine and Experimental Surgery, Department of General, Visceral and Transplant SurgeryHannover Medical SchoolHannoverGermany,German Centre for Infection Researchpartner site Hannover‐BraunschweigHannoverGermany
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mTOR Inhibition Suppresses Posttransplant Alloantibody Production Through Direct Inhibition of Alloprimed B Cells and Sparing of CD8+ Antibody-Suppressing T cells. Transplantation 2017; 100:1898-906. [PMID: 27362313 DOI: 10.1097/tp.0000000000001291] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND De novo alloantibodies (donor-specific antibody) contribute to antibody-mediated rejection and poor long-term graft survival. Because the development of donor-specific antibody is associated with early graft loss of cell transplants and reduced long-term survival of solid organ transplants, we hypothesized that conventional immunosuppressives, calcineurin inhibitors (CNi), and mammalian target of rapamycin inhibitors (mTORi), may not be as effective for suppression of humoral alloimmunity as for cell-mediated immunity. METHODS Wild-type or CD8-depleted mice were transplanted with allogeneic hepatocytes. Recipients were treated with mTORi and/or CNi and serially monitored for alloantibody and graft survival. The direct effect of mTORi and CNi on alloprimed B cell function was investigated in Rag1 mice adoptively transferred with alloprimed IgG1 B cells. The efficacy of mTORi and/or CNi to suppress CD8-mediated cytotoxicity of IgG1 B cells was evaluated in in vitro and in vivo cytotoxicity assays. RESULTS Mammalian target of rapamycin inhibitors, but not CNi, reduced alloantibody production in transplant recipients, directly suppressed alloantibody production by alloprimed IgG1 B cells and delayed graft rejection in both low and high alloantibody producers. Combination treatment with mTORi and CNi resulted in loss of the inhibitory effect observed for mTORi monotherapy in part due to CNi suppression of CD8 T cells which downregulate alloantibody production (CD8 TAb-supp cells). CONCLUSIONS Our data support that mTORi is a potent inhibitor of humoral immunity through suppression of alloprimed B cells and preservation of CD8 TAb-supp cells. In contrast, alloantibody is readily detected in CNi-treated recipients because CNi does not suppress alloprimed B cells and interferes with downregulatory CD8 TAb-supp cells.
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Abstract
BACKGROUND The liver immune environment is tightly regulated to balance immune activation with immune tolerance. Understanding the dominant immune pathways initiated in the liver is important because the liver is a site for cell transplantation, such as for islet and hepatocyte transplantation. The purpose of this study is to examine the consequences of alloimmune stimulation when allogeneic cells are transplanted to the liver in comparison to a different immune locale, such as the kidney. METHODS We investigated cellular and humoral immune responses when allogeneic hepatocytes are transplanted directly to the recipient liver by intraportal injection. A heterotopic kidney engraftment site was used for comparison to immune activation in the liver microenvironment. RESULTS Transplantation of allogeneic hepatocytes delivered directly to the liver, via recipient portal circulation, stimulated long-term, high magnitude CD8 T cell-mediated allocytotoxicity. CD8 T cells initiated significant in vivo allocytotoxicity as well as rapid rejection of hepatocytes transplanted to the liver even in the absence of secondary lymph nodes or CD4 T cells. In contrast, in the absence of recipient peripheral lymphoid tissue and CD4 T cells, CD8-mediated in vivo allocytotoxicity was abrogated, and rejection was delayed when hepatocellular allografts were transplanted to the kidney subcapsular site. CONCLUSIONS These results highlight the CD8-dominant proinflammatory immune responses unique to the liver microenvironment. Allogeneic cells transplanted directly to the liver do not enjoy immune privilege but rather require immunosuppression to prevent rejection by a robust and persistent CD8-dependent allocytotoxicity primed in the liver.
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Najimi M, Defresne F, Sokal EM. Concise Review: Updated Advances and Current Challenges in Cell Therapy for Inborn Liver Metabolic Defects. Stem Cells Transl Med 2016; 5:1117-25. [PMID: 27245366 DOI: 10.5966/sctm.2015-0260] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/22/2015] [Accepted: 03/14/2016] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED : The development of liver cell transplantation (LCT), considered a major biotechnological breakthrough, was intended to provide more accessible treatments for liver disease patients. By preserving the native recipient liver and decreasing hospitalization time, this innovative approach has progressively gained interest among clinicians. LCT initially targets inborn errors of liver metabolism, enabling the compensation of deficient metabolic functions for up to 18 months post-transplantation, supporting its use at least as a bridge to transplantation. The rigorous clinical development and widespread use of LCT depends strongly on controlled and consistent clinical trial data, which may help improve several critical factors, including the standardization of raw biological material and immunosuppression regimens. Substantial effort has also been made in defining and optimizing the most efficient cell population to be transplanted in the liver setting. Although isolated hepatocytes remain the best cell type, showing positive clinical results, their widespread use is hampered by their poor resistance to both cryopreservation and in vitro culture, as well as ever-more-significant donor shortages. Hence, there is considerable interest in developing more standardized and widely accessible cell medicinal products to improve engraftment permanency and post-cell transplantation metabolic effects. SIGNIFICANCE In this therapeutic approach to liver disease, new solutions are being designed and evaluated to bypass the documented limitations and move forward toward wide clinical use. Future developments also require a deep knowledge of regulatory framework to launch specific clinical trials that will allow clear assessment of cell therapy and help patients with significant unmet medical needs.
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Affiliation(s)
- Mustapha Najimi
- Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain and Cliniques Universitaires St Luc, Brussels, Belgium
| | - Florence Defresne
- Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain and Cliniques Universitaires St Luc, Brussels, Belgium
| | - Etienne M Sokal
- Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain and Cliniques Universitaires St Luc, Brussels, Belgium
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Oldhafer F, Bock M, Falk CS, Vondran FWR. Immunological aspects of liver cell transplantation. World J Transplant 2016; 6:42-53. [PMID: 27011904 PMCID: PMC4801804 DOI: 10.5500/wjt.v6.i1.42] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/31/2015] [Revised: 10/21/2015] [Accepted: 12/08/2015] [Indexed: 02/05/2023] Open
Abstract
Within the field of regenerative medicine, the liver is of major interest for adoption of regenerative strategies due to its well-known and unique regenerative capacity. Whereas therapeutic strategies such as liver resection and orthotopic liver transplantation (OLT) can be considered standards of care for the treatment of a variety of liver diseases, the concept of liver cell transplantation (LCTx) still awaits clinical breakthrough. Success of LCTx is hampered by insufficient engraftment/long-term acceptance of cellular allografts mainly due to rejection of transplanted cells. This is in contrast to the results achieved for OLT where long-term graft survival is observed on a regular basis and, hence, the liver has been deemed an immune-privileged organ. Immune responses induced by isolated hepatocytes apparently differ considerably from those observed following transplantation of solid organs and, thus, LCTx requires refined immunological strategies to improve its clinical outcome. In addition, clinical usage of LCTx but also related basic research efforts are hindered by the limited availability of high quality liver cells, strongly emphasizing the need for alternative cell sources. This review focuses on the various immunological aspects of LCTx summarizing data available not only for hepatocyte transplantation but also for transplantation of non-parenchymal liver cells and liver stem cells.
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Scavenger receptor B1, the HDL receptor, is expressed abundantly in liver sinusoidal endothelial cells. Sci Rep 2016; 6:20646. [PMID: 26865459 PMCID: PMC4749959 DOI: 10.1038/srep20646] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2015] [Accepted: 01/07/2016] [Indexed: 02/08/2023] Open
Abstract
Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1. In contrast, the hepatocyte, identified with β-catenin, expressed considerably weaker levels, although optical resolution of SR-B1 was inadequate. Thus, we moved to a different IF strategy, first separating dissociated liver cells by gradient centrifugation into two portions, hepatocytes (parenchymal cells) and LSEC (non-parenchymal cells). Characterizing both portions for the cellular expression of SR-B1 by flow cytometry, we found that LSEC expressed considerable amounts of SR-B1 while in hepatocytes SR-B1 expression was barely perceptible. Assessing mRNA of SR-B1 by real time PCR we found messenger expression in LSEC to be about 5 times higher than in hepatocytes.
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Sana G, Lombard C, Vosters O, Jazouli N, Andre F, Stephenne X, Smets F, Najimi M, Sokal EM. Adult human hepatocytes promote CD4(+) T-cell hyporesponsiveness via interleukin-10-producing allogeneic dendritic cells. Cell Transplant 2015; 23:1127-42. [PMID: 23582182 DOI: 10.3727/096368913x666421] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023] Open
Abstract
The success of liver cell therapy remains closely dependent on how well the infused cells can be accepted after transplantation and is directly related to their degree of immunogenicity. In this study, we investigated the in vitro immunogenic properties of isolated human hepatocytes (hHeps) and adult-derived human liver progenitor cells (ADHLPCs), an alternative cell candidate for liver cell transplantation (LCT). The constitutive expression of immune markers was first analyzed on these liver-derived cells by flow cytometry. Human liver-derived cells were then cocultured with allogeneic human adult peripheral blood mononuclear cells (PBMCs), and the resulting activation and proliferation of PBMCs was evaluated, as well as the cytokine levels in the coculture supernatant. The effect of liver-derived cells on monocyte-derived dendritic cell (MoDC) properties was further analyzed in a secondary coculture with naive CD4(+) T-cells. We report that hHeps and ADHLPCs expressed human leukocyte antigen (HLA) class I and Fas but did not express HLA-DR, Fas ligand, and costimulatory molecules. hHeps and ADHLPCs did not induce T-cell activation or proliferation. Moreover, hHeps induced a cell contact-dependent production of interleukin (IL)-10 that was not observed with ADHLPCs. The IL-10 was produced by a myeloid DC subset characterized by an incomplete mature state. Furthermore, hHep-primed MoDCs induced an antigen-independent hyporesponsiveness of naive CD4(+) T lymphocytes that was partially reversed by blocking IL-10, whereas nonprimed MoDCs (i.e., those cultured alone) did not. hHeps and ADHLPCs present a low immunogenic phenotype in vitro. Allogeneic hHeps, but not ADHLPCs, promote a cell contact-dependent production of IL-10 by myeloid DCs, which induces naive CD4(+) T-cells antigen-independent hyporesponsiveness.
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Affiliation(s)
- Gwenaëlle Sana
- Université Catholique de Louvain and Cliniques Universitaires Saint-Luc, Laboratory of Pediatric Hepatology and Cell Therapy, Brussels, Belgium
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Zimmerer J, Swamy P, Sanghavi P, Wright C, Abdel-Rasoul M, Elzein S, Brutkiewicz R, Bumgardner G. Critical role of NKT cells in posttransplant alloantibody production. Am J Transplant 2014; 14:2491-9. [PMID: 25220596 PMCID: PMC4207222 DOI: 10.1111/ajt.12922] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2014] [Revised: 06/13/2014] [Accepted: 07/01/2014] [Indexed: 01/25/2023]
Abstract
We previously reported that posttransplant alloantibody production in CD8-deficient hosts is IL-4+ CD4+ T cell-dependent and IgG1 isotype-dominant. The current studies investigated the hypothesis that IL-4-producing natural killer T cells (NKT cells) contribute to maximal alloantibody production. To investigate this, alloantibody levels were examined in CD8-deficient WT, CD1d KO and Jα18 KO transplant recipients. We found that the magnitude of IgG1 alloantibody production was critically dependent on the presence of type I NKT cells, which are activated by day 1 posttransplant. Unexpectedly, type I NKT cell contribution to enhanced IgG1 alloantibody levels was interferon-γ-dependent and IL-4-independent. Cognate interactions between type I NKT and B cells alone do not stimulate alloantibody production. Instead, NKT cells appear to enhance maturation of IL-4+ CD4+ T cells. To our knowledge, this is the first report to substantiate a critical role for type I NKT cells in enhancing in vivo antibody production in response to endogenous antigenic stimuli.
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Affiliation(s)
- J.M. Zimmerer
- Department of Surgery, Comprehensive Transplant Center, and the College of Medicine, The Ohio State University, Columbus, OH
| | - P. Swamy
- Medical Student Research Program, College of Medicine, The Ohio State University, Columbus, OH
| | - P.B. Sanghavi
- Medical Student Research Program, College of Medicine, The Ohio State University, Columbus, OH
| | - C.L. Wright
- Department of Surgery, Comprehensive Transplant Center, and the College of Medicine, The Ohio State University, Columbus, OH
| | - M. Abdel-Rasoul
- Center for Biostatistics, The Ohio State University, Columbus, OH 43221
| | - S.M. Elzein
- Department of Surgery, Comprehensive Transplant Center, and the College of Medicine, The Ohio State University, Columbus, OH
| | - R.R. Brutkiewicz
- Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN
| | - G.L. Bumgardner
- Department of Surgery, Comprehensive Transplant Center, and the College of Medicine, The Ohio State University, Columbus, OH
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Zimmerer J, Pham T, Wright C, Tobin K, Sanghavi P, Elzein S, Sanders V, Bumgardner G. Alloprimed CD8(+) T cells regulate alloantibody and eliminate alloprimed B cells through perforin- and FasL-dependent mechanisms. Am J Transplant 2014; 14:295-304. [PMID: 24472191 PMCID: PMC4018729 DOI: 10.1111/ajt.12565] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2013] [Revised: 10/07/2013] [Accepted: 10/27/2013] [Indexed: 01/25/2023]
Abstract
While it is well known that CD4(+) T cells and B cells collaborate for antibody production, our group previously reported that CD8(+) T cells down-regulate alloantibody responses following transplantation. However, the exact mechanism involved in CD8(+) T cell-mediated down-regulation of alloantibody remains unclear. We also reported that alloantibody production is enhanced when either perforin or FasL is deficient in transplant recipients. Here, we report that CD8(+) T cell-deficient transplant recipient mice (high alloantibody producers) exhibit an increased number of primed B cells compared to WT transplant recipients. Furthermore, CD8(+) T cells require FasL, perforin and allospecificity to down-regulate posttransplant alloantibody production. In vivo CD8-mediated clearance of alloprimed B cells was also FasL- and perforin-dependent. In vitro data demonstrated that recipient CD8(+) T cells directly induce apoptosis of alloprimed IgG1(+) B cells in co-culture in an allospecific and MHC class I-dependent fashion. Altogether these data are consistent with the interpretation that CD8(+) T cells down-regulate posttransplant alloantibody production by FasL- and perforin-dependent direct elimination of alloprimed IgG1(+) B cells.
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Affiliation(s)
- J.M. Zimmerer
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
| | - T.A. Pham
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
| | - C.L. Wright
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
| | - K.J. Tobin
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
| | - P.B. Sanghavi
- Medical Student Research Program, College of Medicine, The Ohio State University, Columbus, OH
| | - S.M. Elzein
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
| | - V.M. Sanders
- Department of Molecular Virology, Immunology, and Medical Genetics, The Ohio State University Wexner Medical Center, Columbus, OH
| | - G.L. Bumgardner
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University, Columbus, OH
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Kochat V, Baligar P, Maiwall R, Mukhopadhyay A. Bone marrow stem-cell therapy for genetic and chronic liver diseases. Hepatol Int 2014. [DOI: 10.1007/s12072-013-9499-z] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
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Cytotoxic effector function of CD4-independent, CD8(+) T cells is mediated by TNF-α/TNFR. Transplantation 2013; 94:1103-10. [PMID: 23222736 DOI: 10.1097/tp.0b013e318270f3c0] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
BACKGROUND Liver parenchymal cell allografts initiate both CD4-dependent and CD4-independent, CD8(+) T cell-mediated acute rejection pathways. The magnitude of allospecific CD8(+) T cell in vivo cytotoxic effector function is maximal when primed in the presence of CD4(+) T cells. The current studies were conducted to determine if and how CD4(+) T cells might influence cytotoxic effector mechanisms. METHODS Mice were transplanted with allogeneic hepatocytes. In vivo cytotoxicity assays and various gene-deficient recipient mice and target cells were used to determine the development of Fas-, TNF-α-, and perforin-dependent cytotoxic effector mechanisms after transplantation. RESULTS CD8(+) T cells maturing in CD4-sufficient hepatocyte recipients develop multiple (Fas-, TNF-α-, and perforin-mediated) cytotoxic mechanisms. However, CD8(+) T cells, maturing in the absence of CD4(+) T cells, mediate cytotoxicity and transplant rejection that is exclusively TNF-α/TNFR-dependent. To determine the kinetics of CD4-mediated help, CD4(+) T cells were adoptively transferred into CD4-deficient mice at various times posttransplant. The maximal influence of CD4(+) T cells on the magnitude of CD8-mediated in vivo allocytotoxicityf occurs within 48 hours. CONCLUSION The implication of these studies is that interference of CD4(+) T cell function by disease or immunotherapy will have downstream consequences on both the magnitude of allocytotoxicity as well as the cytotoxic effector mechanisms used by allospecific CD8(+) cytolytic T cells.
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Zimmerer JM, Horne PH, Fiessinger LA, Fisher MG, Jayashankar K, Garcia SF, Abdel-Rasoul M, van Rooijen N, Bumgardner GL. Inhibition of recall responses through complementary therapies targeting CD8+ T-cell- and alloantibody-dependent allocytotoxicity in sensitized transplant recipients. Cell Transplant 2012; 22:1157-69. [PMID: 23069206 DOI: 10.3727/096368912x657350] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Allospecific T memory cell responses in transplant recipients arise from environmental exposure to previous transplantation or cross-reactive heterologous immunity. Unfortunately, these memory responses pose a significant barrier to the survival of transplanted tissue. We have previously reported that concurrent inhibition of CD154 and LFA-1 suppresses primary CD8-dependent rejection responses that are not controlled by conventional immunosuppressive strategies. We hypothesized that CD154- and LFA-1-mediated inhibition, by targeting activation as well as effector functions, may also be efficacious for the control of alloreactive CD8+ T-cell responses in sensitized hosts. We found that treatment with anti-LFA-1 mAb alone enhanced transplant survival and reduced CD8-mediated cytotoxicity in sensitized CD4 KO recipients. However, treatment with anti-CD154 mAb alone did not have an effect. Notably, when both CD4- and CD8-dependent rejection pathways are operative (wild-type sensitized recipients), LFA-1 significantly inhibited CD8-mediated in vivo allocytotoxicity but did not correspond with enhanced hepatocyte survival. We hypothesized that this was due to alloantibody-mediated rejection. When anti-LFA-1 mAb treatment was combined with macrophage depletion, which we have previously reported impairs alloantibody-mediated parenchymal cell damage, in vivo cytotoxic effector function was significantly decreased and was accompanied by significant enhancement of hepatocyte survival in sensitized wild-type recipients. Therefore, LFA-1 is a potent therapeutic target for reduction of CD8-mediated cytotoxicity in sensitized transplant recipients and can be combined with other treatments that target non-CD8-mediated recall alloimmunity.
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Affiliation(s)
- Jason M Zimmerer
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Medical Center, Columbus, OH 43210-1250, USA
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16
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Jorns C, Ellis EC, Nowak G, Fischler B, Nemeth A, Strom SC, Ericzon BG. Hepatocyte transplantation for inherited metabolic diseases of the liver. J Intern Med 2012; 272:201-23. [PMID: 22789058 DOI: 10.1111/j.1365-2796.2012.02574.x] [Citation(s) in RCA: 95] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
Inherited metabolic diseases of the liver are characterized by deficiency of a hepatic enzyme or protein often resulting in life-threatening disease. The remaining liver function is usually normal. For most patients, treatment consists of supportive therapy, and the only curative option is liver transplantation. Hepatocyte transplantation is a promising therapy for patients with inherited metabolic liver diseases, which offers a less invasive and fully reversible approach. Procedure-related complications are rare. Here, we review the experience of hepatocyte transplantation for metabolic liver diseases and discuss the major obstacles that need to be overcome to establish hepatocyte transplantation as a reliable treatment option in the clinic.
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Affiliation(s)
- C Jorns
- Division of Transplantation Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska University Hospital Huddinge, Stockholm, Sweden.
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Bhatt S, Fung JJ, Lu L, Qian S. Tolerance-inducing strategies in islet transplantation. Int J Endocrinol 2012; 2012:396524. [PMID: 22675353 PMCID: PMC3366204 DOI: 10.1155/2012/396524] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/15/2011] [Accepted: 03/08/2012] [Indexed: 12/12/2022] Open
Abstract
Allogeneic islet transplantation is a promising approach for restoring normoglycemia in type 1 diabetic patients. Current use of immunosuppressive therapies for management of islet transplant recipients can be counterintuitive to islet function and can lead to complications in the long term. The induction of donor-specific tolerance eliminates the dependency on immunosuppression and allows recipients to retain responses to foreign antigens. The mechanisms by which tolerance is achieved involve the deletion of donor-reactive T cells, induction of T-cell anergy, immune deviation, and generation of regulatory T cells. This review will outline the various methods used for inducing donor-specific tolerance in islet transplantation and will highlight the previously unforeseen potential of tissue stromal cells in promoting islet engraftment.
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Affiliation(s)
- Sumantha Bhatt
- Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - John J. Fung
- Department of General Surgery, Transplant Center, Digestive Disease Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Lina Lu
- Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
- Department of General Surgery, Transplant Center, Digestive Disease Institute, Cleveland Clinic, Cleveland, OH 44195, USA
| | - Shiguang Qian
- Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA
- Department of General Surgery, Transplant Center, Digestive Disease Institute, Cleveland Clinic, Cleveland, OH 44195, USA
- *Shiguang Qian:
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Teng L, Liu L, Su Y, Yuan X, Li J, Fu Q, Chen S, Wang C. Suppression of Alloimmunity in Mice by Regulatory T Cells Converted with Conditioned Media. J Surg Res 2011; 171:797-806. [DOI: 10.1016/j.jss.2010.03.044] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/23/2009] [Revised: 03/02/2010] [Accepted: 03/19/2010] [Indexed: 02/05/2023]
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Shen Z, Teng X, Qian X, He M, Hu Y, Ye W, Huang H, Yu Y, Chen Y. Immunoregulation effect by overexpression of heme oxygenase-1 on cardiac xenotransplantation. Transplant Proc 2011; 43:1994-7. [PMID: 21693314 DOI: 10.1016/j.transproceed.2011.03.037] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2009] [Revised: 02/11/2011] [Accepted: 03/09/2011] [Indexed: 01/13/2023]
Abstract
OBJECTIVE This study was designed to investigate the effect of the overexpression of heme oxygenase-1 (HO-1) on the immunoregulation in the model of abdominal cardiac xenotransplantation from the guinea pig to the rat. MATERIALS AND METHODS To increase the expression of HO-1, both donors and recipients were injected with heme through the abdomen before the operation. The donors (guniea pigs) and the recipients (Sprague-Dawley [SD] rats) were divided randomly into three groups: group A, the heart from a guinea pig transplanted into the abdomen of an SD rat; group B, the recipients were injected with Chinese cobra venom factor (CVF) into the abdomen (40 μg/kg and 60 μg/kg 24 hours later) prior to transplantation; group C, CVF + HO-1 high-expression group: donors and recipients were abdominally injected with heme (75 μmol/kg for 2 days before transplantation). The mean survival time (MST), pathological changes, the positive area of HO-1 in the grafted hearts, as well as the expressions of C-C chemokine receptor 5 (CCR5), intercellular adhesion molecule-1 (ICAM-1), and natural killer (NK) cell activity in recipients. RESULTS 1. The MST was longest in group C treated with heme. 2. The pathologic changes of hyperacute rejection were showed on the donor heart in group A, while delayed xenograft rejection changes took place on donor heart in other groups. 3. Compared with group B, The HO-1 positive area in the donor hearts of group C was significantly higher. (P < .05). 4. The lever of ICAM-1 and CCR5 in the peripheral blood of recipients (pg/mL) was attenuated in group C injected with heme. 5. Compared with group B, the activity of NK cell in the peripheral blood of recipients was much lower in group C (P < .05). CONCLUSION The MST was prolonged by increasing expressions of HO-1, but acute vascular rejection was not completely overcome. Activation of vascular endothelial cells could be decreased by strengthening the expression of HO-1. NK cell activity was weakened by reinforced expression of HO-1.
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Affiliation(s)
- Z Shen
- Cardiovascular Surgery Department, The First Affiliated Hospital of Soochow University, Suzhou, Jiangsu, PR China.
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20
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Inagaki M, Furukawa H, Satake Y, Okada Y, Chiba S, Nishikawa Y, Ogawa K. Replacement of liver parenchyma in analbuminemic rats with allogenic hepatocytes is facilitated by intrabone marrow-bone marrow transplantation. Cell Transplant 2010; 20:1479-89. [PMID: 21176395 DOI: 10.3727/096368910x547453] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Although hepatocyte transplantation (HCTx) is expected to become a useful therapy for human liver diseases, allogenic hepatocytes still tend to be rejected within a short period due to host immunosurveillance. In the present study, we investigated the effect of prior bone marrow transplantation (BMTx) for the engraftment of allogenic hepatocytes using the analbuminemic rat transplantation model. The hepatocytes of Lewis (LEW) rats were not accepted in the liver of retrorsine (RS)/partial hepatectomy (PH)-treated analbuminemic F344 (F344-alb) rats, which express the disparate major histocompatibility complex (MHC) against that of LEW rats. Prior BMTx with the LEW bone marrow cells (BMCs) after sublethal irradiation achieved acceptance and repopulation of LEW hepatocytes in the liver of the RS/PH-treated F344-alb rats, associated with elevation of serum albumin. The replacement of hepatic parenchyma with albumin positive (Alb(+)) donor hepatocytes and elevation of serum albumin levels were dependent on the bone marrow reconstitution by donor BMCs, which was more efficiently achieved by intrabone marrow (IBM)-BMTx than by intravenous (IV)-BMTx. Our results demonstrate that efficient bone marrow reconstitution by IBM-BMTx enables the replacement of the hepatic parenchyma with allogenic hepatocytes in RS/PH-treated analbuminemic rats without immunosuppressants.
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Affiliation(s)
- Mitsuhiro Inagaki
- Department of Surgery, Asahikawa Medical University, Asahikawa, Japan.
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22
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Horne PH, Zimmerer JM, Fisher M, Lunsford KE, Nadasdy G, Nadasdy T, van Rooijen N, Bumgardner GL. Critical role of effector macrophages in mediating CD4-dependent alloimmune injury of transplanted liver parenchymal cells. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2008; 181:1224-31. [PMID: 18606676 PMCID: PMC3022512 DOI: 10.4049/jimmunol.181.2.1224] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/23/2022]
Abstract
Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection. Host serum alloantibody levels correlated with in vivo allospecific cytotoxic activity in CD8 knockout hepatocyte rejector mice. Host macrophage depletion, but not CD4(+) T cell, NK cell, neutrophil, or complement depletion, inhibited in vivo allocytotoxicity. Recipient macrophage deficiency delayed CD4-dependent hepatocyte rejection and inhibited in vivo allocytotoxicity without influencing alloantibody production. Furthermore, hepatocyte coincubation with alloantibody and macrophages resulted in Ab-dependent hepatocellular cytotoxicity in vitro. These studies are consistent with a paradigm of acute humoral rejection in which CD4(+) T cell-dependent alloantibody production results in the targeting of transplanted allogeneic parenchymal cells for macrophage-mediated cytotoxic immune damage. Consequently, strategies to eliminate recipient macrophages during CD4-dependent rejection pathway may prolong allograft survival.
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Affiliation(s)
- Phillip H. Horne
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Medical Center, Columbus, OH
- Integrated Biomedical Science Graduate Program, College of Medicine, The Ohio State University, Columbus, OH
| | - Jason M. Zimmerer
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Medical Center, Columbus, OH
| | - Mason Fisher
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Medical Center, Columbus, OH
| | - Keri E. Lunsford
- Integrated Biomedical Science Graduate Program, College of Medicine, The Ohio State University, Columbus, OH
| | - Gyongyi Nadasdy
- Department of Pathology, The Ohio State University Medical Center, Columbus, OH
| | - Tibor Nadasdy
- Department of Pathology, The Ohio State University Medical Center, Columbus, OH
| | - Nico van Rooijen
- Dept of Molecular Cell Biology, VUMC, Amsterdam, The Netherlands
| | - Ginny L. Bumgardner
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Medical Center, Columbus, OH
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23
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Horne PH, Lunsford KE, Walker JP, Koester MA, Bumgardner GL. Recipient Immune Repertoire and Engraftment Site Influence the Immune Pathway Effecting Acute Hepatocellular Allograft Rejection. Cell Transplant 2008; 17:829-44. [DOI: 10.3727/096368908786516792] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
As novel acute allograft rejection mechanisms are being discovered, determining the conditions that promote or subvert these distinct rejection pathways is important to interpret the clinical relevance of these pathways for specific recipient groups as well as specific tissue and organ transplants. We have employed a versatile hepatocellular allograft model to analyze how the host immune repertoire and immune locale influences the phenotype of the rejection pathway. In addition, we investigated how peripheral monitoring of cellular and humoral immune parameters correlates with the activity of a specific rejection pathway. Complete MHC mismatched hepatocellular allografts were transplanted into immune competent CD4-deficient, CD8-deficient, or C57BL/6 hosts to focus on CD8-dependent, CD4-dependent, or combined CD4 and CD8-dependent alloimmunity, respectively. Hepatocellular allografts were transplanted to the liver or kidney subcapsular space to investigate the influence of the immune locale on each rejection pathway. The generation of donor-reactive DTH, alloantibody, and allospecific cytotoxicity was measured to assess both cellular and humoral immunity. Graft-infiltrating lymphocytes were phenotyped and enumerated in each recipient group. In the presence of CD8+ T cells, cytolytic cellular activity is the dominant mechanism of graft destruction and is amplified in the presence of CD4+ T cells. The absence of CD8+ T cells (CD8 KO) results in potent humoral immunity as reflected by high levels of cytotoxic alloantibody and graft rejection with similar kinetics. Transplant to the liver compared to the kidney site is distinguished by more rapid kinetics of rejection and alloimmunity, which is predominately cell mediated rather than a mix of both humoral and cell-mediated immunity. These studies define several rejection mechanisms occurring in distinct immune conditions, highlighting the plasticity of acute allograft rejection responses and the need to design specific monitoring strategies for these pathways to allow dynamic immune assessment of clinical transplant recipients and targeted immunotherapies.
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Affiliation(s)
- Phillip H. Horne
- Integrated Biomedical Science Graduate Program, College of Medicine, The Ohio State University Medical Center, Columbus, OH, USA
| | - Keri E. Lunsford
- Integrated Biomedical Science Graduate Program, College of Medicine, The Ohio State University Medical Center, Columbus, OH, USA
| | - Jon P. Walker
- Division of Digestive Diseases, Department of Internal Medicine, The Ohio State University Medical Center, Columbus, OH, USA
| | - Mitchel A. Koester
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Medical Center, Columbus, OH, USA
| | - Ginny L. Bumgardner
- Department of Surgery, Comprehensive Transplant Center, The Ohio State University Medical Center, Columbus, OH, USA
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Lunsford KE, Jayanshankar K, Eiring AM, Horne PH, Koester MA, Gao D, Bumgardner GL. Alloreactive (CD4-Independent) CD8+ T cells jeopardize long-term survival of intrahepatic islet allografts. Am J Transplant 2008; 8:1113-28. [PMID: 18522544 PMCID: PMC3081659 DOI: 10.1111/j.1600-6143.2008.02219.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Despite success of early islet allograft engraftment and survival in humans, late islet allograft loss has emerged as an important clinical problem. CD8+ T cells that are independent of CD4+ T cell help can damage allograft tissues and are resistant to conventional immunosuppressive therapies. Previous work demonstrates that islet allografts do not primarily initiate rejection by the (CD4-independent) CD8-dependent pathway. This study was performed to determine if activation of alloreactive CD4-independent, CD8+ T cells, by exogenous stimuli, can precipitate late loss of islet allografts. Recipients were induced to accept intrahepatic islet allografts (islet 'acceptors') by short-term immunotherapy with donor-specific transfusion (DST) and anti-CD154 mAb. Following the establishment of stable long-term islet allograft function for 60-90 days, recipients were challenged with donor-matched hepatocellular allografts, which are known to activate (CD4-independent) CD8+ T cells. Allogeneic islets engrafted long-term were vulnerable to damage when challenged locally with donor-matched hepatocytes. Islet allograft loss was due to allospecific immune damage, which was CD8- but not CD4-dependent. Selection of specific immunotherapy to suppress both CD4- and CD8-dependent immune pathways at the time of transplant protects islet allografts from both early and late immune damage.
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Affiliation(s)
- Keri E. Lunsford
- Integrated Biomedical Science Graduate Program, College of Medicine and Public Health, The Ohio State University, Columbus, OH
| | - Kartika Jayanshankar
- Department of Surgery, Division of Transplantation, The Ohio State University Medical Center, Columbus, OH
| | - Anna M. Eiring
- Department of Surgery, Division of Transplantation, The Ohio State University Medical Center, Columbus, OH
| | - Phillip H. Horne
- Integrated Biomedical Science Graduate Program, College of Medicine and Public Health, The Ohio State University, Columbus, OH
| | - Mitchel A. Koester
- Department of Surgery, Division of Transplantation, The Ohio State University Medical Center, Columbus, OH
| | - Donghong Gao
- Department of Surgery, Division of Transplantation, The Ohio State University Medical Center, Columbus, OH
| | - Ginny L. Bumgardner
- Department of Surgery, Division of Transplantation, The Ohio State University Medical Center, Columbus, OH
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Gelman AE, Okazaki M, Lai J, Kornfeld CG, Kreisel FH, Richardson SB, Sugimoto S, Tietjens JR, Patterson GA, Krupnick AS, Kreisel D. CD4+ T lymphocytes are not necessary for the acute rejection of vascularized mouse lung transplants. THE JOURNAL OF IMMUNOLOGY 2008; 180:4754-62. [PMID: 18354199 DOI: 10.4049/jimmunol.180.7.4754] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Abstract
Acute rejection continues to present a major obstacle to successful lung transplantation. Although CD4(+) T lymphocytes are critical for the rejection of some solid organ grafts, the role of CD4(+) T cells in the rejection of lung allografts is largely unknown. In this study, we demonstrate in a novel model of orthotopic vascularized mouse lung transplantation that acute rejection of lung allografts is independent of CD4(+) T cell-mediated allorecognition pathways. CD4(+) T cell-independent rejection occurs in the absence of donor-derived graft-resident hematopoietic APCs. Furthermore, blockade of the CD28/B7 costimulatory pathways attenuates acute lung allograft rejection in the absence of CD4(+) T cells, but does not delay acute rejection when CD4(+) T cells are present. Our results provide new mechanistic insight into the acute rejection of lung allografts and highlight the importance of identifying differences in pathways that regulate the rejection of various organs.
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Affiliation(s)
- Andrew E Gelman
- Division of Cardiothoracic Surgery, Department of Surgery, Washington University, St Louis, MO 63110, USA
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26
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Streetz KL, Doyonnas R, Grimm D, Jenkins DD, Fuess S, Perryman S, Lin J, Trautwein C, Shizuru J, Blau H, Sylvester KG, Kay MA. Hepatic parenchymal replacement in mice by transplanted allogeneic hepatocytes is facilitated by bone marrow transplantation and mediated by CD4 cells. Hepatology 2008; 47:706-18. [PMID: 18220289 DOI: 10.1002/hep.22012] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
UNLABELLED The lack of adequate donor organs is a major limitation to the successful widespread use of liver transplantation for numerous human hepatic diseases. A desirable alternative therapeutic option is hepatocyte transplantation (HT), but this approach is similarly restricted by a shortage of donor cells and by immunological barriers. Therefore, in vivo expansion of tolerized transplanted cells is emerging as a novel and clinically relevant potential alternative cellular therapy. Toward this aim, in the present study we established a new mouse model that combines HT with prior bone marrow transplantation (BMT). Donor hepatocytes were derived from human alpha(1)-antitrypsin (hAAT) transgenic mice of the FVB strain. Serial serum enzyme-linked immunosorbent assays for hAAT protein were used to monitor hepatocyte engraftment and expansion. In control recipient mice lacking BMT, we observed long-term yet modest hepatocyte engraftment. In contrast, animals undergoing additional syngeneic BMT prior to HT showed a 3- to 5-fold increase in serum hAAT levels after 24 weeks. Moreover, complete liver repopulation was observed in hepatocyte-transplanted Balb/C mice that had been transplanted with allogeneic FVB-derived bone marrow. These findings were validated by a comparison of hAAT levels between donor and recipient mice and by hAAT-specific immunostaining. Taken together, these findings suggest a synergistic effect of BMT on transplanted hepatocytes for expansion and tolerance induction. Livers of repopulated animals displayed substantial mononuclear infiltrates, consisting predominantly of CD4(+) cells. Blocking the latter prior to HT abrogated proliferation of transplanted hepatocytes, and this implied an essential role played by CD4(+) cells for in vivo hepatocyte selection following allogeneic BMT. CONCLUSION The present mouse model provides a versatile platform for investigation of the mechanisms governing HT with direct relevance to the development of clinical strategies for the treatment of human hepatic failure.
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Affiliation(s)
- Konrad L Streetz
- Department of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA
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Abstract
The liver performs multiple functions that are essential for life, the most crucial being its role in the body metabolism. Impairment of this function, because of liver insufficiency, can be partially restored by medical management but OLT remains the ultimate therapeutic treatment. Because not always indicated or available, other alternatives are proposed such as LCT. Compared to OLT, this procedure is less invasive, less expensive, and fully reversible. More than 50 patients have thus far benefited of this technique and are reviewed here. Indications were multiple including inborn errors of metabolism, FHF, acute on chronic diseases, and decompensated end-stage cirrhosis. Documented results were encouraging, especially for metabolic disorders, with medium-term efficacy up to two yr. Related complications were exceptional. On this basis, LCT has entered its phase of clinical application and current indications and protocols are detailed. Ongoing lines of research are discussed, including cell quality, stem cell field, and rejection prevention. Further improvement of the procedure is therefore expected and should lead to broader applications of LCT.
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Affiliation(s)
- F Smets
- Université Catholique de Louvain et Cliniques Universitaires Saint-Luc, Laboratory of Pediatric Hepatology and Cell Therapy, Brussels, Belgium.
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28
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Horne PH, Koester MA, Jayashankar K, Lunsford KE, Dziema HL, Bumgardner GL. Disparate Primary and Secondary Allospecific CD8+ T Cell Cytolytic Effector Function in the Presence or Absence of Host CD4+ T Cells. THE JOURNAL OF IMMUNOLOGY 2007; 179:80-8. [PMID: 17579024 DOI: 10.4049/jimmunol.179.1.80] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
The role of CD4+ T cells in promoting CD8+ T cell effector activity in response to transplant Ags in vivo has not been reported. We used a hepatocellular allograft model known to initiate both CD4-dependent and CD4-independent rejection responses to investigate the contribution of CD4+ T cells to the development, function, and persistence of allospecific CD8+ T cell effectors in vivo. Complete MHC-mismatched hepatocellular allografts were transplanted into C57BL/6 (CD4-sufficient) or CD4 knockout (CD4-deficient) hosts. The development of in vivo allospecific cytotoxicity was determined by clearance of CFSE-labeled target cells. CD8+ T cell cytotoxic effector activity was enhanced in response to allogeneic hepatocellular grafts with a greater magnitude of allocytotoxicity and a prolonged persistence of CTL effector activity in CD4-sufficient hosts compared with CD4-deficient hosts. Cytolytic activity was mediated by CD8+ T cells in both recipient groups. In response to a second hepatocyte transplant, rejection kinetics were enhanced in both CD4-sufficient and CD4-deficient hepatocyte recipients. However, only CD4-sufficient hosts developed recall CTL responses with an augmented magnitude and persistence of allocytotoxicity in comparison with primary CTL responses. These studies show important functional differences between alloreactive CD8+ T cell cytolytic effectors that mature in vivo in the presence or absence of CD4+ T cells.
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Affiliation(s)
- Phillip H Horne
- Department of Surgery, Comprehensive Transplant Center, Ohio State University Medical Center, 1654 Upham Drive, Columbus, OH 43210, USA
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Stéphenne X, Vosters O, Najimi M, Beuneu C, Dung KN, Wijns W, Goldman M, Sokal EM. Tissue factor-dependent procoagulant activity of isolated human hepatocytes: relevance to liver cell transplantation. Liver Transpl 2007; 13:599-606. [PMID: 17394166 DOI: 10.1002/lt.21128] [Citation(s) in RCA: 61] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Liver cell transplantation (LCT) aims to correct inborn liver function defects by infusing metabolically active cells into the diseased liver. Further improvement in LCT might depend on the prevention of early loss of transplanted cells. As tissue factor (TF)-dependent activation of coagulation was found to contribute to a low rate of beta cell engraftment in islet transplantation, we investigated the potential procoagulant activity (PCA) of hepatocyte preparations. TF expression on hepatocyte preparations was assessed by flow cytometry, reverse-transcription polymerase chain reaction and immunofluorescence. PCA depending on TF was evaluated in human plasma and in whole blood systems. Coagulation parameters were followed by routine techniques in a LCT recipient Crigler-Najjar patient. We determined that hepatocytes express soluble and membrane-bound forms of TF. We showed that hepatocytes exert a TF-dependent PCA. In parallel, delayed increase in D-dimer levels was observed following the hepatocyte infusions in the Crigler-Najjar patient. Furthermore, in vitro experiments demonstrated that TF-dependent PCA of hepatocytes is inhibited by N-acetyl-L-cysteine. In conclusion, hepatocytes exert TF-dependent PCA, which may contribute to early loss of infused cells. Addition of N-acetyl-L-cysteine to the suspensions of hepatocytes might be beneficial in LCT by inhibiting activation of coagulation.
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Affiliation(s)
- Xavier Stéphenne
- Laboratory of Paediatric Hepatology and Cell Therapy, Unité Pé diatrie (PEDI), Université Catholique de Louvain, Brussels, Belgium
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Mashalova EV, Guha C, Roy-Chowdhury N, Liu L, Fox IJ, Roy-Chowdhury J, Horwitz MS. Prevention of hepatocyte allograft rejection in rats by transferring adenoviral early region 3 genes into donor cells. Hepatology 2007; 45:755-66. [PMID: 17326202 DOI: 10.1002/hep.21525] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
UNLABELLED Hepatocyte transplantation is being evaluated as an alternative to liver transplantation for metabolic support during liver failure and for definitive treatment of inherited liver diseases. However, as with liver transplantation, transplantation of allogeneic hepatocytes requires prolonged immunosuppression with its associated untoward effects. Therefore, we explored strategies for the genetic modification of donor hepatocytes that could eliminate allograft rejection, obviating the need for immunosuppression. Products of early region 3 (AdE3) of the adenoviral genome are known to protect infected cells from immune recognition and destruction. In the present study we showed that immortalized rat hepatocytes that had been stably transduced with AdE3 before transplantation into fully MHC-mismatched rats are protected from allograft rejection. Quantitative real-time PCR analysis showed that a similar number of engrafted AdE3-transfected hepatocytes had survived in syngeneic and allogeneic recipients. AdE3 expression did not reduce expression of MHC class I on the surfaces of donor hepatocytes. Consistent with this, the in vivo cytotoxic cell-mediated alloresponse was attenuated but not abolished in recipients of AdE3-transfected allogeneic hepatocytes. In contrast, graft survival correlated with a marked reduction in cell-surface localization of Fas receptor in the transplanted cells and inhibition of Fas-mediated apoptosis, which are related to the antiapoptotic functions of the AdE3 proteins. CONCLUSION AdE3 gene products prevent hepatocyte allograft rejection mainly by protecting the cells from the effector limb of the host immune response and could be used as a tool to facilitate allogeneic hepatocyte transplantation.
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Affiliation(s)
- Elena V Mashalova
- Department of Microbiology and Immunology, Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, NY 10461, USA
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Natural killer cell subsets in allograft rejection and tolerance. Curr Opin Organ Transplant 2007; 12:10-16. [PMID: 27792083 DOI: 10.1097/mot.0b013e3280129f2a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
PURPOSE OF REVIEW To discuss the role of natural killer cells in regulating the survival of transplanted organs. RECENT FINDINGS Natural killer cells have been found to have the dual capacity to promote rejection of transplanted organs and be required for the induction of transplantation tolerance. In murine recipients of bone marrow transplants, or in CD28 recipients of cardiac allografts, different natural killer cell subsets have been shown to promote or delay rejection, depending on their major histocompatibility complex class I specificity. In mouse models of skin and islet allograft acceptance mediated by costimulation-targeting therapies, the presence of natural killer cells was found to be essential for long-term graft acceptance, perhaps due to their ability to eliminate donor or recipient immune cells. SUMMARY Natural killer cells can either accelerate or avert rejection in a manner that is influenced by both donor-recipient major histocompatibility complex disparity as well as the milieu created by costimulation-targeting therapies. In clinical settings, alloreactivity by defined natural killer cell subsets may be important in achieving tolerance, and the outcome of natural killer cell activity may be influenced by specific immunosuppressive regimens.
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Veale JL, Liang LW, Zhang Q, Gjertson DW, Du Z, Bloomquist EW, Jia J, Qian L, Wilkinson AH, Danovitch GM, Pham PTT, Rosenthal JT, Lassman CR, Braun J, Reed EF, Gritsch HA. Noninvasive Diagnosis of Cellular and Antibody-Mediated Rejection by Perforin and Granzyme B in Renal Allografts. Hum Immunol 2006; 67:777-86. [PMID: 17055354 DOI: 10.1016/j.humimm.2006.07.006] [Citation(s) in RCA: 46] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2006] [Revised: 06/30/2006] [Accepted: 07/11/2006] [Indexed: 11/24/2022]
Abstract
A major milestone in transplantation would be the use of biomarkers to monitor rejection. We examined the association between perforin and granzyme-B gene expression detected in the peripheral blood of renal allograft recipients with cellular and antibody-mediated rejection. Furthermore, we judged the appropriateness of assigning negative rejection statuses to persons without a biopsy whose grafts were functioning well clinically. Of the 46 patients who completed the study, recipients with cellular rejection had higher perforin and granzyme-B levels compared with nonrejectors (p = 0.006). Interestingly, recipients with antibody-mediated rejection also had higher perforin and granzyme-B levels compared with nonrejectors (p = 0.04). Patients with high levels of granzyme B had a probability of rejecting that was 26.7 times greater than those patients with low levels of granzyme B. Perforin and granzyme B had sensitivities of 50% and specificities of 95% in predicting rejection (cutoff value = 140). Assigning negative rejection statuses to recipients without a biopsy whose grafts were functioning well did not have a major effect on the direction or significance of covariate values. This study suggests that perforin and granzyme-B gene expressions in peripheral blood are accurate in detecting both cellular and antibody-mediated rejection.
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Affiliation(s)
- Jeffrey L Veale
- Department of Urology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA.
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Lunsford KE, Horne PH, Koester MA, Eiring AM, Walker JP, Dziema HL, Bumgardner GL. Activation and maturation of alloreactive CD4-independent, CD8 cytolytic T cells. Am J Transplant 2006; 6:2268-81. [PMID: 16889609 DOI: 10.1111/j.1600-6143.2006.01479.x] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The goal of this study was to determine the in vivo conditions that promote activation of the (CD4-independent) CD8+ T cell-mediated rejection pathway. We have previously noted that hepatocellular but not islet allografts readily activate this rejection pathway. In the current study, we utilized these two cell transplant models to investigate whether differences in host cell recruitment to the graft site, expression of T-cell activation markers by CD8+ graft infiltrating cells (GICs), and/or development of delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte cell-mediated effector functions could account for the differential transplant outcomes. The collective results demonstrate that recruitment of CD8+ T cells to the site of transplant, CD103 or CD69 expression on CD8+ GICs, and activation of alloreactive DTH responses are insufficient to initiate CD4-independent, CD8-dependent transplant rejection. Instead, rejection by alloreactive (CD4-independent) CD8+ T cells correlated with expression of CD25, CD154 and CD43 by CD8+ GICs, in vitro alloproliferation by recipient CD8+ T cells, and the development of in vivo allospecific cytolytic effector function. These results suggest that tissue-derived factors influence the activation and maturation of (CD4-independent) CD8+ T cells into cytolytic effectors, which correlates with transplant rejection.
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Affiliation(s)
- K E Lunsford
- Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, OH, USA
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Lunsford KE, Koester MA, Eiring AM, Horne PH, Gao D, Bumgardner GL. Targeting LFA-1 and cd154 suppresses the in vivo activation and development of cytolytic (cd4-Independent) CD8+ T cells. THE JOURNAL OF IMMUNOLOGY 2006; 175:7855-66. [PMID: 16339521 DOI: 10.4049/jimmunol.175.12.7855] [Citation(s) in RCA: 30] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Short-term immunotherapy targeting both LFA-1 and CD40/CD154 costimulation produces synergistic effects such that long-term allograft survival is achieved in the majority of recipients. This immunotherapeutic strategy has been reported to induce the development of CD4+ regulatory T cells. In the current study, the mechanisms by which this immunotherapeutic strategy prevents CD8+ T cell-dependent hepatocyte rejection in CD4 knockout mice were examined. Combined blockade of LFA-1 and CD40/CD154 costimulation did not influence the overall number or composition of inflammatory cells infiltrating the liver where transplanted hepatocytes engraft. Expression of T cell activation markers CD43, CD69, and adhesion molecule CD103 by liver-infiltrating cells was suppressed in treated mice with long-term hepatocellular allograft survival compared to liver-infiltrating cells of untreated rejector mice. Short-term immunotherapy with anti-LFA-1 and anti-CD154 mAb also abrogated the in vivo development of alloreactive CD8+ cytotoxic T cell effectors. Treated mice with long-term hepatocyte allograft survival did not reject hepatocellular allografts despite adoptive transfer of naive CD8+ T cells. Unexpectedly, treated mice with long-term hepatocellular allograft survival demonstrated prominent donor-reactive delayed-type hypersensitivity responses, which were increased in comparison to untreated hepatocyte rejectors. Collectively, these findings support the conclusion that short-term immunotherapy with anti-LFA-1 and anti-CD154 mAbs induces long-term survival of hepatocellular allografts by interfering with CD8+ T cell activation and development of CTL effector function. In addition, these recipients with long-term hepatocellular allograft acceptance show evidence of immunoregulation which is not due to immune deletion or ignorance and is associated with early development of a novel CD8+CD25high cell population in the liver.
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Affiliation(s)
- Keri E Lunsford
- Integrated Biomedical Science Graduate Program, The Ohio State University, Columbus, OH 43210, USA
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Horne PH, Lunsford KE, Eiring AM, Wang Y, Gao D, Bumgardner GL. CD4+ T-cell-dependent immune damage of liver parenchymal cells is mediated by alloantibody. Transplantation 2005; 80:514-21. [PMID: 16123727 DOI: 10.1097/01.tp.0000168342.57948.68] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
BACKGROUND Allogeneic hepatocytes initiate both CD4- and CD8-dependent rejection responses. The current studies address the hypothesis that acute damage of allogeneic liver parenchymal cells by the CD4-dependent pathway is alloantibody-mediated and examines immune conditions which promote activation of this pathway. METHODS The role of alloantibody in CD4-dependent hepatocyte rejection was evaluated by assessing hepatocyte (FVB/N, H-2q) survival in CD8-depleted B-cell knockout (KO) (H-2b) recipients and by monitoring hepatocyte survival in C57BL/6.SCID (H-2b) recipients transfused with donor-reactive alloantibody. The development of donor-reactive alloantibody in C57BL/6 (H-2b), CD8-depleted C57BL/6, CD8 KO (H-2b), IFN-gamma KO (H-2b), perforin KO (H-2b), and FasL mutant gld/gld (H-2b) hepatocyte recipients was assessed. RESULTS Hepatocyte rejection in B-cell KO mice was significantly delayed by CD8+ T-cell depletion (median survival time [MST], 35 days) when compared to untreated (MST, 8 days) and CD4-depleted (MST, 10 days) recipient mice. Transfusion of donor-reactive alloantibody into SCID recipients with functional hepatocellular allografts was sufficient to precipitate rejection in a dose-dependent fashion. Donor-reactive alloantibody was minimal in the serum of C57BL/6 hepatocyte recipients, but was produced in significant quantities in hepatocyte recipients genetically deficient in or depleted of CD8+ T cells and in recipients with impaired cytotoxic effector mechanisms. In addition, recipients with defects in Th1 immunity, such as IFN-gamma KO recipients, also produced readily detectable alloantibody. CONCLUSIONS Collectively, these data support the hypothesis that acute immune damage of allogeneic hepatocytes by the CD4-dependent pathway is mediated by alloantibody and that this pathway is favored when Th1- or cell-mediated cytotoxic effector immune mechanisms are impaired.
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Affiliation(s)
- Phillip H Horne
- Integrated Biomedical Science Graduate Program, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210-1250, USA
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Abstract
Glucocorticoids are potent immunosuppressive drugs that are generally withheld from cancer patients receiving immunotherapy. We sought to test the hypothesis that glucocorticoids might interfere with the function of cells after adoptive transfer. We gave dexamethasone, a potent synthetic glucocorticoid, to B16 melanoma-bearing mice receiving the adoptive cell transfer (ACT) of pmel-1 T-cell receptor transgenic CD8+ cells. Dexamethasone caused a profound lymphodepletion but, surprisingly, did not alter the antitumor efficacy of ACT-based regimens whether given before, during, or after ACT. Although dexamethasone radically decreased the number of native CD8+ splenocytes in recipient mice, it did not affect the numbers of CD8+ pmel-1 cells derived from ACT in these mice. In vitro proliferation assays revealed acute inhibition of naive pmel-1 CD8+ cells by dexamethasone without significant effect on activated cells. In vitro interferon (IFN)-gamma release from activated pmel-1 CD8+ cells showed partial inhibition by dexamethasone, but this effect was relatively minor when compared with the near-complete inhibition of naive cells. Thus, glucocorticoids had a profound inhibitory effect on naive CD8+ T cells but had little impact on the proliferation and function of activated CD8+ pmel-1 T cells. Finally, because glucocorticoids had no effect on tumor regression in this model, it may be possible to use glucocorticoids in some patients receiving ACT-based immunotherapy regimens.
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Affiliation(s)
- Christian S. Hinrichs
- From the National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, Maryland
| | - Douglas C. Palmer
- From the National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, Maryland
| | - Steven A. Rosenberg
- From the National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, Maryland
| | - Nicholas P. Restifo
- From the National Cancer Institute (NCI), National Institutes of Health (NIH), Bethesda, Maryland
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Fair JH, Cairns BA, Lapaglia MA, Caballero M, Pleasant WA, Hatada S, Kim HS, Gui T, Pevny L, Meyer AA, Stafford DW, Smithies O, Frelinger JA. Correction of factor IX deficiency in mice by embryonic stem cells differentiated in vitro. Proc Natl Acad Sci U S A 2005; 102:2958-63. [PMID: 15699326 PMCID: PMC548326 DOI: 10.1073/pnas.0409840102] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Murine embryonic stem (ES) cells are pluripotent, but significant functional engraftment does not occur when they are introduced into the liver. However, here we demonstrate that functional liver engraftment does occur if the ES cells (from strain 129 mice) are first differentiated in vitro for 7 days in the presence of FGF. Strikingly, when these differentiated cells, termed putative endodermal precursors (PEPs), were injected into their livers, two of six C57BL/6 and four of eight BALB/c factor IX (F-IX)-deficient mice survived for >7 days, even though the recipients were of a different strain and, in the case of the BALB/c recipients, had a complete MHC mismatch. F-IX was detected in all six of the PEP-injected survivors. Two mice subsequently died of causes unrelated to F-IX; the others survived until death at 38 or 115 days after the transplantation. No uninjected control F-IX-deficient mice survived for >7 days. Large confluent regions of sinusoidal PEP engraftment were demonstrated by immunofluorescence in the long-term BALB/c survivors. The PEP engraftment was not associated with detectable cell fusion, and the transplantation was accompanied with only a low incidence of teratoma formation.
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Affiliation(s)
- Jeffrey H Fair
- Department of Surgery, University of North Carolina, Chapel Hill, NC 27599, USA.
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Lunsford KE, Gao D, Eiring AM, Wang Y, Frankel WL, Bumgardner GL. Evidence for Tissue-Directed Immune Responses: Analysis of CD4- and CD8-Dependent Alloimmunity. Transplantation 2004; 78:1125-33. [PMID: 15502708 DOI: 10.1097/01.tp.0000138098.19429.99] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022]
Abstract
BACKGROUND Transplant rejection has generally been considered a CD4 T-cell-dependent immune process. CD4-independent, CD8 T-cell rejection pathways have recently gained attention because of their relative resistance to immunosuppression. In the current study, the role of the allograft tissue in activation of these distinct pathways was examined by comparing host-immune responses with allogeneic pancreatic islets or hepatocytes transplanted across the same genetic disparity. METHODS To compare activation of CD4-dependent versus CD8-dependent alloimmunity, islets or hepatocytes retrieved from FVB/N (H-2) mice were transplanted into CD8 or CD4 T-cell-reconstituted severe combined immunodeficiency mice, CD4 or CD8 knockout (KO) mice, and anti-CD4 monoclonal antibody (mAb) or anti-CD8 mAb treated C57BL/6 mice (all H-2). The ability to immunomodulate CD4-dependent allograft rejection (in CD8 KO mice) was examined in the context of several mechanistically distinct immunotherapeutic strategies, including anti-CD4 mAb, donor-specific transfusion and anti-CD154 mAb, and anti-lymphocyte function-associated antigen-1 mAb. RESULTS The studies demonstrate that, whereas hepatocytes evoke alloreactive CD4-dependent and (CD4-independent) CD8 T-cell immune responses, allogeneic islets only activate CD4-dependent immune pathways. CD4-dependent host-immune responses initiated by pancreatic islet allografts were readily suppressed by a variety of short-term immunotherapies, whereas hepatocyte-initiated CD4-dependent alloimmune responses were not. CONCLUSIONS These results demonstrate that immune characteristics of the specific allograft tissue uniquely influence the pattern of host immune responses such that the propensity to activate CD4- or CD8-dependent alloimmune responses can be distinguished. Furthermore, CD4-dependent immune responses activated by different tissues from the same donor strain are distinguished by their susceptibility to specific immunotherapy.
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Affiliation(s)
- Keri E Lunsford
- Integrated Biomedical Science Graduate Program, College of Medicine and Public Health, The Ohio State University, Columbus, OH 43210, USA
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Gao D, Lunsford KE, Eiring AM, Bumgardner GL. Critical role for CD8 T cells in allograft acceptance induced by DST and CD40/CD154 costimulatory blockade. Am J Transplant 2004; 4:1061-70. [PMID: 15196062 DOI: 10.1111/j.1600-6143.2004.00490.x] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.
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Affiliation(s)
- Donghong Gao
- Department of Surgery, The Ohio State University Medical Center, Columbus, OH, USA
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Wang Y, Gao D, Lunsford KE, Frankel WL, Bumgardner GL. Targeting LFA-1 synergizes with CD40/CD40L blockade for suppression of both CD4-dependent and CD8-dependent rejection. Am J Transplant 2003; 3:1251-8. [PMID: 14510698 DOI: 10.1046/j.1600-6143.2003.00201.x] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Allogeneic hepatocytes elicit CD4-dependent and (CD4-independent) CD8+ T-cell-initiated graft rejection. The (CD4-independent) CD8+ T-cell pathway is resistant to immunosuppressive strategies that readily and indefinitely suppress CD4+ T-cell-dependent rejection responses. Consequently, successful immunoregulation of hepatocyte-initiated immune responses requires a strategy which regulates both CD4-dependent and CD8-dependent rejection responses. Interference with CD40/CD40 ligand (CD40L) costimulation only transiently suppresses CD4- and CD8-dependent hepatocyte rejection. Interference with CD28/B7 costimulation transiently suppresses CD4-dependent hepatocyte rejection, but is ineffective for suppression of CD8-dependent hepatocyte rejection. To date, hepatocyte survival > 60 days post-transplant has not been achieved by any immunotherapeutic strategy. In the current study, we evaluated a novel immunosuppressive strategy which targets both LFA-1 and CD40L-mediated signals. Targeting LFA-1 suppressed (CD4-independent) CD8+ T-cell-initiated hepatocyte rejection such that allogeneic hepatocyte survival > 60 days was achieved in 70% of CD4 KO mice. Targeting both LFA-1-mediated signals and CD40/CD40L costimulation resulted in synergistic effects, such that hepatocellular survival > 60 days was achieved in 100% of C57BL/6 mice (which have both CD4- and CD8-dependent T-cell pathways available).
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Affiliation(s)
- Yue Wang
- Department of Surgery, The Ohio State University Medical Center, Columbus, OH, USA
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Abstract
Liver repopulation with transplanted cells offers unique opportunities for treating a variety of diseases and for studies of fundamental mechanisms in cell biology. Our understanding of the basis of liver repopulation has come from studies of transplanted cells in animal models. A variety of studies established that transplanted hepatocytes as well as stem/progenitor cells survive, engraft, and function in the liver. Transplanted cells survive life-long, although cells do not proliferate in the normal liver. On the other hand, the liver is repopulated extensively when diseases or other injuries afflict native hepatocytes but spare transplanted cells. The identification of ways to repopulate the liver with transplanted cells has greatly reinvigorated the field of liver cell therapy. The confluence of insights in stem/progenitor cells, transplantation immunology, cryobiology, and liver repopulation in specific models of human diseases indicates that the field of liver cell therapy will begin to reap the promised fruit in the near future.
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Affiliation(s)
- Sanjeev Gupta
- Marion Bessin Liver Research Center, Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
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Bumgardner GL, Gao D, Li J, Bickerstaff A, Orosz CG. MHC-identical heart and hepatocyte allografts evoke opposite immune responses within the same host. Transplantation 2002; 74:855-64. [PMID: 12364867 DOI: 10.1097/00007890-200209270-00020] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
BACKGROUND Purified allogeneic hepatocytes are highly antigenic and elicit immune responses that are not easily controlled. However, it is not clear whether hepatocytes are not capable of protective immune mechanisms or whether they are not to protection by immune mechanisms that permit long-term survival of other allografts. The purpose of the current study was to determine whether donor-matched allogeneic hepatocytes are protected from rejection in mice that have been induced to accept heart allografts. METHODS Transient treatment with anti-CD4 monoclonal antibody (mAb) or gallium nitrate (GN) was used to induce acceptance of heterotopic FVB/N (H-2(q)) heart allografts by C57BL/6 (H-2(b)) mice. Transgenic hA1AT-FVB/N hepatocytes were sequentially transplanted into C57BL/6 mice that had accepted FVB/N heart allografts more than 60 days (heart acceptor mice), CD8 depleted C57BL/6 heart acceptor mice, or B-cell knockout (BCKO, H-2(b)) heart acceptor mice. Hepatocyte survival was determined by the detection of secreted transgenic product hA1AT by enzyme-linked immunosorbent assay (ELISA). RESULTS FVB/N hepatocytes were rejected by day 10-14 posttransplant, while FVB/N heart allografts continued to function in C57BL/6, BCKO, and CD8 depleted heart acceptor mice. When FVB/N hepatocytes and heart allografts were transplanted into C57BL/6 or BCKO mice under short-term cover of anti-CD4 mAb or GN, hepatocyte rejection occurred by day 10 posttransplant, while most heart allografts survived for more than 60 days. CONCLUSIONS Hepatocyte rejection does not appear to interfere with the of mechanisms that permit heart allograft acceptance. However, immune responses to allogeneic hepatocytes are not to regulation by mechanisms induced in heart acceptor mice. The simultaneous rejection of FVB/N allogeneic hepatocytes and continued acceptance of FVB/N-matched heart allografts is independent of host CD8+ T cells and humoral immunity.
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Affiliation(s)
- Ginny L Bumgardner
- The Ohio State University Medical Center, Department of Surgery, Columbus 43210-1250, USA.
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Feinberg MB, Silvestri G. T(S) cells and immune tolerance induction: a regulatory renaissance? Nat Immunol 2002; 3:215-7. [PMID: 11875459 DOI: 10.1038/ni0302-215] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
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