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1
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Luo X, Zhang J, Zou S, Wang X, Chen G, Li Z, Li K, Wang M, Chen Z, Ming C, Zhu X, Gong N. Bone Fragment Co-transplantation Alongside Bone Marrow Aspirate Infusion Protects Kidney Transplant Recipients. Front Immunol 2021; 12:630710. [PMID: 33643315 PMCID: PMC7904687 DOI: 10.3389/fimmu.2021.630710] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2020] [Accepted: 01/22/2021] [Indexed: 12/20/2022] Open
Abstract
Integration of non-vascularized bone grafting and bone marrow aspirate infusion in transplantation may provide clinical benefit. Here we have incorporated bone fragment co-transplantation and bone marrow aspirate infusion (BF-BM) into living kidney transplantation (LKT). Twenty LKT recipients receiving bone fragments and bone marrow aspirates donated from their corresponding donors were enrolled into a retrospective study. A contemporaneous control group was formed of 38 out of 128 conventional LKT recipients, selected using propensity score matching by a 1:2 Greedy algorithm. Ultrasonography, contrast-enhanced ultrasonography (US/CEUS) and SPECT/CT showed that the co-transplanted bone fragments remained viable for 6 months, subsequently shrank, and finally degenerated 10 months post-transplantation. BF-BM resulted in earlier kidney recovery and more robust long-term kidney function. Throughout 5 years of follow-up, BF-BM had regulatory effects on dendritic cells (DCs), T helper (Th1/Th2) cells and regulatory T cells (Tregs). Both alloantigen-specific lymphocyte proliferation and panel reactive antibody levels were negative in all recipients with or without BF-BM. In addition, the BF-BM group experienced few complications during the 5-year follow-up (as did the donors)—this was not different from the controls. In conclusion, BF-BM is safe and benefits recipients by protecting the kidney and regulating the immune response.
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Affiliation(s)
- Xianzhang Luo
- Key Laboratory of the National Health Commission, Institute of Organ Transplantation, Tongji Medical College, The Ministry of Education and Chinese Academy of Medical Sciences, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China.,Key Laboratory for Biorheological Science and Technology of Ministry of Education, Chongqing University Cancer Hospital, Chongqing, China
| | - Ji Zhang
- Key Laboratory of the National Health Commission, Institute of Organ Transplantation, Tongji Medical College, The Ministry of Education and Chinese Academy of Medical Sciences, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Sijuan Zou
- Department of Nuclear Medicine, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Xinqiang Wang
- Key Laboratory of the National Health Commission, Institute of Organ Transplantation, Tongji Medical College, The Ministry of Education and Chinese Academy of Medical Sciences, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Gen Chen
- Department of Radiology, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Zhen Li
- Department of Radiology, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Kaiyan Li
- Department of Medical Ultrasound, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Mengqing Wang
- Key Laboratory of the National Health Commission, Institute of Organ Transplantation, Tongji Medical College, The Ministry of Education and Chinese Academy of Medical Sciences, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Zhishui Chen
- Key Laboratory of the National Health Commission, Institute of Organ Transplantation, Tongji Medical College, The Ministry of Education and Chinese Academy of Medical Sciences, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Changshen Ming
- Key Laboratory of the National Health Commission, Institute of Organ Transplantation, Tongji Medical College, The Ministry of Education and Chinese Academy of Medical Sciences, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Xiaohua Zhu
- Department of Nuclear Medicine, Tongji Medical College, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
| | - Nianqiao Gong
- Key Laboratory of the National Health Commission, Institute of Organ Transplantation, Tongji Medical College, The Ministry of Education and Chinese Academy of Medical Sciences, Tongji Hospital, Huazhong University of Science and Technology, Wuhan, China
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2
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El-Sherbiny YM, El-Jawhari JJ, Moseley TA, McGonagle D, Jones E. T cell immunomodulation by clinically used allogeneic human cancellous bone fragments: a potential novel immunotherapy tool. Sci Rep 2018; 8:13535. [PMID: 30201960 PMCID: PMC6131386 DOI: 10.1038/s41598-018-31979-1] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2017] [Accepted: 08/17/2018] [Indexed: 01/09/2023] Open
Abstract
Multipotential stromal cells (MSCs) demonstrate strong immunomodulation capabilities following culture expansion. We have previously demonstrated that human cancellous bone fragments (CBFs) clinically used as viable allografts for spinal fusion have resident MSCs that exhibit T cell immunomodulation after monolayer expansion. This study investigated the immunomodulatory ability of these CBFs without MSC culture-expansion. CD4 positive T cells were induced to proliferate using CD3/CD28 stimulation and added to CBFs at different ratios of T cells per gram of CBF. A dose-dependent suppressive effect on T cell proliferation was evident and correlated with increased culture supernatant levels of TGF-ß1, but not PGE2. CBF-driven immunosuppression was reduced in co-cultures with TGF-ß neutralising antibodies and was higher in cell contact compared to non-contact cultures. CBF gene expression profile identified vascular cell adhesion molecule-1, bone marrow stromal antigen 2/CD317 and other interferon signalling pathway members as potential immunomodulatory mediators. The CD317 molecule was detected on the surface of CBF-resident cells confirming the gene expression data. Taken together, these data demonstrate that human clinically used CBFs are inherently immunomodulatory and suggest that these viable allografts may be used to deliver therapeutic immunomodulation for immune-related diseases.
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Affiliation(s)
- Yasser M El-Sherbiny
- National Institute of Health Research Leeds Biomedical Research Centre, Leeds Teaching Hospitals NHS Trust, Leeds, UK.,Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt.,Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK
| | - Jehan J El-Jawhari
- National Institute of Health Research Leeds Biomedical Research Centre, Leeds Teaching Hospitals NHS Trust, Leeds, UK.,Clinical Pathology Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt.,Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK
| | | | - Dennis McGonagle
- National Institute of Health Research Leeds Biomedical Research Centre, Leeds Teaching Hospitals NHS Trust, Leeds, UK.,Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK
| | - Elena Jones
- National Institute of Health Research Leeds Biomedical Research Centre, Leeds Teaching Hospitals NHS Trust, Leeds, UK. .,Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK.
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Lin CH, Wang YL, Anggelia MR, Chuang WY, Cheng HY, Mao Q, Zelken JA, Lin CH, Zheng XX, Lee WPA, Brandacher G. Combined Anti-CD154/CTLA4Ig Costimulation Blockade-Based Therapy Induces Donor-Specific Tolerance to Vascularized Osteomyocutaneous Allografts. Am J Transplant 2016; 16:2030-2041. [PMID: 26914847 DOI: 10.1111/ajt.13694] [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: 08/13/2015] [Revised: 12/06/2015] [Accepted: 12/20/2015] [Indexed: 01/25/2023]
Abstract
Tolerance induction by means of costimulation blockade has been successfully applied in solid organ transplantation; however, its efficacy in vascularized composite allotransplantation, containing a vascularized bone marrow component and thus a constant source of donor-derived stem cells, remains poorly explored. In this study, osteomyocutaneous allografts (alloOMCs) from Balb/c (H2(d) ) mice were transplanted into C57BL/6 (H2(b) ) recipients. Immunosuppression consisted of 1 mg anti-CD154 on day 0, 0.5 mg CTLA4Ig on day 2 and rapamycin (RPM; 3 mg/kg per day from days 0-7, then every other day for 3 weeks). Long-term allograft survival, donor-specific tolerance and donor-recipient cell trafficking were evaluated. Treatment with costimulation blockade plus RPM resulted in long-term graft survival (>120 days) of alloOMC in 12 of 15 recipients compared with untreated controls (median survival time [MST] ≈10.2 ± 0.8 days), RPM alone (MST ≈33 ± 5.5 days) and costimulation blockade alone (MST ≈45.8 ± 7.1 days). Donor-specific hyporesponsiveness in recipients with viable grafts was demonstrated in vitro. Evidence of donor-specific tolerance was further assessed in vivo by secondary donor-specific skin graft survival and third-party graft rejection. A significant increase of Foxp3(+) regulatory T cells was evident in tolerant animals. Donor cells populated peripheral blood, thymus, and both donor and recipient bone marrow. Consequently, combined anti-CD154/CTLA4Ig costimulation blockade-based therapy induces donor-specific tolerance in a stringent murine alloOMC transplant model.
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Affiliation(s)
- C H Lin
- Center for Vascularized Composite Allotransplantation, Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, and School of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Y L Wang
- Center for Vascularized Composite Allotransplantation, Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, and School of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - M R Anggelia
- Center for Vascularized Composite Allotransplantation, Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, and School of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - W Y Chuang
- Department of Pathology, Chang Gung Memorial Hospital, Chang Gung Medical College and Chang Gung University, Taoyuan, Taiwan
| | - H Y Cheng
- Center for Vascularized Composite Allotransplantation, Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, and School of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - Q Mao
- Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD
| | - J A Zelken
- Center for Vascularized Composite Allotransplantation, Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, and School of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - C H Lin
- Center for Vascularized Composite Allotransplantation, Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, and School of Medicine, Chang Gung University, Taoyuan, Taiwan
| | - X X Zheng
- Research Center of Translational Medicine, Shanghai East Hospital, Tongji University, Shanghai, China
| | - W P A Lee
- Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD
| | - G Brandacher
- Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD
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Vascularized Skin/Bone Transplantation Model. Plast Reconstr Surg 2015. [DOI: 10.1007/978-1-4471-6335-0_25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/24/2022]
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Barth RN, Rodriguez ED, Mundinger GS, Nam AJ, Ha JS, Hui-Chou H, Jones LS, Panda A, Shipley ST, Drachenberg CB, Kukuruga D, Bartlett ST. Vascularized bone marrow-based immunosuppression inhibits rejection of vascularized composite allografts in nonhuman primates. Am J Transplant 2011; 11:1407-16. [PMID: 21668624 DOI: 10.1111/j.1600-6143.2011.03551.x] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
Vascularized composite allograft (VCA) transplantation (also referred to as composite tissue allotransplantation) has demonstrated clinical success in cases of hand, arm and face transplantation despite prior belief that skin provides an insurmountable barrier to allograft rejection. These overall good outcomes are facilitated by substantial immunosuppressive requirements in otherwise healthy patients, yet still demonstrate frequent rejection episodes. We developed a nonhuman primate model of facial segment allotransplantation to elucidate the unique pathophysiology and immunosuppressive requirements of VCA with addition of concomitant vascularized bone marrow (VBM). Heterotopically transplanted facial segment VCA with VBM treated only with tacrolimus and mycophenolate mofetil (MMF) demonstrated prolonged rejection-free survival, compared to VCA without VBM that demonstrated early rejection episodes and graft loss. While VCA with VBM demonstrated sporadic macrochimerism, acute and chronic rejection and graft loss occurred after discontinuation of immunosuppression. These data support an immunomodulatory role of VBM in VCA that reduces immunosuppressive requirements while providing improved outcomes.
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Affiliation(s)
- R N Barth
- Division of Transplantation, Program for Comparative Medicine Department of Pathology Immunogenetics Laboratory, University of Maryland School of Medicine, Baltimore, MD, USA.
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6
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Wang H, Ge W, Arp J, Zassoko R, Liu W, Ichim TE, Jiang J, Jevnikar AM, Garcia B. Free Bone Graft Attenuates Acute Rejection and in Combination with Cyclosporin A Leads to Indefinite Cardiac Allograft Survival. THE JOURNAL OF IMMUNOLOGY 2009; 182:5970-81. [DOI: 10.4049/jimmunol.0801037] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
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Verbinnen B, Billiau AD, Vermeiren J, Galicia G, Bullens DMA, Boon L, Cadot P, Hens G, Dewolf-Peeters C, Van Gool SW, Ceuppens JL. Contribution of regulatory T cells and effector T cell deletion in tolerance induction by costimulation blockade. THE JOURNAL OF IMMUNOLOGY 2008; 181:1034-42. [PMID: 18606655 DOI: 10.4049/jimmunol.181.2.1034] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Blocking of costimulatory signals for T cell activation leads to tolerance in several transplantation models, but the underlying mechanisms are incompletely understood. We analyzed the involvement of regulatory T cells (Treg) and deletion of alloreactive cells in the induction and maintenance of tolerance after costimulation blockade in a mouse model of graft-vs-host reaction. Injection of splenocytes from the C57BL/6 parent strain into a sublethally irradiated F(1) offspring (C57BL/6 x C3H) induced a GVHR characterized by severe pancytopenia. Treatment with anti-CD40L mAb and CTLA4-Ig every 3 days during 3 wk after splenocyte injection prevented disease development and induced a long-lasting state of stable mixed chimerism (>120 days). In parallel, host-specific tolerance was achieved as demonstrated by lack of host-directed alloreactivity of donor-type T cells in vitro and in vivo. Chimerism and tolerance were also obtained after CD25(+) cell-depleted splenocyte transfer, showing that CD25(+) natural Treg are not essential for tolerance induction. We further show that costimulation blockade results in enhanced Treg cell activity at early time points (days 6-30) after splenocyte transfer. This was demonstrated by the presence of a high percentage of Foxp3(+) cells among donor CD4(+) cells in the spleen of treated animals, and our finding that isolated donor-type T cells at an early time point (day 30) after splenocyte transfer displayed suppressive capacity in vitro. At later time points (>30 days after splenocyte transfer), clonal deletion of host-reactive T cells was found to be a major mechanism responsible for tolerance.
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Affiliation(s)
- Bert Verbinnen
- Division of Clinical Immunology, University Hospital, Catholic University of Leuven, Leuven, Belgium
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8
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Jochum C, Beste M, Stone D, Graves SS, Storb R. Development and in vitro characterization of canine CD40-Ig. Vet Immunol Immunopathol 2008; 123:260-5. [PMID: 18387675 PMCID: PMC2467396 DOI: 10.1016/j.vetimm.2008.02.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2007] [Revised: 02/06/2008] [Accepted: 02/08/2008] [Indexed: 11/21/2022]
Abstract
We recently reported that blockade of the CD40-CD154 ligand interaction with the cross-reacting mouse anti-human CD154 antibody, 5c8, together with donor-specific transfusion led to enhanced but not completely successful engraftment in a canine model of DLA-identical marrow transplantation after 100cGy total body irradiation (TBI). In order to improve the transplantation outcomes, we sought to develop a canine-specific reagent. To that end, we fused the extracellular domain of the canine CD40 with a mouse IgG2a Fc tail and tested the immunosuppressive effectiveness of the fusion protein in mixed leukocyte reactions. The extracellular domain of canine CD40 was fused with the Fc portion of mouse IgG2a in a pcDNA3.1+vector. Dhfr-deficient CHO cells were co-transfected with the CD40-Ig vector and a dhfr-containing vector. Stable, high producing clones were selected under increasing methotrexate concentrations. The fusion protein was purified, tested in mixed leukocyte reactions, and its immunosuppressive effect compared to that of the anti-CD154 antibody 5c8. The transfected cell line produced a CD40-Ig dimer whose identity was confirmed by mass spectroscopy. The purified canine CD40-Ig blocked mixed leukocyte reactions at a concentration of 1nM, which was 10 times more effective than the anti-CD154 antibody. Canine CD40-Ig is more immunosuppressive than the anti-human CD154 antibody 5c8 in canine mixed leukocyte reactions and may be more effective in vivo in a model of marrow transplantation.
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Affiliation(s)
- Christoph Jochum
- Transplantation Biology Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Mechthild Beste
- Transplantation Biology Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Diane Stone
- Transplantation Biology Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Scott S. Graves
- Transplantation Biology Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Rainer Storb
- Transplantation Biology Program, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
- Department of Medicine, University of Washington, Seattle, WA 98195
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Li Y, Ma L, Yin D, Shen J, Chong AS. Long-term control of alloreactive B cell responses by the suppression of T cell help. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2008; 180:6077-84. [PMID: 18424729 PMCID: PMC2605285 DOI: 10.4049/jimmunol.180.9.6077] [Citation(s) in RCA: 15] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/24/2023]
Abstract
Alloantibodies can play a key role in acute and chronic allograft rejection. However, relatively little is known of factors that control B cell responses following allograft tolerance induction. Using 3-83 Igi mice expressing an alloreactive BCR, we recently reported that allograft tolerance was associated with the sustained deletion of the alloreactive B cells at the mature, but not the immature, stage. We have now investigated the basis for the long-term control of alloreactive B cell responses in a non-BCR-transgenic model of C57BL/6 cardiac transplantation into BALB/c recipients treated with anti-CD154 and transfusion of donor-specific spleen cells. We demonstrate that the long-term production of alloreactive Abs by alloreactive B cells is actively regulated in tolerant BALB/c mice through the dominant suppression of T cell help. Deletion of CD25(+) cells resulted in a loss of tolerance and an acquisition of the ability to acutely reject allografts. In contrast, the restoration of alloantibody responses required both the deletion of CD25(+) cells and the reconstitution of alloreactive B cells. Collectively, these data suggest that alloreactive B cell responses in this model of tolerance are controlled by dominant suppression of T cell help as well as the deletion of alloreactive B cells in the periphery.
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Affiliation(s)
- Yijin Li
- Department of Surgery, Section of Transplantation, University of Chicago, Chicago, IL 60637, USA
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10
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Xiang Z, Ma LL, Manicassamy S, Ganesh BB, Williams P, Chari R, Chong A, Yin DP. CD4+ T cells are sufficient to elicit allograft rejection and major histocompatibility complex class I molecule is required to induce recurrent autoimmune diabetes after pancreas transplantation in mice. Transplantation 2008; 85:1205-11. [PMID: 18431243 PMCID: PMC2632575 DOI: 10.1097/tp.0b013e31816b70bf] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
BACKGROUND We characterized the role of T cell subsets and major histocompatibility complex molecules in allograft rejection and recurrence of autoimmune diabetes. METHODS Adoptive cell transfer and vascularized segmental pancreas transplantation were performed in mice. RESULTS In an alloimmune response model, transfer of nondiabetic CD4, but not CD8 T cells, elicited pancreas allograft rejection in streptozotocin (STZ)-induced diabetic NOD/scid mice. Pancreas allografts were acutely rejected in STZ-induced diabetic NOD/beta2m mice (confirmed the absence of major histocompatibility complex [MHC] class I and CD8 T cells) and permanently accepted in NOD/CIIT mice (confirmed the absence of MHC class II and CD4 T cells). The results suggest that rejection of pancreas allograft is CD4-dependent and MHC class I-independent. In the autoimmune diabetes model, whole spleen cells obtained from diabetic NOD mice induced autoimmune diabetes in NOD/scid and NOD/CIIT mice, but the onset of diabetes was delayed in NOD/beta2m mice. However, the purified diabetic T cells failed to elicit autoimmune diabetes in NOD/beta2m mice. NOD/scid and NOD/CIIT pancreas grafts were acutely destroyed whereas four of six NOD/beta2m pancreas grafts were permanently accepted in autoimmune diabetic NOD mice. CONCLUSION CD4 T cells are sufficient for the induction of allograft rejection, and MHC class I molecule is required to induce recurrent autoimmune diabetes after pancreas transplantation in mice.
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Affiliation(s)
- Zhidan Xiang
- Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA
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11
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Ma L, Xiang Z, Sherrill TP, Wang L, Blackwell TS, Williams P, Chong A, Chari R, Yin DP. Bioluminescence imaging visualizes activation of nuclear factor-kappaB in mouse cardiac transplantation. Transplantation 2008; 85:903-10. [PMID: 18360274 PMCID: PMC2632578 DOI: 10.1097/tp.0b013e318166cde1] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
BACKGROUND The purpose of the current study was to evaluate the role of bioluminescence imaging (BLI) in the determination of nuclear factor (NF)-kappaB activation in cardiac allograft rejection and ischemia-reperfusion injury. METHODS To visualize NF-kappaB activation, luciferase transgenic mice under the control of a mouse NF-kappaB promoter (NF-kappaB-Luc) were used as donors or recipients of cardiac grafts. Alternatively, NF-kappaB-Luc spleen cells were adoptively transferred into Rag2 -/- mice with or without cardiac allografts. BLI was performed posttransplantation to detect luciferase activity that represents NF-kappaB activation. RESULTS The results show that luciferase activity was significantly increased in the cardiac allografts when NF-kappaB-Luc mice were used as recipients as well as donors. Luciferase activity was also elevated in the wild-type cardiac allografts in Rag2 -/- mice that were transferred with NF-kappaB-Luc spleen cells. CD154 monoclonal antibody (mAb) therapy inhibited luciferase activity and induced long-term survival of cardiac allografts. toll-like receptor-9 ligand, CpG DNA, enhanced luciferase activity and abrogated tolerance induction by CD154 mAb. Luciferase activity was also increased in ischemia-reperfusion injury of the cardiac grafts. CONCLUSION BLI using Luc-NF-kappaB mice is a noninvasive approach to visualize the activation of NF-kappaB signaling in mouse cardiac allograft rejection and ischemia-reperfusion injury. CD154 mAb can inhibit NF-kappaB activation, which is reversed by toll-like receptor engagement.
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Affiliation(s)
- Lianli Ma
- Department of Surgery, Division of Hepatobiliary Surgery and Liver Transplantation, Vanderbilt University Medical Center, Nashville, TN 37232, USA
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Long-Term Limb Allograft Survival Using a Short Course of Anti-CD45RB Monoclonal Antibody, LF 15-0195, and Rapamycin in a Mouse Model. Transplantation 2007; 84:1636-43. [DOI: 10.1097/01.tp.0000290277.23186.ad] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023]
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Le Blanc K, Rasmusson I, Götherström C, Seidel C, Sundberg B, Sundin M, Rosendahl K, Tammik C, Ringdén O. Mesenchymal stem cells inhibit the expression of CD25 (interleukin-2 receptor) and CD38 on phytohaemagglutinin-activated lymphocytes. Scand J Immunol 2004; 60:307-15. [PMID: 15320889 DOI: 10.1111/j.0300-9475.2004.01483.x] [Citation(s) in RCA: 252] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Mesenchymal stem cells (MSC) are immunomodulatory and inhibit lymphocyte proliferation. We studied surface expression of lymphocyte activation markers and secreted cytokines, when lymphocytes were activated in the presence of MSC. MSC suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated CD3+, CD4+ and CD8+ lymphocytes. MSC significantly reduced the expression of activation markers CD25, CD38 and CD69 on PHA-stimulated lymphocytes. Mixed lymphocyte culture (MLC) supernatants containing MSC suppressed proliferation of MLC and PHA-stimulated lymphocytes dose-dependently. MSC secrete osteoprotegerin (OPG), but not hepatocyte growth factor (HGF) or transforming growth factor-beta (TGF-beta). Stromal-cell-derived factor-1 (SDF-1) is not expressed on the cell surface. A recent report suggested that T-cell suppression by MSC is mediated by HGF and TGF-beta. MSC suppression was not restored by the addition of neutralizing antibodies against SDF-1, OPG, HGF or TGF-beta, alone or in combination. Addition of guanosine to PHA-stimulated lymphocyte cultures containing MSC did not affect lymphocyte proliferation. The immunosuppressive effects of cyclosporine and MSC did not interfere, when present in the cultures of PHA-activated lymphocytes. In summary, human MSC suppress proliferation of both CD4+ and CD8+ lymphocyte and decrease the expression of activation markers.
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Affiliation(s)
- K Le Blanc
- Division of Clinical Immunology, Huddinge University Hospital, Karolinska Institutet, Stockholm, Sweden.
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Abstract
Mesenchymal stem cells (MSC) derived from adult BM or fetal liver form several mesenchymal tissues after appropriate stimulation. Reports indicate that MSC have unique immunologic properties, making them ideal for cellular therapy. MSC are not immunogenic, they do not stimulate alloreactivity, and they escape lysis by cytotoxic T-cells and natural killer (NK)-cells. Thus, MSC may be transplantable between HLA-mismatched individuals without the need for host immunosuppression. Furthermore, adult MSC appear to be immunosuppressive as they reduce alloreactivity and the formation of cytotoxic lymphocytes in vitro. In vivo, adult MSC prolong the time to rejection of mis-matched skin grafts in baboons. The immunosuppressive properties of first trimester fetal MSC are less pronounced, but inducible with IFNgamma. These findings imply a potential role for MSC, not only in the repair of damaged tissues, but also in the manipulation of immune responses.
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Affiliation(s)
- K Le Blanc
- Division of Clinical Immunology, Centre for Allogeneic Stem Cell Transplantation, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden
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Xu MQ, Suo YP, Gong JP, Zhang MM, Yan LN. Prolongation of liver allograft survival by dendritic cells modified with NF-κB decoy oligodeoxynucleotides. World J Gastroenterol 2004; 10:2361-8. [PMID: 15285020 PMCID: PMC4576289 DOI: 10.3748/wjg.v10.i16.2361] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
AIM: To induce the tolerance of rat liver allograft by dendritic cells (DCs) modified with NF-κB decoy oligodeoxynucleotides (ODNs).
METHODS: Bone marrow (BM)-derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF + IL-4 to obtain immature DCs or mature DCs. GM-CSF+IL-4-propagated DCs were treated with double-strand NF-κB decoy ODNs containing two NF-κB binding sites or scrambled ODNs to ascertain whether NF-κB decoy ODNs might prevent DC maturation. GM-CSF-propagated DCs, GM-CSF + NF-κB decoy ODNs or scrambled ODNs-propagated DCs were treated with LPS for 18 h to determine whether NF-κB decoy ODNs could prevent LPS-induced IL-12 production in DCs. NF-κB binding activities, costimulatory molecule (CD40, CD80, CD86) surface expression, IL-12 protein expression and allostimulatory capacity of DCs were measured with electrophoretic mobility shift assay (EMSA), flow cytometry, Western blotting, and mixed lymphocyte reaction (MLR), respectively. GM-CSF-propagated DCs, GM-CSF + IL-4 -propagated DCs, and GM-CSF + NF-κB decoy ODNs or scrambled ODNs-propagated DCs were injected intravenously into recipient LEW rats 7 d prior to liver transplantation and immediately after liver transplantation. Histological grading of liver graft rejection was determined 7 d after liver transplantation. Expression of IL-2, IL-4 and IFN-γ mRNA in liver graft and in recipient spleen was analyzed by semiquantitative RT-PCR. Apoptosis of liver allograft-infiltrating cells was measured with TUNEL staining.
RESULTS: GM-CSF-propagated DCs, GM-CSF+NF-κB decoy ODNs-propagated DCs and GM-CSF+ scrambled ODNs-propagated DCs exhibited features of immature DCs, with similar low level of costimulatory molecule(CD40, CD80, CD86) surface expression, absence of NF-κB activation, and few allocostimulatory activities. GM-CSF + IL-4-propagated DCs displayed features of mature DCs, with high levels of costimulatory molecule (CD40, CD80, CD86) surface expression, marked NF-κB activation, and significant allocostimulatory activity. NF-κB decoy ODNs completely abrogated IL-4-induced DC maturation and allocostimulatory activity as well as LPS-induced NF-κB activation and IL-12 protein expression in DCs. GM-CSF + NF-κB decoy ODNs-propagated DCs promoted apoptosis of liver allograft-infiltrating cells within portal areas, and significantly decreased the expression of IL-2 and IFN-γ mRNA but markedly elevated IL-4 mRNA expression both in liver allograft and in recipient spleen, and consequently suppressed liver allograft rejection, and promoted liver allograft survival.
CONCLUSION: NF-κB decoy ODNs-modified DCs can prolong liver allograft survival by promoting apoptosis of graft-infiltrating cells within portal areas as well as down-regulating IL-2 and IFN-γ mRNA and up-regulating IL-4 mRNA expression both in liver graft and in recipient spleen.
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Affiliation(s)
- Ming-Qing Xu
- Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China.
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Christopher K, Mueller TF, Liang Y, Finn PW, Perkins DL. Modulation of gene expression by alloimmune networks following murine heart transplantation. Mol Genet Genomics 2004; 271:687-96. [PMID: 15138889 DOI: 10.1007/s00438-004-1013-9] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2004] [Accepted: 03/31/2004] [Indexed: 11/24/2022]
Abstract
The objective of our study was to analyze gene expression profiles in a complex in vivo model of solid organ transplantation, and to investigate the effects of single-gene deletions on alloimmunity. Using algorithms to generate dendrograms and self-organizing maps, we differentiated the alloimmune profiles of 16 transgenic knockout mouse strains, and identified subsets of genes that correlate with the duration of graft survival and provide candidates for prognostic and diagnostic indicators following transplantation in our model system.
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Affiliation(s)
- K Christopher
- Laboratory of Molecular Immunology, Renal Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, PBB 170, Boston, MA 02115, USA
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Sun W, Wang Q, Zhang L, Liu Y, Zhang M, Wang C, Wang J, Cao X. Blockade of CD40 pathway enhances the induction of immune tolerance by immature dendritic cells genetically modified to express cytotoxic T lymphocyte antigen 4 immunoglobulin. Transplantation 2004; 76:1351-9. [PMID: 14627915 DOI: 10.1097/01.tp.0000083557.25887.ee] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Immature dendritic cells (DCs) have the tolerogenic potential to induce alloantigen-specific immune tolerance. Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) gene-modified immature DCs have been shown to maintain their tolerogenicity and prolong allograft survival to some extent. We investigated whether blockade of CD40 pathway by anti-CD40 ligand (L) monoclonal antibody (mAb) could enhance the immune tolerance induction by immature DCs genetically modified to express CTLA4Ig (DC-CTLA4Ig). METHODS The tolerogenic properties of DC-CTLA4Ig were analyzed. In the vascularized heterotopic heart transplantation murine model, 2 x 10(6) DC-CTLA4Ig were infused intravenously into recipients, with or without a concomitant administration of anti-CD40L mAb 7 days before transplantation. Host responses to donor alloantigen were quantified by mixed leukocyte reaction and CTL assays. Donor major histocompatibility complex class II (Iab) expression in recipient lymph nodes was detected posttransplantation by semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS The allostimulatory activity of DC-CTLA4Ig was reduced. DC-CTLA4Ig also induced alloantigen-specific T-cell hyporesponsiveness and polarized T helper 2 cytokine production. Pretreatment of the recipients with DC-CTLA4Ig modestly prolonged allograft survival, without long-term allograft acceptance. Combined administration of DC-CTLA4Ig and anti-CD40L mAb significantly prolonged cardiac allograft survival, with long-term (>100 days) survival of 50% of the allografts in the pretreated recipients. More potent donor-specific inhibition of immune response against alloantigens and increased microchimerism were observed in these recipients. CONCLUSIONS Blockade of CD40 pathway with anti-CD40L mAb potentiates the tolerogenic potential of DC-CTLA4Ig and enhances the induction of antigen-specific immune tolerance more effectively.
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Affiliation(s)
- Wenji Sun
- Institute of Immunology, Zhejiang University, Hangzhou, China
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Siemionow M, Ulusal BG, Ozmen S, Ulusal AE, Ozer K. Composite vascularized skin/bone graft model: A viable source for vascularized bone marrow transplantation. Microsurgery 2004; 24:200-6. [PMID: 15160378 DOI: 10.1002/micr.20043] [Citation(s) in RCA: 42] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
In this study, we introduce a new model for vascularized skin and bone marrow transplantation. Twenty-five Lewis (RT1(1)) rats were studied. Anatomic dissection studies were performed in 5 animals. In the experimental group, 10 isograft transplantations were performed between Lewis rats. Combined groin skin and femoral bone flaps were transplanted based on the femoral artery and vein. Transplants were evaluated on a daily basis. All flaps survived without problems over 100 days posttransplant. The skin component remained pink and pliable, and grew new hair. Histological examination of the femoral bone (except the femoral head) revealed active hematopoiesis with a viable compact and cancellous bone components on day 100 posttransplant. This model can be applied to tolerance induction studies across the major Histocompatibility (MHC) barrier, where bone will serve as donor of stem and progenitor cells, and the skin flap will serve as a monitor of graft rejection.
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Affiliation(s)
- Maria Siemionow
- Department of Plastic Surgery, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
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Le Blanc K, Tammik C, Rosendahl K, Zetterberg E, Ringdén O. HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol 2003; 31:890-6. [PMID: 14550804 DOI: 10.1016/s0301-472x(03)00110-3] [Citation(s) in RCA: 1218] [Impact Index Per Article: 55.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
OBJECTIVE Mesenchymal stem cells (MSC) do not elicit alloreactive lymphocyte responses due to immune modulations. We investigated the immunologic properties of MSC after differentiation along three lineages: bone, cartilage, and adipose. METHODS AND RESULTS Flow cytometry showed that undifferentiated MSC express HLA class I but not class II, although HLA class II was present intracellularly as detected by Western blot. Addition of interferon gamma (IFN-gamma) for 48 hours induced greater than 90% of cells to express HLA class II. No lymphocyte response was induced by allogeneic irradiated MSC as stimulators. Results were similar using MSC pretreated with IFN-gamma. After growth of cells in medium to induce differentiation to bone, cartilage, or adipose for 6 or 12 days, the expression of HLA class I increased but no class II was detected on the cell surface. The ability to upregulate HLA class II on the cell surface after exposure to IFN-gamma for 48 hours was clearly diminished after the cells had been cultured in differentiation medium for 6 or 12 days, with only 10% of cells expressing HLA class II. Using MSC grown in osteogenic, chondrogenic, or adipogenic medium as stimulator cells, no lymphocyte alloreactivity was seen, even if differentiated MSC had been pretreated with IFN-gamma. MSC inhibit mixed lymphocyte cultures, particularly after osteogenic differentiation. This suppression was further enhanced by IFN-gamma. CONCLUSIONS Undifferentiated and differentiated MSC do not elicit alloreactive lymphocyte proliferative responses and modulate immune responses. The findings support that MSC can be transplantable between HLA-incompatible individuals.
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Affiliation(s)
- Katarina Le Blanc
- Division of Clinical Immunology, Centre for Allogeneic Stem Cell Transplantation, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden.
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Galactose-α1,3-galactose knockout mouse: a surrogate recipient. Curr Opin Organ Transplant 2003. [DOI: 10.1097/00075200-200303000-00013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Yin D, Dujovny N, Ma L, Varghese A, Shen J, Bishop DK, Chong AS. IFN-gamma production is specifically regulated by IL-10 in mice made tolerant with anti-CD40 ligand antibody and intact active bone. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2003; 170:853-60. [PMID: 12517950 DOI: 10.4049/jimmunol.170.2.853] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
We have developed a strategy to induce tolerance to allografts, involving cotransplantation of allogeneic intact active bone and transient anti-CD40 ligand mAb therapy. Tolerance induced by this approach in C57BL/6 mice receiving BALB/c hearts is not mediated by deletional mechanisms, but by peripheral regulatory mechanisms. Tolerance is associated with diminished ex vivo IFN-gamma production that is donor specific, and a reduction in the frequency of IFN-gamma-producing cells. Splenocytes from mice tolerant to BALB/c grafts, but sensitized to third-party C3H skin grafts, demonstrated normally primed ex vivo IFN-gamma responses to C3H stimulators. Neutralizing anti-IL-10 and anti-IL-10R, but not anti-TGF-beta, anti-IL-4, or anti-CTLA-4, Abs restored the ex vivo IFN-gamma response to BALB/c stimulators. There was no significant difference in IL-2 or IL-4 production between tolerant and rejecting mice, and anti-IL-10 mAbs had no effect on IL-2 or IL-4 production. The Cincinnati cytokine capture assay was used to test whether suppression of IFN-gamma production in vivo was also a marker of tolerance. In naive mice, we observed a dramatic increase in serum IFN-gamma levels following challenge with allogeneic BALB/c splenocytes or hearts. Tolerant mice challenged with allogeneic BALB/c splenocytes or hearts made significantly less or undetectable amounts of IFN-gamma. No IL-4 or IL-10 production was detected in tolerant or rejecting mice. Collectively, our studies suggest that active suppression of IFN-gamma production by IL-10 is correlated with, and may contribute to, tolerance induced with intact active bone and anti-CD40 ligand mAbs.
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Affiliation(s)
- Dengping Yin
- Department of General Surgery, Rush Presbyterian-St. Luke's Medical Center, Chicago, IL 60612, USA
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