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Chimeric Allografts Induced by Short-Term Treatment With Stem Cell-Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats.

ABSTRACT: Transplant tolerance allowing the elimination of lifelong immunosuppression has been the goal of research for 60 years. The induction of mixed chimerism has shown promise and has been extended successfully to large animals and to the clinic; however, it remains cumbersome and requires heavy early immunosuppression. In this study, we reported that four injections of AMD3100, a CXCR4 antagonist, plus eight injections of low-dose FK506 (0.05 mg/kg per day) in the first week after kidney transplantation extended survival, but death from renal failure occurred at 30-90 days. Repeating the same course of AMD3100 and FK506 at 1, 2 and 3 mo after transplant resulted in 92% allograft acceptance (n = 12) at 7 mo, normal kidney function and histology with no further treatment. Transplant acceptance was associated with the influx of host stem cells, resulting in a hybrid kidney and a modulated host immune response. Confirmation of these results could initiate a paradigm shift in posttransplant therapy.

SUBMITTER: Hu X

PROVIDER: S-EPMC4925175 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Chimeric Allografts Induced by Short-Term Treatment With Stem Cell-Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats.

Hu X X Okabayashi T T Cameron A M AM Wang Y Y Hisada M M Li J J Raccusen L C LC Zheng Q Q Montgomery R A RA Williams G M GM Sun Z Z

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons 20160226 7

Transplant tolerance allowing the elimination of lifelong immunosuppression has been the goal of research for 60 years. The induction of mixed chimerism has shown promise and has been extended successfully to large animals and to the clinic; however, it remains cumbersome and requires heavy early immunosuppression. In this study, we reported that four injections of AMD3100, a CXCR4 antagonist, plus eight injections of low-dose FK506 (0.05 mg/kg per day) in the first week after kidney transplanta ...[more]

PMID: 26749344

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Project description:BackgroundCell-based therapies are promising for tolerance induction in bone marrow (BM), solid organs, and vascularized composite allotransplantation (VCA). The toxicity of bone marrow transplantation (BMT) protocols precludes this approach from routine clinical applications. To address this problem, we developed a new therapy of Human Umbilical Di-Chimeric (HUDC) cells for tolerance induction in transplantation. This study established in vitro characterization of the created HUDC cells.MethodsWe performed sixteen ex vivo polyethylene glycol (PEG)-mediated fusions of human umbilical cord blood (UCB) cells from two unrelated donors. Fusion feasibility was confirmed in vitro by flow cytometry (FC) and confocal microscopy (CM). The HUDC cells' genotype was assessed by lymphocytotoxicity test and short tandem repeat-polymerase chain reaction (STR-PCR) analysis, phenotype by FC, viability by LIVE/DEAD® assay, and apoptosis level by Annexin V staining. We used COMET assay to assess HUDC cells' genotoxicity after the fusion procedure. Clonogenic properties of HUDC cells were evaluated by colony forming unit (CFU) assay. Mixed lymphocyte reaction (MLR) assay assessed immunogenic and tolerogenic properties of HUDC cells.ResultsWe confirmed the creation of HUDC cells from two unrelated human donors of UCB cells by FC and CM. Human leukocyte antigen (HLA) class I and II typing, and STR-PCR analysis of HUDC cells confirmed the presence of alleles and loci from both unrelated UCB donors (donor chimerism: 49%±8.3%, n=4). FC confirmed the hematopoietic phenotype of HUDC cells. We confirmed high HUDC cells' viability (0.47% of dead cells) and a low apoptosis level of fused HUDC cells (15.9%) compared to positive control of PKH-stained UCB cells (20.4%) before fusion. COMET assay of HUDC cells revealed a lack of DNA damage. CFU assay confirmed clonogenic properties of HUDC cells, and MLR assay revealed a low immunogenicity of HUDC cells.ConclusionsThis study confirmed creation of a novel HUDC cell line by ex vivo PEG-mediated fusion of UCB cells from two unrelated donors. The unique concept of creating a HUDC cell line, representing the genotype and phenotype of both, transplant donor and the recipient, introduces a promising approach for tolerance induction in BM, solid organs, and VCA transplantation.

Project description:IntroductionTrauma patients are susceptible to coagulopathy and dysfunctional immune responses. Mesenchymal stromal cells (MSCs) are at the forefront of the cellular therapy revolution with profound immunomodulatory, regenerative, and therapeutic potential. Routine assays to assess immunomodulation activity examine MSC effects on proliferation of peripheral blood mononuclear cells (PBMCs) and take 3-7 days. Assays that could be done in a shorter period of time would be beneficial to allow more rapid comparison of different MSC donors. The studies presented here focused on assays for MSC suppression of mitogen-stimulated PBMC activation in time frames of 24 h or less.MethodsThree potential assays were examined-assays of apoptosis focusing on caspase activation, assays of phosphatidyl serine externalization (PS+) on PBMCs, and measurement of tumor necrosis factor alpha (TNFα) levels using rapid ELISA methods. All assays used the same initial experimental conditions: cryopreserved PBMCs from 8 to 10 pooled donors, co-culture with and without MSCs in 96-well plates, and PBMC stimulation with mitogen for 2-72 h.ResultsSuppression of caspase activity in activated PBMCs by incubation with MSCs was not robust and was only significant at times after 24 h. Monitoring PS+ of live CD3+ or live CD4+/CD3+ mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, 2 h, although no increase in the percentage of PS+ cells was seen with time. The ability of MSC in co-culture to suppress PBMC PS+ externalization compared favorably to two concomitant assays for MSC co-culture suppression of PBMC proliferation, at 72 h by ATP assay, or at 96 h by fluorescently labeled protein signal dilution. TNFα release by mitogen-activated PBMCs was dose dependent, reproducible, robust, and evident at the earliest time point taken, with accumulating signal over time. However, suppression levels with MSC co-culture was reliably seen only after 24 h.DiscussionTakeaways from these studies are as follows: (1) while early measures of PBMC activation is evident at 2-6 h, immunosuppression was only reliably detected at 24 h; (2) PS externalization at 24 h is a surrogate assay for MSC immunomodulation; and (3) rapid ELISA assay detection of TNFα release by PBMCs is a robust and sensitive assay for MSC immunomodulation at 24 h.

Dataset Information

Chimeric Allografts Induced by Short-Term Treatment With Stem Cell-Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats.

Publications

Chimeric Allografts Induced by Short-Term Treatment With Stem Cell-Mobilizing Agents Result in Long-Term Kidney Transplant Survival Without Immunosuppression: A Study in Rats.

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