Mixed hematopoietic chimerism and immune tolerance through bone marrow transplantation and infusion of regulatory T cells in a preclinical large animal model

Induction of transplantation tolerance to kidney allografts has been achieved through transient mixed hematopoietic chimerism in a non-myeloablative approach in both, non-human primates and humans. In order to make this approach applicable to other organs less tolerogenic such as lung or heart, we studied an approach to induce long-term mixed chimerism (instead of transient) through bone marrow transplantation (BMT) and infusion of recipient in vitro-expanded regulatory T cells (Tregs) in a non-human primate model (Cynomolgus macaque). Immunosuppression monotherapy was discontinued shortly after BMT. Donor-recipient pairs were major-histocompatibility-complex (MHC) mismatched in order to increase the applicability of this approach. First, we studied the biology of Mauritian Cynomolgus macaque (MCM) Tregs and developed five in vitro Treg expansion protocols for translational studies that included the use of artificial antigen presenting cells (aAPCs), donor peripheral blood mononuclear cells (PBMCs) or a pool from different donors of CD40L-stimulated B cells (CD40L-sBc). Tregs from all protocols suppressed the proliferation of anti-CD2CD3CD28 bead-stimulated autologous PBMCs albeit with different potencies, varying from 1:2-1:4 Treg:PBMC ratios, up to >1:32. Treg expansion varied between protocols but at least 1,000 fold expansion was achieved with all of the them, up to >7,000 folds. Reculture of cryopreserved Tregs permitted reexpansion with improved suppressive activity. Occasionally, CD8 contamination was observed and resolved by resorting. Specificity studies showed suppression of PBMCs from autologous cells, cells from the same donor used for stimulation during the Treg cultures and from a third‐party PBMC responders, suggestive of the polyclonallity of these Tregs. Similar to humans, the Treg–specific demethylated region (TSDR) within the FoxP3 locus correlated with suppressive activity and expression of FoxP3. Contrary to humans, FoxP3 expression did not correlate with CD45RA or CD127 expression.We then investigated the efficacy of ex vivo expanded Tregs to promote the induction of durable mixed chimerism along with BMT. A total of ten recipients received Tregs with different doses of bone marrow (BM) and outcomes were compared to five controls that did not receive Tregs. Prolonged chimerism was observed in Treg-treated recipients that received a high BM dose with infusion of Tregs compared to those that received low-dose BMT or did not received Tregs. Graft-versus-host disease (GVHD) was observed in four recipients, two controls and two animals that received Tregs expanded with CD40L-sBc. In those animals in which prolonged chimerism was observed, a higher number of peripheral Tregs was detected in blood compared to baseline levels and in vitro, the anti-donor response was decreased, suggestive of donor tolerance. BM rejection and chimerism loss was associated with an inversion of the CD4 and CD8 ratios and an increase in the CD8 absolute counts. Cytomegalovirus (CMV) was detected in all recipients post-BMT independently of the administration of Tregs. CMV reactivation was associated with an increase in the CD8 counts and with the loss of the BM graft. Therefore, promptly antiviral treatment was stablished for an early CMV control. In conclusion, Tregs were able to expand the duration of chimerism (albeit transient) when administered with high-dose BMT across MHC barriers without immunosuppression. <br /

Development of a Transplantable GFP+ B-Cell Lymphoma Tumor Cell Line From MHC-Defined Miniature Swine: Potential for a Large Animal Tumor Model

The lack of a reliable and reproducible large animal tumor model for the study of hemolymphatic malignancies limits the ability to explore the underlying pathophysiology and testing of novel therapies. The goal of this study was to develop an aggressive, trackable swine tumor cell line in mice for adoptive transfer into MHC matched swine. Two tumor cell lines, post-transplant lymphoproliferative disease (PTLD) 13271 and chronic myelogenous leukemia (CML) 14736, were previously established from the Massachusetts General Hospital (MGH) miniature swine herd. PTLD 13271 is a swine B-cell lymphoma line originating from an animal that developed PTLD following hematopoietic cell transplantation (HCT), while CML 14736 was generated from a swine that spontaneously developed CML. In order to select for aggressive tumor variants, both lines were passage into NOD/SCID IL-2 receptor γ−/− (NSG) mice. Tumor induced mortality in mice injected with CML14736 was 68% while 100% of mice injected with PTLD 13271 succumbed to PTLD by day 70. Based on aggressiveness, PTLD 13271 was selected for further development and re-passage into NSG mice resulting in increased tumor burden and metastasis. Transduction of the PTLD 13271 cell line with a green fluorescent protein (GFP)-expressing lentivirus facilitated tumor tracking when re-passaged in mice. Utilizing a tolerance induction strategy, GFP+ tumors were injected into an MHC matched miniature swine and successfully followed via flow cytometry for 48 h in circulation, although tumor engraftment was not observed. In summary, we report the development of an aggressive GFP+B-cell lymphoma cell line which has the potential for facilitating development of a large animal tumor model

Differential susceptibility of C57BL/6NCr and B6.Cg-Ptprca mice to commensal bacteria after whole body irradiation in translational bone marrow transplant studies

Abstract Background The mouse is an important and widely utilized animal model for bone marrow transplant (BMT) translational studies. Here, we document the course of an unexpected increase in mortality of congenic mice that underwent BMT. Methods Thirty five BMTs were analyzed for survival differences utilizing the Log Rank test. Affected animals were evaluated by physical examination, necropsy, histopathology, serology for antibodies to infectious disease, and bacterial cultures. Results Severe bacteremia was identified as the main cause of death. Gastrointestinal (GI) damage was observed in histopathology. The bacteremia was most likely caused by the translocation of bacteria from the GI tract and immunosuppression caused by the myeloablative irradiation. Variability in groups of animals affected was caused by increased levels of gamma and X-ray radiation and the differing sensitivity of the two nearly genetically identical mouse strains used in the studies. Conclusion Our retrospective analysis of thirty five murine BMTs performed in three different laboratories, identified C57BL/6NCr (Ly5.1) as being more radiation sensitive than B6.Cg-Ptprca/NCr (Ly5.2). This is the first report documenting a measurable difference in radiation sensitivity and its effects between an inbred strain of mice and its congenic counterpart eventually succumbing to sepsis after BMT.http://deepblue.lib.umich.edu/bitstream/2027.42/112743/1/12967_2007_Article_240.pd

Myeloid Leukemias And Virally Induced Lymphomas In Miniature Inbred Swine: Development Of A Large Animal Tumor Model

The lack of a large animal transplantable tumor model has limited the study of novel therapeutic strategies for the treatment of liquid cancers. Swine as a species provide a natural option based on their similarities with humans and their already extensive use in biomedical research. Specifically, the Massachusetts General Hospital miniature swine herd retains unique genetic characteristics that facilitate the study of hematopoietic cell and solid organ transplantation. Spontaneously arising liquid cancers in these swine, specifically myeloid leukemias and B cell lymphomas, closely resemble human malignancies. The ability to establish aggressive tumor cell lines in vitro from these naturally occurring malignancies makes a transplantable tumor model a close reality. Here, we discuss our experience with myeloid and lymphoid tumors in major histocompatibility characterized miniature swine and future approaches regarding the development of a large animal transplantable tumor model

Donor Lymphocyte Infusion–Mediated Graft-Versus-Host Responses In A Preclinical Swine Model Of Haploidentical Hematopoietic Cell Transplantation

We previously described successful hematopoietic stem cell engraftment across MHC barriers in miniature swine without graft-versus-host disease (GVHD) using novel reduced-intensity conditioning regimens consisting of partial transient recipient T cell–depletion, thymic or low-dose total body irradiation, and a short course of cyclosporine A. Here we report that stable chimeric animals generated with these protocols are strongly resistant to donor leukocyte infusion (DLI)–mediated GVH effects. Of 33 total DLIs in tolerant chimeras at clinical doses, 21 failed to induce conversion to full donor hematopoietic chimerism or cause GVHD. We attempted to overcome this resistance to conversion through several mechanisms, including using sensitized donor lymphocytes, increasing the DLI dose, removing chimeric host peripheral blood cells through extensive recipient leukapheresis before DLI, and using fully mismatched lymphocytes. Despite our attempts, the resistance to conversion in our model was robust, and when conversion was achieved, it was associated with GVHD in most animals. Our studies suggest that delivery of unmodified hematopoietic stem cell doses under reduced-intensity conditioning can induce a potent, GVHD-free, immune tolerant state that is strongly resistant to DLI