Stable Mixed Hematopoietic Chimerism Research Articles

Stability of Chimerism in Non-Obese Diabetic Mice Achieved By Rapid T Cell Depletion Is Associated With High Levels of Donor Cells Very Early After Transplant.

Stable mixed hematopoietic chimerism is a robust method for inducing donor-specific tolerance with the potential to prevent rejection of donor islets in recipients with autoimmune type-1 diabetes. However, with reduced intensity conditioning, fully allogeneic chimerism in a tolerance resistant autoimmune-prone non-obese diabetic (NOD) recipient has rarely been successful. In this setting, successful multilineage chimerism has required either partial major histocompatability complex matching, mega doses of bone marrow, or conditioning approaches that are not currently clinically feasible. Irradiation free protocols with moderate bone marrow doses have not generated full tolerance; donor skin grafts were rejected. We tested whether more efficient recipient T cell depletion would generate a more robust tolerance. We show that a combination of donor-specific transfusion-cyclophosphamide and multiple T cell depleting antibodies could induce stable high levels of fully allogeneic chimerism in NOD recipients. Less effective T cell depletion was associated with instability of chimerism. Stable chimeras appeared fully donor-specific tolerant, with clonal deletion of allospecific T cells and acceptance of donor skin grafts, while recovering substantial immunocompetence. The loss of chimerism months after transplant was significantly associated with a lower level of chimerism and donor T cells within the first 2 weeks after transplant. Thus, rapid and robust recipient T cell depletion allows for stable high levels of fully allogeneic chimerism and robust donor-specific tolerance in the stringent NOD model while using a clinically feasible protocol. In addition, these findings open the possibility of identifying recipients whose chimerism will later fail, stratifying patients for early intervention.

Repeated Injections of IL-2 Break Renal Allograft Tolerance Induced via Mixed Hematopoietic Chimerism in Monkeys.

Tolerance of allografts achieved in mice via stable mixed hematopoietic chimerism relies essentially on continuous elimination of developing alloreactive T cells in the thymus (central deletion). Conversely, while only transient mixed chimerism is observed in nonhuman primates and patients, it is sufficient to ensure tolerance of kidney allografts. In this setting, it is likely that tolerance depends on peripheral regulatory mechanisms rather than thymic deletion. This implies that, in primates, upsetting the balance between inflammatory and regulatory alloimmunity could abolish tolerance and trigger the rejection of previously accepted renal allografts. In this study, six monkeys that were treated with a mixed chimerism protocol and had accepted a kidney allograft for periods of 1-10 years after withdrawal of immunosuppression received subcutaneous injections of IL-2 cytokine (0.6-3 × 10(6) IU/m(2) ). This resulted in rapid rejection of previously tolerated renal transplants and was associated with an expansion and reactivation of alloreactive pro-inflammatory memory T cells in the host's lymphoid organs and in the graft. This phenomenon was prevented by anti-CD8 antibody treatment. Finally, this process was reversible in that cessation of IL-2 administration aborted the rejection process and restored normal kidney graft function.

Stable mixed hematopoietic chimerism permits tolerance of vascularized composite allografts across a full major histocompatibility mismatch in swine.

This study tested the hypothesis that vascularized composite allografts (VCA) could be accepted in a robust model of hematopoietic chimerism by injecting allogeneic bone marrow cells (BMC) into swine fetuses. Outbred Yorkshire sows and boars were screened to ensure the absence of the major histocompatibility (MHC) allele SLA(cc) of inbred MGH miniature swine and then mated. Bone marrow harvested from an SLA(cc) swine donor was T-cell depleted and injected intravenously into the fetuses between days 50-55 of gestation. After birth, the piglets were studied with flow cytometry to detect donor cells and mixed lymphocyte reactions (MLR) and cell-mediated lympholysis (CML) assays to assess their response to donor. Donor-matched VCAs from SLA(cc) donors were performed on four chimeric and two nonchimeric swine. The results showed donor cell engraftment and multilineage macrochimerism after the in utero transplantation of adult BMC, and chimeric animals were unresponsive to donor antigens in vitro. Both control VCAs were rejected by 21days and were alloreactive. Chimeric animals accepted the VCAs and never developed antidonor antibodies or alloreactivity to donor. These results confirm that the intravascular, in utero transplantation of adult BMC leads to donor cell chimerism and donor-specific tolerance of VCAs across a full MHC barrier in this animal model.

Targeting apoptosis to induce stable mixed hematopoietic chimerism and long-term allograft survival without myelosuppressive conditioning in mice

AbstractInduction of mixed hematopoietic chimerism results in donor-specific immunological tolerance by apoptosis-mediated deletion of donor-reactive lymphocytes. A broad clinical application of this approach is currently hampered by limited predictability and toxicity of the available conditioning protocols. We developed a new therapeutic approach to induce mixed chimerism and tolerance by a direct pharmacological modulation of the intrinsic apoptosis pathway in peripheral T cells. The proapoptotic small-molecule Bcl-2 inhibitor ABT-737 promoted mixed chimerism induction and reversed the antitolerogenic effect of calcineurin inhibitors by boosting the critical role of the proapoptotic Bcl-2 factor Bim. A short conditioning protocol with ABT-737 in combination with costimulation blockade and low-dose cyclosporine A resulted in a complete deletion of peripheral donor-reactive lymphocytes and was sufficient to induce mixed chimerism and robust systemic tolerance across full major histocompatibility complex barriers, without myelosuppression and by using moderate doses of bone marrow cells. Thus, immunological tolerance can be achieved by direct modulation of the intrinsic apoptosis pathway in peripheral lymphocytes—a new approach to translate immunological tolerance into clinically applicable protocols.

Vaccinating Female Donors with Y Chromosome-Encoded Disparities in the Pre-Clinical Canine Model of Allogeneic Hematopoietic Cell Transplantation

Abstract 4123Adoptive transfer of tumor antigen-sensitized lymphocytes has been a long-studied approach for the treatment of solid malignancies. Transfer of lymphocytes sensitized against minor histocompatibility (H) antigens, disparate between the marrow donor and recipient, may be a similar approach to enable improved host versus tumor responses in the treatment of hematological malignancies. We have developed a pre-clinical canine transplant model that establishes a state of stable mixed donor-recipient hematopoietic chimerism after a non-myeloablative conditioning regimen, dog leukocyte antigen (DLA)-identical sibling marrow graft, and a short course of post-grafting immunosuppression. Residual recipient hematopoiesis represents a surrogate for relapse from the underlying hematologic malignancy. The only way to eliminate the residual recipient hematopoiesis is by a donor lymphocyte infusion (DLI) from a donor sensitized by minor H antigens though either an infusion of cryopreserved recipient peripheral blood mononuclear cells, a kidney graft, or a skin graft. A DLI from an unsensitized donor does not alter chimerism in the recipient. This model lends itself to testing of vaccines to minor H antigens.We hypothesized that vaccinating donor dogs with autologous antigen presenting cells (APC) expressing recipient minor H antigens followed by DLI will result in conversion to full donor hematopoiesis in the mixed chimerism recipient, providing the experimental evidence in a large animal model of the potential therapeutic benefit for a minor H antigen vaccine to treat relapsed disease. To test our hypothesis, we sensitized two donor dogs with autologous APC’s transfected with poly(A)-selected mRNA from their respective recipients followed by DLI into these mixed chimeric recipients. We performed subcutaneous injections into the donor of 5 to 12 x 10⋀6 CD40-ligand stimulated donor B-cells electroporated with mRNA isolated from PHA blasts from the chimeric recipient every three weeks for a total of three vaccinations. This was followed by a DLI infusion into the chimeric recipient. There was no change in chimerism in the recipient dogs, and we were unable to demonstrate an Elispot response in the donor dogs to the transfected recipient mRNA. Due to the difficulty in isolating sufficient quantities of mRNA and a lack of an ELISpot response, we changed the source of antigen.Utilizing the canine genome sequence database we identified non-synonymous disparities between the × and Y chromosome genes of SMCY, UTY, and SRY. We created ‘minigenes' encoding these disparities flanked by a T7 promoter and a 3’ beta globin untranslated region with a poly(A) tail. These minigenes encoded 80% of the Y chromosome-associated disparities between canine SMCY, UTY, and their × chromosome homologs. The entire SRY gene was included as there is no × homolog for SRY. Minigenes were transcribed in vitro with a cap analogue to produce fully translatable mRNA. These Y chromosome-specific minigenes produced a strongly positive ELISpot in a female dog that had been sensitized with cells from a DLA-identical male sibling. We then performed three consecutive intravenous vaccinations at least one week apart with a combination of 25 to 40 × 106′ CD34-derived donor dendritic cells and CD40 ligand-stimulated B cells transfected with these Y chromosome-specific minigenes. Following the third vaccination, we demonstrated a weak positive ELISpot reaction to the Y chromosome-specific minigenes. However, a DLI into a mixed chimeric recipient failed to convert the recipient to full donor hematopoiesis and did not elicit any graft versus host disease.These studies suggest that in vivo sensitization of the marrow donor against recipient minor H antigens presented by autologous dendritic cells will require additional immune-modulation to accomplish improved graft versus tumor effects in the treatment of hematological diseases. Disclosures:No relevant conflicts of interest to declare.

Allogeneic hematopoietic stem cell transplantation for sickle cell disease: the time is now

AbstractAlthough sickle cell disease (SCD) has a variable clinical course, many patients develop end-organ complications that are associated with significant morbidity and early mortality. Myeloablative allogeneic HSCT (allo-HSCT) is curative but has been historically performed only in children younger than 16 years of age. Modest modifications in the conditioning regimen and supportive care have improved outcome such that the majority of children with a suitable HLA-matched sibling donor can expect a cure from this approach. However, adult patients have been excluded from myeloablative allo-HSCT because of anticipated excess toxicity resulting from accumulated disease burden. Efforts to use nonmyeloablative transplantation strategies in adults logically followed but were initially met with largely disappointing results. Recent results, however, indicate that nonmyeloablative allo-HSCT in adult patients with SCD allows for stable mixed hematopoietic chimerism with associated full-donor erythroid engraftment and normalization of blood counts, and persistence in some without continued immunosuppression suggests immunologic tolerance. The attainment of tolerance should allow extension of these potentially curative approaches to alternative donor sources. Efforts to build on these experiences should increase the use of allo-HSCT in patients with SCD while minimizing morbidity and mortality.

In mixed hematopoietic chimerism, the donor red cells win

In mixed hematopoietic chimerism, the donor red cells win

Canine Bone Marrow-Derived Mesenchymal Stromal Cells Suppress Alloreactive Lymphocyte Proliferation in Vitro but Fail to Enhance Engraftment in Canine Bone Marrow Transplantation

Stable mixed hematopoietic chimerism has been consistently established in dogs who were mildly immunosuppressed by 200 cGy of total body irradiation (TBI) before undergoing dog leukocyte antigen (DLA)-identical bone marrow (BM) transplantation and who received a brief course of immunosuppression with mycophenolate mofetil (28 days) and cyclosporine (35 days) after transplantation. However, when TBI was reduced from 200 to 100 cGy, grafts were nearly uniformly rejected within 3-12 weeks. Here, we asked whether stable engraftment could be accomplished after a suboptimal dose of 100 cGy TBI with host immunosuppression enhanced by donor-derived mesenchymal stromal cells (MSCs) given after transplantation. MSCs were cultured from BM cells and evaluated in vitro for antigen expression. They showed profound immunosuppressive properties in mixed lymphocyte reactions (MLRs) in a cell dose-dependent manner not restricted by DLA. MSC and lymphocyte contact was not required, indicating that immunosuppression was mediated by soluble factors. Prostaglandin E2 was increased in culture supernatant when MSCs were cocultured in MLRs. The addition of indomethacin restored lymphocyte proliferation in cultures containing MSCs. MSCs expressed CD10, CD13, CD29, CD44, CD73/SH-3, CD90/Thy-1, and CD106/VCAM-1. For in vivo studies, MSCs were injected on the day of BM grafting and on day 35, the day of discontinuation of posttransplantation cyclosporine. MSCs derived from the respective BM donors failed to avert BM graft rejection in 4dogs who received DLA-identical grafts after nonmyeloablative conditioning with 100 cGy TBI in a time course not significantly different from that of control dogs not given MSCs. Although the MSCs displayed in vitro characteristics similar to those reported for MSCs from other species, their immunosuppressive qualities failed to sustain stable BM engraftment in vivo in this canine model.

Immunomodulatory Effects of Mixed Hematopoietic Chimerism: Immune Tolerance in Canine Model of Lung Transplantation

Long-term survival after lung transplantation is limited by acute and chronic graft rejection. Induction of immune tolerance by first establishing mixed hematopoietic chimerism (MC) is a promising strategy to improve outcomes. In a preclinical canine model, stable MC was established in recipients after reduced-intensity conditioning and hematopoietic cell transplantation from a DLA-identical donor. Delayed lung transplantation was performed from the stem cell donor without pharmacological immunosuppression. Lung graft survival without loss of function was prolonged in chimeric (n = 5) vs. nonchimeric (n = 7) recipients (p < or = 0.05, Fisher's test). There were histological changes consistent with low-grade rejection in 3/5 of the lung grafts in chimeric recipients at > or =1 year. Chimeric recipients after lung transplantation had a normal immune response to a T-dependent antigen. Compared to normal dogs, there were significant increases of CD4+INFgamma+, CD4+IL-4+ and CD8+ INFgamma+ T-cell subsets in the blood (p < 0.0001 for each of the three T-cell subsets). Markers for regulatory T-cell subsets including foxP3, IL10 and TGFbeta were also increased in CD3+ T cells from the blood and peripheral tissues of chimeric recipients after lung transplantation. Establishing MC is immunomodulatory and observed changes were consistent with activation of both the effector and regulatory immune response.

Investigation of immunological approaches to enhance engraftment in a 1 Gy TBI canine hematopoietic stem cell transplantation model

Stable mixed hematopoietic chimerism can be established in a canine stem cell transplantation model using a conditioning consisting of total body irradiation (TBI; 2 Gy) and postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporin (CSA). Reduction of TBI had resulted previously in graft rejection in this model. We investigated whether postgrafting stimulation of donor T cells against recipient's hematopoietic antigens or graft augmentation with donor monocyte-derived dendritic cells (MoDC) promote engraftment following 1 Gy TBI. All dogs received dog leukocyte-antigen-identical bone marrow transplantation. Dogs were conditioned with either 2 Gy TBI (group 1) or 1 Gy TBI, followed by repetitive recipient hematopoietic cell lysate vaccinations (group 2) or graft augmentation with MoDC (group 3). Immunosuppression consisted of CSA and MMF. In group 1, four animals remained stable chimeras for >110 weeks, and three rejected their grafts (week 10, week 14, week 16). All dogs in groups 2 and 3 rejected their graft (median: week 10 and 11, respectively). Peak chimerism and engraftment duration was shorter in the 1-Gy groups (p < 0.05) compared to group 1. Neither postgrafting vaccination nor graft augmentation with MoDC were effective in supporting durable engraftment. Additional modifications are necessary to improve potential strategies aimed at establishment of early tissue specific graft-vs-host reactions.

Mesenchymal Stem Cells Enhance the Induction of Mixed Chimerism and Tolerance to Rat Hind-Limb Allografts after Bone Marrow Transplantation

Mixed hematopoietic chimerism via bone marrow transplantation has been shown to induce donor specific tolerance to solid organ allografts, but graft versus host disease (GVHD) remains to be a risk. Composite tissue allografts may need a higher percentage of donor chimerism compared with less immunogenic solid organ allografts. In this study, we investigated the potential of mesenchymal stem cells (MSCs) in the induction of stable and high level chimerism and subsequent donor-specific tolerance to composite tissue allograft without the incidence of graft versus host disease (GVHD). Fully mismatched, 4- to 8-wk-old Brown Norway (RT1(n)) and Lewis (RT1(1)) rats were used as cell or hind-limb donors and recipients, respectively. Recipients received a conditioning regimen consisting of antilymphocyte serum, standard immunosuppressive therapy (rapamycin: 0.2 mg/kg/d; days 0 approximately 130) and 3 Gy total body irradiation, followed by an intraportal co-infusion of allogeneic MSCs and bone marrow cells (day 0); then performing hind-limb allotransplants (30 d after BMT); 100 d after hind-limb transplantations, immunosuppressive therapy was stopped and to observe the survival time of the hind-limb allografts. When co-infusion of allogeneic MSCs was administered, 14/15 recipients developed stable and high level chimerism. Subsequently, in the donor specific group, the survival time of hind-limb allografts without RAPA were remarkably prolonged compared with other groups. The results of the histopathological evaluation of skin biopsy and mixed lymphocyte cultures confirmed this operational donor specific tolerance. Without MSCs, only 2/7 of the recipients developed stable chimerism, but GVHD occurred in both of them, and the level of the chimerism was much lower. On the other hand, GVHD was not observed in any of the recipients infused with MSCs (0/15). This study indicates a potential use of MSCs for induction of stable and high level mixed hematopoietic chimerism and subsequent donor specific tolerance in clinical composite tissue allotransplantation.

Hematopoietic chimerism induces renal and skin allograft tolerance in DLA-identical dogs

Hematopoietic chimerism, a state where donor and recipient bone marrow cells coexist, is associated with donor-specific tolerance. Nonmyeloablative bone marrow transplantation (BMT) has been shown to induce stable mixed hematopoietic chimerism in dog leukocyte antigen (DLA)-matched dogs. The potential for inducing renal and skin allograft tolerance with nonmyeloablative BMT was investigated in DLA-identical and DLA-haploidentical dogs in this study. Renal allografts were performed in 8 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with (n = 8) and without (n = 4) simultaneous BMT. Skin allografts were performed in 2 DLA-identical and 4 DLA-haploidentical dogs after stopping CSP and MMF. Two DLA-identical control dogs received renal allografts without TBI, BMT, or immunosuppression with CSP and MMF. Molecular chimerism was determined with a PCR-based DNA microsatellite assay. Serum creatinine (Cr) concentration, urine specific gravity, and sequential renal biopsies were monitored to assess renal allograft function. Donor-type blood cells were first detected 4 weeks posttransplantation in both the myeloid and lymphoid lineages. Donor chimerism was present for at least 76 weeks in the DLA-identical dogs. Mixed chimerism was not observed in the DLA-haploidentical dogs or DLA-identical dogs that did not undergo BMT. The renal allografts were acutely rejected within 14 days in the 2 DLA-identical control dogs. There was long-term (> 5 yrs) renal allograft survival as evidenced by a normal (< 2.0 mg/dL) serum Cr concentration in both the DLA-identical and DLA-haploidentical dogs that underwent 200 cGy TBI and transient immunosuppression with CSP and MMF either with or without simultaneous BMT. Renal allograft inflammation was severe in the control dogs, mild to moderate in the DLA-haploidentical dogs, and minimal in the DLA-identical dogs. Donor-specific skin grafts were accepted in the DLA-identical dogs but rejected in the DLA-haploidentical dogs. Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal and skin allograft tolerance in DLA-identical and permit long-term renal allograft survival in DLA-haploidentical dogs. These findings suggest it may possible to obtain long-term allograft survival in DLA-identical and -haploidentical dogs without the need for chronic immunosuppressive therapy.

Ex Vivo Expanded T Regulatory (Treg) Cells Block Conversion of Mixed Chimeras To Complete Donor Chimerism.

A highly effective method to establish long-term, stable mixed hematopoietic chimerism was developed in the dog model. This involves nonmyeloablative allogeneic hematopoietic cell transplantation (HCT), consisting of 2 Gray (Gy) total body irradiation (TBI), dog leukocyte antigen (DLA)-identical marrow, and short-term post-grafting immunosuppression. We hypothesized that CD4+CD25+ Treg cells may be important regulators for the maintenance of cellular immune tolerance after allogeneic HCT. Previously, we showed in 8 mixed chimeras that naive donor lymphocyte infusion (DLI) did not change the level of donor chimerism. However, reconditioning mixed chimeras with 2 Gy TBI followed by DLI “breaks” tolerance and increases the level of donor chimerism. Seven mixed chimeras were reconditioned with 2 Gy TBI followed by DLI. Within 4 weeks after DLI, conversion to 100% donor chimerism was seen in 5 of 7 dogs and 2 dogs had a > 50% sustained increase in donor chimerism. Four recipients developed graft-versus host disease (GVHD). A control group of 3 mixed chimeras reconditioned with 2 Gy TBI without DLI had no change in donor chimerism. These results suggest that reconditioning with 2 Gy TBI followed by DLI can break the tolerance mechanism established in mixed chimeras. Next we asked if CD4+CD25+ Treg cells obtained from mixed chimeras before reconditioning could block the increase in donor chimerism following 2 Gy TBI and DLI. Peripheral blood mononuclear cells (PBMC) from 8 mixed chimeras were obtained by leukapheresis and cultured in bulk mixed leukocyte culture (MLC) with 3rd party DLA-mismatched, unrelated and irradiated CD34+ derived dendritic cells (10:1 responder: stimulator ratio) or PBMC (1:1). On day 4 of MLC, CD25+ cells were isolated by positive immunomagnetic selection. Next, artificial antigen presenting cells (aAPC, KT32) were added to expand the CD4+CD25+ Treg cells. The aAPC expressed Fcγ receptor CD32, canine CD86, and human IL-15, were loaded with the canine-specific mitogenic anti-CD3ε antibody 17.6F9 and irradiated prior to stimulation of CD4+CD25+ Treg. After 7 days, Treg were expanded a median of 23 (range, 8–36)-fold. Expanded CD4+CD25+ Treg were assessed for phenotype and in vitro function. The Treg cells were generated from 8 mixed chimeras and were infused back into the respective dogs (median dose 1× 107/kg) after reconditioning with 2 Gy TBI and immediately prior to DLI. In 6 of 8 dogs there was no change in the level of donor chimerism at 16–20 weeks follow-up; 2 dogs converted to complete donor chimerism within 6 weeks. Treatment with expanded Treg cells blocked conversion to complete donor chimerism after 2 Gy TBI +DLI in 6 of 8 dogs, compared with significant increases in donor chimerism for all 7 dogs after 2 Gy TBI +DLI not given Treg (p=.007). None of the 8 Treg recipient dogs developed GVHD, compared with 4 of 7 not given Treg, (p=0.02). A control group of 4 mixed chimeras were infused with expanded, non-Treg CD25− T cells. To this end, CD25+ T cells were immunomagneticaly depleted on day 4 of MLC. CD25− T cells were expanded with aAPC. Three of 4 dogs converted to the complete donor chimerism within 7 weeks after 2 Gy TBI, non-Treg and DLI. These results suggest that ex vivo expanded CD4+CD25+ Treg cells have in vivo function in a large animal model and can restore the tolerance mechanism in mixed chimeras that is broken by 2 Gy TBI and DLI.

Partial Donor-Specific Tolerance to Delayed Skin Grafts After Rejection of Hematopoietic Cell Graft

Donor-specific tolerance (DST) is induced after allogeneic hematopoietic cell transplantation (HCT) and is a potential strategy for prolonging survival of solid organ grafts. DST may persist in recipients with transient mixed hematopoietic chimerism (MC) when solid organ transplantation and HCT are done concomitantly. In a canine model of allogeneic HCT after nonmyeloablative conditioning, DST to skin grafts was evaluated in dog leukocyte antigen (DLA)-identical recipients with stable MC (n=11), or after rejection of the hematopoietic cell (HC) graft (n=19). There was significant improvement in the survival of DLA-identical HC donor-derived skin grafts in recipients with MC compared to normal recipients (n=7; P<0.0001). However, HC donor-derived skin grafts in four recipients with MC developed an inflammatory reaction without skin graft loss. This may represent partial DST. Survival of DLA-identical HC donor-derived skin grafts was also significantly prolonged compared to normal recipients even when skin grafting was delayed until after rejection of the HC graft (P=0.002). An inflammatory reaction developed in all nine of the surviving HC donor-derived skin grafts in this group, but there was no graft loss at last follow-up (median, 30 [range, 9-84] weeks). An increased time to rejection of the hematopoietic graft was associated with prolonged survival of the subsequent skin graft (P=0.02). In a model of stable MC, DST to skin grafts may be complete or partial. Partial DST can persist after HC graft rejection even if solid organ transplantation is delayed. Further investigations are required to understand the mechanisms responsible for DST after allogeneic HCT.

Ex vivo expanded T regulatory (T reg) cells block the breaking of tolerance in mixed chimeras

Nonmyeloablative allogeneic hematopoietic cell transplantation (HCT) consisting of 2 Gray (Gy) total body irradiation (TBI), dog leukocyte antigen-identical marrow, and short-term postgrafting immunosuppression with cyclosporine and mycophenolate mofetil was developed in the dog model as an effective way to establish long-term, stable mixed hematopoietic chimerism. We hypothesized that Treg cells are crucial to maintaining stable mixed chimerism. All dogs in the current study were stable mixed chimeras established as described.

Renal Allograft Tolerance in DLA-Identical and Haploidentical Dogs After Nonmyeloablative Conditioning and Transient Immunosuppression With Cyclosporine and Mycophenolate Mofetil

Background Canine models of bone marrow and renal transplantation have provided important preclinical data relevant to developing novel therapeutic protocols for hematopoietic and solid organ transplantation in human beings. Nonmyeloablative transplantation has been shown to induce stable mixed hematopoietic chimerism in normal dogs and correct the phenotype of canine pyruvate kinase deficiency and Glanzman’s thrombasthenia. In this study, we investigated the potential for inducing renal allograft tolerance using a nonmyeloablative bone marrow transplantation strategy that induces mixed chimerism in DLA-identical dogs. Methods Reciprocal renal allografts were performed in 4 DLA-identical and 4 DLA-haploidentical dogs with nonmyeloablative conditioning (200 cGy total body irradiation [TBI]) and transient immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) with and without simultaneous bone marrow transplantation. Two DLA-identical control dogs received reciprocal renal allografts without TBI or immunosuppression with CSP and MMF. Serum creatinine (Cr) concentration was monitored to assess renal allograft function. Results The renal allografts were acutely rejected in the 2 DLA-identical dogs without TBI or immunosuppression. There was long-term (>1 year) renal allograft survival as evidenced by a normal (<2.0 mg/dL) serum Cr concentration in both the DLA-identical and DLA-haploidentical dogs that underwent 200 cGy TBI and transient immunosuppression with CSP and MMF either with or without simultaneous bone marrow transplantation. Conclusions Nonmyeloablative conditioning (200 cGy TBI) and transient immunosuppression with CSP and MMF induce renal allograft tolerance in DLA-identical and DLA-haploidentical dogs without donor/host mixed hematopoietic chimerism. These findings suggest it may be possible to induce tolerance to solid organ transplants without the need for chronic immunosuppressive therapy or stable hematopoietic chimerism in the setting of both DLA-matched and haploidentical transplants.

Donor-Specific Tolerance Is Induced after Nonmyeloablative Conditioning and Allogeneic Hematopoietic Cell Transplantation and May Persist after Graft Rejection.

The association between stable hematopoietic chimerism and donor specific tolerance (DST) for solid organ grafts was demonstrated clinically and in different experimental models. However, the existing data also demonstrate that the establishment of hematopoietic chimerism does not always correlate with the development of DST, especially for skin grafts. As these issues could have major implications for organ transplantation in general, the present study was undertaken to assess DST in recipients with transient or stable hematopoietic cell (HC) chimerism established after nonmyeloablative conditioning regimens. In a canine model of allogeneic hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning, DST to skin grafts was evaluated in DLA-identical recipients with stable mixed hematopoietic chimerism (MC) (n=11), or following transient HC engraftment (n=22). There was a significant improvement in the survival of DLA-identical HC donor-derived skin grafts in recipients with stable MC compared to normal recipients (n=7; P<0.0001). However, HC donor-derived skin grafts in 4 recipients with MC developed an inflammatory reaction without skin graft loss consistent with a chronic rejection process. Survival of DLA-identical HC donor-derived skin grafts was also significantly prolonged in recipients after transient HC engraftment compared to normal recipients (P=0.002). However, a chronic inflammatory process without graft loss developed in all of the HC donor-derived skin grafts from this group. An increased time to rejection of the hematopoietic graft was significantly associated with an improved survival of the subsequent skin graft (P=0.02). The time to rejection of third-party (DLA-nonidentical) skin grafts (n=40) was the same in recipients with stable MC and previous HC graft as well as normals (median- 8 days). Autologous skin grafts (n=40) survived for the duration of follow-up without developing chronic inflammation. Extended HC donor-specific skin graft survival (>15 days) was predictive of subsequent stable engraftment at second HCT in recipients with previous graft rejection after first HCT. Five of 6 dogs with extended survival of the skin graft beyond day 15 became stable MC after second HCT compared to 0/9 dogs with skin grafts surviving less than 11 days (P=0.0008). In this model, DST to skin grafts in recipients with stable MC after nonmyeloablative HCT may not be complete, and chronic inflammation may develop without loss of that graft. Partial DST may persist after rejection of HC grafts established with nonmyeloablative conditioning. Further investigations are required to understand the mechanisms responsible for DST after allogeneic HCT.

Effect of Intravenous Busulfan Versus Total Body Irradiation on Donor Chimerism and Acute Graft-Versus-Host Disease (GVHD) after Allogeneic Stem Cell Transplantation (AlloSCT) in Mice.

The goals of administering transplantation conditioning to leukemia patients undergoing alloSCT are to kill tumor cells, prevent rejection of the donor graft, create “space” for donor hematopoiesis, and provide an overall milieu that favors a donor T cell-mediated “graft-versus-leukemia” effect. However, transplantation conditioning also increases the incidence and severity of acute GVHD by damaging non-hematopoietic tissues and by inducing lymphopenia, which augments the antigen-driven expansion of alloreactive T cells. The purpose of this study was to compare the relative safety and efficacy of transplantation conditioning with intravenous busulfan (Busulfex; ESP Pharma, Edison, NJ) versus total body irradiation (TBI) by administering titrated doses of either agent to mice and comparing their effects on: 1) donor chimerism after syngeneic and semi-allogeneic (parent into F1) SCT; 2) graft rejection after MHC-matched allogeneic SCT; and 3) acute GVHD after semi-allogeneic SCT. Both agents induced titratable donor chimerism after syngeneic BMT, but TBI was more immunosuppressive, since allogeneic marrow engrafted after 700 cGy TBI but not after an equivalently myelotoxic dose of Busulfex (60 mg/kg). When given in equivalently myelotoxic doses, TBI always induced more GVHD than did IV busulfan, as measured by weight loss and the augmentation of donor T cell and myeloid chimerism. As a result, stable mixed hematopoietic chimerism was generated more readily in mice conditioned with nonmyeloablative doses of busulfan than in mice conditioned with equitoxic doses of TBI. These results demonstrate that IV busulfan promotes GVHD to a lesser extent than does an equivalently myelotoxic dose of TBI. IV busulfan may be the preferred agent if stable mixed hematopoietic chimerism is the desired outcome. The relative roles of lymphopenia versus tissue damage in the differential outcomes of TBI versus Busulfex conditioning are being investigated.

Synergy between Nonmyeloablative Doses of Intravenous Busulfan and Post-Transplantation Cyclophosphamide for Induction of Tolerance to MHC-Compatible Stem Cell Allografts.

The goal of conditioning for nonmyeloablative allogeneic stem cell transplantation, or NST, is to permit the stable engraftment of donor cells with a minimum of toxicity to the patient. We have previously developed a minimal conditioning regimen, consisting of low dose total body irradiation (TBI; 100–200 cGy) and post-transplantation cyclophosphamide (Cy), that permits the induction of stable mixed hematopoietic chimerism following the transplantation of modest doses of MHC-identical allogeneic stem cells in mice. The goal of the current study was to determine whether intravenous (IV) busulfan (Busulfex; ESP Pharma, Edison, NJ), an agent with myelotoxic but minimal immunosuppressive activity, could be used in place of TBI in this regimen and still promote allograft tolerance. TBI was more immunosuppressive than Busulfex, because engraftment of allogeneic stem cells could be achieved in mice conditioned with 700 cGy TBI alone but not in mice conditioned with an equivalently myelotoxic dose of 60 mg/kg Busulfex IV. However, when post-transplantation Cy was added, stable mixed hematopoietic chimerism was achieved in mice conditioned with doses as low as 100 cGy TBI or 20 mg/kg Busulfex. Busulfex and post-transplantation Cy were both required for the induction of allograft tolerance, because graft rejection occurred in transplanted mice treated with either agent alone. The capacity of Busulfex to promote allograft tolerance in this model could not be explained by effects of the drug on the host immune system, because the graft rejection response was not restored in Busulfex-conditioned mice reconstituted with mature host lymphocytes at the time of transplantation. These results demonstrate that Busulfex effectively substitutes for TBI in promoting allograft tolerance, and suggests that the creation of marrow “space” promotes alloengraftment in conditions of suboptimal host immunosuppression.

Influence of Hematopoietic Cell Lysate Vaccinations and Dendritic Cell Enriched Marrow Grafts on Engraftment after Nonmyeloablative Hematopoietic Stem Cell Transplantation.

Background: Stable mixed hematopoietic chimerism can be established in a canine marrow transplantation model using nonmyeloablative conditioning consisting of total body irradiation (TBI, 2Gy) and postgrafting immunosuppression with mycophenolate mofetil (MMF) and cyclosporine A (CSA). Reduction of TBI to 1Gy resulted in rejection of marrow grafts in this model even if CD3 depleted donor PBMC were added to the graft. Here, we investigated whether addition of immature dendritic cells or postgrafting in vivo stimulation of donor T cells against recipient derived hematopoietic antigens are capable of enhancing targeted GVH reactions and allow engraftment.Methods: Dog leukocyte antigen-identical littermates received 2Gy TBI (group 1, control, n=7) and 1Gy TBI (group 2, n=7; group 3, n=6) before allogeneic stem cell transplantation (SCT). All recipients were given postgrafting immunosuppression consisting of 15mg/kg CSA BID PO (days -1 to +35) and 10mg/kg MMF BID PO (days 0 to +27). In addition, group 2 was vaccinated ID and SQ with recipient blood cell lysates (3x/week w1-w5; 1x/week w6-w9) together with locally applied granulocyte macrophage-colony stimulating factor (125μg/vaccination). The vaccine consisted of PBMC and granulocytes at a ratio of 1:1 (105 cells/kg before lysis). Dogs of group 3 received ex-vivo generated immature monocyte-derived dendritic cells (MoDC) of donor origin in addition to marrow at day 0. MoDC doses ranged from 6.4–10.9x105 cells/kg (median 7.9x105). Littermate donor marrow was infused IV at a median of 3.4x108 (1.6–11.4x108) nucleated cells/kg.Results: The median platelet and leukocyte nadirs in group 1 were 30x109/l (median at day 11) and 2.4x109/l (median at day 8), respectively. In groups 2 and 3 the median platelet and leukocyte nadirs were 110x109/l (median at day 12) and 4.8x109/l (median at day 7). All dogs in group 1 engrafted and showed mixed hematopoietic chimerism among PBMC and granulocytes (after 4 weeks (w4): median 32% (5–44%) and 68% (10–74%), respectively). 4/7 animals remained stable mixed chimeras >25 weeks, 3/7 rejected their graft (w10, w14, w16). All dogs in group 2 showed initial engraftment and chimerism of donor PBMC and granulocytes (w4: median 12% (10–19%) and 19% (11–24%), respectively), but rejected their grafts a median of 10.3 weeks (w8-w11) after SCT. 4/6 dogs in group 3 had a median w4-chimerism in PBMC and granulocytes of 14% (11–15%) and 20% (16–27%), respectively and 2/6 dogs are too early after SCT to be evaluated. Currently 3/4 evaluable dogs in group 3 remain mixed chimeric. In comparison to group 1 engraftment kinetics of groups 2 and 3 indicate a delayed engraftment combined with significantly lower donor PBMC and granulocytes chimerisms and significantly reduced engraftment duration (group 2). No differences in w4-chimerism patterns between groups 2 and 3 could be observed.Conclusion: Our data show that vaccinations with recipient hematopoietic cell lysates failed to expedite long term bone marrow engraftment following a 1Gy TBI conditioning. Addition of donor-MoDC to the graft might favour sustained engraftment but a longer follow up is needed. Furthermore, equal dosed MMF applied orally seems to be inferior to SQ applications in postgrafting immunosuppression since compared to historical controls less mixed chimeras could be generated following 2Gy TBI.