A preclinical model of double- versus single-unit unrelated cord blood transplantation.

Cord blood transplantation (CBT) with units containing total nucleated cell (TNC) dose >2.5 x 10(7)/kg is associated with improved engraftment and decreased transplant-related mortality. For many adults no single cord blood units are available that meet the cell dose requirements. We developed a dog model of CBT to evaluate approaches to overcome the problem of low cell dose cord blood units. This study primarily compared double- versus single-unit CBT. Unrelated dogs were bred and cord blood units were harvested. We identified unrelated recipients that were dog leukocyte antigen (DLA)-88 (class I) and DLA-DRB1 (class II) allele-matched with cryopreserved units. Each unit contained

Delaying DLA-haploidentical hematopoietic cell transplantation after total body irradiation.

Exposure to accidental or deliberate radiation poses a threat to public health, proving lethal at higher doses in large part because of deleterious effects on marrow. In those cases, allogeneic hematopoietic cell transplantation (HCT) might be required to restore marrow function. Most radiation accident victims will have HLA-haploidentical relatives who could serve as HCT donors. Here, we assessed in a canine HCT model the total body irradiation (TBI) doses after which transplants might be required and successful engraftment would be possible. In an attempt at mimicking the logistical problems likely to exist after radiation accidents, 4-, 8- or 10-day intervals were placed between TBI and HCT. To keep the experimental readout simple, no graft-versus-host disease (GVHD) prevention was administered. All dogs transplanted after a 4-day delay following 700 or 920 cGy TBI successfully engrafted, whereas virtually all those given 450 or 600 cGy rejected their grafts. Transplant delays of 8 and 10 days following 920 cGy TBI also resulted in successful engraftment in most dogs, whereas a delay of 8 days after 700 cGy resulted in virtually uniform graft failure. The time courses of acute GVHD (aGVHD) and rates of granulocyte recovery in engrafting dogs were comparable among dogs regardless of the lengths of delay. In other studies, we showed that most dogs not given HCT survived 700 cGy TBI with intensive supportive care, whereas those given 800 cGy TBI and higher died with marrow aplasia. Thus, DLA-haploidentical HCT was successful even when carried out 4, 8, or 10 days after TBI at or above radiation exposures where dogs survived with intensive care alone.

Intussusception in canine recipients of hematopoietic cell grafts and surgical correction.

Intussusception is a common complication after canine hematopoietic cell transplantation (HCT). The present study was undertaken to evaluate predisposing factors of intussusception and to test whether intussusception can be managed surgically during the period immediately after HCT. We determined the incidence of intussusception after HCT was performed in 325 canine recipients (autologous, n = 43; allogeneic, n = 282) during the interval from January 2002 to May 2005. Intussusception was diagnosed in 16 of 325 dogs (4.9%). Intussusception was not significantly associated with the dose of irradiation, source of hematopoietic graft, use of immunosuppressive agents, gender, or age at transplant. A group of 9 of the affected dogs underwent small-bowel resection after diagnosis, and 7 were managed without surgical intervention. Despite complicating factors such as gastrointestinal toxicity and low neutrophil and platelet counts induced by the marrow conditioning regimen and the use of immunosuppressive agents, successful surgical management of intussusception was achieved in 6 of 9 dogs, as compared with successful management of 0 of 7 without surgery. Intussusception did not recur after surgical intervention in any dog. Recent HCT and post-transplant immunosuppressive therapy are not absolute contraindications to surgical intervention for intussusception in canine recipients of HCT.

RDP58 does not prevent graft-versus-host disease after dog leukocyte antigen-nonidentical canine hematopoietic cell transplantation.

Graft-versus-host disease (GVHD) remains a cause of substantial morbidity for patients undergoing allogeneic hematopoietic cell transplantation (HCT). The present study was undertaken to investigate the effectiveness of RDP58, a peptide derived from the human leukocyte antigen class I heavy chain, in preventing GVHD in the established dog leukocyte antigen (DLA)-nonidentical canine model. Dogs underwent HCT from unrelated DLA-nonidentical donors after conditioning with 920 cGy total body irradiation. Engraftment and achievement of full donor chimerism was seen in five of six dogs, whereas one dog showed rejection and died of marrow aplasia. All five dogs with engraftment developed acute GVHD and were euthanized at an average of 20.6 days after HCT. Compared with historical controls, the Suse of RDP58 neither prevented acute GVHD nor significantly prolonged survival of DLA-nonidentical HCT recipients.

Use of multigeneration-family molecular dog leukocyte antigen typing to select a hematopoietic cell transplant donor for a dog with T-cell lymphoma.

CASE DESCRIPTION: A 7-year-old Golden Retriever was examined because of anorexia, lethargy, vomiting, and gradual weight loss. CLINICAL FINDINGS: Splenomegaly, pancytopenia, high serum calcium concentration, and high alkaline phosphatase activity were detected. Magnetic resonance imaging revealed an enlarged mesenteric lymph node and increased signals from the bone marrow of the ilium and vertebral bodies. Histologic examination and immunophenotyping of biopsy specimens confirmed a stage V (b) T-cell malignant lymphoma. TREATMENT AND OUTCOME: Clinical remission was attained by use of 2 chemotherapy cycles, followed by an allogeneic hematopoietic cell transplant performed at 18 weeks after diagnosis. A donor was identified by molecular dog leukocyte antigen typing methods. The patient was conditioned with 2 fractions of 4 Gy total body irradiation delivered 3 hours apart at 7 cGy/min, followed by an IV infusion of recombinant canine granulocyte colony-stimulating factor mobilized leukapheresis product and postgrafting immunosuppression with cyclosporine. Chimerism analyses revealed full donor engraftment that has been maintained for at least 58 weeks after transplant. Remission has been confirmed by normal results of serum thymidine kinase assays and the absence of peripheral blood clonal T-cell receptor gene rearrangements. CLINICAL RELEVANCE: Systemic chemotherapy induces remissions; however, most dogs succumb to disease recurrence because of multidrug resistance. Outcome of allogeneic hematopoietic cell transplantation in dogs can be excellent because of improved donor-recipient selection by use of molecular dog leukocyte antigen typing, compared with early attempts, and better prevention of graft versus host disease, better supportive care, and substitution of peripheral blood mononuclear cells for bone marrow.

Adoptive immunotherapy against kidney targets in dog-leukocyte antigen-identical mixed hematopoietic canine chimeras.

BACKGROUND: Stable mixed-donor-host-hematopoietic chimerism can serve as a platform for adoptive immunotherapy. Infusions of donor lymphocytes (DLI) sensitized against hematopoietic cells converted mixed hematopoietic into full-donor chimerism in dog-leukocyte antigen (DLA)-identical littermates. Whether sensitization against tissue of solid organs leads to organ-specific immunity that can be transferred by DLI was unknown and was investigated in these experiments using the kidney as target. METHODS: DLA-identical recipients with established stable mixed-donor-host-hematopoietic chimerism were used. In five pairs, hematopoietic stem-cell transplant (HSCT) donors were sensitized by kidney transplantation from the respective chimeras. In a second group, five HSCT donors received vaccinations that were generated from kidney cells of the respective mixed chimeras. Twenty-eight days after sensitization, DLI were administered to the mixed-hematopoietic chimeras. RESULTS: All HSCT donors rejected their kidney grafts from their mixed-chimeric recipients within 22 to 45 days. DLI caused no sustained graft-versus-kidney effects in the mixed-chimeric recipients. However, DLI donors sensitized by kidney transplantation converted 4 of 5 mixed chimeras into virtually complete (>95%) donor-type chimeras, compared with 1 of 5 mixed chimeras given DLI by vaccination from sensitized donors. CONCLUSION: Although DLA-identical kidney grafts from mixed-hematopoietic chimeras were readily rejected by their HSCT donors, subsequent transfusions of sensitized-donor lymphocytes into mixed chimeras converted mixed to all-donor chimerism but failed to induce graft-versus-kidney effects. Vaccination strategies in lieu of kidney grafts failed to convert mixed chimerism.

FTY720 does not abrogate acute graft-versus-host disease in the dog leukocyte antigen-nonidentical unrelated canine model.

BACKGROUND: Acute graft-versus-host disease (GVHD) remains a significant impediment to successful hematopoietic stem-cell transplantation (HSCT). Here, we examined the effectiveness of 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride (FTY720), an immunosuppressant that retraffics activated lymphocytes to secondary lymphoid organs, for the treatment of acute GVHD in an established dog leukocyte antigen-nonidentical unrelated canine HSCT model. METHODS: Dogs were given HSCT after conditioning with 920 cGy total body irradiation. The dogs received methotrexate 0.4 mg/kg/day on days 1, 3, 6, and 11 and FTY720 (5 mg/kg/day orally) after developing GVHD. RESULTS: Five of six dogs achieved engraftment, developed acute GVHD, and were treated with FTY720. FTY720 resulted in a profound decrease in lymphocytes and a temporary mitigation of clinical GVHD; however, GVHD recurred in all dogs. Four of five dogs were euthanized because of severe GVHD and the fifth because of severe inanition associated with moderate GVHD. CONCLUSIONS: Compared with controls, treatment of GVHD with FTY720 did not control this complication or significantly increase survival.

What role for FTY720, a novel immunosuppressive agent, in canine nonmyeloablative hematopoietic stem cell transplantation?

BACKGROUND: Stable mixed donor/host hematopoietic chimerism was almost uniformly achieved in dogs given 200 cGy total body irradiation (TBI) before, and a short course of immunosuppression after, transplantation of marrow from dog leukocyte antigen-identical littermates, but was transient when the TBI dose was decreased to 100 cGy. Here, we examined whether stable engraftment could be achieved in five dogs given FTY720 (days -5 and -4), followed by 100 cGy TBI, dog leukocyte antigen-identical marrow grafts, and mycophenolate mofetil/cyclosporine. RESULTS AND CONCLUSIONS: Although all five dogs showed initial engraftment, four dogs rejected their grafts within 11 weeks, whereas one dog was euthanized on day 17 due to enteritis. This was not different from the control dogs not given FTY720 (P=0.32). Thus, FTY720 failed to enhance allogeneic engraftment in this model, perhaps due to in vivo T-cell depletion of the graft resulting from sequestration of donor lymphocytes in host central lymphoid tissues.

CD154 blockade and donor-specific transfusions in DLA-identical marrow transplantation in dogs conditioned with 1-Gy total body irradiation.

Stable mixed donor/host chimerism has been reliably established in dogs given a sublethal dose (2 Gy) of total body irradiation (TBI) before and immunosuppression with mycophenolate mofetil (MMF) or rapamycin combined with cyclosporine (CSP) after marrow transplantation from dog leukocyte antigen (DLA)-identical littermates (hematopoietic cell transplantation [HCT]). When TBI was reduced to 1 Gy, only transient engraftment was observed. Here we investigated whether stable engraftment after 1-Gy TBI could be accomplished by reducing host-versus-donor immune responsiveness through preceding CD154 blockade and infusion of donor peripheral blood mononuclear cells (PBMCs). We found that the anti-human CD154 antibody, 5c8, cross-reacted with canine lymphocytes and blocked alloimmune responses in vitro. Based on pharmacokinetic studies, 6 dogs received a single intravenous injection of 5 mg/kg anti-CD154 antibody (on day -5), followed 1 day later by donor PBMCs. On day 0, the dogs were given 1 Gy of TBI and underwent DLA-identical marrow grafts. Postgraft immunosuppression consisted of MMF and CSP. All 6 dogs demonstrated initial engraftment; 3 dogs sustained the engraftment for >26 weeks, whereas 3 dogs rejected their grafts, after 9, 22, and 24 weeks, and survived with autologous recovery. Graft survival was significantly improved over that in 11 historical controls conditioned with 1-Gy TBI and given either MMF or rapamycin with CSP after HCT, all of which rejected their grafts between 3 and 12 weeks (P = .03). Preceding donor PBMC infusion and CD154 blockade improved survival of DLA-identical marrow grafts after 1-Gy TBI.

Stable trichimerism after marrow grafting from 2 DLA-identical canine donors and nonmyeloablative conditioning.

Although hematopoietic cell transplantation (HCT) is generally accomplished using a single donor, multiple donors have been used to enhance the speed of engraftment, particularly in the case of umbilical cord blood grafts. Here we posed the question in the canine HCT model whether stable dual-donor chimerism could be established using 2 DLA-identical donors. We identified 8 DLA-identical littermate triplets in which the marrow recipients received 2 Gy total body irradiation followed by marrow infusions from 2 donors and postgrafting immunosuppression. All 8 dogs showed initial "trichimerism," which was sustained in 5 dogs, while 2 dogs rejected one of the allografts and remained mixed chimeras, and 1 dog rejected both allografts. Immune function in one trichimeric dog, as tested by mixed leukocyte culture response and antibody response to sheep red blood cells, was found to be normal. Five dogs received kidney grafts from one of their respective marrow donors at least 6 months after HCT without immunosuppressive drugs, and grafts in 4 dogs are surviving without rejection. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow grafts from 2 DLA-identical littermates could result in sustained trichimerism, and immunologic tolerance could include a kidney graft from one of the marrow donors.

Failure of donor lymphocyte infusion to prevent graft rejection in dogs given DLA-identical marrow after 1 Gy of total body irradiation.

We investigated in a preclinical canine model of hematopoietic cell transplantation (HCT) whether preemptive donor lymphocyte infusion (DLI) given 1 month after HCT could prevent late graft rejection that was the rule in historical dogs given suboptimal conditioning with 1 Gy of total body irradiation (TBI) before and immunosuppression with cyclosporine (CSP) and either mycophenolate mofetil (MMF; n = 6) or rapamycin (n = 5) after dog leukocyte antigen (DLA)-identical marrow transplantation. Nine dogs given DLA-identical marrow after 1 Gy of TBI followed by postgrafting MMF and CSP were studied. A single DLI was given 28-36 days after HCT, either with (n = 5) or without (n = 4) preceding treatment with the immunosuppressive drug pentostatin. Two of the 4 dogs given DLI only maintained stable mixed donor-host chimera beyond 30 weeks after HCT, whereas 2 rejected their grafts, on weeks 10 and 15 after HCT. One of the 5 dogs given pentostatin before DLI maintained a stable mixed donor-host chimera beyond 30 weeks, whereas 4 rejected their grafts, at weeks 8, 12, 12, and 16 after HCT. The 30-week probability of stable mixed chimerism was 33% among dogs given DLI, versus 0% among 11 historical dogs (P = .003). In conclusion, DLI was only moderately effective in preventing graft rejection in this model. Additional immunosuppression with pentostatin did not improve that outcome. The model might be useful in developing potential strategies aimed at preventing graft rejection in patients with low donor chimerism levels.

Postgrafting immunosuppression with sirolimus and cyclosporine facilitates stable mixed hematopoietic chimerism in dogs given sublethal total body irradiation before marrow transplantation from DLA-identical littermates.

We studied the value of postgrafting immunosuppression with sirolimus (SRL) and cyclosporine (CSP) in enhancing engraftment of dog leukocyte antigen-identical littermate marrow after nonmyeloablative conditioning in a canine model. Dogs received either 2 Gy (n=7) or 1 Gy (n=5) total body irradiation (TBI), followed by postgrafting immunosuppression with SRL and CSP. In the first cohort, all 7 dogs showed rapid initial engraftment. One engrafted dog died on day 21 due to hemorrhagic pneumonitis. Durable engraftment was seen in 5 of 6 remaining dogs, with a median follow-up of >48 (range, >32 to >56) weeks. The sixth dog rejected the marrow graft (as assessed by variable number of tandem repeats) at 11 weeks; however, a subsequent skin graft from the same marrow donor did not undergo acute cellular rejection, suggesting donor-specific tolerance. In the second cohort, all 5 dogs rejected the marrow graft at a median of 9 weeks (range, 3-11 weeks). We conclude that SRL/CSP is as effective as a previously studied combination of mycophenolate mofetil and CSP at establishing durable marrow engraftment after sublethal conditioning.

Adoptive immunotherapy to increase the level of donor hematopoietic chimerism after nonmyeloablative marrow transplantation for severe canine hereditary hemolytic anemia.

Severe hemolytic anemia in Basenji dogs secondary to pyruvate kinase deficiency can be corrected by allogeneic hematopoietic cell transplantation (HCT) from littermates with normal hematopoiesis after conventional myeloablative or nonmyeloablative conditioning regimens. If the levels of donor chimerism were low (<20%) after nonmyeloablative HCT, there was only partial correction of the hemolytic anemia. We next addressed whether allogeneic cell therapy after nonmyeloablative HCT would convert mixed to full hematopoietic chimerism, achieve sustained remission from hemolysis, and prevent progression of marrow fibrosis and liver cirrhosis. Three pyruvate kinase-deficient dogs were given HCT from their respective dog leukocyte antigen-identical littermates after nonmyeloablative conditioning with 200 cGy of total body irradiation. Postgrafting immunosuppression consisted of mycophenolate mofetil and cyclosporine. All 3 dogs engrafted and had mixed hematopoietic chimerism with donor levels ranging from 12% to 55% in bone marrow. In 2 of the 3 dogs, there were decreases in the levels of donor chimerism so that at 25 weeks after nonmyeloablative HCT, hemolysis recurred that was associated with increased reticulocyte counts. All 3 dogs then had 2 serial infusions of donor lymphocytes (DLI) from their respective donors at least 20 weeks apart to convert from mixed to full donor chimerism. Both dogs with recurrence of hemolytic anemia after nonmyeloablative HCT achieved higher levels of donor chimerism, with donor contributions ranging from 47% to 62% in the bone marrow and 50% to 69% and 16% to 25% in the granulocyte and mononuclear cell fractions of the peripheral blood, respectively, and with remission of the hemolytic anemia. One dog responded after the first DLI, and 5 weeks after the second DLI, the other dog converted to full donor chimerism. At last follow-up, all these dogs showed clinical improvement, as determined by increasing hematocrits and normal reticulocyte counts. Analysis of the marrow 3 years after HCT showed normal cellularity, a normal myeloid-erythroid ratio, and no or minimal marrow fibrosis. Liver biopsies demonstrated normal histologies with no or minimal fibrosis. We conclude that DLI after nonmyeloablative HCT can increase the levels of donor cells contributing to hematopoiesis in recipients, inducing remissions of the hemolytic process and preventing complications associated with iron overload.

Hematopoietic stem cell transplantation does not restore dystrophin expression in Duchenne muscular dystrophy dogs.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene on the X-chromosome that result in skeletal and cardiac muscle damage and premature death. Studies in mice, including the mdx mouse model of DMD, have demonstrated that circulating bone marrow-derived cells can participate in skeletal muscle regeneration, but the potential clinical utility of treating human DMD by allogeneic marrow transplantation from a healthy donor remains unknown. To assess whether allogeneic hematopoietic cell transplantation (HCT) provides clinically relevant levels of donor muscle cell contribution in dogs with canine X-linked muscular dystrophy (c-xmd), 7 xmd dogs were given hematopoietic cell (HC) transplants from nonaffected littermates. Compared with the pretransplantation baseline, the number of dystrophin-positive fibers and the amount of wild-type dystrophin RNA did not increase after HCT, with observation periods ranging from 28 to 417 days. Similar results were obtained when the recipient dogs were given granulocyte colony-stimulating factor (G-CSF) after their initial transplantation to mobilize the cells. Despite successful allogeneic HCT and a permissive environment for donor muscle engraftment, there was no detectable contribution of bone marrow-derived cells to either skeletal muscle or muscle precursor cells assayed by clonal analyses at a level of sensitivity that should detect as little as 0.1% donor contribution.

Kinetics of engraftment in patients with hematologic malignancies given allogeneic hematopoietic cell transplantation after nonmyeloablative conditioning.

We analyzed the kinetics of donor engraftment among various peripheral blood cell subpopulations and their relationship to outcomes among 120 patients with hematologic malignancies given hematopoietic cell transplantation (HCT) after nonmyeloablative conditioning consisting of 2 Gy total body irradiation (TBI) with or without added fludarabine. While patients rapidly developed high degrees of donor engraftment, most remained mixed donor/host chimeras for up to 180 days after HCT. Patients given preceding chemotherapies and those given granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell (G-PBMC) grafts had the highest degrees of donor chimerism. Low donor T-cell (P = .003) and natural killer (NK) cell (P = .004) chimerism levels on day 14 were associated with increased probabilities of graft rejection. High T-cell chimerism on day 28 was associated with an increased probability of acute graft-versus-host disease (GVHD) (P = .02). Of 93 patients with measurable malignant disease at transplantation, 41 achieved complete remissions a median of 199 days after HCT; 19 of the 41 were mixed T-cell chimeras when complete remissions were achieved. Earlier establishment of donor NK-cell chimerism was associated with improved progression-free survival (P = .02). Measuring the levels of peripheral blood cell subset donor chimerisms provided useful information on HCT outcomes and might allow early therapeutic interventions to prevent graft rejection or disease progression.