Anti-Inducible Costimulator Monoclonal Antibody Treatment of Canine Chronic Graft-versus-Host Disease.

In murine model systems inducible costimulator (ICOS) signaling has been implicated in the formation of chronic graft-versus-host disease (GVHD). Previously, we showed that chronic GVHD can be reproducibly produced in the dog hematopoietic cell transplantation (HCT) model and that ICOS expression is upregulated on T cells in dogs with chronic GVHD. The goal of the present study was to determine whether administration of a short course of anti-canine ICOS mAb could alter the rapid and progressive course of chronic GVHD. Five dogs underwent HCT from dog leukocyte antigen mismatched unrelated donors after total body irradiation. Postgrafting immunosuppression consisted of methotrexate (days 1, 3, 6, and 11) and cyclosporine (days -1 through 78). Anti-ICOS mAb (3 injections, 72 hours apart) was administered upon diagnosis of GVHD. One dog failed to respond to anti-ICOS mAb therapy and succumbed to chronic GVHD in a time course similar to control untreated dogs. Overall, anti-ICOS-treated dogs experienced a significant prolongation in survival from the time of diagnosis of chronic GVHD compared with control dogs. Within the limitations of the number of study dogs we suggest that a short course of anti-ICOS mAb may be useful in the treatment of chronic canine GVHD.

A Canine Model of Chronic Graft-versus-Host Disease.

In long-term survivors of allogeneic hematopoietic cell transplantation (HCT), chronic graft-versus-host disease (GVHD) is the major cause of morbidity and mortality and a major determinant of quality of life. Chronic GVHD responds poorly to current immunosuppressive drugs, and while T cell depletion may be preventive, this gain is offset by increased relapse rates. A significant impediment to progress in treating chronic GVHD has been the limitations of existing animal models. The goal of this study was to develop a reproducible comprehensive model of chronic GVHD in the dog. Ten recipient dogs received 920 cGy total body irradiation, infusion of marrow, and an infusion of buffy coat cells from a dog leukocyte antigen (DLA)-mismatched unrelated donor. Postgrafting immunosuppression consisted of methotrexate (days 1, 3, 6, 11) and cyclosporine. The duration of cyclosporine administration was limited to 80 days instead of the clinically used 180 days. This was done to contain costs, as chronic GVHD was expected to develop at earlier time points. All recipients were given ursodiol for liver protection. One dog had graft failure and 9 dogs showed stable engraftment. Eight of the 9 developed de novo chronic GVHD. Dogs progressed with clinical signs of chronic GVHD over a period of 43 to 164 (median, 88) days after discontinuation of cyclosporine. Target organs showed the spectrum of chronic GVHD manifestations that are typically seen clinically. These included lichenoid changes of the skin, fasciitis, ocular involvement (xerophthalmia), conjunctivitis, bronchiolitis obliterans, salivary gland involvement, gingivitis, esophageal involvement, and hepatic involvement. Peripheral blood lymphocyte surface antigen expression of CD28 and inducible costimulator was elevated in dogs with GHVD compared with those in normal dogs, but not significantly so. Serum levels of IL-8 and monocyte chemotactic protein-1 in GVHD-affected dogs at time of euthanasia were elevated, whereas levels of IL-15 were depressed compared with those in normal dogs. Results indicate that the canine model is well suited for future studies aimed at preventing or treating chronic GVHD.

Long-term Tolerance Toward Haploidentical Vascularized Composite Allograft Transplantation in a Canine Model Using Bone Marrow or Mobilized Stem Cells.

BACKGROUND: The development of safe and reliable protocols for the transplantation of the face and hands may be accomplished with animal modeling of transplantation of vascularized composite allografts (VCA). Previously, we demonstrated that tolerance to a VCA could be achieved after canine recipients were simultaneously given marrow from a dog leukocyte antigen-identical donor. In the present study, we extend those findings across a dog leukocyte antigen mismatched barrier. METHODS: Eight recipient dogs received total body irradiation (4.5 cGy), hematopoietic cell transplantation (HCT), either marrow (n = 4) or granulocyte-colony stimulating factor mobilized peripheral blood stem cells (n = 4), and a VCA transplant from the HCT donor. Post grafting immunosuppression consisted of mycophenolate mofetil (28 days) and cyclosporine (35 days). RESULTS: In 4 dogs receiving bone marrow, 1 accepted both its marrow transplant and demonstrated long-term tolerance to the donor VCA (>52 weeks). Three dogs rejected both their marrow transplants and VCA at 5 to 7 weeks posttransplant. Dogs receiving mobilized stem cells all accepted their stem cell transplant and became tolerant to the VCA. However, 3 dogs developed graft-versus-host disease, whereas 1 dog rejected its stem cell graft by week 15 but exhibited long-term tolerance toward its VCA (>90 weeks). CONCLUSIONS: The data suggest that simultaneous transplantation of mobilized stem cells and a VCA is feasible and leads to tolerance toward the VCA in a haploidentical setting. However, there is a higher rate of donor stem cell engraftment compared with marrow HCT and an increase in the incidence of graft-versus-host disease.

Anti-CD28 Antibody-Initiated Cytokine Storm in Canines.

BACKGROUND: CD28 signal blockade following T cell receptor activation is under intense investigation as a tolerance-inducing therapy for transplantation. Our goal is to produce a CD28-specific reagent as a therapy for the prevention of graft rejection and graft-versus-host disease in the canine model of allogeneic hematopoietic cell transplantation (HCT). METHODS: We infused a monoclonal mouse anti-canine CD28 antibody (1C6 mAb) into four dogs and a fragment of antigen-binding (1C6 Fab) into two dogs. Pharmacokinetics, pathology, cytokine release, and the crystal structure of 1C6 Fv were evaluated. RESULTS: Within an hour of an IV injection of the 1C6 mAb, the dogs became leukopenic and developed a steroid-refractory cytokine storm. Two of the dogs developed high fevers, one experienced diffuse alveolar hemorrhage, and another developed gastrointestinal hemorrhage. The cytokine storm was characterized by elevated plasma levels of MCP-1, IP-10, IL-10, IL-6, and TNF-α. In addition, one dog showed elevated levels of IL-2, IL-8, and IL-18. In contrast, infusion of 1C6 Fab was well tolerated without any side effects. Dry-coating 1C6 mAb onto tissue culture plates induced CD3-independent proliferation and TNF-alpha production. Crystal structure analysis revealed that 1C6 binds to canine CD28 in a manner different than previously reported for conventional agonistic or superagonistic antibodies. CONCLUSIONS: These results indicate that dogs and humans develop a similar cytokine storm following infusion ofanti-CD28 mAb, providing an appropriate large animal for further study. 1C6 Fab warrants evaluation as a tolerance-inducing reagent in the canine model of allogeneic HCT.

Simultaneous transplantation of hematopoietic stem cells and a vascularized composite allograft leads to tolerance.

BACKGROUND: We have previously demonstrated that tolerance to a vascularized composite allograft (VCA) can be achieved after the establishment of mixed chimerism. We test the hypothesis that tolerance to a VCA in our dog leukocyte antigen-matched canine model is not dependent on the previous establishment of mixed chimerism and can be induced coincident with hematopoietic cell transplantation (HCT). METHODS: Eight dog leukocyte antigen-matched, minor antigen mismatched dogs received 200 cGy of radiation and a VCA transplant. Four dogs received donor bone marrow at the time of VCA transplantation (group 1), whereas a second group of four dogs did not (group 2). All recipients received a limited course of postgrafting immunosuppression. All dogs that received HCT and VCA were given donor, third-party, and autologous skin grafts. RESULTS: All group 1 recipients were tolerant to their VCA (>62 weeks). Three of the four dogs in group 2 rejected their VCA transplants after the cessation of immunosuppression. Biopsies obtained from the muscle and skin of VCA from group 1 showed few infiltrating cells compared with extensive infiltrates in biopsies of VCA from group 2. Compared with autologous skin and muscle, elevated levels of CD3+ FoxP3+ T-regulatory cells were found in the skin and muscle obtained from the VCA of HCT recipients. All group 1 animals were tolerant to their donor skin graft and promptly rejected the third-party skin grafts. CONCLUSION: These data demonstrated that donor-specific tolerance to all components of the VCA can be established through simultaneous nonmyeloablative allogeneic HCT and VCA transplantation protocol.

Inducible costimulator (ICOS) up-regulation on activated T cells in chronic graft-versus-host disease after dog leukocyte antigen-nonidentical hematopoietic cell transplantation: a potential therapeutic target.

BACKGROUND: Inducible costimulator (ICOS), a member of the CD28 family of costimulatory molecules, is induced on CD4 and CD8 T cells after their activation. ICOS functions as an essential immune regulator and ICOS blockade is a potential approach to immune modulation in allogeneic transplantation. Here, we describe the expression profile of ICOS in dogs and determine whether ICOS expression is up-regulated during chronic graft-versus-host disease (GVHD) and host-versus-graft reactions in the canine hematopoietic cell transplantation model. METHODS: Monoclonal antibodies (mAbs) against cell surface-expressed ICOS were produced and tested in vitro for suppression of canine mixed leukocyte reactions (MLR). Expression of ICOS on CD3 cells was evaluated by flow cytometry using peripheral blood, lymph nodes, and splenocytes obtained from dogs undergoing graft-versus-host and host-versus-graft reactions. RESULTS: Canine ICOS was expressed in an inducible pattern on T cells activated by concanavalin A, anti-CD3 mAb in combination with anti-CD28 mAb, and alloantigen stimulation. Immunosuppressive effects of ICOS blockade were observed in MLR using peripheral blood mononuclear cells from dog leukocyte antigen-nonidentical dogs. Immunosuppressive effects of ICOS blockade were observed in MLR when anti-ICOS was combined with suboptimal concentrations of cytotoxic T-lymphocyte antigen 4-Ig or cyclosporine. ICOS expression was significantly up-regulated on T cells in dogs undergoing graft rejection or chronic GVHD after allogeneic hematopoietic cell transplantation. CONCLUSIONS: These studies suggest that ICOS plays a role in graft rejection and GVHD in an outbred animal model, and ICOS blockade may be an approach to prevention and treatment of chronic GVHD.

Reduced graft-versus-host disease in C3-deficient mice is associated with decreased donor Th1/Th17 differentiation.

Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation is mediated by the activation of recipient dendritic cells and subsequent proliferation of donor T cells. The complement system was recently shown to modulate adaptive immunity through an interaction of the complement system and lymphocytes. Complement proteins participate in the activation of dendritic cells, antigen presentation to T cells, and proliferation of T cells. Our studies with a murine model of bone marrow transplantation demonstrate that complement system regulates alloimmune responses in GVHD. Mice deficient in the central component of the complement system (C3(-/-)) had significantly lower GVHD-related mortality and morbidity compared with wild-type recipient mice. The numbers of donor-derived T cells, including IFN-γ(+), IL-17(+), and IL-17(+)IFN-γ(+) subsets, were decreased in secondary lymphoid organs of C3(-/-) recipients. Furthermore, the number of recipient CD8α(+)CD11c(+) cells in lymphoid organs was reduced. We conclude that C3 regulates Th1/17 differentiation in bone marrow transplantation, and define a novel function of the complement system in GVHD.

Keratinocyte dysplasia in hematopoietic stem cell transplantation recipients in the day-28-to-84 posttransplantation period.

Severe keratinocyte dysplasia (SKD) has been reported as a common event in the early posttransplantation period of hematopoietic stem cell transplantation patients. The purpose of our study is to determine the possible causes of SKD during the intermediate posttransplantation period and to ascertain its prevalence in skin biopsies. Skin biopsy slides, obtained from hematopoietic stem cell transplantation recipients who were days 28 to 84 posttransplantation, were evaluated for SKD. Forty-four examples of SKD were identified in 467 slides, or 9%. Thirty-seven patients were evaluated as cases in a case-control design. SKD was strongly associated with a conditioning regimen containing busulfan with an odds ratio of 7.25 (P = .0002). In a multivariate-adjusted analysis, SKD was not associated with cyclophosphamide, fludarabine, total-body irradiation, or a nonmyeloablative conditioning regimen. SKD was not associated with clinical acute graft-versus-host disease. SKD histology gradually resolved, reaching a normal histology after an average of 241 days. This study finds that severe keratinocyte dysplasia in the period 28 to 84 days post-HSCT is strongly associated with a busulfan-conditioning regimen.

Randomized trial of hydroxychloroquine for newly diagnosed chronic graft-versus-host disease in children: a Children's Oncology Group study.

The Children's Oncology Group conducted a multicenter Phase III trial for chronic graft-versus-host disease (cGVHD). The double-blind, placebo-controlled, randomized study evaluated hydroxychloroquine added to standard therapy for children with newly diagnosed cGVHD. The study also used a novel grading and response scoring system and evaluated clinical laboratory correlates of cGVHD. The primary endpoint was complete response (CR) after 9 months of therapy. Fifty-four patients (27 on each arm) were enrolled before closure because of slow accrual. The CR rate was 28% in the hydroxychloroquine arm versus 33% in the placebo arm (odds ratio [OR] = 0.77, 95% confidence interval [CI]: 0.20-2.93, P = .75) for 42 evaluable patients. For 41 patients with severity assessment at enrollment, 20 (49%) were severe and 18 (44%) moderate according to the National Institutes of Health Consensus Conference global scoring system. The CR rate was 15% for severe cGVHD and 44% for moderate cGVHD (OR = 0.24, 95% CI: 0.05-1.06, P = .07). Although the study could not resolve the primary question, it provided important information for future cGVHD study design in this population.

Durable donor engraftment after radioimmunotherapy using α-emitter astatine-211-labeled anti-CD45 antibody for conditioning in allogeneic hematopoietic cell transplantation.

To reduce toxicity associated with external γ-beam radiation, we investigated radioimmunotherapy with an anti-CD45 mAb labeled with the α-emitter, astatine-211 ((211)At), as a conditioning regimen in dog leukocyte antigen-identical hematopoietic cell transplantation (HCT). Dose-finding studies in 6 dogs treated with 100 to 618 µCi/kg (211)At-labeled anti-CD45 mAb (0.5 mg/kg) without HCT rescue demonstrated dose-dependent myelosuppression with subsequent autologous recovery, and transient liver toxicity in dogs treated with (211)At doses less than or equal to 405 µCi/kg. Higher doses of (211)At induced clinical liver failure. Subsequently, 8 dogs were conditioned with 155 to 625 µCi/kg (211)At-labeled anti-CD45 mAb (0.5 mg/kg) before HCT with dog leukocyte antigen-identical bone marrow followed by a short course of cyclosporine and mycophenolate mofetil immunosuppression. Neutropenia (1-146 cells/µL), lymphopenia (0-270 cells/µL), and thrombocytopenia (1500-6560 platelets/µL) with prompt recovery was observed. Seven dogs had long-term donor mononuclear cell chimerism (19%-58%), whereas 1 dog treated with the lowest (211)At dose (155 µCi/kg) had low donor mononuclear cell chimerism (5%). At the end of follow-up (18-53 weeks), only transient liver toxicity and no renal toxicity had been observed. In conclusion, conditioning with (211)At-labeled anti-CD45 mAb is safe and efficacious and provides a platform for future clinical trials of nonmyeloablative transplantation with radioimmunotherapy-based conditioning.

Tolerance to vascularized composite allografts in canine mixed hematopoietic chimeras.

BACKGROUND: Mixed donor-host chimerism, established through hematopoietic cell transplantation (HCT), is a reproducible strategy for the induction of tolerance toward solid organs. Here, we ask whether a nonmyeloablative conditioning regimen establishing mixed donor-host chimerism leads to tolerance of antigenic vascularized composite allografts. METHODS: Stable mixed chimerism was established in dogs given a sublethal dose (1-2 Gy) total body irradiation before and a short course of immunosuppression after dog leukocyte antigen-identical marrow transplantation. Vascularized composite allografts from marrow donors were performed after a median of 36 months (range, 4-54 months) after HCT. RESULTS: All marrow recipients maintained mixed donor-host hematopoietic chimerism and accepted vascularized composite allografts for periods ranging between 52 and 90 weeks; in turn, marrow donors rejected vascularized composite allografts from their respective marrow recipients within 18 to 29 days. Biopsies of muscle and skin of vascularized composite allografts from mixed chimeras showed few infiltrating cells compared with extensive infiltrates in biopsies of vascularized composite allografts from marrow donors. Elevated levels of CD3+ FoxP3+ T-regulatory cells were found in skin and muscle of vascularized composite allografts of mixed chimeras compared with normal tissues. In mixed chimeras, increased numbers of T-regulatory cells were found in draining compared with nondraining lymph nodes of vascularized composite allografts. CONCLUSIONS: These data suggest that nonmyeloablative HCT may form the basis for future clinical applications of solid organ transplantation and that T-regulatory cells may function toward maintenance of the vascularized composite allograft.

Inhibition of IL-32 activation by α-1 antitrypsin suppresses alloreactivity and increases survival in an allogeneic murine marrow transplantation model.

Interleukin (IL)-32 was originally identified in natural killer cells and IL-2-activated human T lymphocytes. As T cells are activated in allogeneic transplantation, we determined the role of IL-32 in human mixed lymphocyte cultures (MLCs) and GVHD. In allogeneic MLCs, IL-32 increased two-fold in responding T cells, accompanied by five-fold increases of TNFα, IL-6, and IL-8. After allogeneic hematopoietic cell transplantation, IL-32 mRNA levels in blood leukocytes were statistically significantly higher in patients with acute GVHD (n = 10) than in serial samples from patients who did not develop acute GVHD (n = 5; P = .02). No significant changes in IL-32 levels were present in patients with treated (n = 14) or untreated (n = 8) chronic GVHD, compared with healthy controls (n = 8; P = .5, and P = .74, respectively). As IL-32 is activated by proteinase-3 (PR3), we determined the effect of the serine protease inhibitor α-1 antitrypsin (AAT) on IL-32 levels and showed suppression of IL-32 and T-lymphocyte proliferation in MLCs. In an MHC-minor antigen disparate murine transplant model, preconditioning and postconditioning treatment with AAT resulted in attenuation or prevention of GVHD and superior survival compared with albumin-treated controls (80% vs 44%; P = .04). These findings suggest that AAT modulates immune and inflammatory functions and may represent a novel approach to prevent or treat GVHD.

Long-term regulation of genetically modified primary hematopoietic cells in dogs.

We report long-term results from a large animal model of in vivo selection. Nine years ago, we transplanted two dogs (E900 and E958) with autologous marrow CD34(+) cells that had been transduced with a gammaretrovirus vector encoding a conditionally activatable derivative of the thrombopoietin receptor. Receptor activation through administration of a chemical inducer of dimerization (CID) (AP20187 or AP1903) confers a growth advantage. We previously reported responses to two 30-day intravenous (i.v.) courses of AP20187 administered within the first 8 months post-transplantation. We now report responses to 5-day subcutaneous (s.c.) courses of AP20187 or AP1903 at months 14, 90, and 93 (E900), or month 18 (E958), after transplantation. Long-term monitoring showed no rise in transduced cells in the absence of drug. Retroviral insertion site analysis showed that 4 of 6 (E958) and 5 of 12 (E900) transduced hematopoietic cell clones persisted lifelong. Both dogs were euthanized for reasons unrelated to the gene therapy treatment at 8 years 11 months (E958) and 11 years 1 month (E900) of age. Three clones from E900 remained detectable in each of two secondary recipients, one of which was treated with, and responded to, AP1903. Our results demonstrate the feasibility of safely regulating genetically engineered hematopoietic cells over many years.

Evaluation of posttransplant methotrexate to facilitate engraftment in the canine major histocompatibility complex-haploidentical nonmyeloablative transplant model.

BACKGROUND: Posttransplant cyclophosphamide has been shown to control graft-versus-host disease and facilitate engraftment in the major histocompatibility complex-haploidentical transplant setting. Here, we hypothesized that methotrexate (MTX) could be used in a similar fashion. In patients with genetic diseases, the use of MTX rather than an alkylating agent such as cyclophosphamide would be preferable due to its reduced risk of promoting secondary malignancies. METHOD: Using our standard conditioning regimen consisting of a specific anti-CD44 mAb (S5) and 200 cGy total body irradiation followed by postgrafting immunosuppression with cyclosporine and mycophenolate mofetil as a control group, we compared outcomes with experimental animals receiving the same regimen with the addition of a single, large dose of posttransplant MTX on day +3 (50-400 mg/m2). RESULTS: Adding MTX at all dose levels did not abrogate initial engraftment and controlled graft-versus-host disease in most cases. Dogs receiving MTX at the first dose level (50 mg/m2) improved time to rejection compared with controls (P=0.03) but did not decrease overall rates of rejection (P=0.56). However, increasing the dose of MTX beyond 50 mg/m2 seemed to have detrimental effects in both average (P=0.04) and peak (P=0.002) donor chimerism. Increasing the dose of MTX also promoted more profound lymphopenia. Finally, delaying cyclosporine and mycophenolate mofetil until after MTX administration did not seem to significantly improve engraftment kinetics. CONCLUSION: Adding high-dose MTX seemed to benefit the duration of donor chimerism at the lowest dose studied, but there was no benefit when escalating MTX doses to toxicity.

Chemokine receptor CCR5 mediates alloimmune responses in graft-versus-host disease.

Allogeneic bone marrow transplantation (BMT) is an effective therapy for hematologic malignancies. However graft-versus-host disease (GVHD) is a major limiting factor for a successful patient outcome. GVHD is a result of alloimmune responses of donor T lymphocytes attacking the recipient's cells and tissues. Chemokine receptor CCR5 plays a role in solid organ allograft rejection and mediates murine GVHD pathogenesis. Herein, we report that infiltrating lymphocytes in the skin of human acute GVHD (aGVHD) samples are predominantly CCR5(+) T cells. In addition, we characterized the features of the CCR5 expression on alloreactive T lymphocytes. We found that the CCR5(+) population exhibits the characteristics of the activated effector T cell phenotype. CCR5 expression is upregulated upon allogenic stimulation, and CCR5(+) cells are proliferating with coexpression of T cell activation markers. Furthermore, the activated T cells producing inflammatory cytokine tumor necrosis factor (TNF)alpha, interleukin (IL)-2, or interferon (IFN)-gamma, are positive for CCR5. Thus, CCR5 is a marker for GVHD effector cells and CCR5(+) T cells are active participants in the pathogenesis of human aGVHD.

Blocking LFA-1 activation with lovastatin prevents graft-versus-host disease in mouse bone marrow transplantation.

Graft-versus-host disease (GVHD) following bone marrow transplantation (BMT) is mediated by alloreactive donor T lymphocytes. Migration and activation of donor-derived T lymphocytes play critical roles in the development of GVHD. Leukocyte function-associated antigen-1 (LFA-1) regulates T cell adhesion and activation. We previously demonstrated that the I-domain, the ligand-binding site of LFA-1, changes from the low-affinity state to the high-affinity state on LFA-1 activation. Therapeutic antagonists, such as statins, inhibit LFA-1 activation and immune responses by modulating the affinity state of the LFA-1 I-domain. In the present study, we report that lovastatin blocked mouse T cell adhesion, proliferation, and cytokine production in vitro. Furthermore, blocking LFA-1 in the low-affinity state with lovastatin reduced the mortality and morbidity associated with GVHD in a murine BMT model. Specifically, lovastatin prevented T lymphocytes from homing to lymph nodes and Peyer's patches during the GVHD initiation phase and after donor lymphocyte infusion (DLI) after the establishment of GVHD. In addition, treatment with lovastatin impaired donor-derived T cell proliferation in vivo. Taken together, these results indicate the important role of lovastatin in the treatment of GVHD.

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.

Transmission and expansion of HOXB4-induced leukemia in two immunosuppressed dogs: implications for a new canine leukemia model.

OBJECTIVE: There are currently no large animal models to study the biology of leukemia and development of novel antileukemia therapies. We have previously shown that dogs transplanted with homeobox B4 (HOXB4)-transduced autologous CD34(+) cells developed myeloid leukemia associated with HOXB4 overexpression. Here we describe the transmission, engraftment, and expansion of these canine leukemia cells into two genetically unrelated, immunosuppressed dogs. MATERIALS AND METHODS: Two dogs immunosuppressed after major histocompatibility complex-haploidentical hematopoietic cell transplantation and exhibiting mixed donor-host chimerism were accidentally infused trace amounts of HOXB4-overexpressing leukemia cells from a third-party dog. RESULTS: Six weeks after infusion of HOXB4-overexpressing leukemia cells, these two dogs rapidly developed myeloid leukemia consisting of marrow and organ infiltration, circulating blasts, and, in one dog, chloromatous masses. Despite neither of these dogs sharing any dog leukocyte antigen haplotypes with the sentinel case, the HOXB4-transduced clones engrafted and proliferated without difficulty in the presence of immunosuppression. Chimerism studies in both dogs confirmed that donor and, in one case, host hematopoietic cell engraftment was lost and replaced by third-party HOXB4 cells. CONCLUSIONS: The engraftment and expansion of these leukemia cells in dogs will allow studies into the biology of leukemia and development and evaluation of novel antileukemia therapies in a clinically relevant large animal model.