ALBERT

Description: Sustained engraftment after marrow transplantation from major histocompatibility complex matched donors depends on the intensities of both pretransplant conditioning and postgrafting immunosuppression. Here we asked, both in a preclinical canine model and in the clinical setting, whether, additionally, the number of transplanted marrow cells had a role in the success of engraftment after minimally intensive conditioning. Thirty-four dogs received marrow from dog leukocyte antigen (DLA)-matched related donors after 1 Gy total body irradiation (TBI). Postgrafting immunosuppression included cyclosporine with or without added drugs, most often mycophenolate mofetil, for no more than 5 weeks. Median total nucleated cell (TNC) doses administered were 3.6 (range 1.7–7.98) x 108/kg. Donor chimerism reached median (range) peaks of 15 (2–75) % for granulocytes and 9 (2–55) % for mononuclear cells (MNC), respectively. Eventually, 31 dogs rejected their grafts, after a median of 10 (range 3–20) weeks. TNC doses had significant impacts on outcomes, after adjustment for immunosuppressive regimens received (Table 1); each increase by 1x108/kg decreased the hazard ratio (HR) of rejection by 0.7, prolonged time to rejection by 2 weeks, and increased maximal percentages of donor chimerism by 8% in granulocytes and by 5% in MNC. Eighteen patients with hematological malignancies received marrow grafts from 10/10 human leukocyte antigen (HLA)-matched unrelated donors, after conditioning with Fludarabine, 90 mg/m2, and 2 Gy TBI, followed by postgrafting mycophenolate mofetil and cyclosporine. Median (range) TNC doses were 3.3 (1.3–4.6) x 108/kg, CD34 cells were 2.8 (0.9–7.2) x 106/kg, and CD3 cells were 31 (16–62) 106/kg. Donor chimerism reached median peaks of 95 (0–100) % in granulocytes and 43 (0–100) % in T cells. Eight patients rejected their grafts after a median of 1 (range 1–4.5) month. The numbers of CD34 and CD3 cells infused were not significantly associated with the risk of rejection (Table 2). However, TNC doses significantly impacted on engraftment; each increase in TNC dose by 1x108/kg decreased the HR of rejection by a factor of 0.3 and increased percentages of donor T-cell chimerism by 18% (p=0.05); there was no correlation with granulocyte chimerism. This effect was not negated by adjustment for patient age, diagnosis group, and numbers of preceding cycles of chemotherapy. In conclusion, while allogeneic marrow grafts are possible following minimal intensity conditioning in dogs and human patients, eventual graft rejection is a major problem, the risk of which can be minimized by transplanting the largest number of nucleated marrow cells possible. Impact of an increase in TNC dose by 108 cells/kg on outcomes of allogeneic canine marrow transplantation Outcome p-value 95% CI Rejection HR = 0.7 0.04 0.5–1.0 Time to rejection Prolonged by 2 weeks 0.02 0.4–4.0 Granulocyte chimerism Increased by 8% 0.006 2.4–13.0 Mononuclear cell chimerism Increased by 5% 0.003 1.9–8.6 Impact of marrow cell doses on rejection after allogeneic human marrow transplantation Cell populations and doses Hazard Ratio of Rejection p-value 95% CI TNC 108/kg increase 0.3 0.02 0.1–0.8 CD34 106/kg increase 0.6 0.21 0.3–1.3 CD3 0.6 0.44 0.2–2.2

Description: 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.

Description: Cord blood (CB) is increasingly used for hematopoietic cell transplantation (HCT) due to its rapid availability and less stringent HLA matching requirements. However, low cell dose of available CB units and delayed engraftment remain significant obstacles for increased use of CBT in adults. Recent clinical results with 2-unit CBT may help overcome these problems. We sought to develop a large animal model of CBT to improve the understanding of engraftment across histocompatibility barriers and cell dose limitations of multiple units of CBT. Since the outbred dog is a very well-established preclinical model of HCT, our first aim was to establish feasibility of canine CBT. We harvested and cryopreserved individual units of canine CB obtained from litters following Caesarian section at day 56 to 60 of gestation. We asked if multiple units of previously cryopreserved, DLA-identical sibling CB could engraft in DLA-identical recipient dogs after myeloablative total body irradiation (TBI). Five adult dogs (8–18 months of age) received 920 cGy TBI (7 cGy/min) followed by intravenous infusion of 2 to 4 units of thawed CB with a combined total nucleated cell dose range 0.4x107 − 2.6x107/kg. Transplanted total CD34+/kg cell dose range was 0.5−2.5x105/kg. Postgrafting immunosuppression was combined cyclosporine and mycophenolate mofetil for 35 and 28 days, respectively. G-CSF was given until recovery of neutrophil counts, and dogs received blood product transfusion support and intravenous and oral antibiotics. Four dogs engrafted and survived; they are currently 35 to 212 days after CBT. One dog died on day 13 due to neutropenic sepsis. All 4 surviving dogs engrafted with sustained neutrophil recovery 〉1000/mL between 29 – 35 days after CBT. Bi-weekly chimerism analysis was assessed by PCR using informative microsatellite markers. For each of the 4 surviving recipients all transplanted donor CB units were detected after engraftment. However, among the 3 dogs currently at 3 to 7 months after CBT, only 1 of the multiple transplanted CB units dominated hematopoiesis with 75–95% donor chimerism. For 2 of the 3 dogs, the CB unit with the highest CD34+ dose was the dominant donor. There was no evidence of acute or chronic graft-versus-host disease (GVHD). Among the 3 dogs at 3 to 7 months after CBT, immune reconstitution studies included normal T cell proliferation stimulation index (mixed lymphocyte reaction) and gradual recovery of the absolute number of CD4 and CD8 T-cell subsets. In summary, cryopreserved DLA-matched CB successfully engrafted and provided durable hematopoietic recovery in lethally irradiated dogs. The cell dose of transplanted CB units was comparable with CB units for adult human transplantation. These results establish that multiple units of canine CB with limited cell dose can engraft and initiate immune reconstitution without GVHD in DLA-identical dogs transplanted after marrow-lethal irradiation.

Description: Although hematopoietic cell transplantation is generally accomplished utilizing a single donor and recipient pair, multiple donors have been used to mediate engraftment, particularly in the case of low donor cell counts as in umbilical cord blood transplantation. The general engraftment outcome of HLA nonidentical cord blood transplantation is the predominance of one of the units over the other. Here we pose the question whether in the canine hematopoietic cell transplantation model, can we establish multiple donor chimerism using two DLA-identical donors and a single recipient. We identified 8 triplets of DLA-identical littermates by matching highly polymorphic microsatellite markers within DLA class I and class II regions and confirmed by DLA-DRB1 gene sequencing. The marrow recipients received 2 Gy total body irradiation followed by intravenous infusions of marrow cells from both donors 1 and 2. The median number of donor cells injected was 4.1 (range = 2.0–7.0) X 108 cells/kg. Post grafting immunosuppression consisted of cyclosporine (CSP) and mycophenolate mofetil (MMF) given for 35 and 28 days, respectively. The median time to hematological recovery (blood counts equivalent to pre-HCT levels) was 38 (range = 30–42) days. The degrees of chimerism were determined using variable number tandem repeat-polymerase chain reaction (VNTR-PCR) methods. As shown in the Table, sustained trichimerism occurred in 4 out of 8 dogs with engraftment for a period grater than 26 weeks. For G631, both donor grafts were rejected shortly following discontinuation of CSP and MMF. Dog G513 developed graft versus host disease (GvHD) which was successfully treated with a short course of CSP. Five dogs received kidney allografts from one of the respective HCT donors 6 months after HCT to assess donor specific immune tolerance. Chimerism Analysis of Dogs Receiving Marrow from Two Donors Duration of Engraftment in Weeks (% Chimerism) Recipient Donor 1 Donor 2 Recipient GVHD Accept Donor 1 Kidney Graft (wks) ND = not determined; [R] = rejection G158 〉43 (5%) 37 (0%) [R] 〉43 (95%) No Yes (20) G193 〉44 (72%) 16 (0%) [R] 〉44 (28%) No Yes (10) G362 〉47 (28%) 〉47 (55%) 〉47 (17%) No Yes (16) G513 〉47 (53%) 〉47 (22%) 〉47 (25%) Yes Yes (16) G551 〉32 (35%) 〉32 (20%) 〉32 (45%) No ND G631 8 (10%) [R] 8 (12%) [R] 8 (〉88%) No ND G643 〉30 (28%) 〉30 (40%) 〉30 (23%) No Yes (4) G664 〉26 (50%) 6 (2%) [R?] 〉26 (48%) No ND Kidney allografts were found to be essentially free of inflammation when assessed histologically on biopsy. Dog G643, was tested for immune function by a mixed leukocyte reaction and found to be competent against DLA nonidentical stimulator cells but nonresponsive against either of the donor stimulator cells. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow stem cells from two DLA-identical littermates can result in trichimerism. Furthermore, immunological tolerance to multiple hematopoietic cell donors can include a solid organ graft from one of the marrow donors.

Description: 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.

Description: 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.

Description: Genetically modified donor T cells with an inducible “suicide” gene have the potential to improve the safety and availability of allogeneic hematopoietic stem cell transplantation by enhancing engraftment and permitting control of graft-versus-host disease (GVHD). However, several clinical studies of gene-modified T cells have shown limited to no in vivo function of the ex vivo expanded T cells. Using the well-established dog model of allogeneic marrow transplantation, the question was asked if retrovirally transduced, donor derived, ex vivo expanded cytotoxic T lymphocytes (CTLs) that are recipient specific could enhance engraftment of dog leukocyte antigen (DLA)–haploidentical marrow following a single dose of 9.2 Gy total body irradiation and no postgrafting immunosuppression. In this setting, only 4 of 11 control recipients of DLA-haploidentical marrow without added CTLs engrafted. CTLs did not enhance engraftment of CD34+ selected peripheral blood stem cells. However, recipient-specific CTLs enhanced engraftment of DLA-haploidentical marrow in 9 of 11 evaluable recipients (P = .049). All dogs that engrafted developed multiorgan GVHD. To facilitate in vivo tracking, 8 dogs received CTLs transduced with a retroviral vector encoding green fluorescent protein (GFP) and neomycin phosphotransferase (neo). Recipients that engrafted had sharp increases in the numbers of circulating GFP+ CTLs on days +5 to +6 after transplantation. GFP+ CTLs isolated from blood were capable of recipient-specific lysis. At necropsy, up to 7.1% of CD3+ cells in tissues were GFP+ and polymerase chain reaction in situ hybridization for neoshowed infiltration of transduced CTLs in GVHD-affected organs. These results show that ex vivo expanded, transduced T cells maintained in vivo function and enhanced marrow engraftment.

Description: Stable mixed chimerism can be established in dogs given a sublethal dose of 200cGy total body irradiation (TBI) before and immunosupression with mycophenolate mofetil (MMF) and Cyclosporine (CSP). When the TBI dose is reduced to 100 cGy, only transient engraftment is observed. Here we asked whether stable engraftment after 100 cGy TBI could be accomplished by first reducing the intensity of host immune responsiveness with the help of an anti-CD154 antibody which blocks the CD40-CD154 pathway. Accordingly, recipients were given a single i.v. injection of 5 mg/kg anti-CD154 antibody (day -5) followed one day later by combined i.v./sc. injections of 107 peripheral white blood cells/kg from their intended DLA-identical littermate marrow donors. TBI, 100cGy (delivered at 7 cGy/min), was given on day 0 followed by infusion of a median of 4.22x108 marrow cells/kg. Postgrafting immunosuppression consisted of MMF (10mg/kg, BID, days 0 to 28) and CSP (15 mg/kg BID, days -1 to 35). Six dogs have been transplanted. Two dogs are too early after transplant for evaluation, while four dogs have been followed for 〉9, 12, 〉18 and 〉26 weeks. All four dogs had initial engraftment. Three of four dogs have continued to show stable mixed donor/host hematopoietic chimerism among lymphocytes and granulocytes from the peripheral blood. One dog rejected the graft 12 weeks after transplantation. All six transplanted dogs are alive and well. Besides mild allergic reactions to the antibody in five of the six dogs (redness of the eyes, skin rashes and dry mucous membranes), no additional side effects attributed to the pretransplant treatment were observed. Data were consistent with previous findings in dogs, in which the costimulatory signal between B7 and CD28 was blocked, and support the hypothesis, that stable marrow allografts would be established by combining non-myeloablative pretransplant host immunosuppression and posttransplant immunosupression of both host and donor cells using MMF and CSP.