The influence of pretransplantation conditioning on graft-vs.-leukemia effect in mice.

OBJECTIVE: The therapeutic potential of allogeneic hematopoietic stem cell transplantation relies on the graft-vs.-leukemia (GVL) effect to eradicate residual tumor cells immunologically. The relationship of conditioning intensity to GVL effect was investigated. MATERIALS AND METHODS: Lethally irradiated (either 900 or 1300 cGy) B6D2F1 (H-2(b/d)) recipients were transplanted from B6 (H-2(b)) donors. P815 or L1214 (H-2(d)) tumor cells were injected intravenously or subcutaneously on day 1 post-transplantation to generate a GVL model. RESULTS: Tumors in allogeneic mice treated with 1300 cGy exhibited markedly delayed subcutaneous tumor growth in vivo as compared with mice treated with 900 cGy, while intravenous tumor growths were comparable between the two radiation doses. Serum levels of tumor necrosis factor-α or interferon-γ were similar and the percentages of donor T-cell proliferation or apoptosis early after hematopoietic stem cell transplantation were comparable. The numbers of CD8(+) T cells from recipients that received 1300 cGy were significantly elevated in skin and tumor tissues. CD62L(low) and CD44(high) CD8(+) T-cell fractions were also elevated in 1300 cGy. After the higher radiation dose, P815-specific interferon-γ responses of splenic CD8(+) T cells were markedly enhanced and the fractions of T cells of interferon-γ-producing T cells in tumor tissues but not in spleen were increased. The protein concentrations of chemokines in skin and tumor tissues were substantially elevated in 1300 cGy compared to 900 cGy. CONCLUSIONS: The higher radiation dose (1300 vs. 900 cGy) resulted in significantly enhanced GVL effect against an extramedullary tumor and the alterations in effector T-cell trafficking into tumor tissue are the most likely mechanism.

Treatment with GM-CSF secreting myeloid leukemia cell vaccine prior to autologous-BMT improves the survival of leukemia-challenged mice.

Vaccination with irradiated autologous tumor cells, engineered to secrete granulocyte macrophage-colony stimulating factor (GM-CSF) (GM tumor), can generate potent antitumor effects when combined with autologous bone marrow transplantation (BMT). That notwithstanding, the post-BMT milieu, characterized by marked cytopenia, can pose a challenge to the implementation of vaccine immunotherapies. To bypass this problem, partial post-BMT immune reconstitution has been allowed to develop prior to vaccination. However, delaying vaccination can also potentially allow the expansion of residual tumor cells. Other approaches have used reinfusion of "primed" autologous lymphocytes and multiple administrations of GM tumor cells, which required the processing of large amounts of tumor. Utilizing the MMB3.19 murine myeloid leukemia model, we tested whether a single dose of GM tumor cells, 7 days prior to syngeneic BMT, could be a curative treatment in MMB3.19-challenged recipient mice. This vaccination protocol significantly improved survival of mice by eliciting long-lasting host immune responses that survived lethal irradiation, and were even protective against post-BMT tumor rechallenge. Furthermore, we demonstrated that mature donor lymphocytes can also play a limited role in mounting the antitumor response, but our pre-BMT vaccination strategy obviated the need for either established de novo immune reconstitution or the use of multiple post-BMT immunizations.

Prevention of acute graft-vs-host disease by a single low-dose cyclophosphamide injection following allogeneic bone marrow transplantation.

OBJECTIVE: Previously, we documented that conditioning based on donor-specific cell transfusion (DST) and subsequent selective depletion of activated donor-reactive cells by cyclophosphamide (CY) facilitates alloengraftment in a murine transplantation model. Transplantation event represents a strong immunogenic stimulus for host-reactive donor T cells that induce graft-vs-host disease (GVHD). Therefore, in this study, we addressed the question of whether a single posttransplantation CY administration (CY2) can prevent acute GVHD-related mortality without compromising engraftment of allogeneic transplant. MATERIALS AND METHODS: Splenocyte-enriched C57BL/6 bone marrow was transplanted to BALB/c recipients after mild irradiation, and conditioning with DST and 100 mg/kg CY. Following transplantation, recipients were left untreated or given on a specified day a single CY2 injection (50 mg/kg). All animals were monitored for survival, chimerism, and clinical signs of GVHD. Experimental mice that received BCL1 leukemia cells before transplantation were monitored for leukemia-related mortality as well. RESULTS: Animals that received no CY2 after transplantation died of acute GVHD. A single low-dose CY2 treatment within the first 5 days after transplantation prevented mortality in most recipients. However, only CY2 administration on days +1 or +5 preserved chimerism. Most chimeras survived GVHD-free for >200 days. Prolonged persistence of host-reactive T cells in mice (CY2 on day +5) permitted a reduction to be made in engraftment-essential irradiation dose and preserved a strong graft-vs-leukemia effect of transplantation. CONCLUSION: Acute GVHD can be prevented in mice by a single properly timed posttransplantation low-dose CY administration.

Myeloablative allografting for chronic lymphocytic leukemia: evidence for a potent graft-versus-leukemia effect associated with graft-versus-host disease.

In all, 30 patients with CLL proceeded to myeloablative allogeneic BMT using related (n=20, 67%) or unrelated (n=10) donors, at the Princess Margaret Hospital (Toronto) (n=20) or the Leukemia/BMT Program of BC (Vancouver) (n=10), from 1989 to 2001. Median (range) interval from diagnosis to BMT was 4.8 (0.3-13) years, median number of prior therapies was three and median age 48 years. The preparative regimen included total body irradiation in 15 (50%). In all, 14 of 30 patients (47%) are alive, with median (range) follow up of 4.3 (2.4-10.5) years. All are in complete remission, two following therapy for post-BMT progression. Actuarial overall (OS) and event-free survival (EFS) at 5 years is 39% (OS 48% for related donor and 20% for unrelated donor BMT); cumulative incidence of nonrelapse mortality (NRM) and relapse is 47 and 19%, respectively. Both acute (RR=0.008, P=0.01) and chronic (RR=0.006, P=0.02) Graft-versus-host disease (GVHD) were associated with markedly decreased risk of relapse. Patients receiving grafts from unrelated donors had increased NRM (RR=3.6, P=0.02) and decreased OS (RR of death=3.4, P=0.002). Allogeneic BMT has resulted in long-term EFS in approximately 40% of patients with CLL. There is evidence for a strong graft-versus-leukemia effect associated with acute and chronic GVHD, resulting in near complete protection from relapse.

Unrelated bone marrow transplantation for high-risk anaplastic large cell lymphoma in pediatric patients: a single center case series.

OBJECTIVES: The use of allogeneic stem cell transplantation in NHL patients is not yet clearly defined, especially in children and adolescents, but this option offers the advantages of a tumor-free graft and the possible induction of a graft-vs.-tumor effect. PATIENTS AND METHODS: We report the results of four consecutive pediatric patients affected by anaplastic large cell lymphoma (ALCL) and treated with allogeneic stem cell transplantation from an unrelated donor. The conditioning regimen was based on total body irradiation given in association with etoposide in three patients, and with thiotepa and cyclophoshamide in one patient. Graft-vs.-host disease (GVHD) prophylaxis consisted of cyclosporin, a short course of methotrexate and rabbit antithymocyte globulin. RESULTS: All patients had rapid engraftment within 3-4 wk for neutrophils and platelets, and achieved a stable full donor chimerism that has been maintained to the last follow-up visit. One patient later developed a restrictive pneumonopathy. This patient had been heavily pretreated during the course of the disease having suffered four relapses and had received a cumulative dose of bleomycin of 160 mg/m(2). After a follow-up of 11-42 months, all patients are alive in complete hematological and molecular remission; and three of them without any chronic GVHD. CONCLUSIONS: The increasing number of volunteer bone marrow donors and the reduced toxicity of unrelated stem cell transplantation, especially in children, make this therapeutic option worth more extensive investigation in the treatment of high-risk failure ALCL, although more data is needed to evaluate the long-term benefits. In this regard, the presence of factors predictive of worst outcome such as an early relapse (within 12 months from diagnosis), a refractory or relapsing ALCL and the persistent detection on blood or bone marrow of nucleophosmin-anaplastic lymphoma kinase protein (NPM-ALK) transcript may help select the patients eligible to allogeneic related or unrelated stem cell transplantation.

Prevention of graft-versus-host disease while preserving graft-versus-leukemia effect after selective depletion of host-reactive T cells by photodynamic cell purging process.

In this study, we investigated the possibility of selective depletion of donor alloantigen-specific T cells from C57BL/6 (H-2(b)) mice to prevent graft-versus-host disease (GVHD). These cells were first activated with irradiated BALB/c (H-2(d)) host spleen cells in a 5-day mixed lymphocyte culture. Following this activation, a photoactive rhodamine derivative called 4,5-dibromorhodamine 123 (TH9402), was added. This compound is selectively retained in the mitochondria of activated host-reactive cells but not tumor- or third-party-specific resting cells. The treated cells were subsequently exposed to visible light (514 nm) to deplete the TH9402-enriched activated host-reactive cells. Treatment with photodynamic cell purging process (PDP) inhibited antihost responses measured by cytotoxic T lymphocytes (CTL) by 93%, and interferon-gamma production by 66%. By contrast, anti-BCL1 (BALB/c-origin leukemia/lymphoma) and anti-third-party C3H/HeJ (H-2(k)) responses were preserved. PDP-treated primed C57BL/6 cells were further tested in vivo. All lethally irradiated BALB/c mice inoculated with BCL1 cells and T-cell-depleted bone marrow cells developed leukemia by day +30, with 50% mortality by 100 days. All mice died of GVHD after addition of 5 x 10(6) untreated primed C57BL/6 cells. However, addition of same numbers of PDP-treated cells allowed 90% of the recipients to survive more than 100 days without detectable BCL1 tumor cells and free of GVHD. Moreover, PDP-treated primed C57BL/6 cells retained the ability to induce GVHD in the third-party C3H/HeJ mice. These data suggest that PDP can selectively deplete host alloantigen-specific T cells for GVHD prevention and immune and antileukemia function preserve.

Transforming growth factor-beta inhibits lymphokine activated killer cytotoxicity of bone marrow cells: implications for the graft-versus-leukemia effect in irradiation allogeneic bone marrow chimeras.

BACKGROUND: We have previously shown that allogeneic bone marrow (BM) chimeras preconditioned with total lymphoid irradiation and low-dose total body irradiation (TLI/TBI) develop a stronger graft-versus-leukemia (GVL) effect than chimeras preconditioned with high-dose total body irradiation only (TBI). Here, we report on the possible role of cytokines in the mechanism underlying this GVL effect. METHODS: Splenic mRNA levels of the cytokines interleukin (IL)-1, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta (TGF-beta), and of inducible nitric oxide synthetase were determined by reverse transcription-polymerase chain reaction in TLI/TBI- or TBI-conditioned C3H/AKR BM chimeras challenged with AKR-type BW5147.3 leukemia cells. Ex vivo TGF-beta protein production by splenocytes was determined using ELISA. The possibility that cytokines influence the GVL effect by modulating the activity of IL-2-activated lymphocytes (LAK cells) was investigated by in vitro assays on donor-type BM cells. RESULTS: Of all cytokine mRNA levels studied, those of TGF-beta and IL-7 were different between groups; both were significantly more elevated in TBI- than in TLI/ TBI-conditioned or normal mice. Differences were apparent after conditioning and were not influenced by additionally injected BM or leukemia cells. Cultured splenocytes of TBI-conditioned animals produced significantly more TGF-beta protein than those of TLI/TBI-conditioned ones or normal controls. r-TGF-beta but not r-IL-7 suppressed in vitro LAK activity of donor-type BM cells against BW5147.3 cells in a dose-dependent way. CONCLUSIONS: High-dose TBI-induced, host-derived splenic TGF-beta may inhibit generation of LAK cells from subsequently transplanted donor BM cells, suppressing their capacity to generate cytotoxicity upon injection of leukemia cells. The cytokine profile, induced by irradiation in host hematopoietic organs, can significantly modify posttransplant immunological processes such as the GVL effect and graft-versus-host disease (GVHD).