Allogeneic haematopoietic cell transplantation for multiple myeloma: reducing transplant-related mortality while harnessing the graft-versus-myeloma effect

Allogeneic haematopoietic cell transplantation (allo-HCT) provides effective therapy for patients with various haematological malignancies. In multiple myeloma (MM) this approach can induce response rates in 35-75% of patients. However, the outcome is hampered by high rates of treatment-related mortality (TRM). Reduced intensity conditioning to lower TRM has been successfully applied. The fact that previous clinical reports have documented graft-versus-myeloma (GVM) activity without graft-versus-host disease (GVHD) suggests that at least two distinct immunocompetent cell populations mediating GVHD and/or GVM may exist. Further characterization of effectors after allo-HCT and their targets may help to clarify the immune response that mediates the GVM effect. This review considers the clinical results with myeloablative and reduced intensity conditioning prior to allo-HCT for MM, with emphasis on attempts to prevent GVHD while preserving the GVM effect. Strategies including donor lymphocyte infusions as part of the allogeneic protocol and antigenic targets for GVM effect are reviewed.     

Unrelated donor leukocyte infusions to treat relapse after unrelated donor bone marrow transplantation

Allogeneic stem cell transplantation (SCT) may cure many patients with hematologic malignancies due to both the intensive conditioning therapy, and in many patients, the potent graft-vs-leukemia (GVL) effect of the donor graft. The GVL effect is mediated in large part by mature T-cells contained in the donor graft and has been defined in detail in animal models of transplantation. The GVL activity has been observed in the clinical setting after SCT from both matched siblings and unrelated donors. The best demonstration and most direct evidence of GVL activity in humans come from the use of donor leukocyte infusions (DLI). For patients who relapse with chronic myelogenous leukemia after matched sibling SCT, infusions of leukocytes collected from the original transplant donor will re-establish complete and durable remission in 60-80% of patients. DLI is less effective for more advanced phases of CML and for patients who relapse with diseases other than CML. DLI after matched sibling SCT is complicated primarily by graft-vs-host disease (GVHD), marrow aplasia, and unfortunately, relapse in some cases. There has been little information regarding the use of unrelated DLI (UDLI). Available data now shows that despite initial concerns that UDLI would result in excessive toxicity, it is an effective approach to relapse after unrelated donor marrow grafting. Response rates are similar to those seen after the use of matched sibling DLI, and many remissions remain durable. Graft-vs-host disease is a frequent complication after UDLI though the incidence and severity of GVHD is also similar to the use of matched sibling DLI. It is not clear that the GVL and GVHD effects can be separated, since the majority of responding patients also develop GVHD. The most effective cell dose for UDLI has not been established, though there does not appear to be either a dose-response or dose-toxicity relationship from UDLI. Although second unrelated donor bone marrow transplantation (BMT) may cure a small minority of patients, GVL induction with UDLI offers a safer and potentially more effective therapy for relapsed leukemia, and offers insights in methods to manipulate the human immune system for therapeutic benefit.     

Nonmyeloablative stem cell transplantation for lymphoma

High-dose chemotherapy and allogeneic stem cell transplantation is a potentially curative therapy for younger patients with non-Hodgkin's lymphoma (NHL). The benefits of this therapy, however, are largely offset by the high rate of treatment-related mortality, exceeding 40% in many studies. Risks increase with comorbidities, advanced age, histocompatibility, and disease-related prognostic factors. Given the potential efficacy of graft-versus-malignancy effects against many lymphoid malignancies, we evaluated an alternative strategy utilizing less toxic, nonmyeloablative conditioning regimens to allow engraftment of donor cells, and then exploit the graft-versus-lymphoma (GVL) effects of allogeneic transplantation as the primary therapy. This strategy involved fludarabine-based preparative regimens +/- high-dose rituximab, graft-versus-host disease (GVHD) prophylaxis for 6 months, and donor lymphocyte infusion (DLI) only for progressive or nonresponding disease. Results from these trials confirm the full potential on nonmyeloablative transplantation for patients with NHL.     

Graft-Versus-Host Disease in Multiple Myeloma Patients Treated With Daratumumab After Allogeneic Transplantation

IntroductionAllogeneic hematopoietic cell transplantation (allo-HCT) represents an adoptive immunotherapy strategy for eliciting a graft-versus-myeloma, the effect for high-risk or relapsed multiple myeloma (MM). Allo-HCT recipients are at risk for graft-versus-host disease (GVHD) as well as associated increases in morbidity and mortality. Daratumumab, an anti-CD38 human immunoglobulin G1 kappa humanized monoclonal antibody, is used for treatment of MM. Daratumumab also affects CD38⁺ nonmyeloma cells, including T cells, which mediate GVHD. The use of daratumumab after allo-HCT has not been well described, and its potential impact on GVHD is unknown.Patients and MethodsIn a multicenter retrospective study, we evaluated incidence of GVHD in 34 patients with relapsed MM treated with daratumumab after allo-HCT.ResultsOverall response to daratumumab (partial response or better) was 41% (95% confidence interval, 24-59). Five patients (15%) developed acute GVHD after daratumumab therapy; no chronic GVHD events were observed after daratumumab therapy. One of these 5 patients had a history of chronic GVHD and developed a flare of acute GVHD during daratumumab therapy. The remaining 4 patients did not have a history of GVHD before daratumumab.ConclusionThe incidence of GVHD after daratumumab was low and did not result in increased exacerbation of GVHD in patients with a history of GVHD.     

NK细胞的生物学特性及临床应用研究进展

 目前NK细胞的临床治疗主要通过利用细胞因子体内扩增、激活NK细胞和体外产生LAK、CIK细胞杀伤自体肿瘤细胞,但并未取得实质性进展。随着自然杀伤细胞抑制性受体（KIR）及其他NK细胞受体（NKR）的发现,对NK细胞受体在识别和裂解肿瘤细胞的重要作用的不断了解,为肿瘤的免疫治疗提供了新的治疗策略。由KIR独特型不相容引发的NK细胞异源反应性在异基因造血干细胞移植中促进移植物植入,预防GVHD发生以及增强GVL作用,已经成为国内外学者研究的热点。就目前关于NK细胞的生物学特性及临床应用研究进展予以综述。     

Experimental and clinical approaches for optimization of the graft-versus-leukemia effect

The goal of allogeneic (allo)-hematopoietic stem-cell transplantation (HSCT) in the treatment of hematologic malignancies is to harness the graft-versus-leukemia (GVL) effect, while minimizing the risk of graft-versus-host disease (GVHD). Allo-HSCT research has focused on the GVL target antigens and effector mechanisms, and on potential approaches to exploit GVL independently of GVHD. Donor lymphocyte infusion (DLI) achieves the most powerful anti-leukemic responses, and this approach is often used in combination with nonmyeloablative transplant regimens to optimize GVL and reduce GVHD. Serial, dose-escalating, and CD8(+) T-cell-depleted DLI have been introduced into clinical practice, while other variants of DLI have so far been explored only in animal models. The role of naturally occurring regulatory T cells in transplantation tolerance is being increasingly acknowledged, and murine studies indicate the potential ability of T cells to regulate GVHD while maintaining GVL. Experimental and clinical studies have demonstrated the importance of host-type chimerism, particularly for antigen-presenting cells, in determining the occurrence of DLI-induced GVL. Murine studies could assist in the development of clinical strategies targeted at antigen-presenting cells. Clinical studies exploiting natural killer-cell-mediated antitumor reactivity in the context of killer inhibitory receptor-ligand-mismatched allo-HSCT have provided promising results.     

Alloreactive donor lymphocytes (DLI) after allogeneic hematopoietic stem cell transplantation (HSCT): study of toxicity and efficacy

Adoptive immunotherapy represents a new therapeutic tool able to eradicate or to improve the course of various hematological malignancies and solid tumors. Alloreactive lymphocytes present within the hematopoietic transplant have been shown to be responsible for the occurrence of acute and chronic graft-versus-host-disease (GVHD). Retrospective analysis of survival curves after allogeneic bone marrow transplantation performed for leukemia have shown that GVHD was accompanied with lower relapse rates suggesting graft-versus leukemia (GVL) affect. T cell-depletion demonstrated the inverse balance existing between GVHD/GVL and relapse. Allogeneic bulk lymphocyte infusions were further used for successful treatment of relapse after transplantation in CML patients. However they were responsible for some cases of lethal acute GVHD and myelosuppressions in some patients. The concept of allogeneic transplantations after reduced intensity conditioning regimen (RICT) has recently emerged as a new strategy to treat cancer. This strategy exploited the immunological properties of the graft followed or not by donor lymphocyte infusions (DLI) while reducing the toxicity of the preparative regimen. Different means to perform RICT have been described, however the systematic use of DLI (bulk or escalating doses) after these transplantations remains controversial. Hereby, we report a summary of historical data that lead to the concept of adoptive immunotherapy and its applications.     

Immunoablative reduced-intensity stem cell transplantation: potential role of donor Th2 and Tc2 cells

Allogeneic reduced-intensity stem cell transplantation (RISCT) decreases regimen-associated morbidity and mortality, but it is unfortunately still constrained by the same immune T-cell reactions that limit myeloablative transplantation, including graft rejection, graft-versus-host disease (GVHD), and suboptimal graft-versus-leukemia (GVL) or graft-versus-tumor (GVT) effects. Graft rejection is mediated by host T cells, whereas GVHD and GVL/GVT effects are initiated by donor T cells, and to this extent, future advances in RISCT will likely benefit from an ability to modulate both donor and host T-cell immunity. As a step in this direction, we have developed a RISCT approach that first involves chemotherapy-induced host T-cell ablation, and second involves administration of allogeneic inocula enriched for donor CD4(+) Th2 and CD8(+) Tc2 T-cell subsets that in murine studies mediate reduced GVHD. In a pilot clinical trial, "immunoablative" RISCT with human leukocyte antigen (HLA)-matched related allografts resulted in rapid and complete donor chimerism and GVL effects early post-transplant, with GVHD being the primary toxicity. Using this immunoablative RISCT approach, we are now evaluating the feasibility and safety of augmenting allografts with additional donor CD4(+) Th2 cells that are generated in vitro via CD3/CD28 costimulation in the presence of interleukin (IL)-4. We review the biology of host and donor T-cell immunity during allogeneic RISCT and discuss the strategies of host immunoablation and donor Th2 and Tc2 cell therapy as potential means to improve the clinical results in RISCT.     

A pilot study of allogeneic cellular therapy for patients with advanced hematologic malignancies

Allogeneic hematopoietic stem cell transplantation provides curative therapy for some patients with advanced hematologic malignancies. Disease response after allogeneic transplant is, at least in part, mediated by donor immune cells. In this report we describe a cellular therapy using haploidentical peripheral blood stem cells administered after very low dose total body irradiation (TBI) (100 cGy). The donor cells were anticipated to be rejected, so no graft-versus-host (GVHD) prophylaxis was used. Patients with persistent disease beyond 8 weeks could be further treated with infusions of irradiated haploidentical donor cells. Of the 10 patients enrolled in the study, durable engraftment of allogeneic cells was seen in one patient. Two patients with resistant relapsed acute myelogenous leukemia (AML) had a disease response. Analysis of T cell reactivity from one patient who achieved a complete response but did not have durable engraftment of donor cells indicated that disease response was associated with the generation of host-derived anti-leukemic cytotoxic CD8+ T cells that reacted with an AML-associated proteinase 3 epitope. Results from this patient suggest that allogeneic therapy induced a host anti-tumor response associated with cytotoxic T cells reactive with a low affinity self-antigen.     

Relapse of preB-ALL after rituximab treatment for chronic graft versus host disease. Implications for its use?

In acute lymphatic leukaemia (ALL) treated with allogenic stem cell transplantation limited chronic graft versus host disease (cGVHD) is associated with a higher 5 year disease free survival. This indicates an important graft-versus-leukemia (GVL) effect, although this effect is less pronounced in ALL than in other malignancies. B-cell depletion using Rituximab is a treatment option in therapy refractory cGVHD that has been used successfully in a limited number of patients with an approximate response rate of 70%. Only two ALL patients with cGVHD treated with Rituximab are found in the literature. Here we describe a pre-B-ALL patient with therapy refractory cGVHD who relapsed only one month after receiving Rituximab therapy for cGVHD. The Rituximab therapy may have triggered the relapse by targeting the shared cGVHD and GVL mechanisms, diminishing the GVL effect. Although no definite conclusions can be drawn from one case this might have implications for the use of Rituximab in cGVHD in ALL patients.     

Th2 and Tc2 cells in the regulation of GVHD, GVL, and graft rejection: considerations for the allogeneic transplantation therapy of leukemia and lymphoma

Allogeneic stem cell transplantation (SCT) represents a curative treatment option for patients with leukemia and lymphoma. T lymphocytes contained in the allograft mediate a graft-versus-leukemia (GVL) effect and prevent graft rejection; however, T cells also initiate graft-versus-host disease (GVHD). Identification of T cell populations which mediate a GVL effect and prevent rejection with reduced GVHD will likely improve transplantation outcome. T cells exist in four functionally-defined populations, the CD4+, Th1/Th2 and CD8+, Tc1/Tc2 subsets. Th1-type CD4 cells primarily secrete type I cytokines (IL-2 and IFN-gamma), whereas Th2 cells secrete type II cytokines (IL-4, IL-5, and IL-10). Similarly, the CD8+ Tc1 and Tc2 cells differentially secrete the type I and type II cytokines, respectively. In addition to cytokine secretion, Tc1 and Tc2 populations mediate cytolytic effects, with Tc1 cells utilizing both perforin- and fas-based killing pathways, whereas Tc2 cells primarily utilize perforin-mediated cytolysis. In murine transplantation models of graft rejection, GVHD, and GVL effects, we have evaluated such functional T cell subsets for their ability to differentially mediate and regulate transplantation responses. These studies demonstrate that donor Th2 cells do not initiate acute GVHD, and can regulate the GVHD mediated by unmanipulated donor T cells without impairing alloengraftment. Additional experiments have shown that allospecific donor Tc2 cells result in reduced GVHD, and mediate a significant GVL effect. Thirdly, we have demonstrated that non-host reactive Tc2 cells with veto-like activity can potently abrogate marrow rejection independent of GVHD. Together, these results demonstrate that functionally-defined donor Th2 and Tc2 populations play an important role in the regulation of GVHD, the prevention of graft rejection, and the mediation of GVL effects, and suggest that utilization of Th2 and Tc2 cells in clinical allogeneic SCT may have potential for improving treatment outcome.     

减低强度预处理的异基因造血干细胞移植拯救性治疗难治性白血病

 讨论减低强度预处理的异基因造血干细胞移植（RIC-HSCT）治疗难治性白血病的治疗时机和病例选择、预处理方案设计、移植后嵌合状态监测、移植物抗宿主病等问题。已经有越来越多的学者认为,对于难治性白血病患者,不应以牺牲患者的整体状况为代价,强求在完全缓解状态下进行造血干细胞移植。RIC-HSCT的本质属于一种过继性免疫治疗,所以必须考虑到免疫攻击的靶点和免疫反应的潜伏期。相对缺乏免疫攻击靶点和生长过快的肿瘤可能难以用RIC-HSCT控制。RIC-HSCT目前并无统一的预处理方案,但是对于难治复发白血病患者,预处理剂量的强度起着十分重要的作用,很多研究者采用了介于"非清髓"的剂量标准和传统清髓方案之间的剂量。RIC-HSCT早期往往难以达到完全供者嵌合。稳定的完全供者嵌合状态以发挥移植物抗白血病（GVL）效应清除微小残留病变,是确保患者长期生存的关键,故建议对于采用RIC-HSCT的患者,应当采用敏感的方法（如聚合酶链反应检测短串联重复序列,PCR-STR）更频繁的（2～4周）监测移植后嵌合状态,并应当对特异的细胞系列进行检测（如T细胞）。同传统移植相比,RIC-HSCT时急性和慢性移植物抗宿主病（GVHD）的发生率是相似的。GVHD的发生同GVL效应有相关性,早期减、停免疫抑制剂和供者淋巴细胞输注,可以促进向完全供者嵌合状态的转化,可能对于预防未缓解期行RIC-HSCT的白血病患者复发有一定益处,但最大的并发症就是诱发GVHD。     

异基因造血干细胞移植治疗血液肿瘤5例

目的；探讨基因造血干细胞移植对血液肿瘤的疗效。方法：采用异基因造血干细胞移植治疗血液肿瘤５例，包括２例第一次完全缓解（ＣＲ１）急性粒细胞白血病，１例ＣＲ１急性淋巴细胞白血病，１例急性粒细胞白血产现任昨发和１例非霍奇金氏淋巴瘤Ⅳ期ＣＲ１。其中异基因骨髓移植４例，异基因外周血造血干细胞移植１例。所有病例均采用ＴＢＩ＋ＣＹ＋ＣＣＮＵ作预处理。结果：所有病例均移植成功。２例发生急性移植抗宿主病，２例发生慢     

Donor Lymphocyte Infusion for the Treatment of Relapse after Allogeneic Hematopoietic Stem Cell Transplantation

The results of donor lymphocyte infusion (DLI) for treatment of relapse after bone marrow transplantation (BMT) are reviewed. Durable complete remission can be achieved at the molecular level for a majority (more than 70%) of patients with CML, when treated at early relapse. Results are less favourable for acute leukemias, although useful responses have been reported. Data are scarce though promising for myelodysplastic syndromes and multiple myeloma. Major treatment-associated toxicities are GVHD and bone marrow aplasia. The latter complication can be predicted by evaluating the level of residual donor-derived hematopoiesis. Modification of infused cells (CD8 negative selection or transduction with a suicide gene), addition of peripheral blood stem cells, and early implementation of escalating doses may counteract the complications and increase the response rate. Response rate is variably influenced by the presence of chronic GVHD after initial BMT, T-cell depleted BMT, underlying disease and stage at relapse, and the level of mixed chimerism. DLI is a direct demonstration of the graft-versus-leukemia effect (GVL). Because GVL after BMT is sometimes the predominant cause of cure, it may be advisable in such situations to redirect the conditioning regimens for BMT towards engraftment and less immediate cytotoxicity.     

Prevention of graft-versus-host disease by selective depletion of CD6-positive T lymphocytes from donor bone marrow.

PURPOSE: Acute and chronic graft-versus-host disease (GVHD) continues to be the major causes of morbidity and mortality after allogeneic bone marrow transplantation (BMT). In this study, we have evaluated the clinical effects of selective in vitro T-cell depletion of donor allogeneic bone marrow by using a single monoclonal antibody ([MoAb] anti-T12, CD6) and rabbit complement. This antibody recognizes mature T cells, but not other cellular elements such as natural-killer (NK) cells, B cells, and myeloid precursors. PATIENTS AND METHODS: From August 1983 to April 1991, 112 consecutive adult patients with hematologic malignancies underwent BMT with bone marrow from HLA-identical sibling donors. Marrow was harvested and depleted of mature T lymphocytes ex vivo by the use of three rounds of incubation with an anti-T12 antibody and rabbit complement. The preparative regimen consisted of cyclophosphamide and fractionated total body irradiation (TBI) in 108 patients. No patients received prophylactic immune suppression post-BMT. Purgation by anti-T12 was used as the only method for the prevention of GVHD. RESULTS: Twenty patients (18%) developed acute GVHD (grade 2 to 4); only eight patients developed chronic GVHD. The incidence of GVHD did not increase significantly with age. Only three of 112 patients (2.7%) exhibited acute graft failure. One patient developed late graft failure that was associated with cytomegalovirus (CMV) infection. Within the subset of 50 patients who had not previously undergone unsuccessful conventional therapy (acute leukemia in first remission or chronic myelogenous leukemia [CML] in stable phase), we estimated by the Kaplan-Meier method that the probability of disease-free survival was 50% at 3 years post-BMT, with a median follow-up of 44 months. The treatment-related mortality rate in this group was only 14% and was independent of patient age. CONCLUSIONS: We conclude that selective in vitro T-cell depletion with an anti-T12 monoclonal antibody effectively reduces the incidence of both acute and chronic GVHD after allogeneic BMT without compromising engraftment. Moreover, depletion of CD6-positive cells from donor marrow obviates the need to administer immune suppressive medications to the majority of patients. This approach reduces the morbidity and mortality of allogeneic BMT and permits the BMT of older patients.     

半相合骨髓移植的动物实验研究

目的：观察半相合骨髓移植对GVHD或GVL的影响。方法：以C57，BL-6小鼠急性放射病为模型，进行半相合小鼠骨髓移植，同时设异基因骨髓移植为对照，观察移植后小鼠存活率、造血重建、GVHD或GVL作用。结果：GVHD中，半相合骨髓移植组30天死亡率低于基因骨髓移植。半相合骨髓移植与异基因骨髓移植比较，外周血象或骨髓CFU-GM恢复快，以9天以后明显，脾结节数及嵌合体测定高于异基因组。体內混合淋巴细胞反应证实半相合骨髓移植较异基因骨髓移植GVHD小。在GVL中，异基因骨髓移植组主要死于GVHD。半相合骨髓移植组30天生存率仍为56.3％，表现出明显GVL作用。结论：半相合骨髓移植在减轻GVHD同时，产生明显的GVL作用。     

Unrelated cord blood transplantation after myeloablative conditioning regimen in adolescent and young adult patients with hematologic malignancies: a single institute analysis

We report the results of unrelated cord blood transplantation (CBT) after myeloablative conditioning regimen in 16 patients with hematologic malignancies from 15 to 20 years old. The median times of myeloid and platelet engraftment were 21 and 38 days, respectively. The cumulative incidences of acute graft-vs-host disease (GVHD) was 62.0%, all of which were grade I or II, and that of extensive-type chronic GVHD was 12.5%. The probabilities of overall and disease-free survival at 3 years were 68.2% and 48.6%, respectively, comparable to adult or childhood cases. Adolescents and young adult patients with hematologic malignancies who have no HLA-matched adult donors could be considered as candidates for CBT.     

The role of interleukin-12 in preserving the graft-versus-leukemia effect of allogeneic CD8 T cells independently of GVHD.

Interleukin (IL)-12 is a potent immunostimulatory cytokine and inducer of Th1 cell activity and of cytotoxic T lymphocyte and natural killer cell function. This cytokine also has anti-tumor activity. Although IL-12 has been shown to be an important pathogenic cytokine in the induction of graft-versus-host disease (GVHD), injection of exogenous IL-12 to murine allogeneic bone marrow transplantation (BMT) recipients paradoxically leads to a significant delay in the onset of GVHD mortality in fully MHC plus multiple minor antigen-mismatched strain combinations, and to complete inhibition of GVHD in a single haplotype-mismatched murine BMT model. IL-12-induced inhibition of GVHD is associated with reduced donor T cell activation and expansion, in part through an interferon (IFN)-gamma-mediated mechanism. Fas-mediated apoptosis of donor T cells also plays a significant role in IL-12-induced GVHD protection. Importantly, IL-12 preserves the graft-versus-leukemia (GVL) effect of allogeneic CD8 T cells against EL4, a host-type leukemia/lymphoma, while inhibiting GVHD. Like the protective effect against GVHD, the GVL effect in IL-12-treated mice is dependent on IFN-gamma. Thus, treatment with IL-12 leads to separation of GVHD-promoting and GVL effects of allogeneic BMT via an IFN-gamma-dependent mechanism.     

Graft-versus-Leukemia Effect and Its Clinical Implications

Graft-versus-leukemia (GVL) effect is an immunologically important phenomenon which decreases the relapse rate of leukemia after allogeneic bone marrow transplantation. GVL effect is sometimes associated with the occurrence of graft-versus-host disease (GVHD). Analyses of GVL effect and GVHD showed that these two phenomena were separable in some conditions. Although we cannot yet completely control the development of the GVL effect without inducing GVHD in humans, basic analyses using animal models show potential benefits of the GVL effect for clinical applications. Autologous GVHD is another important phenomenon which can help to eradicate minimal residual disease. Interleukin 2 and/or cyclosporin A are extensively used in animal models and in humans to induce autologous GVHD, showing beneficial effects. In the future, cytokine usage and allogeneic stem cell transplantation or leukocyte infusion appear to be promising in the control of minimal residual disease. Further studies on the mechanisms of GVL effects and GVHD may well open a new era for cell transplantation.     

Induction of early post-transplant graft-versus-leukemia effects using intentionally mismatched donor lymphocytes and elimination of alloantigen-primed donor lymphocytes for prevention of graft-versus-host disease

Graft-versus-leukemia (GVL) effects can be induced in tolerant mixed chimeras prepared with nonmyeloablative conditioning. GVL effects can be amplified by post-grafting donor lymphocyte infusion (DLI). Unfortunately, DLI is frequently associated with graft-versus-host disease (GVHD). We investigated the feasibility of induction of potent GVL effects by DLI using intentionally mismatched lymphocytes followed by elimination of alloreactive donor T cells by cyclophosphamide for prevention of lethal GVHD following induction of very short yet most potent GVL effects. Mice inoculated with B-cell leukemia (BCL1) and mismatched donor lymphocytes were treated 2 weeks later with low-dose or high-dose cyclophosphamide. All mice receiving cyclophosphamide 2 weeks after DLI survived GVHD, and no residual disease was detected by PCR; all control mice receiving DLI alone died of GVHD. Analysis of host (female) and donor (male) DNA showed that cyclophosphamide treatment eradicated most alloreactive donor cells, yet mixed chimerism was converted to full donor chimerism following transient self-limited GVHD. Our working hypothesis suggests that short-term yet effective and safe adoptive immunotherapy of leukemia may be accomplished early post-transplantation using alloreactive donor lymphocytes, with prevention of GVHD by elimination of GVL effector cells.