Synergism of interleukin-2 and cyclosporine A in induction of a graft-versus-tumor effect without graft-versus-host disease after syngeneic bone marrow transplantation.

Interleukin-2 (IL-2) therapy generates killer cells with major histocompatibility complex (MHC)-unrestricted cytotoxicity against most tumors but not normal tissues. Cyclosporine A (CsA) has been reported to break tolerance to self and to induce killer cells with specificity against class II MHC (Ia) antigens both on the host and the tumor cells, resulting in a mild graft-versus-host disease (GVHD) in an autologous bone marrow transplantation (BMT) setting in the rat. We used these two agents in a syngeneic BMT model in a strain of mice that does not develop GVHD with CsA. Therapy with either agent alone was ineffective, whereas a combination of CsA plus IL-2 after BMT induced a potent graft-versus-tumor (GVT) effect against a melanoma and an acute myeloid leukemia. The antitumor effect could be adoptively transferred by infusing spleen cells harvested from mice treated with CsA plus IL-2 into secondary recipients that received chemoradiotherapy. The cytotoxicity of these cells was not influenced by treatment of tumor cells with gamma-interferon or Ia antibody. The cytotoxic effect was mediated by Thy 1+ and asialo GM 1+ cells. There was no GVHD either in the primary recipients of CsA and IL-2 or in those receiving the adoptively transferred spleen cells. Our findings show that combination therapy with CsA and IL-2 after syngeneic BMT induces a potent GVT effect in a non-MHC-restricted manner, and point to the existence of differences between the mechanisms of GVT and GVHD.     

Syngeneic graft-versus-host disease: a report of two cases and literature review

Rappeport et al first reported the clinical syndrome of graft-versus-host disease (GVHD) in syngeneic bone marrow transplant patients. Recently, there have been more reports of a GVHD-like syndrome in syngeneic bone marrow transplant patients (SGVHD) that may result in significant clinical morbidity. A total of 17 cases of SGVHD in syngeneic bone marrow transplant patients have been reported to date in the medical literature. The current report reviews these cases and presents two additional cases of severe SGVHD that have occurred at our institution.     

Combined syngeneic bone marrow transplantation and immunotherapy of a murine B-cell lymphoma: active immunization with tumor-derived idiotypic immunoglobulin

Recurrence of the underlying malignancy remains a major cause of treatment failure after autologous bone marrow transplantation (BMT) for patients with lymphoma. In this regard, we have developed an immunotherapeutic approach designed to induce resistance against residual tumor cells persisting after BMT. Previous studies in the model system of 38C13, a lethal B-cell lymphoma of C3H origin, have shown that active immunization with purified tumor-derived surface immunoglobulin (Id), as a tumor-associated antigen, produces resistance to tumor growth. Id immunization of lethally irradiated mice at 3 or 5 weeks after reconstitution with syngeneic bone marrow resulted in significantly prolonged survival after tumor challenge compared with nonspecifically immunized controls. Low levels of idiotype-specific antibody were also demonstrated in the sera of specifically immunized mice at this early time, when other functional studies in the literature of immunocompetence after syngeneic reconstitution might have predicted incomplete recovery. Immunization of mice before lethal irradiation and syngeneic marrow reconstitution also induced significant resistance to tumor challenge, suggesting the persistence of established host antitumor immunity through total body irradiation. These studies demonstrate the feasibility of id immunization in conjunction with bone marrow transplantation.     

Successful treatment of disseminated human Hodgkin's disease in SCID mice with deglycosylated ricin A-chain immunotoxins

To evaluate the effects of deglycosylated ricin A-chain (dgA) immunotoxins against disseminated Hodgkin's lymphoma, we used RFT5.dgA (CD25) and IRac.dgA (70 kD) to treat L540Cy Hodgkin cells in severely immunodeficient SCID mice. In this model, more than 90% of the animals developed multiple lymphomas in various organs such as the lymph nodes, liver, bone marrow, and extranodal sites that killed untreated animals after a mean survival time (MST) of 36.3 days. A single intraperitoneal injection of 8 micrograms of either immunotoxin rendered 95% (RFT5.dgA) and 93% (IRac.dgA), respectively, of mice tumor-free when applied 1 day after tumor challenge. The MST of the RFT5.dgA-treated group was extended by more than 80 days (P < .00001). SCID mice treated 12 days after tumor challenge had lower remission rates (46%), suggesting that the antitumor effect of the immunotoxins depends on the number of tumor cells present. We conclude that ricin A-chain immunotoxins have potent antitumor effects against disseminated Hodgkin's tumors in SCID mice and that this model is ideally suited for the evaluation of different immunotoxin treatment modalities.     

NK-cell purging of leukemia: superior antitumor effects of NK cells H2 allogeneic to the tumor and augmentation with inhibitory receptor blockade

Natural killer (NK) cells are composed of subsets characterized by the expression of inhibitory or activating receptors, or both, specific for different major histocompatibility complex (MHC) class I determinants. We have previously shown that inhibitory receptor blockade of syngeneic NK cells was an effective means of ex vivo purging of leukemia-contaminated bone marrow and that the transplantation of mice with the purged bone marrow cells (BMCs) resulted in long-term, relapse-free survival. We have extended the investigation to assess the antitumor effects mediated by NK cells H2-allogeneic to tumor cells. We demonstrate that various tumor cell lines are more susceptible to lysis by H2-allogeneic NK cells than by syngeneic NK cells in vitro even though comparable percentages of Ly49 NK cells were present. Using allogeneic NK cells to purge leukemia-contaminating BMCs before transplantation resulted in a higher proportion of mice with long-term survival than using syngeneic NK cells. Allogeneic NK cells did not suppress hematopoietic reconstitution as measured by granulocyte/monocyte-colony-forming unit (CFU-GM), complete blood count (CBC), and donor chimerism at various days after transplantation. Inhibitory receptor blockade of allogeneic NK cells also significantly increased these antitumor effects at lower NK/tumor ratios compared with those of syngeneic NK cells. These results demonstrate that H2-allogeneic NK cells mediate more potent antitumor effects than syngeneic NK cells without adverse hematologic effects and thus may be useful in cancer therapy.     

Rapid generation of a functional NK-cell compartment

Bone marrow transplants are an important therapeutic tool for treating certain types of cancer as well as genetic diseases affecting the hematopoietic system. Until the transferred stem cells differentiate and reconstitute the immune system, recipients are at increased risk from opportunistic infections. We report the rapid generation of a functional natural killer (NK) compartment in lethally irradiated mice that received bone marrow cells from a syngeneic donor by treatment with IL-2/anti-IL-2 antibody complexes. We demonstrate that IL-2 complexes specifically expand the donor but not the host NK population and discuss the implications of this finding in the context of graft-versus-host disease and tumor relapse. Finally, we show that NK cells rapidly generated by IL-2 complexes kill MHC class I-deficient cells effectively in vivo. These data underline the unique therapeutic potential of IL-2 complexes.     

Successful in vitro graft-versus-tumor effect against an Ia-bearing tumor using cyclosporine-induced syngeneic graft-versus-host disease in the rat

Lethally irradiated LouM rats reconstituted with syngeneic bone marrow and then treated with cyclosporine (CsA) for 40 consecutive days following transplant developed a graft-v-host disease (GVHD)-like syndrome after CsA cessation. This model of GVHD was used to define and characterize a graft-v-tumor (GVT) effect against a syngeneic plasmacytoma CRL1662 cell line which expresses class II major histocompatibility (MHC) antigen (Ia). Nylon wool-nonadherent spleen cells from animals who developed syngeneic GVHD were capable of significant lysis against chromium-labeled tumor target cells in a four-hour chromium released cell mediated lympholysis assay; maximum lysis occurred five days following cessation of CsA when clinical signs first appeared. Cytolytic activity declined to baseline as GVHD symptoms resolved. Fractionation of splenocytes into lymphocyte subsets demonstrated that cytolytic lymphocytes (CTLs) of the OX8 phenotype (non-helper T) were capable of significant lysis against tumor target cells. Lysis of tumor cells was blocked by preincubation with monoclonal antibodies (MoAb) specific for the rat anti-class II MHC antigen but not with MoAb against class I. Incubation of tumor cells with gamma-interferon increased expression of tumor class II MHC antigens and significantly increased their susceptibility to lysis by nylon wool-nonadherent splenocytes from animals with syngeneic GVHD. These studies have demonstrated an in vitro GVT of syngeneic GVHD against an Ia-bearing tumor; the effector cell is a CTL of the OX8 phenotype specific for the class II MHC antigen.     

Potential use of interleukin-6 in bone marrow transplantation: effects of recombinant human interleukin-6 after syngeneic and semiallogeneic bone marrow transplantation in mice

The potential of recombinant glycosylated human interleukin-6 (rhIL-6) for enhancing immunohematopoietic reconstitution and survival after syngeneic and semiallogeneic bone marrow transplantation (BMT) in BALB/c mice subjected to total body irradiation (TBI) was investigated. rhIL-6 produced enhanced reconstitution of white blood cells as assessed on days 8 and 14 after syngeneic BMT and of platelets as assessed on day 10. Moreover, rhIL-6 treatment produced significant improvement of survival in lethally irradiated mice receiving either syngeneic or semiallogeneic BMT with limiting number of BM cells. This effect of IL-6 was not seen with large BM cell inocula producing high survival by themselves. rhIL-6 showed no toxic effects and did not affect the survival of mice that were lethally irradiated but not reconstituted by BM cells. However, the sensitivity of mice to sublethal irradiation was increased by rhIL-6 in the absence of BM cell transplantation. In experimental conditions inducing graft-versus-host disease (GVHD), in which lethally irradiated (BALB/c x C57BL/6)F1 mice received mixtures of BM and spleen cells from C57BL/6 donors, rhIL-6 was found to enhance GVHD manifestations. No consistent enhancement of T-cell in vitro proliferative responses to allogeneic spleen cells or T- and B-cell-dependent mitogens were seen in the splenocytes obtained from recipients of syngeneic or semiallogeneic BMT. Our data suggest that rhIL-6 may be useful in BMT procedures to enhance thrombopoiesis and hematologic recovery, as well as to increase overall survival rates. In addition, the potentiation of GVHD, which is considered to correlate with graft-versus-leukemia effects, may be of interest in enhancing GVHD-dependent antitumor effects in protocols combining radiochemotherapy with BMT.     

Interleukin 2 prevents graft-versus-host disease while preserving the graft-versus-leukemia effect of allogeneic T cells.

We have recently demonstrated that interleukin 2 (IL-2), when administered in high doses for several days beginning on the day of allogeneic bone marrow transplantation (BMT), markedly diminishes graft-versus-host disease (GVHD) mortality in lethally irradiated mice. An optimal anti-GVHD effect was attained by coadministering T-cell-depleted (TCD) syngeneic marrow. We demonstrate here that the full graft-versus-leukemia effect of allogeneic T lymphocytes is obtained even when GVHD is markedly diminished by the coadministration of IL-2 and TCD syngeneic marrow. This methodology represents an approach to the treatment of leukemia in which the beneficial effects of allogeneic T cells can be exploited while their major deleterious effect, GVHD, is avoided. These results may thus have an impact on the clinical use of BMT for the treatment of hematologic malignancies.     

CD3/CD28-costimulated T1 and T2 subsets: differential in vivo allosensitization generates distinct GVT and GVHD effects

Adoptive T-cell therapy using CD3/CD28 co-stimulation likely requires in vivo generation of antigen specificity. Because CD28 promotes TH1/TC1 (T1) or TH2/TC2 (T2) differentiation, costimulation may generate donor T1 or T2 cells capable of differentially mediating allogeneic graft-versus-tumor (GVT) effects and graft-versus-host disease (GVHD). Costimulation under T1 or T2 conditions indeed generated murine TH1/TC1 cells secreting interleukin-2/interferon-gamma (IL-2/IFN-gamma) or TH2/TC2 cells secreting IL-4/IL-5/IL-10. In vivo, allogeneic T1 cells expanded, maintained T1 secretion, and acquired allospecificity involving IFN-gamma and IL-5. In contrast, allogeneic T2 cells expanded less and maintained T2 secretion but did not develop significant allospecificity.Allogeneic, but not syngeneic, T1 cells mediated a GVT effect against host-type breast cancer cells, as median survival time (MST) increased from 25.6 +/- 2.6 (tumor controls) to 69.2 +/- 5.9 days (P < 1.2 x 10(-9)). This T1-associated GVT effect operated independently of fasL because T1 cells from gld mice mediated tumor-free survival. In contrast, allogeneic T2 cells mediated a modest, noncurative GVT effect (MST, 29 +/- 1.3 days; P <.0019). T1 recipients had moderate GVHD (histologic score, 4 of 12) that contributed to lethality after bone marrow transplantation; in contrast, T2 recipients had minimal GVHD (histologic score, 1 of 12). CD3/CD28 co-stimulation, therefore, generates T1 or T2 populations with differential in vivo capacity for expansion to alloantigen, resulting in differential GVT effects and GVHD.     

Dual targeting of Bruton's tyrosine kinase and Janus kinase 3 with rationally designed inhibitors prevents graft-versus-host disease (GVHD) in a murine allogeneic bone marrow transplantation model

The purpose of the present study was to evaluate the effectiveness of targeting Bruton's tyrosine kinase (BTK) with a specific BTK inhibitor, alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13), for prevention of acute fatal graft-versus-host disease (GVHD) in a murine model of allogeneic bone marrow transplantation (BMT). Vehicle-treated control C57BL/6 mice receiving bone marrow/splenocyte grafts from allogeneic BALB/c donors developed severe multi-organ acute GVHD and died after a median survival time (MST) of 40 d. LFM-A13 treatment (25 mg/kg/d) significantly prolonged the MST of the BMT recipients to 47 d. The probability of survival at 2 months after BMT was 2 +/- 2% for vehicle-treated control mice and 22 +/- 6% for mice treated with LFM-A13 (P = 0.0008). Notably, the combination regimen of LFM-A13 plus the standard anti-GVHD drug methotrexate (MTX) (10 mg/m(2)/d) was more effective than LFM-A13 alone, while the combination regimen of LFM-A13 plus the novel anti-GVHD drug JANEX-1 (60 mg/kg/d), targeting Janus kinase 3, was more effective than LFM-A13, JANEX-1 or MTX alone. More than 70% of recipients receiving this most effective GVHD prophylaxis (LFM-A13 + JANEX-1) remained alive throughout the 80-d observation period with an MST of >80 d. Taken together, these results indicate that targeting BTK with the chemical inhibitor LFM-A13 may attenuate the severity of GVHD, especially when it is combined with other anti-GVHD drugs, such as MTX and JANEX-1.     

Pre-treatment of transplant bone marrow cells with hydrocortisone and cyclosporin A alleviates graft-versus-host reaction in a murine allogeneic host-donor combination.

The aim of the study was to alleviate graft-versus-host reaction (GVHR) by pre-treatment of the bone marrow (BM) transplant with hydrocortisone (HC) and cyclosporin A (CsA) in C57BL/6J (donor) --> CBA/J (recipient) mouse combination. BM cells were exposed to HC and CsA for 1 h at 37 degrees C and then injected into lethally irradiated (9.5 Gy) mice at a dose of 2 x 10(6) BM cells/mouse. Haematopoietic recovery was assessed on day 12, and survival was followed for 100 days. Combinations of 1000 microg/ml HC and 100 microg/ml CsA, and 100 microg/ml HC and 10 microg/ml CsA significantly reduced MLR and additively mitigated GVHR in vivo, achieving 40% and 26% survival rates, respectively. However, HC and CsA altered neither the peripheral blood cell counts nor in vitro and in vivo BM cell clonogenic potential. Additional studies have shown that HC and CsA blocked con A-driven differentiation of CD8+ and CD4+ CD8+ lymph node cells (LNC) and progression of LNC to S + G2/M cell cycle phases, and inhibited IL-1, IL-2 and TGF-beta while enhancing GM-CSF gene expression in BM cells. Taken together, these data indicate that the pre-treatment of the BM transplant with HC and CsA results in inactivation of GVHR effector cells and mitigation of GVHR while sparing BM repopulating capacity.     

Interleukin-2 in bone marrow transplantation: preclinical studies.

Interleukin-2 (IL-2) promotes the generation and proliferation of killer cells in the peripheral blood and bone marrow (BM) both in vitro and in vivo. When employed in a syngeneic bone marrow transplantation (BMT) setting and followed by IL-2 therapy, murine BM cells activated with IL-2 in vitro (ABM) demonstrate potent graft-versus-leukemia (GVL) and anticytomegalovirus effects. ABM cells retain the capacity to reconstitute the hemopoietic system both in normal and leukemic mice. This therapy does not cause graft-versus-host disease (GVHD). Human ABM cells carry out purging of leukemia without loss of progenitor cell activity in vitro. The purging ability of ABM can be augmented by interleukin-1, interferon, and tumor necrosis factor. IL-2 therapy stimulates the veto suppressor cell activity of T cell-depleted BM, and has reduced GVHD and permitted engraftment of mismatched allogeneic BM in murine models. Future studies should determine the optimum treatment schedules with IL-2 for improving the GVL effect in autologous BMT, and for abolishing GVHD in allogeneic BMT settings.     

A two-phase pathogenesis of graft-versus-host disease in mice

Activation of donor T cells is required for the development of graft-versus-host disease (GVHD), a major complication of bone marrow transplantation. We investigated a murine model of GVHD across major and minor histocompatibility barriers. BALB/c recipients were lethally irradiated and transplanted with 10(7) bone marrow and 5 x 10(6) spleen cells from C57BL/6 donors. There were two separate phases of clinical disease. The first phase was most severe on day 7 after transplant. Weight and condition improved until day 12 and then a second phase of clinical GVHD commenced, which persisted until euthanasia. IL-2 mRNA expression, as a measure of T cell activation, was determined by quantitative PCR. The two phases of clinical GVHD were preceded by two separate peaks of IL-2 mRNA in the spleen. Host MHC class II(+) cells became undetectable by flow cytometry 7 days after transplantation, whereas donor MHC class II(+) cells increased during the first 9 days after transplantation. Removal of donor MHC class II(+) cells from the graft had no effect on the first phase. Possible roles for host and donor antigen-presenting cells (APC) in the two phases of the disease are discussed.     

Bone marrow transplantation with interleukin-2-activated bone marrow followed by interleukin-2 therapy for acute myeloid leukemia in mice

We have investigated approaches to induce graft-versus-leukemia (GVL) effect in autologous bone marrow transplantation (ABMT) without graft-versus-host disease to improve survival and cure in leukemia. The present study shows that bone marrow transplantation (BMT) using syngeneic bone marrow activated with interleukin-2 (ABM) for 24 hours in vitro, followed by interleukin-2 (IL-2) therapy, was superior to BMT with fresh, syngeneic bone marrow (FBM) in terms of survival and cure in mice with acute myeloid leukemia (P less than .001) and led to normal hematopoietic reconstitution. Addition of IL-2 therapy after BMT with FBM did not improve the results over BMT with FBM alone (P = .98). These results suggest that the GVL effect of ABMT can be enhanced by using ABM for BMT followed by IL-2 therapy without compromising engraftment.     

Alloengraftment in IL-2-treated mice.

Despite major advances, graft-versus-host disease (GVHD) remains a major obstacle to clinical bone marrow transplantation. Prophylaxis by T cell depletion is associated with increased rates of engraftment failure and leukemic relapse. Treatment with high-dose IL-2 can markedly protect lethally irradiated mice from GVHD-related mortality, especially when T cell-depleted (TCD) syngeneic bone marrow cells (BMC) are co-administered. In these IL-2-protected animals, allogeneic reconstitution is observed, and a graft-versus-leukemia effect of allogeneic T cells is preserved. To determine whether IL-2 might increase alloresistance under conditions in which alloengraftment is more difficult to achieve, we have now evaluated the possible effect of IL-2 on: (1) competitive repopulation of lethally irradiated mice by mixtures of TCD allogeneic and TCD syngeneic BMC; (2) radiation protection by TCD allogeneic BMC; (3) timing of hematologic recovery; and (4) allogeneic engraftment in sublethally irradiated recipients. The results show that IL-2 has only a limited and strain-restricted effect on alloengraftment. This effect may reflect activation of alloresistant host natural killer cells, a cell population which is not essential for the protective effect of IL-2 against GVHD.     

Induction of in vitro graft-versus-leukemia activity following bone marrow transplantation for chronic myeloid leukemia

We studied the in vitro effects of lymphokine-activated killer (LAK) cells from the peripheral blood of chronic myeloid leukemia (CML) patients after allogeneic and syngeneic bone marrow transplantation (BMT). LAK cells were generated by incubating peripheral blood mononuclear cells from patients post-BMT with recombinant interleukin-2 (IL-2) (500 U/mL) in 10% AB serum for 7 days. They were phenotyped and tested for activity in a standard 4-hour 51Cr release assay (n = 37) and in a CFU-GM assay (n = 24). We found that the LAK cells were mainly activated natural killer cells, but some were CD3+ T cells. In the 51Cr release assay LAK cells from 20 of 33 (61%) allogeneic and 2 of 4 syngeneic recipients killed recipient CML cells and in 22 of 37 (60%) cases also killed the HLA disparate CML cells. In the CFU-GM assay the LAK cells incubated together with the CML cells in liquid culture before plating inhibited (P less than .05) colony growth in 16 of 22 allogeneic and 2 of 2 syngeneic recipients. Cell-cell contact was necessary for optimal effect. There was little or no inhibition of proliferation of donor marrow CFU-GM. This in vitro graft-versus-leukemia (GVL) effect could also be demonstrated after LAK effectors were depleted of CD3+ T cells. It was inducible in recipients of both T cell-depleted and T cell-replete donor marrow and in recipients with or without graft-versus-host disease. These results suggest that a major histocompatibility complex-unrestricted GVL effect is inducible following allogeneic and syngeneic BMT. The use of IL-2/LAK cells after BMT could reduce the risk of relapse.     

Promotion of murine marrow alloengraftment and hematopoietic recovery across the major histocompatibility barrier by administration of recombinant human interleukin-1 alpha.

Irradiated C57BL/6 (H-2b) recipients of T-cell-depleted (TCD) BALB/c (H-2d) bone marrow (BM) and recombinant interleukin-1 alpha (IL-1 alpha) (1 microgram/d) had a significantly (P less than or equal to .006) higher 100-day actuarial survival rate, accelerated hematopoietic recovery, and higher levels of alloengraftment than a group of transplanted control mice treated identically, but given phosphate-buffered saline (PBS). To elucidate the mechanisms involved with IL-1 alpha-induced promotion of alloengraftment and hematopoietic recovery, we performed sequential splenic FACS studies on transplanted mice and secondary transfer studies in syngeneic mice given IL-1 alpha or PBS. Splenic phenotyping showed that recipients of IL-1 alpha had a higher proportion of donor granulocytes (52% v 19%) as compared with PBS controls as early as 7 days after bone marrow transplantation (BMT). On day 11 post-BMT, recipients of IL-1 alpha had a more than fourfold increase in splenocyte number, which included a higher percentage (90% v 59%) of donor cells, especially donor granulocytes (52% vs 32%), and a sevenfold increase in donor T cells as compared with controls. Host T-cell numbers were not affected. Taken together, these data suggest that IL-1 alpha stimulated bipotential (myeloid and lymphoid) donor cell engraftment. In a syngeneic BMT system, administration of IL-1 alpha resulted in a higher incidence of survival when recipients were transplanted with BM cells, indicating that IL-1 alpha administration probably either expanded or potentiated engraftment of a committed progenitor cell pool. Secondary transfer experiments using marrow from IL-1 alpha-treated mice showed that the number of day 12 colony-forming unit-spleen (CFU-S) cells was unaltered compared with untreated control mice, suggesting that more primitive, albeit committed, hematopoietic progenitor cells were not affected. We also examined the potential additive effects of IL-1 alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) administered in combination (for 14 days). Mice receiving a suboptimal amount of IL-1 alpha along with GM-CSF had significantly higher levels of donor alloengraftment (92%) with superior hematopoietic recovery, as compared with mice receiving either IL-1 alpha (57%) or GM-CSF (18%) alone.     

Activated dendritic cells from bone marrow cells of mice receiving cytokine-expressing tumor cells are associated with the enhanced survival of mice bearing syngeneic tumors.

Dendritic cells (DCs), which phagocytose antigens and subsequently proliferate and migrate, may be the most powerful antigen-presenting cells that activate naive T cells. To determine their role in the immune response to tumors, we used WEHI-3B murine leukemia cells transduced with adenovirus vectors expressing cytokines. We found that mixtures of irradiated cells expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) plus those expressing interleukin-4 (IL-4) or tumor necrosis factor alpha (TNFalpha) protected mice against WEHI-3B-induced leukemias. When bone marrow mononuclear cells (BMMNCs) obtained from mice that had been injected with irradiated, cytokine-expressing tumor cells were injected into tumor-bearing mice, the survival of the latter was significantly prolonged; the longest survival was observed in mice receiving BMMNCs containing an increased number of DCs from animals injected with a mixture of tumor cells expressing GM-CSF with those expressing IL-4. Assay for antileukemic effects in spleen of the latter animals showed specific antitumor cytotoxicity against WEHI-3B, suggesting that DCs from donor mice activate specific T cells in the tumor-bearing recipients. These results suggest that the infusion of syngeneic BMMNCs stimulated with cytokine-expressing tumor cells may be effective in treating certain types of tumors.