T cells recognizing leukemic CD34(+) progenitor cells mediate the antileukemic effect of donor lymphocyte infusions for relapsed chronic myeloid leukemia after allogeneic stem cell transplantation.

Adoptive immunotherapy with donor lymphocyte infusions (DLI) is an effective treatment for relapsed chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. To identify the effector and target cell populations responsible for the elimination of the leukemic cells in vivo we developed an assay to measure the frequency of T lymphocyte precursor cells capable of suppressing leukemic progenitor cells. Target cells in this assay were CML cells that were cultured in the presence of stem cell factor, interleukin 3, granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, and erythropoietin. [3H]thymidine incorporation at day 7 represented the proliferation of the progeny of the CD34(+) CML progenitor cells, and not of the more mature CD34(-) CML cells. Effector cells were mononuclear cells, which were used in a limiting dilution analysis to measure the frequencies of CML progenitor cell-inhibitory lymphocyte precursors (PCILp) in peripheral blood of seven patients before and after DLI for relapsed CML. In the six patients who entered complete remission, a 5- to 100-fold increase of PCILp was found during the clinical response. In the patient with resistant relapse the frequency of PCILp was <10 per ml before and after DLI. Leukemia-reactive helper T lymphocyte precursor frequencies remained unchanged after DLI. A significant increase in cytotoxic T lymphocyte precursor frequency against more mature leukemic cells was found in only two responding patients. These results indicate that T cells specifically directed against CD34(+) CML progenitor cells mediate the antileukemic effect of DLI.

Generation of leukemia-reactive cytotoxic T lymphocytes from HLA-identical donors of patients with chronic myeloid leukemia using modifications of a limiting dilution assay.

Donor leukocyte transfusions (DLT) have an anti-leukemic effect in most patients with a relapse of chronic myeloid leukemia (CML) after allogeneic stem cell transplantation. However, DLT are often complicated by graft-versus-host disease. Selection of donor lymphocytes with a relative specificity for leukemic cells is desirable. The generation of leukemia-reactive cytotoxic T lymphocyte (CTL) responses between HLA-identical donors and patients in bulk cultures showed major variations, and false negative results were observed. In a modification of a limiting dilution analysis (LDA) two-fold serial dilutions of HLA-identical donor mononuclear cells (MNC) were cultured in the presence of CML cells. The anti-leukemic CTL precursor frequencies in these donors varied between <1 and 9 per 106 MNC. HLA-restricted CD4+ or CD8+ lymphocytes as well as MHC non-restricted gammadelta T cells were responsible for the anti-leukemic responses. A positive correlation between cytotoxicity in the various wells after 3, 4 and 5 weeks of culture could be found. The LDA may be superior to bulk cultures in selecting stable immune responses and in separating multiple different anti-leukemic T cell responses in each donor-patient combination.

Generation of dendritic cells expressing bcr-abl from CD34-positive chronic myeloid leukemia precursor cells.

Patients with a relapse of chronic myeloid leukemia (CML) after allogeneic bone marrow transplantation can be successfully treated with blood mononuclear cells from the original bone marrow donor. However, the antileukemic effect of this treatment is often accompanied by graft-versus-host disease (GVHD). Treatment with cytotoxic T-lymphocyte (CTL) lines or clones that are specifically generated against leukemic antigen-presenting cells from the patient, may separate antileukemic effects from GVHD. In this report we demonstrate that after culturing CD34-positive cells purified from bone marrow of patients with chronic phase CML in medium containing human serum, GM-CSF, TNF alpha, and IL-4 up to 28% of the cultured cells were dendritic cells, characterized by morphology, phenotypic analysis, and their efficient capacity to stimulate allogeneic T lymphocytes. The expression of HLA and costimulatory molecules and the stimulatory capacity of the dendritic cell-enriched cell suspensions were optimal between days 7 and 10 after onset of the cultures. Fluorescence in situ hybridization revealed that all cultured dendritic cells contained the CML specific t(9;22) translocation. PCR analysis showed expression of the translocation specific bcr-abl mRNA. These leukemic dendritic cells may enhance the induction and proliferation of CTL lines and clones with more specificity for the leukemic cells.

Interleukin-10, interleukin-12, and tumor necrosis factor-alpha differentially influence the proliferation of human CD8+ and CD4+ T-cell clones.

Activation and proliferation of human T lymphocytes in vitro can be obtained by various stimuli including specific antigens, mitogens, and cytokines. Here we describe the effect of interleukin-10, interleukin-12 and tumor necrosis factor-alpha on the interleukin-2 dependent proliferation and function of established human CD4+ and CD8+ alloreactive T-cell clones in the absence of antigen presenting cells. IL-12 and TNF-alpha both demonstrated an inhibitory effect on the proliferation of CD8+ cytotoxic T lymphocyte clones, whereas IL-10 enhanced the proliferation. IL-12-induced inhibition of CD8+ CTL clones was not mediated by the endogenous production of TNF-alpha by these clones. The strong inhibitory effect of IL-12 and TNF-alpha did not result in apoptosis. These cytokines did not alter the cytotoxicity of CD8+ CTL clones. When CD4+ T-cell clones were tested in the absence of APC, no significant change in IL-2-dependent proliferation due to IL-10, IL-12, and TNF-alpha could be measured. Since these effects on established CTL clones are in contrast to the effects of IL-10, IL-12, and TNF-alpha during the induction phase of immune responses, a dichotomy of immunomodulatory cytokines such as IL-10, IL-12, and TNF-alpha early and late in the immune response is suggested.

Minor histocompatibility antigen-specific, leukemia-reactive cytotoxic T cell clones can be generated in vitro without in vivo priming using chronic myeloid leukemia cells as stimulators in the presence of alpha-interferon.

At present, allogeneic bone marrow transplantation (BMT) is the only curative treatment for chronic myeloid leukemia (CML) in chronic phase (CP). The graft-vs.-leukemia (GVL) effect appears to play an important role in this treatment. Direct evidence for a GVL effect has been reported in Ph1-positive CML patients who relapsed after allogeneic BMT and who were treated with leukocyte transfusion from the original marrow donor. Alpha-interferon (alpha-IFN) may have facilitated this GVL effect since many patients were treated with it also. We investigated whether leukemia-reactive cytotoxic T lymphocytes (CTLs) can be generated from human leukocyte antigen (HLA)-genotypically identical sibling bone marrow (BM) donors who donated marrow for two patients with Ph1-positive CML in CP and one patient with Ph1-positive acute lymphoblastic leukemia (ALL). We also investigated alpha-IFN's ability to facilitate the generation of CTLs. In the absence of alpha-IFN, CTL lines with only low cytotoxicity and no CTL clones could be generated. In the presence of alpha-IFN, however, alloreactive, leukemia-reactive CTL lines with high cytotoxicity could be generated, and CD8+ CTL clones could be established with HLA class I restricted minor histocompatibility antigen (mHa)-specific recognition. In a cell-mediated clonogenic cytotoxicity assay, the CTL clones showed specific growth inhibition of leukemic precursor cells from the recipient and a second CML patient, but the clones did not inhibit growth of hematopoietic precursor cells (HPCs) from the donor. The normal HPCs from an unrelated donor with the HLA class I restriction molecule were also recognized by the CTL clones, illustrating that the antigen recognized is not leukemia-specific. The mechanism of the immunomodulating effect by alpha-IFN is not clear. Addition of alpha-IFN to medium did not alter the expression of HLA or adhesion molecules on CML cells. In the treatment of CML, administration of alpha-IFN as adjuvant immunotherapy after allogeneic BMT may increase GVL reactivity.