Effective presentation of tumor antigens is fundamental to strategies aimed at enrolling the immune system in eradication of residual disease after conventional treatments. Myeloid malignancies provide a unique opportunity to derive dendritic cells (DCs), functioning antigenpresenting cells, from the malignant cells themselves. These may then co-express leukemic antigens together with appropriate secondary signals and be used to generate a specific, antileukemic immune response. In this study, blasts from 40 patients with acute myeloid leukemia (AML) were cultured with combinations of granulocyte-macrophage colony-stimulating factor, interleukin 4, and tumor necrosis factor ␣, and development to DCs was assessed. After culture, cells from 24 samples exhibited morphological and immunophenotypic features of DCs, including expression of major histocompatibility complex class II, CD1a, CD83, and CD86, and were potent stimulators in an allogeneic mixed lymphocyte reaction (MLR). Stimulation of autologous T-cell responses was assessed by the proliferative response of autologous T cells to the leukemic DCs and by demonstration of the induction of specific, autologous, antileukemic cytotoxicity. Of 17 samples, 11 were effective stimulators in the autologous MLR, and low, but consistent, autologous, antileukemic cytotoxicity was induced in 8 of 11 cases (mean, 27%; range, 17%-37%). This study indicates that cells with enhanced antigen-presenting ability can be generated from AML blasts, that these cells can effectively prime autologous cytotoxic T cells in vitro, and that they may be used as potential vaccines in the immunotherapy of AML. (Blood. 2001; 97:2764-2771)

The in vitro effect of pegylated recombinant human megakaryocyte growth and development factor (PEG rHuMGDF) on megakaryopoiesis in normal subjects and patients with myelodysplasia and acute myeloid leukaemia Summary. Mpl ligand is a recently cloned haemopoietic growth factor that stimulates megakaryopoiesis in vitro and in vivo. We describe the in vitro effect of a truncated form of Mpl ligand, recombinant human megakaryocyte growth and development factor (rHuMGDF), on megakaryopoiesis in bone marrow from normal subjects and patients with myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). We used both semi-solid and suspension culture techniques to assess the effect of pegylated (PEG) rHuMGDF on megakaryocyte colony growth (CFU-Mk) and on the production of CD61 þ cells in 7 d suspension cultures. PEG rHuMGDF increased CFU-Mk growth and CD61 þ cell production in a dose-dependent fashion in all normal marrows tested. Normal CFU-Mk growth was increased threefold with the addition of 10 ng/ml PEG rHuMGDF to cultures and CD61 þ cells were increased 8-10-fold by the

Recombinant human megakaryocyte growth and development factor (rHuMGDF), a truncated form of the Mpl ligand, stimulates megakaryopoiesis both in vitro and in vivo. We describe the in vitro effect of pegylated recombinant human MGDF (PEGrHuMGDF) alone and in combination with other haemopoietic growth factors (G‐CSF, GM‐CSF, IL3, IL6, erythropoietin, SCF) on megakaryopoiesis in bone marrow from 11 normal subjects and 19 patients with aplastic anaemia (AA). We used semi‐solid cultures to assess megakaryocyte colony growth (CFU‐Mk) and 7 d suspension cultures to assess production of platelet glycoprotein IIIa (CD61) positive cells. CFU‐Mk growth from normal marrow increased 3–4‐fold and CD61+ve cells in suspension culture increased 8–10‐fold with the addition of 10 ng/ml PEGrHuMGDF. In normal subjects growth factor combinations further increased responses in suspension culture, PEGrHuMGDF + SCF, PEGrHuMGDF + IL3 and PEGrHuMGDF + SCF + IL3 + Epo (P < 0.05). IL6, GM‐CSF, G‐CSF or Epo added with PEGrHuMGDF did not consistently give this increase. CFU‐Mk growth from AA marrow remained very low in the presence of PEGrHuMGDF, with or without the addition of other growth factors. CD61+ve cells in suspension culture were, however, increased in the presence of PEGrHuMGDF alone in 12/19 AA cases. Of the 12 patients responsive to PEGrHuMGDF, nine were tested with additional growth factors and further responses were seen in six. In the AA cases PEGrHuMGDF+SCF and PEGrHuMGDF+SCF+IL3+Epo gave the highest responses. These data suggest that PEGrHuMGDF, alone or in combination with SCF and/or IL3, can enhance megakaryocyte proliferation in some patients with aplastic anaemia and may therefore have a role in the treatment of thrombocytopenia in these cases.

Pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) can stimulate megakaryopoiesis in vitro in some myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) patients. We assessed PEG-rHuMGDF combined with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), interleukin 3 (IL-3), IL6, stem cell factor (SCF) or erythropoietin in 40 MDS, 33 AML and 16 normal bone marrow samples. CD61-positive cells in suspension cultures increased with PEG-rHuMGDF alone in 20/25 RA + RAS, 11/14 RAEB + RAEBt and 29/33 AML cases. Further increases when IL-3 and/or SCF were added to PEG-rHuMGDF occurred in 14/20 RA + RAS, 8/13 RAEB + RAEBt and 18/26 AML cases. CFU-Mk growth was poor overall, but could be enhanced by PEG-rHuMGDF combinations in some patients. Stimulation of megakaryopoiesis by PEG-rHuMGDF can be augmented by IL-3 and SCF in many MDS and AML patients.

Summary. We have studied paired peripheral blood progenitor cells (PBPC) and bone marrow (BM) samples from 12 acute myeloid leukaemia (AML) patients following intensive chemotherapy, and assessed direct granulocyte± macrophage colony-forming units (CFU-GM), erythroid burst-forming units (BFU-E), megakaryocyte CFU (CFUMk) numbers and the production of CD61 1 (platelet glycoprotein IIIa) cells in suspension culture in response to various haemopoietic growth factor combinations. We found that CFU-GM and BFU-E numbers per 10 5 mononuclear cells were similar in both AML PBPC and BM harvests; CFU-Mk numbers, however, were significantly higher in PBPC than BM. In addition, the higher total white cell count of the PBPC harvests meant that PBPC have much higher numbers of total progenitors per collection. CD611 cell numbers in suspension cultures of AML PBPC and BM were lower than those of harvested normal marrow. However, response to pegylated recombinant human megakaryocyte growth and development factor (PEGrHuMGDF) both alone and in combination with other growth factors was qualitatively similar to that of normal BM. As with normal BM, response to PEGrHuMGDF alone did not increase further with addition of granulocyte colonystimulating factor (G-CSF), granulocyte±macrophage CSF (GM-CSF), interleukin 6 (IL-6) or erythropoietin (EPO) in the AML PBPC and BM. Further responses over PEGrHuMGDF alone were seen when added with stem cell factor (SCF) or with a combination of SCF 1 IL-3 1 EPO in both AML PBPC and BM cultures; however, the magnitude of the response was greater in the PBPC cultures. Response to PEGrHuMGDF 1 IL-3 was seen in the PBPC cultures but not in the AML BM. These data suggest that, in AML patients, there are proportionally more megakaryocyte progenitor cells in the mobilized PBPC than in the BM harvests, which would explain the more rapid platelet recovery following PBPC autografts.