Post-chemotherapy and cytokine pretreated marrow stromal cell layers suppress hematopoiesis from normal donor CD34+ cells.

Marrow stromal layers were used to investigate the potential role of negative regulators produced by the marrow microenvironment as one potential cause of hematopoietic suppression after chemotherapy and cytokines. Stromal layers were established from marrow of normal or prechemotherapy donors and breast cancer patients after hematological recovery from one cycle of 5-fluorouracil, leucovorin, doxorubicin, and cyclophosphamide and GM-CSF or PIXY321 (GM-CSF/IL-3 fusion protein). Normal donor CD34+ cells were placed in contact with stromal layers, and the number of colony-forming units for granulocytes and macrophages (CFU-GM) was determined. There were 25-79% fewer CFU-GM in post-chemotherapy stromal layer cocultures than in no chemotherapy cocultures. With neutralizing antibody to TNF-alpha the number of CFU-GM in no chemotherapy and post-chemotherapy stromal cocultures was, respectively, 96 +/- 7% (n = 5) and 142 +/- 8% (n = 5) of the number with no antibody treatment. PIXY321 and GM-CSF pretreated stromal layers also suppressed production of CFU-GM. Anti-TNF-alpha promoted an increase in CFU-GM numbers from GM-CSF, but not PIXY321, pretreated stromal cocultures. The results demonstrate that post-chemotherapy marrow stromal layers were deficient in supporting in vitro hematopoiesis and suggest that negative regulators induced by chemotherapy and cytokines may be one cause for this defect.

In vitro generation of allospecific human CD8+ T cells of Tc1 and Tc2 phenotype.

We have previously shown that allospecific murine CD8+ T cells of the Tc1 and Tc2 phenotype could be generated in vitro, and that such functionally defined T-cell subsets mediated a graft-versus-leukemia (GVL) effect with reduced graft-versus-host disease (GVHD). To evaluate whether analogous Tc1 and Tc2 subsets might be generated in humans, CD8+ T cells were allostimulated in the presence of either interleukin-12 (IL-12) and transforming growth factor-beta (TGF-beta) (Tc1 culture) or IL-4 (Tc2 culture). Tc1-type CD8 cells secreted the type I cytokines IL-2 and interferon gamma (IFN-gamma), whereas Tc2-type cells primarily secreted the type II cytokines IL-4, IL-5, and IL-10. Both cytokine-secreting populations effectively lysed tumor targets when stimulated with anti-T-cell receptor (TCR) antibody; allospecificity of Tc1- and Tc2-mediated cytolytic function was demonstrated using bone marrow-derived stimulator cells as targets. In addition, both Tc1 and Tc2 subsets were capable of mediating cytolysis through the fas pathway. We therefore conclude that allospecific human CD8+ T cells of Tc1 and Tc2 phenotype can be generated in vitro, and that these T-cell populations may be important for the mediation and regulation of allogeneic transplantation responses.

Early suppressive effects of chemotherapy on recovery of bone marrow megakaryocyte precursors: possible relationship to platelet recovery.

This study utilized a recently developed culture and quantitation system to detect megakaryocyte precursors in CD34+ bone marrow cells from normal donors and breast cancer patients treated with 5-fluorouracil, leucovorin, adriamycin and cyclophosphamide (FLAC). Bone marrow was obtained from patients before and then after their first cycle of FLAC once blood cell counts had recovered. CD34+ cells were isolated and placed in liquid culture with growth factors to stimulate proliferation and lineage commitment. Absorbance values from an enzyme-linked immunosorbent assay were used to quantitate expression of platelet glycoprotein GPIIb/IIIa. There was an increase in absorbance with increasing numbers of cells seeded per culture that was associated with an increase in the number of megakaryocyte lineage cells produced. After 10 days in liquid culture, absorbance values for expression of GPIIb/IIIa from 2,000 normal donor and pre-chemotherapy CD34+ marrow cells were > or = 1.0. Absorbance values from cultures of post-chemotherapy CD34+ cells from four patients were similar to values from pre-chemotherapy CD34+ cells. In contrast, absorbance values from cultures of post-chemotherapy CD34+ cells from two other patients were low (absorbance < 0.5). Low absorbance values for GPIIb/IIIa expression indicate that megakaryocyte production from those CD34+ cells was reduced. Both of those patients developed prolonged thrombocytopenia and platelet nadirs of less than 20,000/microl during FLAC chemotherapy. In contrast, only one out of four patients whose cultures of post-chemotherapy CD34+ cells had absorbance values > or = 1.0 developed platelet nadirs less than 20,000/microl. These results suggest that low platelet nadirs and delayed platelet recovery may be associated with suppressive effects of chemotherapy on recovery of megakaryocyte precursors.