Interleukin-2 activation of chemotherapy and growth factor-mobilized peripheral blood stem cells for generation of cytotoxic effectors.

Based on our previous studies demonstrating marked anti-tumor activity of interleukin-2 (IL-2)-activated bone marrow in vitro and in vivo, we studied the generation of anti-tumor cytotoxic effectors from chemotherapy- and growth factor-mobilized PBSC from 12 patients with different solid tumors. As chemotherapy and growth factor priming could lead to important qualitative and quantitative alterations of lymphoid cells, we also looked at the distribution of lymphoid cells contained in primed PBSC. In addition, different variables were defined for successful application of the technique to clinical protocols. The cells were placed in culture at varying cell densities in either serum-containing or serum-free culture medium, supplemented with IL-2, 100 or 1000 Cetus units/ml at 37 degrees C for 24 or 72 h, in flasks or in culture bags. Anti-tumor cytotoxicity was tested against A375 (melanoma), K562 (CML) and Daudi (Burkitt's lymphoma) cell lines in standard 4 h 51Cr release assay. Marked cytotoxicity was seen against all cell lines tested (A375: 32.7% +/- 5.2; K562: 52.8% +/- 4.8; Daudi: 50.5% +/- 7.2). Cytotoxicity was comparable in serum-containing and serum-free culture conditions and in tissue culture flasks and bags. Cell density up to 10 x 10(6)/ml was not associated with any significant decline in cytotoxicity. IL-2 activation of PBSC after thawing led to the generation of cytotoxicity comparable to that obtained with fresh PBSC. On flow cytometric analysis, the proportion of CD8+ T cells and NK cells (CD56+) was found to be higher in primed PBSC than in control peripheral blood mononuclear cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of a licensed electrolyte solution as an alternative to tissue culture medium for bone marrow collection.

Bone marrow for transplantation is traditionally collected into tissue culture medium with heparin. A licensed electrolyte solution (Plasma-Lyte A [PLA]) was used as a substitute for tissue culture medium in the harvesting of 28 bone marrows, 17 autologous and 11 allogeneic, which were subsequently transplanted. Data that were analyzed from the 25 evaluable patients consisted of the numbers of cells and colony-forming units in the transplanted marrow as well as the time to neutrophil and platelet engraftment. These results were compared with those in the 30 (26 evaluable) preceding transplanted marrows that were collected into a tissue culture medium (RPMI-1640 [RPMI]). The autologous marrow transplant patients in both the PLA and RPMI groups reached a neutrophil count of > or = 0.5 x 10(9) per L a mean of 19 days following transplantation. The patients who underwent transplantation with allogeneic bone marrow collected in RPMI achieved > or = 0.5 x 10(9) per L of neutrophils an average of 20 days following transplantation, while those who received marrow collected in PLA achieved engraftment of neutrophils to that level in a mean of 21 days. Because in vitro and in vivo results with RPMI and PLA are similar in this study, further work using a licensed solution for clinical bone marrow transplantation is indicated.

A novel approach to immunomodulation of frozen human bone marrow with interleukin-2 for clinical application.

Interleukin-2 (IL-2) activation of fresh or frozen bone marrow (BM) in vitro generates killer cells with potent anti-tumor effect both in vitro and in vivo. The IL-2-activated BM (ABM) retains the capacity to reconstitute the hematopoietic system in an autologous bone marrow transplantation (ABMT) setting. The killer cells lose their cytotoxicity if the ABM undergoes the procedures of freezing and thawing. Therefore, for clinical application, the ABM has to be generated after thawing a frozen stock of BM before ABMT. The thawed BM cells are fragile and may undergo lysis, resulting in clump formation and cell loss. The frozen autograft also contains components of cryoprotectant mixture whose effects on the generation of ABM have not been defined. The present studies have been carried out to optimize a technique of handling the frozen BM for immunomodulation with IL-2 for 24 h at 37 degrees C prior to ABMT, with minimal loss of cells. IL-2-activation of BM was carried out in bags containing serum free medium which were designed to permit gaseous exchange. Addition of deoxyribonuclease (DNAse) (100 micrograms/ml of BM concentrate) immediately after thawing and the presence of heparin (20 units/ml) in the medium completely abrogated immediate or delayed clumping of cells. The presence of DNAse and/or heparin during in vitro culture did not affect the cell viability, cytotoxicity against tumor cells or the progenitor cell activity of the ABM; all these functions were well maintained even when BM was placed in culture immediately after thawing (without washing). There was no microbial contamination.(ABSTRACT TRUNCATED AT 250 WORDS)