Immunotherapeutic Potential of Dendritic Cells Generated in Long Term Stroma-Dependent Cultures

Abstract

Long term cultures (LTC) producing dendritic cells (DC) have been established from spleen. A well developed stromal cell layer supported production of DC in numbers suitable for experimentation. Cells had obvious membrane pseudopodia and could be collected from culture every 2-3 days. Large cells produced in LTC stained with fluorescently labelled monoclonal antibodies specific for DC such as 33D1, and M1/70 which is specific for DC and myeloid cells. These staining patterns confirmed the presence of DC within the LTC population. LTC-DC were tested and shown capable of migration in vivo in B10.A(2R) mice following footpad inoculation. Most cells entered the spleen and a small number entered popliteal lymph node. LTC-DC have migratory capacity comparable with control spleen lymphocytes. LTC-DC were tested for capacity to induce an anti-tumour immune response after exposing cells to tumour cell membranes. LTC-DC pulsed with BL/VL3 tumour antigens were able to induce a BL/VL3-specific primary cytotoxic T lymphocyte (CTL) response detectable in popliteal lymph nodes and spleen of C57BL/6J mice within 6 days of priming. BL/VL3 tumour cells grew in sublethally irradiated C57BL/6J mice giving 100% mortality. Adoptive transfer of spleen cells from mice given BL/VL3 antigen-pulsed LTC-DC, two weeks previously, significantly slowed the growth of BL/VL3 tumour cells in mice. DC produced in LTC can function as antigen presenting cells (APC) when adoptively transferred into animals. Their capacity to migrate effectively, to induce a CTL response and to reduce tumour load suggests that DC grown using this in vitro system may have valuable clinical potential in humans.

Identification of progenitor cells in long-term spleen stromal cultures that produce immature dendritic cells

“…The antigenpresenting capacity of cells produced in LTC has been confirmed at numerous time points (14). LTC-DC can stimulate both allogeneic and syngeneic naive T cells as well as present antigen to antigen-specific T helper cells (11,15). In this report, surface marker expression, function, and differentiative capacity have been used to characterize two major DC subsets produced in LTC.…”

“…Cultures were established and maintained in supplemented DMEM from 6-to 8-week-old female mice as described in detail previously (15). They contain a stromal cell layer of fibroblasts and endothelial cells.…”

Targeting Dendritic Cells with Antigen-Containing Liposomes

“…Murine fetal skin DCs (FSDC) [C57BL/6-DBA/2J F1 (H-2 b/d )] were cultured in the same medium but without geneticin. Murine long-term culture DCs (LTC-DC) [B10.A(2R; H-2 k/b )], isolated and cultured as described (28), were a gift from Dr. Helen O'Neill (School of Biochemistry and Molecular Biology, Australian National University).…”

Identification of differentially expressed genes representing dendritic cell precursors and their progeny

“…LTCs were established from whole spleen of 6-to 8-week-old female mice and were maintained in supplemented Dulbecco modified Eagle medium (sDMEM) as described previously. 23 Nonadherent LTC-DCs were collected from culture flasks without disturbance to the stroma and were filtered through a 200-gauge nylon filter to remove cell aggregates.…”