Ontogeny of the BMSC plasmablast-supporting capacity. (A) The capacity of BM cells to support the recovery of TT-specific ASCs was evaluated in coculture experiments. Fresh total BM cells from 5 individual mice were seeded immediately after harvesting into 96-well plates at a density of 5 × 104 cells/well. Enriched BMSCs generated from a pool of 10 10-week-old mice were generated by 5 days of culture as described in “BMSC cultures and plasmoblast survival assay” and reseeded at the same density. TT-specific splenic PBs (5 × 103) purified by anti-CD138 AutoMACS (Miltenyi Biotech, Bergisch Gladbach, Germany) sorting were added 24 hours later. Control wells contained PBs incubated in medium alone or with 10 ng/mL recombinant murine IL-6. TT-specific IgG ASCs were enumerated by ELISpot at the onset and after 48 hours of culture. The proportion of surviving PBs was calculated (recovered TT-specific ASCs/initial TT-specific ASCs × 100) and expressed as the mean (± SD) of 6 independent cultures. *P < .05 versus coculture of PB with adult total BM cells. (B) Similar experiments were performed with enriched BMSCs generated from a pool of fifteen 2- or 10-week-old (control) mice. Results are expressed as the mean (± SD) of 6 independent cultures per age group. One representative experiment of 3 is shown. *P < .05 versus adult enriched BMSCs. (C) Similar experiments were performed with enriched BMSCs generated from a pool of fifteen 4- or 10-week-old (control) mice. Results are expressed as the mean (± SD) of 6 independent cultures per age group. One representative experiment of 3 is shown. Error bars represent SD.
