Greatly improved recovery of in vitro B-memory cell function after enzymic dispersion of immunized mouse spleens.

We have used the protease dispase to disperse the fine clumps that persist after mechanical disruption of spleens from immunized mice. After 4-8 days in culture, the resulting 'D/C' cells spontaneously generated many more IgG plaque-forming cells (PFC) against sheep erythrocytes (SRBC) than did conventional (CONV) suspensions. The difference averaged 12-fold and was consistently high after a wide range of immunization protocols. The major difference between the two cell preparations proved to be in the B-cell lineage rather than in antigen-presenting cells or T cells and, indeed, the response was largely T-cell independent. Antigen-driven culture responses to SRBC were also more than 10-fold higher with D/C than with CONV suspensions, and again there was apparently an improved recovery of B-memory cells. However, when fresh cell preparations were assayed immediately for PFC, there was no D/C:CONV difference--just as we have previously reported for memory responses on cell transfer to irradiated recipients. One simple interpretation is that germinal centres tend to remain as fine clumps on mechanical disruption, and their constituent B-memory cells are enriched by our procedure. If so, their responses are much more evident in vitro than after cell transfer.