Measurement of daughter cell accumulation during lymphocyte proliferation in vivo.
We describe a method to characterize the effects that immunological adjuvants have on in vivo lymphocyte proliferation at the level of daughter cell accumulation. We used standard 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeling techniques to follow T cells through multiple rounds of division in experimentally treated mice and measured both the fold-increase in cell number and average number of divisions undergone by each cell. These data were then incorporated into a calculation to determine the average number of daughter cells that accumulated from each round of mitosis, termed the daughter cell accumulation index. In vivo proliferation of T cells that had been stimulated by antigen in the absence of adjuvant was associated with an index value of 1.2, far below the theoretical maximum of 2.0. Low index values indicate poor daughter cell accumulation during proliferation, either because the newly produced cells died or persisted without dividing again. Inclusion of the natural adjuvant lipopolysaccharide (LPS) led to average accumulation of 1.75 daughter cells per cell cycle and an exponential increase in peak clonal expansion. Adjuvant-induced increases in average daughter cell accumulation appeared to account for more of the enhancement in clonal expansion than did adjuvant-induced increases in the number of cell divisions undergone by each cell. Therefore measurement of changes in daughter cell accumulation can be important to understanding how adjuvants influence the yield of proliferating lymphocytes. Measurement of average daughter cell accumulation is likely to be helpful in any cellular context in which it is useful to characterize strictly mitogenic versus accumulative effects.
Thompson
Bruce S
BS
Institute for Cellular Therapeutics and the Department of Microbiology and Immunology, University of Louisville, School of Medicine, Louisville, KY 40202, USA.
Mitchell
Thomas C
TC
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D002452:Cell Count / Q000379:methods*; D049109:Cell Proliferation / Q000187:drug effects; D005434:Flow Cytometry; D005452:Fluoresceins / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology; D008214:Lymphocytes / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D012680:Sensitivity and Specificity; D013388:Succinimides / Q000494:pharmacology
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000276:Adjuvants, Immunologic; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides