Analyzing T Cell Responses Using Measurements Of Mitotic Number

A Wells and LA Turka, University of Pennsylvania, Philadelphia, Pennsylvania

Most analyses of T cell proliferative function are performed on bulk populations. The DNA analog BrdU enables determinations of T cell proliferation at the single cell level, but only distinguishes between cells which have never divided and those which have done so one or more times. Here we report on the characterization and use of carboxyfluorescein-diacetate-succinimidyl ester (CFSE), to analyze T cell immune responses as a function of mitotic number.

CFSE is a lipophilic fluorescent dye which labels membranes of living cells. Labeled cells are detected by FACS, and the fluorescent intensity decreases by half with each round of division, making it possible to determine the number of times a T cell has divided. CFSE can be used with surface stains and with viability dyes in multicolor flow cytometry. Here, unfractionated murine spleen cells were labeled ex vivo with CFSE (5 µg/ml) for 10 min at 20ºC, washed and stimulated for 4 days with 1 µg/ml soluble anti-CD3 mAb. Cells were stained with anti-Thy1 to gate on T cells and the CFSE profile was determined. The numerals indicate the number of cell divisions represented by each peak.

(See original Abstract for Graph)

Our series of our studies using CFSE are notable for the following: (1) Even using hi dose anti-CD3 as a stimulus, cells proliferate asynchronously, with first mitoses occurring from day 2 to day 5; (2) By day 5, 80% of T cells have divided at least once, and 25% have gone through 5-7 mitoses; (3) As expected, there are many dead cells in late cultures, but surprisingly, dead cells have a strong "proliferative history" and have typically undergone more divisions than viable cells; (4) When CD28-mediated costimulation is blocked with CTLA4Ig, most cells are still able to divide, however division is abruptly halted after 2-3 mitoses, and there is an excess number of dead cells; The implication of these findings will be discussed.