A kinetic study of the murine mixed lymphocyte reaction by 5,6-carboxyfluorescein diacetate succinimidyl ester labeling 11 Abstract 12Alternatives to the use of radioisotopes to measure cell proliferation in mixed lymphocyte reactions (MLR) are desirable to 13 avoid the hazards and costs associated with radioisotope use. The versatile fluorochrome 5,6-carboxyfluorescein diacetate 14 succinimidyl ester (CFSE) has been used to measure MLR and provides the opportunity to measure several different growth 15 parameters. This study was aimed at determining which growth parameter is most practical and suitable for measuring murine 16 MLR. The parameters measured were: the relative number of daughter T-cells, the relative number and frequency of reactive T-17 cell precursors and the relative number of mitotic events. Responder cells were CFSE-labeled unfractionated splenocytes from 18 C57BL/6 mice. Stimulator cells included irradiated splenocytes from C57BL/6 (control), B6D2F 1 (haplo-allogeneic) or FVB/N 19 (allogeneic) mice. Cultures were harvested daily for 1 week. Stimulator T-cells rapidly declined to less than 0.2 -0.3% of the 20 mixed population by day 2 of culture. Experimental groups had a significantly higher number of daughter T-cells and mitotic 21 events after 2 days of culture with the number of daughter T-cells climbing exponentially after 5 days of culture. The number 22 and frequency of reactive T-cell precursors were significantly higher in experimental groups on days 2 -3, but this difference 23 became insignificant by day 4. Among all the parameters, the relative number of daughter T-cells was the most practical for 24 measuring MLR, after 5 days of culture, based upon the growth kinetics of responder T-cells and the survival of the stimulator 25 cells. 26 D

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SummaryThe effects of donor T cells, or their CD8 + subset, on engraftment and tolerance induction in fetal transplantation were evaluated using an F 1 -intoparent mouse-model that does not permit a graft-versus-host effect.