Background. Direct measurement of the precursor frequency of alloreactive C
D4(+) T cells has been impossible due to the lack of a specific means of de
termining the absolute number of daughter cells generated with each divisio
n in a repertoire of stimulated T cells.
Methods. Responder lymphocytes were fluorescently labeled and adoptively tr
ansferred into irradiated allogeneic stimulator mice or incubated in vitro
with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation pe
riod, responder cells were analyzed by flow cytometry.
Results. The precursor frequency of dividing CD4(+) T cells was determined
both in vivo and in vitro. The observed number of alloreactive daughter cel
ls generated with each round of division was used to calculate the frequenc
y of alloantigen-specific CD4(+) T cells.
Conclusions. A novel method for the direct calculation of the frequency of
alloreactive CD4(+) T cells is described. This technique allows the determi
nation of changes in the frequency of alloreactive T cells that might under
lie tolerance 60 alloantigens.