Background. Direct measurement of the precursor frequency of alloreactive CD4+ T cells has been impossible due to the lack of a specific means of determining the absolute number of daughter cells generated with each division in a repertoire of stimulated T cells. Methods. Responder lymphocytes were fluorescently labeled and adoptively transferred into irradiated allogeneic stimulator mice or incubated in vitro with irradiated stimulator splenocytes. After a 65- to 70-hr stimulation period, responder cells were analyzed by flow cytometry. Results. The precursor frequency of dividing CD4+ T cells was determined both in vive and in vitro. The observed number of alloreactive daughter cells generated with each round of division was used to calculate the frequency 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 determination of changes in the frequency of alloreactive T cells that might underlie tolerance to alloantigens.
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