Flow cytometric analysis of lymphocyte activation in the mixed lymphocyte response

Interleukin 2 receptor (IL2R) expression may be a useful parameter for assessing lymphocyte activation in the mixed lymphocyte response (MLR). However, the contribution of irradiated stimulator (S*) cells to the levels of IL2R+ cells recovered must first be defined. We have used a flow cytometric parameter termed the sip count to assess this potential contribution of S* cells. This parameter, which is the number of cells within a defined cell gate, is a reflection of the viable cell number per culture well, since (a) a constant number of cells were plated per well on day 0, (b) cells recovered from a well were resuspended in a constant volume, (c) the flow cytometer aspirated (sipped) a constant volume of cell suspension, and (d) nonviable cells were not included in the gate. Sip count assessment showed that only 5% of S* cells were recoverable by day 4 of culture; in contrast, 70% or more of unstimulated responder cells were recoverable. Sip counts of MLR cultures identified an increase in cell number beginning on day 5, reflecting DNA synthesis and cell division. We then used the sip count to assess changes in the levels of IL2R+ cells in MLR cultures. The number of IL2R+ cells continued to increase up to day 7, even though maximal DNA synthesis occurred on day 5. Further, dual color analysis revealed that the proportion of CD4 cells expressing IL2R was maximal on day 5, whereas the proportion of CD8 cells expressing IL2R continued to increase until day 9. These findings show that flow cytometry can be used to study lymphocyte activation by alloantigens.