PHENOTYPE TRANSITION OF CD4(+) T-CELLS FROM CD45RA TO CD45R0 IS ACCOMPANIED BY CELL ACTIVATION AND PROLIFERATION

An investigation of proliferation and activation events in subsets of human CD4(+) cells, defined by their expression of CD45RA and CD45RO, is reported. A single-laser based assay for the study of multiple surface antigens and two-parameter cell cycle analysis was used for sorting of and subsequent analysis of proliferation in CD4(+) CD45RA(+) CD45RO(-), CD4(+)CD45RB(-)CD45RO(+) subsets and phenotypically intermediate stages. After labelling with BrdUrd, cells were sorted with now cytometry on the basis of light-scattering properties and staining with anti-CD45RA, anti-CD45RO, and anti-CD4 markers. Sorted cells were double stained with anti-Brdurd-antibodies and PI, and the frequencies of proliferating cells were determined. After 48 h, the highest rate of proliferation was found among cells with a phenotype intermediate between CD4(+) CD45RA(+)CD45RO(-) and CD4(+)CD45RA(-)CD45RO(+). After 72 h of culture, the situation was changed insofar as the point of highest proliferation had shifted towards the CD4(+)CD45RA(-)CD45RO(+) population. These findings were further corroborated by four-colour staining with anti-CD4, anti-CD45RA, anti-CD45RO, and Hoechst 33342. This indicates that the phenotype transition is accompanied by cell proliferation. The correlated temporal expression of antigens related to activation (HLA-DR, CD25, CD69, CD71) and cell adhesion (CD11a, CD54, L-selectin) in each of the different subsets was also investigated. Ah the activation markers CD25, CD69, and CD71 show a more heterogeneous pattern of expression among the CD4(+) CD45RA(-)CD45RO(+) cells than the CD4(+) CD45RA(+)CD45RO(-) cells, indicating a subpopulation of CD4(+)CD45RA(-)CD45RO(+) cells responding more slowly to the mitogenic stimulation. (C) 1995 Wiley-Liss, Inc.