Flow cytometric comparison of CD25⁺ versus CD25^- cell lines. After 3 to 4 weeks of culture expansion, antigen expression was profiled by FACS analysis. Shown are representative plots of CD25^--derived cell lines compared with potent suppressor cell lines and weakly suppressive cell lines. (A) Cell lines derived from CD25^- cells express low levels of CD25 as they revert to a more quiescent state. (B) Potent suppressor cell lines maintain high levels of CD25 expression. (C) Cell lines derived from CD4⁺CD25⁺ cells that have weak suppressor function express intermediate levels of CD25. (D) CD4⁺CD25^--derived cell lines express minimal intracellular CTLA4. (E) Potent suppressor cell lines maintain high intracellular expression of CTLA4. (F) Weak suppressor lines express intermediate levels. (G) CD25^--derived cell lines express variable levels of CD62L and diminished CD27. (H) Potent suppressor cell lines contain a higher percentage of cells that express both CD62L and CD27. (I) Weak suppressor cell lines contain a lower percentage of cells that express CD62L and CD27. (J-K) Cell sorting of suppressor cell line subsets reveals potent suppressor cells to express CD62L and CD27. (J) FACS plot showing sorting gates for CD62L and CD27 subsets. (K) Functional analysis in MLRs reveals suppressor activity solely within the CD62L and CD27 double-positive subset (▤). Control MLR cultures (▦) and suppressed MLR (▪). The CD62L⁺/CD27^- subset (brick bar) and the CD62L^-/CD27^- subset (weaved bar) both augment the MLR. Error bars represent 1 SD above and below the mean.