Measurement of T-cell CD69 expression: a rapid and efficient means to assess mitogen- or antigen-induced...

We have analyzed the expression of the activation antigen CD69 on normal human T cells by flow cytometry following stimulation with mitogens and recall antigen. These data were compared to parallel studies assessing the proliferative response using the 3H-thymidine (3H-TdR) incorporation assay. Three different mitogens (PHA, ConA, and CD2/CD2R) induced maximal expression of CD69 at 24 h, which remained stable throughout the 72 h culture period. The mitogen-stimulated cells initiated DNA synthesis as determined by the 3H-TdR assay (72 h) while nonstimulated cells failed to upregulate CD69 or incorporate 3H-TdR. We next compared T cell CD69 expression (n = 12) following stimulation with either CD2/CD2R (5 micrograms/ml) or the recall antigen, tetanus toxoid (1:1500). Cell proliferation was determined by the 3H-TdR assay at 72 h (CD2/CD2R) or 120 h (tetanus toxoid). Evaluation of CD69 expression at 6 h predicted CD2/CD2R but not tetanus responder status as defined by 3H-TdR incorporation. However, when 4 known tetanus responders (3H-TdR) were evaluated over time, it was found that at 48 h the fluorescence intensity (of CD69) on tetanus-stimulated CD3+ cells increased markedly compared with nonstimulated cells (range of increase 43-850%). One individual whose cells failed to respond to tetanus toxoid (3H-TdR and CD69) did respond normally to CD2/CD2R. These observations suggest that flow cytometric evaluation of T cell CD69 expression following mitogen (6 h) or antigen (48 h) stimulation may provide an accurate screen of T-cell responsiveness in normals.