Immediate STAT5A phosphorylation (pSTAT5A) upon T cell receptor stimulation is critical event in T cells proliferation. Here we present a simple and sensitive flow cytometric – based assay to assess T cell proliferation. Given the critical role STAT5A phosphorylation in T cell proliferation, we decided to investigate a phosphorylation of STAT5A as an indicator of T cell proliferation.
We determined pSTAT5A in T cell from 19 adult healthy donors stimulated with either CD3/CD28 or PHA.
After stimulation, T cells displayed a strong long-lasting phosphorylation of STAT5A, reaching a peak value after 24 hours. The median fluorescence intensity (MFI) of pSTAT5A increased from 112 ± 17 to 512 ± 278 (CD3/CD28) (24 h) and to 413 ± 123 (PHA) (24 h), the IL-2 receptor-α (CD25) expression was greatly enhanced and after 72 h T cell proliferation amounted to 52.3 ± 10.3 % (CD3/CD28) and to 48.4 ± 9.7 % (PHA). Treatment with specific STAT5 and JAK3 inhibitors resulted in a complete blockage of phosphorylation of STAT5A, CD25 expression, and suppression of T cell proliferation.
Compared with currently available methods, pSTAT5A is well suited to predict T cell proliferation. Moreover, due to its simplicity and robustness, the flow cytometric based pSTAT5 assay is especially appropriate to rapidly assess primary immune deficiencies (PIDs) associated with STAT5 defects including autoimmune diseases, CD25 deficiency and T cells proliferation defects.