Comparative dose-responses of recombinant human IL-2 and IL-7 on STAT5 phosphorylation in CD4+FOXP3- cells versus regulatory T cells: a whole blood perspective

Interleukin(IL)-2 and IL-7 are cytokines with important functions related to CD4(+) lymphocyte proliferation, differentiation and survival. Depending on doses, they theoretically activate regulatory (Treg) and/or effector T cells (Teff) and thus may be indicated with different therapeutic objectives. In this study we assessed ex vivo the differential dose-responses of CD4(+) T cell subsets (Treg versus CD4(+)FOXP3(-) cells) to recombinant human (rh) IL-2 and rhIL-7. Fresh whole blood from healthy donors was stimulated with increasing doses of cytokines. By using a novel flow cytometry procedure of intracellular signaling pathway staining (e.g., detection of STAT5 phosphorylation; a pivotal marker of cytokine-induced activation; in combination with intracellular FOXP3 staining), we were able to specifically measure Treg and CD4(+)FOXP3(-) cell responses in the same tube. Half maximal effective concentrations (EC50) were calculated. We observed a dose-response effect on Treg and CD4(+)FOXP3(-) cells for both cytokines. Interestingly, low doses of hIL-2 preferentially activated Treg (EC50 Treg = 0.15 pg/ml versus CD4(+)FOXP3(-) cells = 750 pg/ml - p < 0.0001) whereas low doses of rhIL-7 preferentially induced CD4(+)FOXP3(-) cell activation (EC50 Treg = 25 pg/ml and CD4(+)FOXP3(-) cells = 2.5 pg/ml - p < 0.0001). To our knowledge, this work is the first to show differential dose-response effects on CD4(+)FOXP3(-) cells versus Treg of rhIL-7 and rhIL-2 in one ex vivo whole blood single tube assay including two intracellular stainings (i.e., pSTAT5 and FOXP3). Beyond the confirmation of the dose-dependent differential effects of IL-2 versus IL-7 on CD4(+)FOXP3(-) cells/Treg, our results illustrate the value of this approach for monitoring drugs' activities by flow cytometry in daily clinical practice.