Fig. 6.
Calcineurin Aβ (CNAβ) deficiency increases spontaneous activation of T cells in vivo and effector differentiation in vitro. (a, b) Splenocytes from 2-month-old Cnab−/−, Cnab−/−Tgfb1+/− and control mice were prepared and surface-stained for CD4, CD8, CD62L and CD44 and analysed as described in Fig. 4. For forkhead box P3 (FOXP3) staining, cells were surface-stained with CD4 and CD25 antibodies and then fixed/permeabilized and stained with FOXP3 antibody, as described in the Methods. For in vitro stimulation experiments, 1 × 106 splenocytes from 7-week-old Cnab−/− and control mice were cultured in the presence of anti-CD3/CD28 microbeads from Invitrogen (T cell expander beads) for 3 days and cells were stained for CD4 and CD25 as described above and analysed by flow cytometry (c). Supernatants were collected and assayed for cytokines by sandwich enzyme-linked immunosorbent assay using kits from BD-Biosciences (d, e). Interleukin-6 levels in unstimulated (medium) cultures were below detection levels.