Requirements for cell activation for the upregulation of α4β7 expression by RA treatment. (A) Aliquots of PBMCs from normal healthy RM, PM, and SM were cultured for 48 h in complete RPMI 1640 media alone (green), complete media containing 1 μM RA alone for the last 24 h (orange), complete media containing anti-CD3/28 immunobeads + IL-2 alone (red), and the anti-CD3/28 immunobeads + IL-2 along with 1 μM RA for the last 24 h (blue). (C) The same experiment as outlined in (A) except an additional culture containing the immunobeads + IL-2 and 1 μM RA was set up that included the addition of the inhibitor FK866 that was added at the initiation of the culture. The cells were washed and the flow-cytometric profile of CD4+ T cells that expressed α4β7 is shown in (A) and (C). The experiment was repeated three times, and the data obtained were expressed as fold change (mean ± SD) of MFI relative to baselines (B and D). **p < 0.001.