Fig 1. Thawing of frozen Tregs reduces CD62L expression and inhibits the binding to MADCAM1.

(A) Relative number of CD4⁺ T cells among live splenocytes and Tregs among CD4⁺ T cells before freezing and after thawing. (B) Representative dot plots illustrating CD62L expression on Tregs before freezing and after thawing. Dot plots are gated on live CD4⁺Foxp3⁺ T cells. Relative number of CD62L expressing cells among Tregs. Mean fluorescence intensity (MFI) of fluorescein isothiocyanate coupled to Anti-CD62L of CD62L⁺ Tregs. Shown are 4 mice per group from one of at least two independent experiments. (C) Representative histograms showing CD62L expression on human CD4⁺Foxp3⁺ Tregs before and after cryopreservation. Shown are 4 single donor samples per group. (D) Representative bioluminescence images of isolated Tregs that adhere to plate-bound MADCAM1 in vitro using Cell Titer Glo. Shown is one of at least two independent experiments performed in triplicates. (E) Representative bioluminescence images of luc ⁺ Tcons in mice receiving Tcons alone or Tcons with fresh Tregs incubated without or with CD62L-blocking antibody (Mel14) prior to transplantation. Shown are five animals per group.