An intrinsic low level of calpain is associated with the high strength binding by LFA-1 on T reg cells. (A, left) Mean forces of wild-type or LFA-1 T–DC–deficient T reg, or T conv cells adhering to DC2.4 cells. (right) Mean forces of wild-type or LFA-1–deficient T reg cells adhering to wild-type or ICAM-1–deficient BMDCs. Representative of four independent experiments. (B) Levels of LFA-1 expression on T reg, resting (CD25⁻), and activated (CD25⁺) T conv cells. Representative of three independent experiments. (C, left) Schematic diagram. (right) Adhesion forces between wild-type or Cd11a^−/− T conv or T reg cells and recombinant Fc-ICAM-1–coated on a glass slide. B6 T reg cell/blank glass contact was recorded as the background. Representative of three independent experiments. (D) ADP ribosylation of LFA-1, which is induced by NAD treatment, is known to reduce 2D7 staining intensities. (left) T reg and T conv cells were stained with 2D7 antibody without NAD treatment. (middle) The NAD treatment–induced down-regulation of 2D7 staining in comparison with the untreated control. The comparable 2D7 staining intensities on T reg cells indicate no difference in ADP-ribosylation (left). (right) Similar expression of ART2b (ART2.2), the enzyme responsible for surface LFA-1 ADP ribosylation, between T reg and T conv cells. Representative of three independent experiments. (E) Adhesion forces between IL-2–treated or untreated human T reg cells adhering to PMA-stimulated THP-1 cells grown on the disk. (left) Scheme. Bar graph (middle left) shows mean adhesion forces of IL-2–treated or untreated human T reg cells. Levels of total (middle) or open conformation (middle right) LFA-1 expression on IL-2-treated human T reg cells from the peripheral blood. Gray: isotype control. Human T reg cells treated with Mg²⁺ and EGTA showed elevated expression of open LFA-1 (right). Representative of three independent experiments. (F, left) T reg and T conv cells were incubated with calpain substrate CMAC; calpain activities as fluorescence signals from the digested substrate were determined by FACS after a 5-min incubation. (right) The remaining substrates were quantified. Representative of at least 10 independent experiments. (G) Mean forces of untreated or calpeptin-treated T reg cell (left), T reg or T conv (middle), and wild-type, LFA-1-deficient T reg cell, or T conv cells (right) adhering to DC2.4. Representative of five independent experiments. (H) Mean forces of vector control, m-calpain, constitutively active m-calpain, or µ-calpain–overexpressing T reg adhering to DC2.4. Representative of three independent experiments. (I) T reg cell–mediated suppression of OT-II T cell division, as measured by CellTrace dilution, stimulated by OVA-pulsed DC2.4 cells as described in the Materials and methods. (left to right) Division in the presence or absence of OVA without T reg cells; in the presence of vector transfection control or m-calpain–overexpressing T reg cells; vector or CA (constitutively active) m-calpain–overexpressing T reg cells; and vector or µ-calpain–overexpressing T reg cells. Statistical results (far right) are pooled from three independent experiments. *, P < 0.05; **, < 0.01; ***, < 0.001; NS, not significant.