LFA-1 on human Tregs is essential for cell-contact mediated suppression. A, Proliferation of mouse CD4⁺CD25⁻ T cells alone (black bar), or with a 1:1 ratio of fresh hTregs (hCD25^hi, gray bar) or B, pre-activated hTregs (act.hCD25^hi) in the presence of isotype control or anti-hCD11a (efalizumab), -hCD18 (TS1/18), or -hICAM-1/2/3 blocking mAbs. C, Proliferation of mouse CD4⁺CD25⁻ T cells alone (black bar), or with a 1:1 ratio of fresh hTregs (hCD25^hi, gray bar) in the presence of isotype control or different clones of anti-hCD11a or -hCD18 mAbs. For the above assays, the mouse responders were stimulated with mouse APCs and soluble anti-mCD3 while the fresh hTregs were optimally activated in the cocultures with plate-bound anti-hCD3/CD28. The pre-activated hTregs were not restimulated. D, Suppression of human CD4⁺CD25⁻ T cells with FACS-sorted hTregs (hCD25^hi), mTregs (mCD25⁺), or pre-activated mTregs (act.mCD25⁺). In this assay, the human responders and Tregs were stimulated with human APCs and soluble anti-hCD3, while the fresh mTregs were optimally activated in the cocultures with plate-bound anti-mCD3/CD28. The pre-activated mTregs were not restimulated. Data are representative of three independent experiments. Asterisk (*) represents p < 0.05 for the difference between the black and gray bars in each group.

Human Tregs target mouse DCs and not T cells via an LFA-1/ICAM-1 dependent interaction. A, Proliferation of CD4⁺CD25⁻ T cells from wild-type (WT) C57BL/6 or ICAM-1^−/− (KO) mice stimulated with soluble anti-mCD3 and T-depleted splenocytes or B, splenic DCs alone (black bar) or cocultured with 1:1 ratio of pre-activated hTregs (act.hCD25^hi, gray bar) with or without blocking anti-mICAM-2 or -hCD11a (efalizumab) mAbs. The pre-activated hTregs were not restimulated. Asterisk (*) represents p < 0.05 for the difference between the black and gray bars in each group. C, Level of CD80/CD86 expression on splenic DCs from wild-type (WT) C57BL/6 or ICAM-1^−/− mice at 0 h (shaded histogram) and after 18 h cultured alone (opened histogram) or with pre-activated human (dashed histogram) hCD25^hi or hCD25⁻ T cells from healthy donor. D, CD80/CD86 expression on splenic DCs from wild-type (WT) C57BL/6 mice at 0 h (shaded histogram) and after 18 h cultured alone (opened histogram) or with pre-activated human (dashed histogram) hCD25^hi from healthy donor, hCD25^hi from LAD-1 patient or hCD25⁻ T cells from healthy donor. Data are representative of three (A, B and C) and two (D) independent experiments.

Human Tregs from LAD-1 patients lack suppressive function. A, Proliferation of mouse CD4⁺CD25⁻ T cells alone (black bar), with 1:1 ratio of fresh hTregs (hCD25^hi, gray bar) from healthy donors (D1/D2), or LAD-1 patients (P1/P2). White bar is the hTregs stimulated alone. Upper panel represents post-sort flow cytometric FOXP3 staining on the hTregs. Asterisk (*) represents p < 0.05 for the difference between the black and gray bars in each group. B, Proliferation of mouse CD4⁺CD25⁻ T cells alone, or cultured with varying numbers of pre-activated hTregs (act.hCD25^hi) from healthy donor D1, or LAD-1 patient P1. Upper panel represents FOXP3 staining of 48 h pre-activated hTregs. In these assays, the mouse responders were stimulated with mouse APCs and soluble anti-mCD3 while the fresh hTregs were optimally activated with plate-bound anti-hCD3/CD28. The pre-activated hTregs were not restimulated. C, Proliferation of CD4⁺CD25⁻ or D, CD8⁺CD25⁻ T cells from a normal donor alone (black bar) or with 1:1 ratio of fresh hTregs (gray bar) from healthy donors (D1/D2), or LAD-1 patients (P1/P2). In this assay, the human responders and Tregs were stimulated with human APCs and soluble anti-hCD3. Data are representative of two independent experiments. Asterisk (*) represents p < 0.05 for the differences in suppression between healthy donors and LAD-1 patients.

Human Tregs modulate splenic DCs to have decreased capacity to present antigen and activate mouse responder T cells. A, Proliferation of mouse CD4⁺CD25⁻ T cells from HA-TCR Tg mice in the presence of HA peptide and re-isolated splenic DCs that had been previously cultured for 18 h alone (mDC), with Tregs from healthy donor (hCD25^hi), with Tregs from LAD-1 patient (hCD25^hi LAD-1), or with CD4⁺CD25⁻ T cells from healthy donor (hCD25⁻). Upper panel represents the purity of splenic DCs repurified from the cocultures based on staining with mouse CD11c and human CD4. B, Expression of CD69 and CD25 on mouse CD4⁺CD25⁻ from the cocultures stimulated with splenic DCs that had been cultured for 18 h alone (dashed histogram) or with the three human T cell populations (solid histograms). Data are representative of two independent experiments. Asterisk (*) represents p < 0.05 for the differences in suppression between mDCs from hCD25^hi vs. mDCs from hCD25^hi LAD-1 or hCD25⁻.

IL-2 is required for Treg suppressor function under suboptimal stimulatory conditions, but IL-2 consumption plays no role in hTreg-mediated suppression. A, Proliferation of mouse CD4⁺CD25⁻ T cells alone (black bar) or with 1:1 ratio of fresh > 95% FOXP3⁺ hTregs (hCD25^hi, gray bar) in the absence (none) or presence of 0.5 U/ml of recombinant human IL-2 (rhIL-2) under suboptimal stimulatory condition for hTregs. B, Proliferation of mouse CD4⁺CD25⁻ T cells alone (black bar) or with 1:1 ratio of fresh < 90% FOXP3⁺ hTregs (hCD25^hi, gray bar) in the presence of isotype control, daclizumab (anti-hCD25) or 0.5 U/ml of human IL-2 under suboptimal stimulatory condition. C, Proliferation of mouse CD4⁺CD25⁻ T cells alone (black bar) or with 1:1 ratio of fresh > 95% FOXP3⁺ hTregs (hCD25^hi, gray bar) in the presence of isotype, daclizumab or 0.5 U/ml of rhIL-2 under optimal stimulatory condition. Top panel represents post-sort FOXP3 purity for hTregs. D, Proliferation of mouse CD4⁺CD25⁻ T cells alone (black bar) or with 1:1 ratio of pre-activated hTregs (act.hCD25^hi, gray bar) in the presence of isotype or daclizumab (anti-hCD25). In these assays, the mouse responders were stimulated with mouse APCs and soluble anti-mCD3, while the fresh hTregs were stimulated either under suboptimal (3 μg/ml anti-hCD3 and 1 μg/ml anti-hCD28 plate-bound mAbs) or optimal stimulatory condition (5 μg/ml anti-hCD3 and 2.5 μg/ml anti-hCD28 plate-bound mAbs) in the cocultures. The pre-activated hTregs were not restimulated in the cocultures. Data are representative of three independent experiments. Asterisk (*) represents p < 0.05 for the difference between the black and gray bars in each group.

Human Treg suppression of mouse T cell activation and proliferation. A, In vitro suppression of CD4⁺CD25⁻ T cells from BALB/c mice with FACS-sorted human CD4⁺CD127⁻ CD25^hi (hCD25^hi), hCD25^int, hCD25⁻ or mouse mCD25⁺ T cells. In the assay, the mouse CD25⁻ and CD25⁺ were activated with mouse APCs and soluble anti-mCD3, while the human cells were activated with plate-bound anti-hCD3 (5 μg/ml) and anti-hCD28 (2.5 μg/ml) in the cocultures. Top panel represents post-sort level of FOXP3 and CD25 in the three human T cell populations. B, Expression of CD69 and CD25 on mouse CD4⁺CD25⁻ T cells cocultured for 3 days at 1:1 ratio with hCD25⁻ (solid line histogram), hCD25^int (dashed line histogram), and hCD25^hi (shaded histogram). C. In vitro suppression of mouse, CD4⁺CD25⁻ or D, CD8⁺CD25⁻ T cells activated with mouse APCs and soluble anti-mCD3 alone or with fresh mouse Tregs (mCD25⁺) or pre-activated human Tregs (act.hCD25^hi) without restimulation. E, In vitro suppression of mouse CD4⁺CD25⁻ T cells with pre-activated human Tregs at 1:0, 1:1 and 4:1 ratios of mouse responder to hTreg in the presence of isotype control or neutralizing mAbs to TGFβ (anti-TGFβ), human IL-10 (anti-hIL10), mouse IL-10 receptor (mIL10R), and human CTLA-4 (anti-hCTLA4). Recombinant human latency associated peptide (rhLAP) also was used to neutralize TGFβ. Data are representative of three independent experiments.