Fig. S1 Production of Fc-GITR-L fusion protein

1. 98 64 50 36 Non-red. Red. Mw in kD Purified Fc-GITR-L Fig. S1 Production of Fc-GITR-L fusion protein S1A Diagram of Fc-GITR-L fusion protein. Human IgG1 Fc Fragment Mouse GITR Ligand Transmemberance and intracellular domain Ig light chain leader sequence Extracellular domain signal peptide Fc fragment S1B Analysis molecular weight and purity of Fc-GITR-L by SDS-PAGE.

2. CD4+ T Cells Resting Activated GITR+/+ GITR  Fc-GITR-L Fig. S2 The Fc-GITR-L fusion protein binds to its receptor GITR. S2A Fc-GITR-L binds to 293F cells transfected with GITR. GITR Fc-GITR-L S2B Fc-GITR-L binds to GITR in both resting and activated CD4+ T cells.

3. CD4+CD25– CD4+CD25+ Fc-GITR-L GITR S2C Fc-GITR-L binds to CD4+CD25+ Treg and CD4+CD25 Teff cells.

4. Fig. S3 Fc-GITR-L induces in vivo proliferation of CD4+FoxP3+ but not CD4+FoxP3 cells S3A Administrating Fc-GITR-L for 2 weeks induces proliferation of Treg but not Teff cells. 2 weeks ns 500 400 ns 300 Cell number(x106) 200 ns 100 0 ns 50 40 ns 30 CD4+(%) ns 20 10 0 IgG IgG IgG Fc-GITR-L Fc-GITR-L Fc-GITR-L Thymus Spleen MLN

5. S3B Administrating Fc-GITR-L for 4 weeks induces proliferation of Treg but not Teff cells. 4 weeks 500 ns 400 ns 300 Cell number(x106) 200 ns 100 0 ns 50 40 ns 30 CD4+(%) ns 20 10 0 IgG IgG IgG Fc-GITR-L Fc-GITR-L Fc-GITR-L Thymus MLN Spleen

6. Fig. S4 Fc-GITR-L induces in vivo proliferation of CD4+FoxP3+ cells in both C57BL/6 and BALB/c mice strain S4A Administrating Fc-GITR-L induces proliferation of Treg cells in C57BL/6 mice. *** 20 16 12 8 4 0 C57BL/6 FoxP3+/CD4+(%) IgG Fc-GITR-L S4B Administrating Fc-GITR-L induces proliferation of Treg cells in BALB/c mice. ** 12 8 4 0 BALB/c FoxP3+/CD4+(%) IgG FcGITRL