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

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

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

4. ThymusSpleenMLN Fc-GITR-L IgG Fc-GITR-L IgG Fc-GITR-L IgG 0 10 20 30 40 50 CD4 + (%) ns 0 100 200 300 400 500 Cell number(x10 6 ) ns Fig. S3 Fc-GITR-L induces in vivo proliferation of CD4 + FoxP3 + but not CD4 + FoxP3  cells 2 weeks S3A Administrating Fc-GITR-L for 2 weeks induces proliferation of Treg but not Teff cells.

5. Thymus Spleen MLN Fc-GITR-L IgG Fc-GITR-L IgG Fc-GITR-L IgG 0 10 20 30 40 50 CD4 + (%) ns Cell number(x10 6 ) 0 100 200 300 400 500 ns 4 weeks S3B Administrating Fc-GITR-L for 4 weeks induces proliferation of Treg but not Teff cells.

6. IgGFc-GITR-L *** FoxP3 + /CD4 + (%) 20 16 12 8 4 0 C57BL/6 IgGFcGITRL 12 8 4 0 ** FoxP3 + /CD4 + (%) BALB/c 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. S4B Administrating Fc-GITR-L induces proliferation of Treg cells in BALB/c mice.