A. ICOS-L gene expression analysis in mRNA from melanoma cell lines, wild type L cells and L cells expressing ICOS-L. RT-PCR was performed for ICOS-L and β-actin genes. Data was normalized to the β-actin gene (Mean ± SD). Three separate graphs are shown to clearly illustrate the different levels of ICOS-L expression in the different sets of all lines. The expression of L cells was considered 1 in all the analysis. B. Protein expression of ICOS-L on melanoma cell lines. White histograms show anti ICOS-L and gray histograms isotype control. Normal melanocytes do not express ICOSL (not shown).

A–C. ICOS-L, B7-H1 and HLA class II expression analysis from fresh melanoma samples. A are melanomas that did not express ICOS-L, B shows the samples that have low expression of ICOS-L, and C the high ICOS-L expressing melanomas. White histograms show specific antibody staining and gray histograms show isotype. D. Summary of ICOS-L expression in fresh melanomas (n= 12).

Sorted CD4⁺CD25⁻ (CD25−) and CD4⁺CD25⁺ (CD25+) cells were cultured with irradiated melanoma cell lines and anti-CD3. On day 5 the CD4⁺ cells were analyzed for the expression of Foxp3, CD25 and ICOS. ICOS-L⁺ tumors are WM35 and A684 and ICOS-L- cells are WM793. Foxp3 expression on sorted CD25⁻ and CD25⁺ cells are shown in the left panels. ICOS expression on Foxp3⁺ (blue gate) and Foxp3^hi cells (red gate) on CD25⁺ cells are shown in the right panels. B. Percentage of CD4⁺CD25⁺ cells expressing high levels of Foxp3 from different donors (n= 10) cultured with melanoma lines that express (+) or do not express ICOS-L (−). C. Sorted CD4⁺CD25⁻ and CD4⁺CD25⁺ cells were labeled with CFDA-SE and activated with anti-CD3 over a monolayer of irradiated melanoma lines. After 5 days, the expression of Foxp3 and CFDA-SE dilution was analyzed. Where indicated the TGF-β-R1 kinase inhibitor SB431542 was added on day 1. D. Patients with melanoma have high levels of Foxp3⁺ ICOS ⁺ T cells in the tumor infiltrates. Tumor infiltrating lymphocytes (TIL) and PBMC from patients with stage IV metastatic melanoma were analyzed for the expression of CD4, CD25, Foxp3 and ICOS by flow cytometry. Shown are the percentages of CD25⁺Foxp3⁺ in the CD4⁺ population and the Foxp3⁺ ICOS⁺ cells in the CD4⁺CD25⁺ population.

A–B. Sorted CD4⁺CD25⁺ cells from normal donors or melanoma patients were labeled with CFDA-SE and activated with anti-CD3 over a monolayer of irradiated melanoma lines, ICOS-L⁺ WM35 or ICOSL⁻ WM793. On day one mouse IgG or anti-ICOS-L antibody (MIH12) was added to the cultures. A. After 5 days cells were analyzed for Foxp3 expression and CFDA-SE dilution. B. Percentage of CD25⁺ cells expressing high levels of Foxp3 and CFDA-SE^low from different donors (n=5) cultured with ICOS⁺ or ICOSL⁻ melanomas and mouse IgG or anti ICOSL antibodies. C. Blockade of ICOS-L in vivo reduces the number of Foxp3⁺ ICOS⁺ T regs in the tumor tissue. Rat IgG or anti-ICOS-L was administered i.p. to C57BL/6 mice every 2 days for 16 days. Mice were i.v. injected with B16-OVA melanoma cells on day 2 and on day 7 mice were injected with 2 million OT-II-Ly5.1 CD4⁺ T cells. On day 17 lymphocytes from the lung were analyzed for the expression of CD45.1, CD4, CD25, Foxp3 and ICOS. Numbers in the plots are the percentage of Foxp3⁺ Tregs from Ly5.1⁻ (recipient Treg) and Ly5.1⁺ (OT-II T reg) CD4⁺ T cells. Numbers in the histogram show the percentage of ICOS⁺ T cells from CD4⁺Foxp3⁺ cells. Graphs on the right show the percentage and total cell number of Foxp3⁺ cells from CD45.1⁻ and CD45.1⁺ cells in the lungs of all mice analyzed (n=5).

A. Tregs activated with ICOS-L-expressing WM793 cells or L cells can suppress effector T cells similarly as those activated over ICOS-L⁻ WM793 and L cell controls. Sorted CD4⁺CD25⁺ T cells that were co-cultured with irradiated ICOS-L⁺ and ICOSL⁻ WM793 or L cells were rested in media with no IL-2 and used at 1:1 ratio in a suppression assay with isolated autologous CD4⁺CD25⁻ T cells pre-labeled with CFDA-SE. After 6 days, cell division by CFDA-SE dilution was analyzed. B. Tregs activated with ICOS-L-expressing melanoma cells produce IL-10. Activated CD4⁺CD25⁺ and CD4⁺CD25⁻ cells co-cultured with the indicated cell line were intracellular stained for IL-10 and IFN-γ.