Supplementary Figure 2. Expression of CD73 and Adenosine Receptors on Tumor Cell Lines

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Supplementary Figure 1. Neither A2A nor PD-1 blockade modulates the cytotoxic activity of OT-I splenocytes cocultured with MC38 ova tumor cells in vitro

(A-B) Naive OT-I splenocytes were cultured with Mitomycin C (5 μM) treated MC38ovadim tumor cells and SIINFEKL peptide (10 ng/ml) in the presence or absence of anti-PD-1 (50 μg/ml), SCH58261 (1 μM), NECA (1 μM) and/or 2A3 isotype control (50 μg/ml) for four days. (A) Supernatants were analysed by ELISA for IFNγ content (B) The MFI of Granzyme B staining for the TCRβ+CD8+ cells was determined by flow cytometry. (A-B) Data pooled from four independent experiments and presented as mean ± SEM. * p < 0.05. (C-D) SIINFEKL pulsed OT-I T cells were cocultured with MC38ovadim tumor cells in the presence or absence of either anti-PD-1 (50 μg/ml), 2A3 isotype control (50 μg/ml), NECA (1 μM) or SCH58261 (1 μM) (C) MC38ovadim tumor cells were labelled with 51Cr (100 μCi/ 1x106 cells) prior to coculture with OT-I cells. After 4 hours the percentage 51Cr release was determined. Data represented as the mean ± SEM of 3 independent experiments. (D) OT-I splenocytes were cocultured with MC38ovadim tumor cells for 16 hours and then CD107a and GolgiStop were added to the assay. The percentage of TCRβ+CD8+ cells that were positive for CD107a was then quantified. Results shown are the mean ± SD for a representative experiment of n= 3. * p<0.05. *** p<0.001.

Supplementary Figure 2. Expression of CD73 and adenosine receptors on tumor cell lines

(A) CD73 expression on MC38ovadim Cherry, MC38ovadim-Cherry-CD73, AT3ovadim-Cherry and AT-3ovadim Cherry-CD73 was determined by flow cytometry. Data represented as isotype control (shaded histogram) or anti-CD73 (open histogram). Representative of two experiments performed. (B) Expression of A1, A2A, A2B and A3 receptors on MC38ovadim Cherry, MC38ovadim-Cherry-CD73, AT3ovadim-Cherry and AT-3ovadim Cherry-CD73 and 4T1.2 tumor cells was determined by qRT-PCR. Results are presented as mean ± SD of triplicates and presented as arbitrary units normalised to the expression of actin in each tumor cell line. ND- Not detected.

Supplementary Figure 3 Adenosine receptor stimulation does not modulate the proliferation or immunogenic phenotype of AT-3 ova dim, MC38 ova dim or 4T1.2 tumor lines

(A) 1 x 104 tumor cells were seeded onto a 96 well plate and then treated with indicated concentrations of NECA or SCH58261 cells for 72 hours. After 72 hours cell viability was determined by Resazurin assay. (B) 1 x 105 tumor cells were cultured for 16 hours in the presence or absence of NECA (10µM) and IFNγ (10ng/ml). Expression of indicated markers was determined by flow cytometry.

Supplementary Figure 4. PD-1 is only expressed on a minor subset of CD8+ T cells in the 4T1.2 tumor model.

(A) Wild Type C57BL/6 mice were injected subcutaneously with 5 x 105 AT-3 ovadim CD73+ tumor cells. 16 days post tumor injection, tumors were excised and analyzed by flow cytometry for the expression of PD-1 on CD8+ T cells, CD4+ T cells or CD3-NK1.1+ NK cells. (B) Wild Type BALB/C mice were injected with 5 x 104 4T1.2 tumor cells into the fourth mammary fat pad. 10 days post tumor injection, tumors were excised and analyzed by flow cytometry for the expression of PD-1 on CD8+ T cells, CD4+ T cells or CD3-DX5+ NK cells. Representative flow cytometry histograms are shown for each tumor model. Shaded histogram indicates expression of PD-1 on the splenic counterparts of each population.

Supplementary Figure 5. PD-1 blockade enhances the expression of A2A specifically on CD8+ tumor-infiltrating lymphocytes (A-C) TCRβ+CD8+ or TCRβ+CD4+ lymphocytes were FACS sorted from AT-3ovadim CD73+ tumors (A-C), spleen (A) or draining lymph nodes (B) of mice two days after the mice were treated with either isotype control or anti-PD-1 mAb. (A-C) Cells were isolated from five mice per group and then pooled and lysed. IFNγ and A2A expression was analyzed by qRT-PCR. Data is expressed relative to expression of naïve splenic CD8+ or CD4+ cells respectively and presented as mean of triplicates ± SD. n.s- not significant * p < 0.05, ** p < 0.01, ***p <0.001

Supplementary Figure 6. Effect of PD-1 and A2A blockade on the frequency and PD-1 expression of CD8+, CD4+foxp3- and CD4+foxp3+ tumor infiltrating lymphocytes.

C57BL/6 wild type mice were injected subcutaneously with 5 x 105 AT-3ovadim CD73+ tumor cells. Mice were treated i.p at day 14 and 18 with either an isotype control antibody (2A3, 200 μg/mouse) or anti-PD-1 mAb (RMP1-14, 200 μg/mouse) and either SCH58261 (1 mg/kg) or vehicle control (days 14-20). Tumor infiltrating lymphocytes were isolated from AT-3ovadim CD73+ tumors 1, 2 or 7 days post treatment. The proportion of (A) TCRβ+CD8+ (B) TCRβ+CD4+foxp3- and (C) TCRβ+CD4+foxp3+ subsets from within the CD45+ gate was determined along with their expression of PD-1. Data shown as mean ± SEM of 8 - 11 mice per group pooled from two separate experiments, n.s.- not significant.

Supplementary Figure 7. Combination of PD-1 and A2A blockade enhances IFNγ production of CD8+ tumor-infiltrating lymphocytes 7 days post treatment

C57BL/6 wild type mice were injected subcutaneously with 5 x 105 AT-3ovadim CD73+ tumor cells. Mice were treated i.p at day 14 and 18 with either an isotype control antibody (2A3, 200 μg/mouse) or anti-PD-1 mAb (RMP1-14, 200 μg/mouse) and either SCH58261 (1 mg/kg) or vehicle control (days 14-20). Tumors were excised 7 days post treatment and TCRβ+CD8+ cells analyzed for the expression of IFNγ (A) and Granzyme B (B) by flow cytometry. * p<0.05 , n.s.- not significant.

Supplementary Figure 8. Analysis of phenotype of tumor-infiltrating lymphocytes following therapy with anti-PD-1 alone or in combination with SCH58261.

(A-D) C57/BL6 wild type mice were injected subcutaneously with 5 x 105 AT-3ovadim CD73+ tumor cells. Mice were treated i.p at days 14 and 18 with either an isotype control antibody (2A3, 200 μg/mouse) or anti-PD-1 mAb (RMP1-14, 200 μg/mouse) and either SCH58261 (1 mg/kg) or vehicle control (days 0-6 post anti-PD-1 therapy). Tumor infiltrating lymphocytes were isolated 1, 2 or 7 days post treatment as indicated. (A) Proportion of CD4+ T cells expressing IFNγ (B) Expression of Granzyme B on the TCRβ+CD8+PD-1hi and TCRβ+CD8+PD-1low cells. (C) Expression of IFNγ on the CD8+PD-1hi and CD8+PD-1low T cells. (D) Proportion of CD8+ tumor-infiltrating lymphocytes reactive to the ova tetramer. (A-C). Data shown as mean ± SEM for 11 mice per group pooled from two separate experiments. (D) Data shown as mean ± SEM for 5-6 mice per group. * p<0.05, ** p<0.01, ***p<0.001 , n.s. not significant.

Supplementary Figure 9. A2A blockade does not modulate TIL phenotype or function in the absence of anti-PD-1

(A) C57/BL6 wild type mice were injected subcutaneously with 5 x 105 AT-3ovadim CD73+ tumor cells. Mice were treated i.p at days 14 and 15 with SCH58261 (1 mg/kg) or vehicle control. Tumor infiltrating lymphocytes were analysed at day 2 post treatment (day 16) by flow cytometry. (A) Expression of PD-1 on TCRβ+CD8+ T cells, (B) Expression of PDL-1 on tumor cells, (C-D) Expression of IFNγ and Granzyme B in TCRβ+CD8+ T cells. (A, C, D) Data is represented as the mean ± SEM of 4-6 mice. (B) A representative histogram plot is shown.