Table 1 Case # Age at primary Biopsy (year) Age at metastasis Primary Tumor site Metastasis Biopsy decalcification PD-L1 metastatic expression PD-1+ TIL metastatic 1 7 9 R radius L iliac bone & soft tissue No + 2 8 L distal femur Mediatinum, thymus Yes 3 14 18 R distal femur Lung - 4 12 ++ 5 15 6 13 R femur L proximal tibia 10 L humerus 21 R shoulder 11 N/A Unknown 16 Table 1

Metastatic Osteosarcoma Primary Osteosarcoma Metastatic Osteosarcoma A. 50um 50um 50um PD-L1 C. 50um 50um Isotype B. 50um 50um 50um 50um PD-1 Figure 1A. PDL1 staining 50um 50um 50um Isotype

Days post implantation %Survival P<0.0001 Figure 2, CD8 depleted (dotted), control (solid)

A. C. B. D. PD-L1 % of Maximum 30% 4073 1848 CD8 PD-1 SPLEEN TIL Pre Implantation Tumor C. CD8 PD-1 SPLEEN TIL 6% 87% 30% 94% 13% B. D. Isotype PD1 Healthy Lung Late Disease Lung Early Disease Lung 50 µm * %PD1+ CD8 Figure 3 50 µm SPLEEN TIL

A. C. B. D. * * * * * * * * * * * * * * Figure 4A, TIL CD8 p=0.0046 TIL + 4T1 TIL + 4T1 TIL + K7M2 TIL + K7M2 B. TIL + K7M2 PD-L1 shRNA TIL + 4T1 K7M2 lysate TIL + K7M2 PD-L1 shRNA TIL + 4T1 K7M2 lysate D. * * * * * * Figure 4A, TIL CD8 p=0.0046 TIL CD8, tumor lysate p=0.021 TIL + Antigen in tumor versus lysate p=0.0437 TIL + K7M2 compared to TIL +K7m2 PD-L1- p=0.001 B. TIL CD8 differences p=0.009 TIL CD8+ K7M2 PDL1- differences p=0.05 Tumor lysate differences p= 0.1865 Tumor lysate compared to TIL k7m2 p=0.02 TIL K7M2 compared to TIL K7M2 PDL1 – p=0.002 C. tIL K7M2 differences p= 0.009 K7M2 PD-L1- differences = .109 Tumor lysate differences =.8718 TIL K7M2 compared to tumor lysate p=0.009 TIL K7M2 compared to K7M2 PD-L1- p=0.0107 Open bars=TIL only Black bars= TIL+Antigen D. K7M2 compared to 4T1 with K7M2 p<0.0001 K7M2 compared to K7M2 PD-L1- p<0.0001 TIL TIL TIL + 4T1 TIL + K7M2 TIL + K7M2 PD-L1 shRNA TIL + 4T1 K7M2 lysate

A. B. %Survival P=0.0005 Days post implantation Figure 5A. Solid line= controls Dashed= PD-L1 blockade Volume of largest metastasis (mm3) # of metastases Control α-PD-L1 Ab Control α-PD-L1 Ab

A. C. B. %IFNγ+ Control PD-L1 Ab %IL-2+ Control PD-L1 Ab %TNF+ Control * %IL-2+ * Control PD-L1 Ab %TNF+ * B. Figure 6 Control PD-L1 Ab