1. Successful Immunotherapy against a Transplantable Mouse Squamous Lung Carcinoma with Anti–PD-1 and Anti-CD137 Monoclonal Antibodies 
Arantza Azpilikueta, BSc, Jackeline Agorreta, PhD, Sara Labiano, BSc, José Luis Pérez-Gracia, MD, PhD, Alfonso R. Sánchez-Paulete, BSc, M. Angela Aznar, PhD, Daniel Ajona, PhD, Ignacio Gil-Bazo, MD, PhD, Marta Larrayoz, PhD, Alvaro Teijeira, PhD, María E. Rodriguez-Ruiz, MD, PhD, Ruben Pio, PharmD, PhD, Luis M. Montuenga, PhD, Ignacio Melero, MD, PhD 
Journal of Thoracic Oncology 
Volume 11, Issue 4, Pages (April 2016)
DOI: /j.jtho
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

2. Figure 1
Generation of a transplantable lung squamous cell carcinoma cell line model. (A) Schematic representation of the generation of the transplantable cell line UN-SCC680AJ. Squamous cell carcinoma cells were retrieved from N-nitroso-tris-chloroethylurea (NTCU)-induced tumors that were serially passaged in culture and subcutaneously grafted into immunodeficient Rag2-/-IL2Rγ-/- mice. Those tumors were disaggregated, and a second tumor cell line was derived (UN-SCC680X) from a cell suspension; it was finally successfully engrafted in immunocompetent A/J mice, and the cell line UN-SCC680AJ was derived. A cell line derived from an engrafted tumor in syngeneic mice almost constantly gave rise to progressively growing tumors in A/J immunocompetent mice. (B) Microphotographs of hematoxylin and eosin (HE) and the indicating immunohistochemistry stainings (pancytokeratins: cytokeratin [CK], thyroid transcription factor 1 [TTF1], and P40). Scale bar = 50 μm.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

3. Figure 2
UN-SCC680AJ was amenable to treatment by programmed cell death 1 blockade and agonist anti-CD137 monoclonal antibodies. (A-C) Subcutaneous tumors derived from UN-SCC680AJ were treated on the indicated days after tumor cell inoculation with doses of 100 μg of the corresponding monoclonal antibodies or their combinations by the intraperitoneal route. The graphs on the left show individual follow-up of tumor size. On the right graphs, each column represents the average tumor size (mean ± SD) of all treated mice with statistical comparisons between the indicated treatments. ***p < 0.001, **p < Data on panel C were replicated in an identical experiment shown in Supplementary Figure 1. PD-1, programmed cell death 1; PD-L1, programmed cell death ligand 1; IgG, immunoglobulin G.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

4. Figure 3
CD8 T-cell depletion abrogates the immunotherapeutic activity of anti-CD137 plus anti–programmed cell death 1 treatment on UN-SCC680AJ-derived tumors. Tumor-engrafted mice were treated on days 15, 18, and 21 with the combined immunostimulatory regimen. The indicating depleting antibodies were administered intraperitoneally on days 14, 17, and 20. Data represent the individual follow-up of tumor diameters. The fraction of mice macroscopically rejecting their tumors is also provided in each graph.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

5. Figure 4
The molecular targets for immunostimulatory monoclonal antibodies are expressed in the tumor microenvironment. (A) Flow cytometry experiments on UN-SCC680AJ cells cultured with and without 1000 U/mL of interferon-γ. Surface expression of CD80, programmed cell death ligand 1 (PD-L1), programmed cell death ligand (PD-L2), and interferon-γRα (IFN-γRα) is represented as histograms with the mean fluorescence intensity. Results are representative of four tumors analyzed independently. Supplementary Figure 2 shows data on major histocompatibility complex I and major histocompatibility complex II immunostaining analyzed in identical samples. (B) Representative images of immunohistochemical analysis of CD3, CD137, programmed cell death 1 (PD-1), and PD-L1 on tumors derived from the UN-SCC680AJ cells. Positive lymphocytes are indicated by arrows. Scale bar = 50 μm.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

6. Figure 5
CD8 and CD4 T lymphocytes coexpress CD137 and programmed cell death 1 (PD-1) in the tumor microenvironment. Cell suspensions from excised UN-SCC680AJ–derived tumors on days 17 (A) and 29 (B) were immunostained and analyzed by fluorescence-activated cell sorting. Data represent the percentage of double-positive PD-1+CD137+ CD8 and CD4 T lymphocytes from mice treated as indicated in the legend. A representative contour plot from control treated tumors is included. IgG, immunoglobulin G.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

7. Figure 6
Relative quantification of lymphoid and myeloid cells infiltrating UN-SCC680AJ tumors under treatment with immunostimulatory monoclonal antibodies. (A) Comparisons of the indicated lymphoid subsets given as percentages of total cells in the cell suspensions of day 17 tumors treated on days 12 and 15. (B) Similar analysis of the lymphoid subsets in the cell suspensions of day 29 tumors treated on days 23 and 26. These experiments were performed on six tumors per experimental treatment group. *p < 0.05 and ∗∗p < 0.01 using Mann-Whitney U tests. Abbreviations: IgG, immunoglobulin G; PD-1, programmed cell death 1.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

8. Supplementary Figure 1
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

9. Supplementary Figure 2
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions

10. Supplementary Figure 3
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2016 International Association for the Study of Lung Cancer Terms and Conditions