1. PD-1 Modulates Radiation-Induced Cardiac Toxicity through Cytotoxic T Lymphocytes 
Shisuo Du, MD, Lin Zhou, MD, Gregory S. Alexander, BA, Kyewon Park, PhD, Lifeng Yang, MD, Nadan Wang, MD, Nicholas G. Zaorsky, MD, Xinliang Ma, MD, Yajing Wang, PhD, Adam P. Dicker, MD, PhD, Bo Lu, MD, PhD 
Journal of Thoracic Oncology 
Volume 13, Issue 4, Pages (April 2018)
DOI: /j.jtho
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

2. Figure 1
CD8 lymphocytes mediate acute mortality from cardiac irradiation (CIR) plus anti–programmed anti–programmed death 1 (anti–PD-1). (A). Immune cell infiltration was analyzed by immunofluorescence. CD45 was used as a marker for lymphocytes. CD4 and CD8 were used to identify CD4-positive T cells and CD8-positive T cells, respectively. F4/80 was used a marker for macrophages. (B) Schema of the T-lymphocyte subset deletion experiment. Mice receiving anti–PD-1 (black arrow) and 20 Gy of CIR in a single fraction (lightning bolt) were randomized to receive either anti-CD4 or anti-CD8 (black triangle). Survival curves for T-cell depletion experiment. Anti-CD8 reversed mortality from CIR plus anti–PD-1. A log-rank test was used for all statistical analyses (p < 0.05 [n = 10]). IgG, immunoglobulin G; mAb, monoclonal antibody; NS, not significant; H&E, hematoxylin and eosin; DAPI, 4′,6-diamidino-2-phenylindole.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

3. Figure 2
Establishment of a mouse model for studying the toxicities from cardiac irradiation (CIR). The dose distributions of transverse, sagittal, and coronal sections and dose-volume histogram (DVHs) of different CIR plans with a 10 × 10-mm circular collimator. (A) Two diagonal crossing beams. (B) Two anterior-posterior (AP-PA) beams. (C) One partial arc (from 120 degrees to –120 degrees clockwise. (D) One single arc (from –179 degrees to 179 degrees clockwise). Plan A was used in this clinical experiment.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

4. Figure 3
Increased acute mortality and reduced cardiac output after cardiac irradiation (CIR) with concurrent anti–programmed death 1 (anti–PD-1). (A) The schedule of the experiment. (groups 1–4) Anti–PD-1 monoclonal antibody (mAb) or immunoglobulin G (IgG) control was intraperitoneally administrated 24 hours before of CIR (black lightning bolt) with a loading dose of 200 μg, then every other day with 100 μg per mouse. (B) Survival curves for each group. Mice receiving combined treatment had an inferior survival (p = 0.023). (C) Ejection fraction and fractional shortening decreased significantly in the group receiving CIR plus anti–PD-1, whereas LVID-s increased significantly in the same group. **p < 0.01, *p < 0.05. NS, not significant.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

5. Figure 4
Increased cytotoxic T-cell response and acute cardiac mortality from fractionated cardiac irradiation (fCIR) plus anti–PD-1. (A) Mice were subjected to 30Gy delivered over 5 fractions with either control immunoglobulin G (IgG) or anti–programmed death 1 (anti–PD-1). (B) Survival curves were determined by a log-rank test (p < 0.05 [n = 10]). (C) Immune infiltration was analyzed by immunofluorescence. CD45 was used as a marker for lymphocytes. CD4 and CD8 were used to identify CD4-positive T cells and CD8-positive T cells, respectively. F4/80 was used a marker for macrophages. H.E., hematoxylin and eosin; IFNγ, interferon gamma; TNFα, tumor necrosis factor-α.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

6. Figure 5
(A and B) Flow cytometry analyses of cardiac tissue. (C) Tumor necrosis factor-α (TNF-α) and interferon gamma (IFN-γ) expression by real-time polymerase chain reaction using irradiated cardiac tissue. **p < 0.01, *p < 0.05 (n = 3 or 4 in each group). anti–PD-1 (anti–programmed death 1); fCRT, fractionated conformal radiotherapy.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions

7. Figure 6
Acute fibrosis in cardiac tissues treated with fractionated cardiac irradiation (fCIR) concurrently with programmed death 1 (PD-1) blockade. (A) Cardiac tissue samples were stained with Masson’s trichrome. (B) Hydroxyproline was measured by using the same cardiac tissue samples with the Hydroxyproline Assay Kit (SigmaAldrich) per the manufacturer's instruction. **p < 0.01, *p < (n = 3). IgG, immunoglobulin G.
Journal of Thoracic Oncology  , DOI: ( /j.jtho )
Copyright © 2017 International Association for the Study of Lung Cancer Terms and Conditions