PD-1 Modulates Radiation-Induced Cardiac Toxicity through Cytotoxic T Lymphocytes

Abstract

Introduction: Combined immune checkpoint blockade has led to rare autoimmune complications, such as fatal myocarditis. Recent approvals of several anti-programmed death 1 (anti-PD-1) drugs for lung cancer treatment prompted ongoing clinical trials that directly combine PD-1 inhibitors with thoracic radiotherapy for locally advanced lung cancer. Overlapping toxicities from either modality have the potential to increase the risk for radiation-induced cardiotoxicity (RICT), which is well documented among patients with Hodgkin's disease and breast cancer.

Methods: To investigate cardiotoxicity without the compounding pulmonary toxicity from thoracic radiotherapy, we developed a technique to deliver cardiac irradiation (CIR) in a mouse model concurrently with PD-1 blockade to determine the presence of cardiac toxicity by using physiological testing and mortality as end points along with histological analysis.

Results: We observed an acute mortality of 30% within 2 weeks after CIR plus anti-PD-1 antibody compared with 0% from CIR plus immunoglobulin G (p = 0.023). Physiological testing demonstrated a reduced left ventricular ejection fraction (p < 0.01) by echocardiogram. Tissue analyses revealed increased immune cell infiltrates within cardiac tissue. Depletion of CD8-positive lymphocytes with anti-CD8 antibody reversed the acute mortality, suggesting that the toxicity is CD8-positive cell-mediated. To validate these findings using a clinically relevant fractionated radiotherapy regimen, we repeated the study by delivering five daily fractions of 6 Gy. Similar mortality, cardiac dysfunction, and histological changes were observed in mice receiving fractionated radiotherapy with concurrent anti-PD-1 therapy.

Conclusions: This study provides strong preclinical evidence that radiation-induced cardiotoxicity is modulated by the PD-1 axis and that PD-1 blockade should be administered with careful radiotherapy planning with an effort of reducing cardiac dose.

Keywords: CD8; Immunotherapy; PD-1; Radiation-induced cardiac toxicity; Radiotherapy; Toxicity.

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.

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 Awas used in this clinical experiment.

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.

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.

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.

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 < 0.05. (n = 3). IgG, immunoglobulin G.