Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Pathway in High-Risk Lymphocytic Acute Myocarditis

Figures

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  Figure 1. Flowchart of the Study The flowchart illustrates the comparisons made among groups assessing cardiac PD-1 and PD-L1 expression. ACR = acute cellular rejection; AM = acute myocarditis; CS = cardiac sarcoidosis; ICI = immune checkpoint inhibitor; LAM = lymphocytic acute myocarditis; Infl-CMP = inflammatory cardiomyopathy; PD-1 = programmed cell death-1; PD-L1 = programmed cell death-ligand 1.

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  Figure 2. Positive and Negative Tissue Controls for Programmed Cell Death-1 and Programmed Cell Death Ligand 1 Staining (A to C) Staining for PD-1 of a human tonsil at different magnifications (from 4× to 20×, indicating magnification of 40- to 200-fold, respectively) marks germinal center-associated T cells that express PD-1 (arrows). In contrast, few T cells in the interfollicular T-cell zone express PD-1. (D and E) Staining for PD-L1 of a human tonsil at different magnifications (from 4× to 10×, indicating 40- to 100-fold magnification) marks the epithelium of the tonsillar crypts highly expressing PDL-1 (arrows). In the tonsil germinal centers, macrophages show low-intensity PD-L1 positivity, whereas T cells are negative for PD-L1 (red rectangle in D). (F) Staining of PD-L1 in endomyocardial biopsies (EMB) and a human tonsil on the same slide as a positive control. (G to I) Staining of an EMB from a patient admitted with heart failure served as a negative control, showing no cardiac inflammatory infiltrates on hematoxylin and eosin (H&E) stain (G), and no expression for PD-1 (H) or PD-L1 (I) (10×, indicating 100-fold magnification). Abbreviations as in Figure 1.

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  Figure 3. Cardiac PD-L1 Expression in Patients With Lymphocytic Acute Myocarditis Compared With Patients With Cardiac Sarcoidosis and Non-Inflammatory Cardiomyopathy, and in Patients With Fulminant Myocarditis vs Non-Fulminant Myocarditis (A to F) Endomyocardial biopsies (EMB) from 3 representative patients with LAM, CS, and non-Infl-CMP stained with hematoxylin and eosin (H&E) and PD-L1. (A and B) In the patient with LAM (case AM24, a 30-year-old man), the H&E stain shows cardiac necrosis and inflammatory infiltrates, consistent with active myocarditis; the PD-L1 stain reveals high PD-L1 expression on cardiomyocytes (grade 4, indicating 50% to 74% of cardiomyocytes were positive for PD-L1). Note that in this case, the positive PD-L1 staining of the plasma membrane is also in non-gap-junction regions of cardiomyocytes, while in low-intensity cases, PD-L1 is mainly or exclusively seen in gap-junction regions. (C and D) In the patient with CS (case CS25, 53-year-old man), H&E shows myocardium without (w/o) granulomas and inflammatory infiltrates associated with dim-junctional PD-L1 expression on cardiomyocytes (grade 1 of the visual semiquantitative scale). (E and F) In the patient with…

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  Figure 4. Expression of PD-1 and PD-L1 in a Representative Patient With Cardiac Sarcoidosis (A) H&E stain of an endomyocardial biopsy (EMB) showing an area of normal myocardium and a non-caseating granuloma (in the inset, the granuloma at higher magnification reveals a giant cell). The sample was from a 53-year-old man presenting with dyspnea, high-degree atrioventricular block, and an LVEF of 35% (case CS25). (B) At the top, PD-L1 stain shows dim/junctional PD-L1 expression in cardiomyocytes unaffected by granuloma (grade 1). In contrast, in the granuloma (inset), PD-L1 expression is moderate in inflammatory cells, likely macrophages/histiocytes, based on a visual semiquantitative scale (grade 2 expression on inflammatory cells). On the bottom, the PD-1 stain shows no expression of PD-1 in the myocardium unaffected by the granuloma. In contrast, in the granuloma (inset), there is grade 2 PD-1 expression on inflammatory cells, likely T cells (grade 2 on the visual semiquantitative scale represents 11% to 25% expression in inflammatory cells). H&E = hematoxylin and eosin; LVEF = left ventricular ejection fraction; other abbreviations as in Figure 1.

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  Figure 5. Cardiac PD-L1 Expression in Heart Transplant Recipients With Acute Cellular Rejection or Without Acute Cellular Rejection (A to F) Endomyocardial biopsy (EMB) from 2 representative patients with ACR of grade ≥2R and 1 patient with no ACR stained with H&E and showing PD-L1. At the top, the H&E stain shows different grades of ACR: 3R (case HT15) in A and 2R (case HT55) in B, with no ACR (case HT56) in C. On the bottom, high PD-L1 expression on cardiomyocytes in the patient with ACR of grade 3 (D), grade 2 PD-L1 expression in the patient with ACR of grade 2 (E), and no PD-L1 expression on cardiomyocytes of the patient without ACR (F). (G) In heart transplant recipients with ACR ≥2R, the PD-L1 expression level on cardiomyocytes was significantly higher than in recipients with no ACR. Abbreviations as in Figure 1.

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  Figure 6. Cardiac PD-1 and PD-L1 Expression in Patients With Lymphocytic Acute Myocarditis Compared With Heart Transplant Recipients With Acute Cellular Rejection (A and B) In a representative patient with LAM (case AM15, a 56-year-old woman), staining for PD-1 shows no PD-1 expression on inflammatory cells, whereas staining for PD-L1 shows PD-L1 expression in cardiomyocytes. (C and D) In a representative patient with ACR of grade 3R (ACR ≥2R; case HT15, 48-year-old woman), staining for PD-1 shows grade 2 PD-1 expression on inflammatory cells, and staining for PD-L1 shows PD-L1 expression in cardiomyocytes. (E) In patients with LAM (n = 39), PD-L1 expression on cardiomyocytes was significantly higher than in heart transplant recipients with ACR ≥2R. Arrows indicate PD-L1 expression in the 2 representative patients (B and D). (F) In patients with LAM, the PD-1 expression level on inflammatory cells infiltrating the myocardium was significantly lower than in ACR ≥2R. Arrows indicate the PD-1 expression of the 4 representative patients (A, C, H, and J). Mann-Whitney test was used. (G and H) In a representative patient with LAM (case AM11, 51-year-old woman), H&E stain reveals myocardial…

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  Figure 7. Cardiac PD-1 and PD-L1 Expression in Patients With Immune Checkpoint Inhibitor Acute Myocarditis vs Lymphocytic Acute Myocarditis (A to D) Two representative patients with ICI-AM, stained for PD-1 and PD-L1 showing grade 3 PD-L1 expression in cardiomyocytes and grade 3 PD-1 expression on inflammatory mononuclear cells. (E) In patients with LAM (n = 39), PD-L1 expression on cardiomyocytes was similar than in patients with ICI-AM. Arrows indicate PD-L1 expression in the 2 representative patients (A and C). (F) In patients with LAM, the PD-1 expression level on inflammatory mononuclear cells infiltrating the myocardium was significantly lower than in ICI-AM. Arrows indicate the PD-1 expression in the 2 representative patients (B and D). Mann-Whitney test was used. Abbreviations as in Figure 1.

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  Central Illustration. Cardiac PD-1 and PD-L1 Expression in High-Risk Lymphocytic Acute Myocarditis Compared With Other Myocardial Inflammatory Forms PD-L1 is overexpressed in cardiomyocytes of patients with lymphocytic acute myocarditis (LAM). Other forms of myocardial inflammation, such as acute cellular rejection and immune checkpoint inhibitor (ICI)-associated acute myocarditis, driven by T-cell-mediated injury, also express PD-L1 on cardiomyocytes, which is generally not expressed by cardiomyocytes. Cardiac PD-1 expression in inflammatory mononuclear cells is blunted in LAM compared with ACR and ICI-AM, suggesting a potential disruption of the PD-L1/PD-1 axis in LAM that can favor T-cell infiltration in severe forms of myocarditis. Abbreviations as in Figure 1.