Fig 1: The Cellular Source of TWEAK Expression in Cardiac Allografts and Circulating EVs During ACR(A) Coimmunofluorescence staining for (left) TWEAK/CD31, (middle) TWEAK/CD45, and (right) TWEAK/ACTA2 in EMBs from patients with ACR. (B) Fraction of cells/field classified as TWEAK+ (n = 80 fields total for non-ACR, and n = 79 for ACR). ∗∗∗P < 0.001 using Mann-Whitney U test. (C) Fraction of cells/field classified as TWEAK+ and ACTA2+ (n = 37 fields total), CD31+ (n = 35), or CD45+ (n = 43). ∗P < 0.05, ∗∗∗P < 0.001 using unpaired Student’s t-tests. (D) Coimmunofluorescence staining for (left) TWEAK/CD3 and (right) TWEAK/CD68 in EMBs from patients with ACR. (E) Fraction of cells/field classified as TWEAK+ and CD3+ (n = 23 fields total) or CD68+ (n = 21). ∗P < 0.05 using an unpaired Student’s t-tests. (F) Nanoscale flow cytometric quantification of TWEAK+ EVs in cell supernatant from primary monocytes (MON), T cells (TC), and primary human cardiac endothelial cells (HCAEC) with and without stimulation with 100 ng/mL IFN-γ. ∗P < 0.05 using an unpaired Student’s t-test. (G) Nanoscale flow cytometric quantification of TWEAK+ EVs in plasma from patients with transplants with (n = 16) and without (n = 33) ACR. EVs were costained with TWEAK and cellular markers for T-cells (CD3), monocytes (CD16), and endothelial cells (CD31). ∗P < 0.05, ∗∗∗P < 0.001 using unpaired Student’s t-tests. DAPI = 4′,6-diamidino-2-phenylindole; EMB = endomyocardial biopsy; IFN = interferon; other abbreviations as in Figures 1 and 2.