Figure 1: Persistence of Donor Macrophages after Heart Transplantation.

A-D) At baseline, all macrophages co-express GFP and CD68. A subset of GFP⁺ CD68⁺ macrophages express RFP and are identified as donor CCR2⁺ macrophages. Donor CCR2⁻ macrophages are identified as CD68⁺ GFP⁺ RFP⁻. At post-transplant day 1 (d1), donor CCR2⁻ and CCR2⁺ macrophages co-exist with recipient monocyte derived macrophages (CD68⁺ only). By post-transplant day 14 (d14), only rare donor macrophages are identified. B) Donor CCR2⁻ macrophages significantly decrease over time from baseline to d14 (Kruskal-Wallis; p < 0.0003). Specifically, there is a decrease in donor CCR2⁻ macrophages between baseline and post-transplant day 7 (d7, n = 7) (Dunn’s test for multiple comparisons; p = 0.0015) and baseline and d14 (Dunn’s test for multiple comparisons; p = 0.0014) (baseline n = 8, d1 = 7, d3 = 7, d 7 = 7, d14 = 3). C) There is a significant decrease of donor CCR2⁺ macrophages over time from baseline to d14 (Kruskal-Wallis; p = 0.0111) and specifically between baseline and d7 (Dunn’s test for multiple comparisons; p = 0.0197) and baseline and d14 (Dunn’s test for multiple comparisons; p = 0.0215) (baseline n = 8, d1 = 7, d3 = 7, d 7 = 7, d14 = 3). D) There is a significant increase in recipient CD68⁺ cells during the course of rejection (Kruskal-Wallis; p < 0.0001) and specifically between post-transplant day 1(d1) and d7 (Dunn’s test for multiple comparisons; p = 0.0134) and baseline and d14 (Dunn’s test for multiple comparisons; p = 0.0015) (d1 = 5, d3 = 5, d 7 = 5, d14 = 3). E) DAPI⁺ donor macrophages (GFP⁺) were considered to have undergone cell death. There was a significant increase in donor macrophages cell death after transplant (Kruskal-Wallis; p = 0.0010) and from baseline to d7 (Dunn’s test for multiple comparisons; p = 0.002) (baseline n = 6, d1 = 4, d 7 = 6, d14 = 10). Scale bar = 100 μm.