Fig 2: Phenotypic and functional characteristics of circulating monocytes in heart transplant recipients compared with healthy individuals. Expression levels of (A) HLA-DR, (B) CD54 (ICAM-1) are increased in CD16+ monocyte pool during rejection compared to non-rejection. The percentages of IL-1ß-, IL-6-, TNF-a-, IFN-?-, IL-12-, and IL-10-producing monocytes of both healthy individual group (n = 9-14) and heart transplant recipients (n = 10) are shown at both NR and R time points after LPS stimulation (C) (mean ± SEM) (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: CD16+ cells in graft tissue are monocytes and accumulate upon rejection. The expression of CD14 and CD16 was analyzed in biopsies at two time points; non-rejection (NR; n = 25) and rejection (R; n = 25) and compared intra-individually. The absolute number of cells is depicted for (A) CD14+CD16-, (B) CD14+CD16+, and (C) CD14-CD16+. The expression of (D) CD56 was measured using immunohistochemistry, analyzed by ImageJ and depicted as mean % positive stained area. The absolute numbers of cells of (E,F) CD56+CD16- and CD56+16+ were determined. A representative overview of rejecting endomyocardial tissue stained for (G) CD56 (green) and CD16 (red) at 20× magnification is shown. A representative overview of rejecting endomyocardial tissue stained for (H) CD14 (green), (I) CD16 (green) and CD68 (red) at 20× magnification is given (*p < 0.05, ***p < 0.001).

Fig 4: Expression of Tie2 on peripheral blood CD14+CD16+ monocytes.(A) Purified peripheral blood monocytes were stained with conjugated control antibodies (IgG-FITC, IgG-PE) or with CD16-PE antibody alone (B) or with CD14-FITC antibody alone (C) to determine the presence of CD14+CD16+ monocytes (D). (E) TEMs were distinguished by using IgG-APC control and Tie2-APC antibodies. The percentage of TEMs in peripheral blood CD14+CD16+ monocytes for this representative sample was 41.2%.

Fig 5: Western blot of human neutrophil lysate before and after Fc?RIII immunoprecipitation. The identity of Fc?RIIIb was confirmed with an anti-CD16 antibody. The flow-through lanes show the diluted unbound fraction of the immunoprecipitation, while the eluate lanes present the purified Fc?RIIIb protein. The neutrophils lanes represent ~25 µg of total protein content from the neutrophil cell lysate from donor 2. Apparent differences in the western blot are due to concentration differences. The weaker signal of extremely low and high MW proteoforms is lost at lower concentrations after IP. The cropping area is indicated by dashed lines. The complete gel and blot are presented in Figures S1 and S2.