Additional Supporting Information may be found in the online version of this article.


Figure S1: Breakdown of TV-DTH responses within Col V reactive patients. The Col V reactive group is comprised of three CAD patients, two lung transplant patients, and one heart transplant patient. The TV-DTH responses to Col V and TT and the sensitivity to P2X7R inhibition is indicated for each of the three groups. Data represent 2–4 replicates per sample/per group.

Figure S2: LPS stimulation of IL-1β and TNFα occurs independent of P2X7R activity. LPS (10 ng/ml) stimulated PBMCs from Col V reactive patients were treated with or without Suramin or AZD9056 for 16 h and analyzed for IL-1β and TNFα production by ICCS. (A) Representative flow plots indicate neither Suramin nor AZD9056 inhibit LPS stimulated IL-1β or TNFα production in our model. (B) Quantified flow cytometry data of IL-1β or TNFα positive cells following LPS stimulation. Data are from all six Col V reactive patients, repeated 2–3 times. All points are significant from respective media controls at a p < 0.001.

Figure S3: Col V stimulated production of IL-1β and TNFα in isolated monocytes from Col V reactive patients is inhibited by P2X7R antagonists. Monocytes from Col V reactive patients were isolated by MACS separation, and stimulated overnight with Col V (5 μg) in the presence or absence of Suramin or AZD9056. Quantified flow cytometry staining for IL-1β (Suramin p < 0.04; AZD9056 p < 0.03) and TNFα (Suramin p < 0.03; AZD9056 p < 0.03) from six Col V reactive patients is shown.

Figure S4: Representative H&E staining (×20) from cardiac biopsy of a Col V-reactive patient shows clear infiltration in the large and small (inset) vessel, characteristic of cardiac allograft vasculopathy.

Figure S5: Inhibition of Col V, but not TT TV-DTH responses with the MEK1/2 Inhibitor, U0126. PBMCs from Col V reactive patients were stimulated with Col V (5 μg) or TT (25 μg) in the presence or absence of 10 μM U0126 and used in the TV-DTH assay. Data from AZD9056 treatment on Col V or TT stimulated swelling is shown for comparison. Values represent the observed TV-DTH response for the respective group minus the PBS control. Data are from six individual patients.

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