Differential Requirement for P2X7R Function in IL-17 Dependent vs. IL-17 Independent Cellular Immune Responses
Version of Record online: 27 MAY 2014
© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons
American Journal of Transplantation
Volume 14, Issue 7, pages 1512–1522, July 2014
How to Cite
Sullivan, J. A., Jankowska-Gan, E., Shi, L., Roenneburg, D., Hegde, S., Greenspan, D. S., Wilkes, D. S., Denlinger, L. C. and Burlingham, W. J. (2014), Differential Requirement for P2X7R Function in IL-17 Dependent vs. IL-17 Independent Cellular Immune Responses. American Journal of Transplantation, 14: 1512–1522. doi: 10.1111/ajt.12741
- Issue online: 20 JUN 2014
- Version of Record online: 27 MAY 2014
- Manuscript Accepted: 10 MAR 2014
- Manuscript Revised: 24 FEB 2014
- Manuscript Received: 3 JAN 2014
- NIH. Grant Number: PO1AI084853
- EU-Sponsored One Study
- R01. Grant Number: HL118115
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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