Receptor-interacting protein kinase 3 (RIP3) is critical for necroptosis in the setting of inflammation. It has been shown that kidney and heart allografts deficient in RIP3 have prolonged graft survival, making inhibition of RIP3 kinase activity an attractive therapeutic target. However, the role of the RIP3 kinase function, rather than RIP3 deficiency, in graft rejection is unknown. Here we utilize RIP3 kinase-dead (RIP3K51A / RIP3KD) and knock-out (RIP3KO) mice to evaluate the role of donor RIP3 kinase activity in allograft survival.

Methods: CBA mice (H-2k) received cardiac allografts from B6 (H-2b) WT, RIP3KO or RIPK3KD donors. Graft mean survival time (MST) was determined in the setting of co-stimulation blockade (CTLA4-Ig 200 mcg i.p. POD0, 2, 4, 6). In some experiments, mice were sacrificed on POD14 and PBMCs, splenocytes, and graft infiltrating lymphocytes (GILs) were collected for FACS analysis for markers of activation, proliferation and cytokine expression. T cell memory phenotypes were examined by CD62L and CD44 expression.

Results: In the absence of costimulation blockade, WT, RIP3KO and RIP3KD heart transplants had similar rejection rates (WT MST 8 d, n=6; RIP3KO MST 7 d, n=3; RIP3KD MST 8 d, n=5). In the setting of costimulation blockade, RIP3KO hearts had prolonged allograft MST compared to WT, while RIP3KD hearts had decreased MST (WT MST 60 d, n=5; RIP3KO MST>100 d; n=3; RIP3KD, MST=27 d, n=6). Splenic CD4 T cells had increased TNFα expression in recipients of RIP3KD vs WT grafts, but not between RIP3KO vs WT grafts (%TNFα+: RIP3KD 36±2.1% vs WT 28.3±3.8%, p=0.045; RIP3KO 23±2.0% vs. WT, p=0.4). IFNγ production by splenic CD8 T cells was also increased in recipients of RIP3KD vs WT grafts, but not between RIP3KO and WT grafts (%IFNγ+: RIP3KD 30±1.1% vs WT 22.3±1.3%, p=0.04; RIP3KO 24±1.2% vs WT 22.3±1.3%, p=0.4). Similar differences were observed in CD4 and CD8 T cells from PBMCs and GILs. More CD4 and CD8 T cells had effector memory phenotype in PBMCs from RIP3KD vs WT donors (CD4+: 27±4.0% vs 10.3±1.1%, p=0.038; CD8+: 19.5%±0.9 vs 7.3%±0.6, p=0.02), and vs RIP3KO donors (CD4+: 27±4.0% vs 11.5±2.0%, p=0.047; CD8+: 19.5±0.9% vs 7.8±2.5%, p=0.05).

Conclusion: Though donor RIP3 deficiency extends allograft survival in the setting of costimulation blockade, deficiency of the RIP3 kinase activity leads to more rapid graft loss accompanied by increased T cell memory and effector cytokine expression.

CITATION INFORMATION: Zhu M., Samy K., Lin L., Wang J., Cendales L., Kirk A., Barbas A., Brennan T. Donor Deficiency of RIP3 Phosphokinase Activity Shortens Cardiac Allograft Survival in the Setting of Costimulation Blockade Am J Transplant. 2017;17 (suppl 3).

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