Mitochondria released in the setting of tissue injury are a source of damage associated molecular patterns (DAMPs) that initiate innate immune responses. We hypothesized that exposure of donor allografts to circulating mitochondrial DAMPs (mtDAMPs) prior to organ procurement would increase rates of graft rejection.

Methods: MHC-mismatched BALB/c to B6 heterotopic cardiac allografts transplants were performed in the setting of costimulation blockade with murine CTLA4-Ig (200 mcg, i.p., POD0, 2, 4, & 6). Donor mice were treated with murine mitochondria (300 mpg, iv) or vehicle solution one day prior to organ procurement. Mean survival time (MST) of allografts was determined and graft histology, immunohistochemistry and FACS analysis of infiltrating lymphocytes (GILs) was performed on POD7. To follow the allospecific T cell response, Thy1.1+ CD4 and CD8 T cells from TCR-tg transgenic (TCR-tg) mice with direct allospecificity (4C and 2C mice, respectively) were adoptively transferred on POD2. On POD7, splenic and GIL TCR-tg and non-tg CD4 and CD8 T cells were FACS analyzed for proliferation (Ki-67) and effector cytokine production (IFN-gamma).

Results: In the setting of costimulation blockade, donor treatment with mtDAMPs prior to organ procurement resulted in more rapid rejection compared to vehicle treated donors (mtDAMPs: MST 18±6.1 days, n=7; vehicle: MST >65 days, n=8). The total number of CD4 and CD8 GIL T cells was significantly greater on POD7 in allografts from mtDAMP-treated donors vs control donors (14.3- and 32.6-fold, respectively). Similarly, GIL allospecific TCR-tg CD4 and CD8 T cells were increased in mtDAMP vs control donor allografts (4.5- and 32-fold, respectively). Splenic T cells of recipients of mtDAMP-treated allografts had increased Ki-67+ allospecific T cell frequency (CD4: mtDAMP 38.8±2.4%, control 24.3±1.2%, P=0.0059; CD8: mtDAMP 26.6±1.6% vs control 16.3±1.3%; P=0.0077). IFN-gamma expression was also increased in allospecific splenic T cells (CD4: mtDAMP 55.5±5.0% vs control 17.1±1.9%, P<0.0001; CD8: mtDAMP 25.2±4.4% vs control 5.6±2.2%, P=0.0026) and CD8+ GIL T cells (mtDAMP 6.1±0.2% vs control 0.67±0.67%, P=0.0013).

Summary: Donor allograft exposure to circulating mtDAMPs increases allograft rejection by increasing recipient allospecific CD4 and CD8 T cell activation, proliferation and graft infiltration. Mitochondrial DAMPs released by tissue injury in the setting of deceased organ donation predisposes allografts to rejection.

CITATION INFORMATION: Lin L., Wang J., Cendales L., Kirk A., Brennan T. In Vivo Allograft Exposure to Mitochondrial DAMPs Prior to Organ Procurement Increases Graft Rejection in Mice Am J Transplant. 2017;17 (suppl 3).

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