EXPERIMENTAL IMMUNOBIOLOGY

K. Shek¹, Z. Zhang², K. Khan³, S. Wang², A. Lau⁴, Z. Yin², W. Liu⁵, B. Garcia⁵, B. Singh³, H. Wang⁶, A.M. Jevnikar^7
1Microbiology And Immunology, University Of Western Ontario, London/ON/CANADA, ²Department Of Medicine, University of Western Ontario, London/CANADA, ³, University of Western Ontario, London/CANADA, ⁴Pathology, University of Western Ontario, London/ON/CANADA, ⁵Department Of Pathology, University of Western Ontario, London/CANADA, ⁶Department Of Surgery, London Health Sciences Center-University Hospital, London/CANADA, ⁷Department Of Medicine, Multi-Organ Transplant Program-London Health Sciences Centre, London/CANADA

Body: Introduction: PI-9 is an intracellular serpin that inhibits Granzyme B (GrB), a serine protease found in the cytosolic granules of cytotoxic T (CTL), Natural Killer (NK) cells and dendritic cells. PI-9 may function to prevent "misdirected" apoptosis by GrB expressing cells during inflammation. The murine homolog of PI-9 is serpin protease inhibitor 6 (SPI-6). Kidney tubular epithelial cells (TEC) are a principal target for cytotoxic cells following transplant. Therefore TEC resistance or sensitivity to injury and rejection regulates graft function and long term survival. Expression of PI-9 has been observed in kidneys in cortical areas with subclinical rejection, suggesting a potential protective role in graft homeostasis. The expression and regulation of SPI-6 in TEC has not been studied. We demonstrate for the first time that murine TEC express SPI-6. In primary culture TEC, SPI-6 protein but not mRNA increases rapidly in response to IFNγ, LPS and also 24-48 hours after IRI, suggesting SPI-6 may require rapid up-regulation for its as yet undefined role in vivo. Methods/Results: We have previously shown that NK cells are important effector cells in ischemia-reperfusion injury (IRI) and can lyse TEC primarily through a perforin/GrB pathway. To test the role of SPI-6 in NK mediated TEC lysis in vitro, IL-2 activated NK cells (1:10) purified from Rag1-/- mice were added to CFSE labeled TEC from B6 and B6-SPI-6 null kidneys. TEC death was determined by Annexin V-PI staining/FACS after 5 hours. NK mediated TEC death was consistently greater in SPI-6 null TEC (30% vs 24% in wild type TEC). We then tested the role of SPI-6 in vivo in a mouse transplant model, using donor kidneys from C57BL/6 or B6 SPI-6 null mice (H-2^b) and BALB/c (H-2^d) recipients. Recipients were nephrectomized to clearly identify the contribution of the donor kidney to function and survival. SPI-6 deficient kidney recipients had a clear decrease in function by day 8 post transplantation compared to wild type controls (serum creatinines:113+51 µmol/l vs. 28+6 µmol/l, n=5, P<0.01) and on kidney histology had greater tubular injury and extensive infiltration. Furthermore in survival studies, loss of kidney SPI-6 shortened graft survival time (20+19 vs 66+33 days, n=8-10, P<0.001). Conclusion: Taken together, our data demonstrates for the first time that resistance of kidney TEC to GrB killing is mediated by the expression of the serpin protease inhibitor SPI-6. This may represent a critical endogenous form of protection from CD8+T and NK cytotoxic cells. These data also suggest that perforin/GrB effectors such as NK cells may be relevant to chronic injury. Augmenting or maintaining expression of PI-9/SPI-6 by TEC may enhance protection from IRI and rejection injury and promote long term allograft survival.

Disclosure: All authors have declared no conflicts of interest.