Background: Kidney tubular epithelial cells (TEC) may negatively regulate NK cell activation and cytotoxic capacity by surface expression of a novel class of C-type lectin-related ligand proteins (Clr) which may be exploited to prevent ischemia reperfusion and alloimmune transplant injury.

Methods: Expression of Clr-b/-f was confirmed in wild type (WT) and transformed mouse TEC using RT-PCR. IRI was induced by renal artery clamping (30-45m) in uni-nephrectomized mice, and changes in Clr-b/-f mRNA expression were determined. TEC were treated in vitro with Clr-b and –f siRNA, and silencing was confirmed by RT-PCR and flow cytometry. Cell death was measured in NK-TEC co-cultures by target cell ⁵¹Cr release.

Results: NK cells kill β2m null TEC more efficiently than WT TEC in vitro (n=4, p<0.05), confirming MHC class I is an inhibitory mechanism of NK killing of TEC. MHC-independent mechanisms such as binding of NKRP1 receptors to Clr family members, may also regulate NK-induced TEC death. Clr-b and f were expressed by WT TEC and upregulated by TNFα+IFNγ in vitro. Clr-b surface expression was increased for >48hr in B6 kidneys following IRI. Elimination of either Clr-b or Clr-f by TEC did not increase NK mediated killing. However, simultaneous silencing of both Clr-b and Clr-f expression resulted in increased NK killing of TEC compared to silenced Clr-b or Clr-f TEC (p<0.01), or WT control TEC (p<0.001). The use of combined Clr-b/Clr-f fusion proteins may thus be effective to attenuate NK cytotoxicity and these studies are in progress.

Conclusion: TEC may increase their expression of Clr-b/-f with ischemia and alloimmune renal injury as an endogenous mechanism of protection from NK cells during inflammation. The augmented killing of TEC following loss of Clr-b and Clr-f strongly supports that NKRP1-Clr binding is an important inhibitory pathway in NK mediated kidney injury. As no current drugs target NK cells effectively, Clr-b and Clr-f fusion proteins that bind to NK cells bearing NKRP1 receptors may represent a novel strategy to protect organs from diverse forms of NK mediated inflammation and cytotoxicity.

CITATION INFORMATION: Fuhrmann B., Lian D., Yin Z., Ma S., Arp J., Zhang Z., Jevnikar A. Regulation of NK Cell Cytotoxicity by TEC Expression of Clr Proteins in Kidney Ischemia Reperfusion Injury (IRI) Am J Transplant. 2017;17 (suppl 3).

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