Soluble C Type Lectin Receptor Protein Inhibition of Natural Killer Cell Mediated Tubular Epithelial Cell Death
1Physiology, Western University, London, ON, Canada, 2Microbiology and Immunology, Western University, London, ON, Canada, 3London Health Sciences Centre, London, ON, Canada
Meeting: 2019 American Transplant Congress
Abstract number: A88
Keywords: Epithelial cells, Immunosuppression, Kidney transplantation, Natural killer cells
Session Information
Session Name: Poster Session A: Ischemia Reperfusion & Organ Rehabilition
Session Type: Poster Session
Date: Saturday, June 1, 2019
Session Time: 5:30pm-7:30pm
Presentation Time: 5:30pm-7:30pm
Location: Hall C & D
*Purpose: We have previously shown that Natural Killer (NK) cells can mediate kidney tubular epithelial cell (TEC) death and kidney ischemia-reperfusion injury (IRI) as well as chronic transplant rejection. MHC-independent mechanisms such as binding of NKRP1 receptors to C-type lectin receptor (Clr) family proteins, may regulate NK cell function.TEC can regulate NK cell activation and cytotoxicity by their expression of inhibitory Clrs, which we have now produced in genetically altered tobacco to test in our models.
*Methods: Expression of Clr-b and Clr-f was confirmed in TEC using RT-PCR. WT and Clr-b-/- TEC were treated in vitro with Clr-f siRNA; silencing was confirmed by RT-PCR and flow cytometry. Cell death was measured in NK-TEC co-cultures by 51Cr release assay. To obtain Clr-b, Clr-f, and Clr-b+f fusion protein, we designed and synthesized a codon-optimized Clr-b, -f, and fusion genes according to the frequency of codon usage in tobacco. The genes were then cloned into plant viral expression vectors and transiently expressed in tobacco plants. The expression of proteins was confirmed by immunoblotting and the expressed Clr proteins were purified using nickel affinity chromatography.
*Results: Clr-b and f expression in TEC was upregulated by TNFα+IFNγ treatment in vitro. Elimination of either Clr-b or Clr-f alone in TEC did not increase NK mediated killing. However, silencing of both Clr-b and Clr-f expression resulted in increased NK killing of TEC (n=3, p<0.01), or WT control TEC (p<0.001). Addition of soluble plant expressed Clr-b/Clr-f proteins to NK cell-TEC cocultures decreased NK-mediated TEC death(n=3, p<0.05).
*Conclusions: Our data support that NKRP1-Clr binding is an important inhibitory pathway in NK mediated kidney injury. As no current drugs target NK cells effectively, Clr-b/Clr-f soluble proteins expressed by plants may represent a novel strategy to inhibit NK-mediated injury of kidneys in IRI and post-transplant. Indeed, plant-expressed Clr proteins may represent a novel source of NK directed therapeutics to protect organs from diverse forms of NK mediated inflammation and cytotoxicity.
To cite this abstract in AMA style:
Balaji S, Fuhrmann B, Lian D, Diao H, Ma S, Zhang Z, Jevnikar AM. Soluble C Type Lectin Receptor Protein Inhibition of Natural Killer Cell Mediated Tubular Epithelial Cell Death [abstract]. Am J Transplant. 2019; 19 (suppl 3). https://atcmeetingabstracts.com/abstract/soluble-c-type-lectin-receptor-protein-inhibition-of-natural-killer-cell-mediated-tubular-epithelial-cell-death/. Accessed May 9, 2025.« Back to 2019 American Transplant Congress