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Abstract: TH-PO543

Generation of CX3CR1-Expressing T Regulatory Cells Using Retroviral Transduction

Session Information

Category: Glomerular Diseases

  • 1401 Glomerular Diseases: From Inflammation to Fibrosis

Authors

  • Diefenhardt, Paul, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Puetz, David L., Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Trinsch, Bastian, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Nies, Jasper Friedrich, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Sierra Gonzalez, Claudio, University of Cologne Center for Molecular Medicine Cologne, Cologne, Nordrhein-Westfalen, Germany
  • Schermer, Bernhard, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Brinkkoetter, Paul T., Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Benzing, Thomas, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
  • Braehler, Sebastian, Universitatsklinikum Koln, Koln, Nordrhein-Westfalen, Germany
Background

T cells play a central role in the pathogenesis of acute glomerulonephritis. Studies in rodent models as well as observations in patients suggest an imbalance of pro-inflammatory T effector cells and anti-inflammatory T regulatory cells (Treg), resulting in autoimmune-mediated glomerular injury. Increasing the numbers of T regulatory cells in the inflamed kidney might restore the T cell balance and, thus, attenuate disease. This could be achieved by transfusion of Tregs equipped with specific chemokine receptors to increase their migration into the inflamed kidney. In this regards, the CX3CR1-CX3CL1 axis represents a promising target, as it has been implicated in a variety of inflammatory diseases in the kidney.

Methods

The nephrotoxic nephritis model (NTN) is used to assess the distribution of CX3CL1 in the kidney using IHC stainings. The expression of CX3CR1 on renal and splenic immune cells during homeostasis and NTN is analysed using flow cytometry. Standard cloning techniques are used to design a retroviral vector for transduction of naïve murine T cells.

Results

Flow cytometry analysis after 7 days of nephrotoxic nephritis reveal CX3CR1 expression mostly on macrophages and classical dendritic cells Type 2 in the kidney and spleen. Renal and splenic T cells, on the other hand, express less CX3CR1, with about 20% of cytotoxic T cells and 10-15% of Tregs and Th17 cells expressing the receptor. Immunohistochemistry reveals increased Fractalkine expression in nephritic kidneys compared to healthy controls. To generate induced Tregs expressing CX3CR1, the protein coding sequences for CX3CR1 or eGFP were linked with a P2A site to the sequence for FOXP3 and introduced into a MSCV retroviral vector system. This vector will be used for transfection of the Pheonix-Eco packaging cell line to produce virions for subsequent transduction.

Conclusion

The expression of CX3CL1 is upregulated in nephritic kidneys and might therefore represent a promising point of action to increase renal migration of ex vivo induced T regulatory cells. A viral vector was successfully cloned to generate CX3CR1 expressing Tregs. Future experiments will validate the suppressive function of these Tregs in vitro and in vivo. Ultimately, the therapeutic potential in attenuating NTN will be assessed.