Kidney Week

Abstract: FR-PO1104

Ex Vivo Exposure to IL-6 and TNFa Improves Proliferation of Regulatory T Cells Without Impairing Their Function and Lineage Stability

Session Information

• Transplantation: Basic
  November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Transplantation

• 1901 Transplantation: Basic

Authors

• Skartsis, Nikolaos, University of California, San Francisco, California, United States

Nikolaos Skartsis,

• Peng, Yani, University of California, San Francisco, California, United States

Yani Peng,

• Muller, Yannick, University of California, San Francisco, California, United States

Yannick Muller,

• Ferreira, Leonardo M., University of California, San Francisco, California, United States

Leonardo M. Ferreira,

• Vincenti, Flavio, University of California, San Francisco, California, United States

Flavio Vincenti,

• Tang, Qizhi, University of California, San Francisco, California, United States

Qizhi Tang,

Background

Clinical trials testing the efficacy of regulatory T cell (Treg) therapy in kidney and liver transplantation are underway. The survival and function of the infused Tregs in the inflammatory environment of the recipient are key determinants of the safety and efficiency of Treg therapy. We sought to investigate the impact of ex vivo exposure to IL-6 and TNFa on Treg proliferation, phenotype, and function.

Methods

First, we isolated CD4⁺CD25⁺CD62L⁺ Tregs from C57BL/6J mice lymph nodes using fluorescence activated cell sorting (FACS). We stimulated the Tregs with anti-CD3/CD28 beads in the presence of IL-2, with or without IL-6 and TNFa and monitored Treg proliferation over a 10-day period. In addition, we adoptively transferred IL-6 and TNFa exposed NOD.BDC2.5 TCR transgenic Tregs into NOD.CD28KO mice. Similarly, we setup ex-vivo cultures of human CD4+CD25^hiCD127^lo Tregs isolated from peripheral blood mononuclear cells of healthy donors. Finally, we profiled both murine and human Tregs using flow cytometry, luminex, bisulfite conversion and pyrosequencing.

Results

We observed that C57BL/6J mouse Tregs exposed to IL-6 and TNFa have increased proliferation (136+/-28 versus 1106+/-505 fold; n=3; p=0.02), expressed Foxp3 and Helios and remained demethylated at the Treg specific demethylated region (TSDR). Adoptive transfer of IL-6 and TNFa exposed NOD.BDC2.5 TCR transgenic Tregs protected NOD.CD28KO recipients from diabetes. Similarly, ex-vivo exposure to IL-6 and TNFa increased proliferation of human CD4⁺CD25⁺CD127^lo/- Tregs (24+/-13 versus 53+/-19 fold; n=4; p=0.04). IL-6 and TNFa exposed human Tregs remained FOXP3^hiHELIOS^hi, did not produce pro-inflammatory cytokines such as IL-2, IL-4, IFNg or IL-17, and maintained demethylated TSDR. Finally, IL-6 and TNFa exposed human Tregs maintained their suppressive function against pre-activated CD4⁺ Teff cells, similar to their non-exposed Treg counterparts.

Conclusion

Our results demonstrate that Treg exposure to IL-6 and TNFa enhances their proliferation without negatively impacting their lineage stability and suggests that Tregs positively respond to these cytokines by increasing their proliferation as a way to scale to inflammation. This property may be exploited to improve therapeutic Treg manufacturing for transplantation.

Funding

• Other NIH Support