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Abstract: FR-PO1128

Calcium Release-Activated Calcium (CRAC) Channel Inhibitor BTP2 Blocks IL2 and CD25 Expression in Human Peripheral Blood Mononuclear Cells

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

  • Shankaranarayanan, Divya, Weill Cornell Medicine, New York, New York, United States
  • Lagman, Milagros T., Weill Cornell Medicine, New York, New York, United States
  • Sharma, Vijay K., Weill Cornell Medicine, New York, New York, United States
  • Li, Carol Y., Weill Cornell Medicine, New York, New York, United States
  • Cassidy, Michael F., Weill Cornell Medicine, New York, New York, United States
  • Botticelli, Brittany, Weill Cornell Medicine, New York, New York, United States
  • Snopkowski, Catherine, Weill Cornell Medicine, New York, New York, United States
  • Yang, Hua, Weill-Cornell , New York, New York, United States
  • Muthukumar, Thangamani, Weill Cornell Medicine, New York, New York, United States
  • Machaca, Khaled, Weill Cornell Medicine, Doha, Qatar
  • Suthanthiran, Manikkam, Weill Cornell Medicine, New York, New York, United States

Group or Team Name

  • Weill Cornell Medicine, New York City
Background

CRAC channels are essential for the signaling of cells via calcineurin. BTP2, a tri-fluoro methyl pyrazole has been found to be a selective blocker of CRAC channels, prevents calcineurin activation and subsequent import of dephosphorylated NFAT into the nucleus in cell lines. In a search for a new type of calcineurin inhibitor, we investigated whether BTP2 inhibits calcineurin activation dependent expression of IL-2 and IL-2R alpha (CD25) by normal human peripheral blood mononuclear cells (PBMC).

Methods

PBMC isolated from healthy volunteers (N=4), were incubated alone, with 2ug/ml phytohemagglutinin (PHA) or with PHA + 1000nM BTP2 for 16 hours at 37C in 5% CO2. The cells were retrieved and analyzed by two methods: (i) Single cell Flow cytometry by labelling the cells with CD3-PE and CD25-FITC antibodies and (ii) Pre-amplification enhanced real time quantitative PCR (RT qPCR) to quantify the absolute copy numbers of mRNA encoding IL2 and CD25.

Results

BTP2 was a potent inhibitor of PHA induced expression of IL2 mRNA (Figure 1) and CD25 mRNA (Figure 2). In accord with its effect on CD25 mRNA, BTP2 inhibited the induced expression of CD25 protein as assessed at the single cell level by flow cytometry (Figure 3A & 3B).

Conclusion

To date, the effect of CRAC channel blockade with BTP2 has been studied in rat models and cell lines. We established normal human PBMC cell culture to investigate the effect of BTP2 and demonstrate that BTP2 reduced IL2 and CD25 expression in human PBMC. These inhibitory effects would effectively block T cell clonal expansion, a prerequisite for allograft rejection. CRAC channel blockade represents a novel strategy for immunosuppression.