Activated T Regulatory Cells Escape Death by Increasing Cytoplasmic Serine Protease Inhibitor 6 (SPI6)
Renal/ Transplantation Research Center, Brigham and Women's Hospital, Boston, MA.
Meeting: 2015 American Transplant Congress
Abstract number: 132
Session Information
Session Name: Concurrent Session: Regulatory T Cells
Session Type: Concurrent Session
Date: Sunday, May 3, 2015
Session Time: 4:00pm-5:30pm
Presentation Time: 4:00pm-4:12pm
Location: Room 122-AB
Regulatory T cells (Tregs) can employ a granzyme B (GrB)-dependent mechanism to mediate suppression of effector cells by inducing their apoptosis. In this study we show that activated Tregs increase intracellular Spi6 expression that partially inhibits the intracellular activity of leaking GrB, contributing to its protection from self-inflicted damage. Further upregulation of Spi6 expression in wild type (WT) Tregs using a small molecule agonist of Spi6 gene promoter improved Tregs homeostasis.
Generated Spi6-/- mice were used in heart transplant models in addition to a Graft versus host disease (GVHD) model. Multiple experiments were done using FACS sorting, Flow cytometry, confocal microscopy, Luminex and Western Blot assays.
Our study shows that induced Treg in vitro, or in a GVHD model in vivo, increased their intracellular expression of Spi6 and GrB over time. Using the GrantoxiLux assay, and confocal microscopy, we observed intracellular leaking of GrB from granules of stimulated natural Tregs in vitro. Our data was confirmed with subcellular fractionation and GrB activity measurement on the cytoplasm and granules of Treg. In a Treg generation assay in vitro, we observed by flow cytometry analysis that generated Spi6-/- Treg, similar to WT Treg, increased production of GrB over time but had higher rate of apoptosis than WT T cells. Hence, stimulated WT CD4+ T cells generated 4 to 5 times more Treg in vitro than Spi6-/- CD4+ T cells. This Spi6-/- Treg generation defect was rescued by adding Granzyme B inhibitor (Z-AAD-CMK) at 1 μM.
Furthermore, Spi6-/- natural Tregs were less efficient in suppressing T cell proliferation in vitro using a Tregs suppression assay and in vivo using a GVHD model and went more into apoptosis compared to WT Tregs.
Moreover, SPI6-/- recipients of fully mismatched vascularized heart transplant showed resistance to tolerance induction by MR1 and demonstrated defects in Tregs survival.
Interestingly, upregulating Spi6 expression using a small molecule agonist of Spi6 gene promoter that is estrogen receptor agonist, increased Spi6 expression and reduced the apoptosis of stimulated WT Tregs in vitro.
This novel discovery sheds light on a previously unknown activated Tregs survival pathway, with many therapeutic applications in Tregs cell therapy.
To cite this abstract in AMA style:
Radwan A, Uehara M, Lukyanchykov P, Riella L, Chandraker A, Abdi R, Azzi J. Activated T Regulatory Cells Escape Death by Increasing Cytoplasmic Serine Protease Inhibitor 6 (SPI6) [abstract]. Am J Transplant. 2015; 15 (suppl 3). https://atcmeetingabstracts.com/abstract/activated-t-regulatory-cells-escape-death-by-increasing-cytoplasmic-serine-protease-inhibitor-6-spi6/. Accessed May 10, 2025.« Back to 2015 American Transplant Congress