Regulatory T cells (Tregs) can employ a granzyme B (GrB)-dependent mechanism to mediate suppression of effector cells by inducing their apoptosis. Herein, we show that activated Tregs express SPI6, a GrB inhibitor that protects Tregs from self-inflicted damage induced by intra-cytoplasmic leaking of GrB.

Our study shows that both induced Tregs (iTregs) and natural Tregs (nTregs) increase their intracellular expression of SPI6 and GrB over time, either upon activation in vitro, or in GVHD model in vivo. Using the GrantoxiLux assay and confocal microscopy, we observed intracellular leaking of GrB from granules of stimulated nTregs in vitro Our data was confirmed with subcellular fractionation and measurement of GrB activity in the cytoplasm and granules of Tregs. Activated SPI6-/- Tregs had higher cytoplasmic GrB activity, went more into apoptosis and were less efficient than WT Tregs in suppressing T cell proliferation in a Treg suppression assay in vitro, and in a GVHD model in vivo. Furthermore, The higher rate of apoptosis of activated SPI6-/- Tregs in vitro was reversed by the addition of a GrB inhibitor (Z-AAD-CMK).

To study the survival of nTregs in live animals over time, we transduced SPI6-/- and WT Tregs with a lentivirus vector encoding Gluc, a highly sensitive, naturally secreted luciferase from the marine copepod Gaussia princeps. Irradiated BALB/c mice were reconstituted with WT bone marrow and T cells to induce GVHD, and received either SPI6-/- or WT transduced Tregs; Gluc secretion by live Tregs in the BALB/c mice was then analyzed at days 2, 8 and 16. The level of Gluc activity in blood was similar in the two groups at day 2 but significantly dropped at later time points in mice recipient of Gluc+ SPI6-/- Tregs rather than WT Tregs; furthermore, whole body bioluminescent imaging at day 10 showed lower signal intensity in mice that received Gluc+ SPI6-/- Tregs rather than WT Tregs.

To study the role of SPI6 in Treg generation in alloimmunity, we used the MHC class II-mismatched bm12⇒B6 vascularized cardiac transplant model. Allograft rejection was remarkably accelerated in SPI6-/- recipients, and was, furthermore, associated with a significant reduction in the ratio of Tregs in allografts and DLN, and a higher rate of Treg apoptosis compared to WT recipients. This novel discovery illuminates a previously unknown activated Treg survival pathway, with many potential therapeutic applications in alloimmunity.

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