title: Serine protease inhibitor 6 plays a critical role in protecting murine granzyme B-producing regulatory T cells.
abstract: Regulatory T cells (Tregs) play a pivotal role in the maintenance of immune tolerance and hold great promise as cell therapy for a variety of immune-mediated diseases. However, the cellular mechanisms that regulate Treg maintenance and homeostasis have yet to be fully explored. Although Tregs express granzyme-B (GrB) to suppress effector T cells via direct killing, the mechanisms by which they protect themselves from GrB-mediated self-inflicted damage are unknown. To our knowledge, we show for the first time that both induced Tregs and natural Tregs (nTregs) increase their intracellular expression of GrB and its endogenous inhibitor, serine protease inhibitor 6 (Spi6) upon activation. Subcellular fractionation and measurement of GrB activity in the cytoplasm of Tregs show that activated Spi6(-/-) Tregs had significantly higher cytoplasmic GrB activity. We observed an increase in GrB-mediated apoptosis in Spi6(-/-) nTregs and impaired suppression of alloreactive T cells in vitro. Spi6(-/-) Tregs were rescued from apoptosis by the addition of a GrB inhibitor (Z-AAD-CMK) in vitro. Furthermore, adoptive transfer experiments showed that Spi6(-/-) nTregs were less effective than wild type nTregs in suppressing graft-versus-host disease because of their impaired survival, as shown in our in vivo bioluminescence imaging. Finally, Spi6-deficient recipients rejected MHC class II-mismatch heart allografts at a much faster rate and showed a higher rate of apoptosis among Tregs, as compared with wild type recipients. To our knowledge, our data demonstrate, for the first time, a novel role for Spi6 in Treg homeostasis by protecting activated Tregs from GrB-mediated injury. These data could have significant clinical implications for Treg-based therapy in immune-mediated diseases.
authors.affiliation: Transplantation Research Center, Renal Division, Brigham and Women's Hospital and Children's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Jamil
authors.initials: J
authors.lastname: Azzi
authors.forename: Nikolaos
authors.initials: N
authors.lastname: Skartsis
authors.forename: Marwan
authors.initials: M
authors.lastname: Mounayar
authors.forename: Ciara N
authors.initials: CN
authors.lastname: Magee
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Batal
authors.forename: Christopher
authors.initials: C
authors.lastname: Ting
authors.forename: Robert
authors.initials: R
authors.lastname: Moore
authors.forename: Leonardo V
authors.initials: LV
authors.lastname: Riella
authors.forename: Shunsuke
authors.initials: S
authors.lastname: Ohori
authors.forename: Rozita
authors.initials: R
authors.lastname: Abdoli
authors.forename: Brian
authors.initials: B
authors.lastname: Smith
authors.forename: Paolo
authors.initials: P
authors.lastname: Fiorina
authors.forename: Dean
authors.initials: D
authors.lastname: Heathcote
authors.forename: Tannous
authors.initials: T
authors.lastname: Bakhos
authors.forename: Philip G
authors.initials: PG
authors.lastname: Ashton-Rickardt
authors.forename: Reza
authors.initials: R
authors.lastname: Abdi
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000276:immunology; D005434:Flow Cytometry; D006086:Graft vs Host Disease / Q000276:immunology; D053804:Granzymes / Q000276:immunology* / Q000378:metabolism; D016027:Heart Transplantation / Q000276:immunology; D006706:Homeostasis / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D012697:Serine Endopeptidases / Q000276:immunology* / Q000378:metabolism; D015843:Serpins / Q000276:immunology* / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D015843:Serpins; D053804:Granzymes; D012697:Serine Endopeptidases; C106610:Serpinb9 protein, mouse
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