title: Residual methylprednisolone suppresses human T-cell responses to spleen, but not islet, extracts from deceased organ donors.
abstract: Pancreatic islets, transplanted into recipients with type 1 diabetes, are exposed to allogenic and auto-immune T-cell responses. We set out to develop an assay to measure these responses using PBMC. Our approach was to prepare spleen extract from the islet donors (allo-antigen) and islet extracts (auto-antigen). To our surprise, we found that spleen extracts potently inhibited the proliferation of human T cells driven by antigen (tetanus toxoid) and mitogen (anti-CD3 mAb, OKT3), whereas extracts prepared from pancreatic islets from the same donor did not suppress T-cell proliferation. Suppression mediated by spleen extracts was unaffected by blocking mAbs against the IL-10R, transforming growth factor-β or CD152 (CTLA-4). It was also unaffected by denaturing the spleen extracts by heating, exposing to reducing agents or protease digestion. Because deceased organ donors are commonly given the immunosuppressive glucocorticoid methylprednisolone prior to death, we hypothesized that suppression was due to residual methylprednisolone in the spleen extracts. Methylprednisolone could be detected by mass spectrometry in spleen extracts at concentrations that suppress T-cell proliferation. Finally, the glucocorticoid receptor antagonist mifepristone completely reversed the suppression caused by the spleen extracts. We conclude that extracts of human spleen, but not islets, from deceased organ donors contain sufficient residual methylprednisolone to suppress the proliferation of T-cells in vitro.
authors.affiliation: Immunology and Diabetes Unit, St Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.
authors.forename: Max
authors.initials: M
authors.lastname: Joffe
authors.forename: Andra S
authors.initials: AS
authors.lastname: Necula
authors.forename: Rochna
authors.initials: R
authors.lastname: Chand
authors.forename: Brett C
authors.initials: BC
authors.lastname: McWhinney
authors.forename: Balasubramanian
authors.initials: B
authors.lastname: Krishnamurthy
authors.forename: Tom
authors.initials: T
authors.lastname: Loudovaris
authors.forename: David
authors.initials: D
authors.lastname: Goodman
authors.forename: Helen E
authors.initials: HE
authors.lastname: Thomas
authors.forename: Thomas W H
authors.initials: TW
authors.lastname: Kay
authors.forename: Stuart I
authors.initials: SI
authors.lastname: Mannering
mesh_terms: D001324:Autoantigens / Q000276:immunology; D002457:Cell Extracts / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology / Q000628:therapy*; D006358:Hot Temperature; D006801:Humans; D007165:Immunosuppression Therapy; D007515:Islets of Langerhans / Q000737:chemistry / Q000276:immunology*; D016381:Islets of Langerhans Transplantation / Q000276:immunology*; D007519:Isoantigens / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D013058:Mass Spectrometry; D008775:Methylprednisolone / Q000032:analysis / Q000494:pharmacology*; D015735:Mifepristone / Q000494:pharmacology; D013154:Spleen / Q000737:chemistry / Q000276:immunology*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000276:immunology; D014019:Tissue Donors
chemical_list: D001324:Autoantigens; D002457:Cell Extracts; D007519:Isoantigens; D015735:Mifepristone; D008775:Methylprednisolone