TY - JOUR
T1 - Residual methylprednisolone suppresses human T-cell responses to spleen, but not islet, extracts from deceased organ donors
AU - Joffe, Max
AU - Necula, Andra S.
AU - Chand, Rochna
AU - Mcwhinney, Brett C.
AU - Krishnamurthy, Balasubramanian
AU - Loudovaris, Tom
AU - Goodman, David
AU - Thomas, Helen E.
AU - Kay, Thomas W.H.
AU - Mannering, Stuart I.
N1 - Funding Information:
Australian National Health and Medical Research Council (NHMRC#559007); Juvenile Diabetes Research Foundation (JDRF 4-2006-1025); Diabetes Australia Research Trust; Operational Infrastructure Support Program of the Victorian Government.
PY - 2012/7
Y1 - 2012/7
N2 - 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.
AB - 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.
KW - Deceased organ donors
KW - Human spleen
KW - Methylprednisolone
KW - Mifepristone
KW - T-cells
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U2 - 10.1093/intimm/dxs042
DO - 10.1093/intimm/dxs042
M3 - Article
C2 - 22378502
AN - SCOPUS:84863998008
SN - 0953-8178
VL - 24
SP - 447
EP - 453
JO - International Immunology
JF - International Immunology
IS - 7
ER -