Autoimmune Diabetes Onset Results From Qualitative Rather Than Quantitative Age-Dependent Changes in Pathogenic T-Cells

Sylvaine You 1 2 Me riam Belghith 1 2 Stephen Cobbold 0 Marie-Alexandra Alyanakian 1 2 Christine Gouarin 1 2 Samia Barriot 1 2 Corinne Garcia 1 Herman Waldmann 0 Jean-Fran cois Bach 1 2 Lucienne Chatenoud 1 2 0 Sir William Dunn School of Pathology, Oxford, U.K. INSERM U580, Ho pital Necker , 161 rue de Se` vres, 75015 Paris , France 1 Faculte de Medicine , Rene Descartes Paris 5, Paris, France; and the 2 Institut National de la Sant e et de la Recherche M e dicale (INSERM) U580 , Paris , France; the Diabetogenic T-cells can be detected in pre-diabetic nonobese diabetic (NOD) mice after transfer in NODSCID recipients. Here we demonstrate that 6-week-old pre-diabetic NOD mice, >2 months before disease onset, already harbor pathogenic T-cells in equal numbers to overtly diabetic animals. The delay in diabetes appearance is explained by the presence of regulatory CD4 CD25 T-cells that control diabetogenic effectors and that are, in our hands, transforming growth factor (TGF)- dependent. Our present results suggest, however, that diabetes onset is only partly explained by a decline in this regulatory T-cell activity. Another major factor appears to be the progressive resistance of diabetogenic cells to TGF-- dependent mediated inhibition. We propose that progression to overt disease correlates with the pathogenic T-cell's escape from TGF-- dependent T-cell-mediated regulation. Diabetes 54:1415-1422, 2005 - Iby selective destruction of insulin-secreting -cells nsulin-dependent diabetes (type 1 diabetes) is a genetically controlled autoimmune disease caused by pathogenic T-cells (1). In nonobese diabetic (NOD) mice, a spontaneous model of type 1 diabetes, pathogenic T-cells are easily assessed by adoptive transfer into syngeneic immunodeficient recipients (15). In NOD mice, rupture of self-tolerance is first evidenced at 3 4 weeks of age when the initial wave of mononuclear cells infiltrates pancreatic islets that is 3 4 months before the first signs of overt disease, as assessed by glycosuria and hyperglycemia (1,6,7). During this quite long phase (i.e., pre-diabetes), insulitis progresses until 1214 weeks of age as a benign process that is not associated with massive -cell destruction (1). Although major progress has been made in our knowledge on the candidate autoantigens (8 10) and the forces driving the emergence of diabetogenic T-cells (1,1117), there are still uncertainties about the mechanisms underlying the progression to irreversible -cell destruction. There is compelling evidence to show that regulatory T-cells mediating transferable tolerance actively control diabetogenic effectors (18 24). However, is this just a game of numbers? Does diabetes onset exclusively result from either the decrease in regulatory T-cells over time or from the progressive expansion of -cellspecific T-cell effectors overriding the control afforded by regulatory T-cells? Our current results point to an alternative possibility, which is that there are key age-dependent differences in effector T-cells that are qualitative rather than quantitative and render them progressively insensitive to T-cellmediated regulation. This insensitivity involves progressive unresponsiveness of effector T-cells to the immunoregulatory cytokine transforming growth factor (TGF)-. RESEARCH DESIGN AND METHODS NOD mice (Kd, I-Ag7, Db) and NOD-SCID mice were bred in our animal facility under specific pathogen-free conditions. Glycosuria and glycemia were monitored using colorimetric strips (Glukotest and Hemoglukotest; BoehringerMannheim). Fluorescence-activated cell sorting analysis. Lymphocytes were stained for surface markers and analyzed by flow cytometry. Antibodies to TGF(2G.7) were purified and fluoresceinated in our laboratory. Anti glucocorticoid-induced tumor necrosis factor receptor (GITR) antibodies were kindly provided by Dr. Cobbold (Sir William Dunn School of Pathology, Oxford, U.K.). CD62L, CD25, CD4, and CD103 ( E integrin subunit) antibodies were obtained from PharMingen-BD (San Diego, CA). Cell preparations. Splenocytes were isolated from 4- or 6-week-old or diabetic NOD mice. Splenocytes were then purified on the basis of CD62L, CD25, or CD4 expression using magnetic bead cell sorting (Miltenyi Biotech, Bergisch-Gladbach, Germany). Purity of the sorted cells was 90 97%, and recovery ranged from 50 to 70%. Adoptive cell transfers. Recipients were adult 6-week-old NOD-SCID mice. Animals were injected intravenously with either a single cell population or, in the case of cotransfer experiments, a mixture of two distinct populations. The precise cell numbers used varied depending on the experiments and are detailed in the RESULTS section. In vitro proliferation assays. Cells were cultured in complete RPMI 1640. CD4 CD25 and CD4 CD25 T-cells were seeded in 96-well microplates (2 104 cells/well) and stimulated with soluble anti-CD3 antibody (2.5 g/ml 145-2C11; provided by J.A. Bluestone) in the presence of mitomycin-treated antigen-presenting cells (APCs). Neutralizing antibodies to TGF- were added to the cocultures (10 or 50 g/ml 2G.7). For other experiments, CD4 CD25 and CD4 CD25 CD62L T-cells were incubated with APCs, anti-CD3 antibody, and recombinant active TGF- (0 4,000 pg/ml; R&D Systems, Abingdon, U.K.). For criss-cross cocultures, CD4 CD25 T-cells from 6-week-old animals were coincubated with CD4 CD25 T-cells isolated from diabetic NOD mice, and, inversely, CD4 CD25 T-cells from 6-week-old animals were coincubated with CD4 CD25 T-cells isolated from diabetic NOD mice. After 72 h at 37C, cells were pulsed with [3H]thymidine (Amersham). Data were expressed as the percent inhibition, deduced as: percent inhibition {1 [cpm (CD4 CD25 plus CD4 CD25 )/cpm CD4 CD25 ]} 100, where cpm is the counts per minute. Similar assays were performed in 24-well plates using transwells (Costar). 5,6-carboxyfluorescein diacetate-succinimidyl esterlabeled CD4 CD25 Tcells from 6-week-old NOD mice (2 105 cells/well) were stimulated with anti-CD3 antibody and APCs. CD4 CD25 T-cells (2 105 cells/well) were added in the transwell with APCs. After 4 days, cells were stained with CD4 antibodies, and the CD4 CD25 T-cell proliferation was analyzed by fluorescence-activated cell sorting. Cytokine production. CD4 CD25 and CD4 CD25 T-cells from 6-week-old or diabetic mice were used and cultured as described in the proliferation assay. Age-matched or crossed cocultures were performed, and supernatants were recovered after 24, 48, and 72 h of culture. Interleukin (IL)-4, IL-10, and -interferon (IFN-) were measured by enzyme-linked immunosorbent assays, and TGF-1 was measured using a DuoSet kit (R&D Systems). Real-time quantitative RT-PCR. DNase Itreated total RNA from tissues was prepared using the SV Total RNA isolation system (Promega, Madison, WI). Reverse transcription was performed using a proStar kit with random hexamers (Stratagene, Cedar Creek, TX). Real-time quantification was performed using gene-specific (...truncated)