Mice with blood glucose levels above 300mg/dl were considered diabetic

Mice with blood glucose levels above 300mg/dl were considered diabetic. amino acid substitutions, respectively. NIHMS236870-supplement-01.eps (286K) GUID:?F6776D05-B55A-4A58-B4C6-2AC3AD3344C0 02: Supplementary Table 1. RIP-GP Berlin mice show increased and more aggressive CD8 infiltrations resulting in earlier and stronger insulin loss RIP-GP Armstrong (RIP-GP Arm) and RIP-GP Berlin mice received the peptide/adjuvant treatment as depicted in Figure 1B. On days 0, 4, 7, 9, and 11 after P14 transfer, pancreata were isolated and stained for CD8 and insulin. The 3-Cyano-7-ethoxycoumarin table shows a summary of all mice analyzed (n=5 for each group). All RIP-GP Berlin mice were diabetic by day 11. In contrast, only 2 out of 5 RIP-GP Armstrong mice were diabetic on d11. Numbers in red indicate diabetes onset. PI, peri-insulitis; I, Insulitis; DI, destructive Insulitis. NIHMS236870-supplement-02.eps (281K) GUID:?234AC3F1-3404-4954-8266-498CD09BAC33 Abstract It is not fully understood how the expression level of autoantigens in beta cells impacts autoimmune diabetes (T1D) development. Earlier studies using ovalbumin and also insulin had shown that secreted antigens could enhance diabetes development through facilitated presentation by antigen presenting cells. Here we sought to determine how the expression level of a membrane bound, non-secreted or cross-presented neo-antigen, the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), would influence T1D. We found that a RIP-LCMV transgenic mouse line exhibiting higher levels of beta cell GP expression developed more severe diabetes after LCMV infection 3-Cyano-7-ethoxycoumarin or transfer of high numbers of activated autoreactive T cells. Importantly, all beta cells were lost and a significant increase in morbidity and mortality from T1D was noted. Insulitis and accumulation of autoaggressive CD8 cells was more profound in the RIP-LCMV-GP high-expressor line. Interestingly, the additional introduction of neo-antigen-specific CD4+ MMP2 helper or regulatory T cells was able to influence diabetogenesis positively or negatively. We conclude that a higher degree of autoantigen expression results in increased diabetes susceptibility. Therefore, autoantigens such as insulin that are expressed at higher levels in beta cells might have a more profound impact on diabetes pathogenesis. for three days in 24-well-plate wells with 1g/ml gp61 peptide (Abgent, NH2-GLNGPDIYKGVYQFKSVEFD-COOH) and 100U/ml rhIL-2 in 2ml RPMI containing 10% FCS. After three days in culture (CD4+ fraction was confirmed CD69+CD44hiCD62Lint-lo, activated phenotype), 1107 total cells were administered intravenously into RIP-GP Armstrong recipients four days after P14 transfer. For the generation of Smarta CD4+ regulatory T cells (Tregs), 3107 MACS-purified CD4+CD25? Smarta spleen and lymph node cells were cultured for seven days with 3107 T cell-depleted B6 spleen cells as antigen-presenting cells (APCs), 1g/ml gp61 peptide, 50U/ml rhIL-2, 110?7M Vitamin D3 and 310?8M Dexamethasone in 40ml RPMI containing 10% FCS. 1106 cultured cells were subsequently administered intravenously into RIP-GP Berlin recipients eight days after P14 transfer. 2.4. Flow cytometry Flow cytometry 3-Cyano-7-ethoxycoumarin antibodies were purchased from BD Pharmingen and Biolegend (San Diego, CA). For intracellular stains, single-cell suspensions from pancreatic draining lymph node (PDLN) and pancreas were restimulated for 5 C 6 h with gp33 peptide in the presence of brefeldin A (Sigma) followed by staining for surface expression of CD8, V2 and V8.1/2 (specific for P14 TCR), CD44 and CD62L. Cells were then fixed with 3% formaldehyde, permeabilized with 0.05% saponin, and stained for intracellular IFN- and TNF. Samples were acquired on a LSRII flow cytometer and analyzed using FlowJo software (Tree Star, Ashland, OR). 2.5. Immunohistochemistry Pancreata were immersed in Tissue-Tek OCT compound (Sakura Finetek USA, Torrance, CA) and quick-frozen on dry ice. 6C10-m pancreatic tissue sections were acetone-fixed and stained with CD8-specific and insulin-specific antibodies. Immunohistochemical detection was done using biotinylated secondary antibodies and Avidin-D coupled to HRP, followed by enzymatic development with DAB or AEC chromogen (Vector Laboratories), or using AP-labeled secondary antibodies followed by enzymatic development with Vector Blue (Vector Laboratories). 2.6. Immunofluorescence Acetone-fixed 3-Cyano-7-ethoxycoumarin pancreatic tissue sections were stained with insulin-specific antibody (guinea pig anti-swine insulin, 1:250-diluted, DAKO) and LCMV-GP-specific ascites (WE33.6, mouse IgG2a, 1:50 dilution, kind gift of Dr. Michael Buchmeier). Immunofluorescent detection was done using AF568-labeled goat anti-guinea pig and AF488-labeled goat anti-mouse polyclonal IgG (both 1:400 dilution, Invitrogen) secondary antibodies. After the samples were mounted with Immuno-Fluore mounting medium (MP Biomedicals Inc.), coverslips were visualized by fluorescence microscopy with a Nikon Eclipse E800 fluorescence microscope. 2.7. RT-PCR Organs were snap-frozen, homogenized in Trizol reagent (Invitrogen) and total RNA was extracted. To prevent its degradation, pancreatic RNA was re-extracted a second time to ensure complete removal of RNases. Residual genomic DNA was eliminated by DNase digestion for 20 min with the TURBO DNA-free kit (Ambion). Reverse transcription was carried out with 15g total RNA using the first-strand cDNA synthesis kit (Amersham Biosciences) with the oligo dT-primer provided in the kit according to the manufacturers instructions..