Biotinylated antibodies

Blood samples were obtained from Hoxworth Blood bank. Samples were de-identified, and the study was conducted under an exemption provided by the Cincinnati Children’s Hospital Medical Center (CCHMC) IRB. PBMCs were depleted of CD45RO⁺, CD8⁺, and CD25⁺ cells using biotinylated antibodies (BioLegend) and IMag streptavidin beads (BD Biosciences) and stimulated in 6-well plates at 8x10⁶ cells/2 ml with soluble αCD3 (2 μg/ml, OKT-3, BioXCell) and αCD28 (1 μg/ml, BD Biosciences) or CTLA4-Ig (7.5 μg/ml, BioXCell) in RPMI 1640 supplemented with 10% FBS, penicillin/streptomycin, L-glutamine, and β-mercaptoethanol. To generate regulatory T cells (Treg cells), αCD3, αCD28, IL-2 (100 U/ml, Roche), and TGF-β (10 ng/ml, Pepro Tech.) were added. Cells were harvested after 4 days and washed in PBS, and CD4⁺ T cells were purified by negative selection using the human CD4 EasySep kit (STEMCELL Technologies). Purified CD4⁺ cells were rested for 4–5 days in medium and then re-stimulated with soluble αCD3 (2 μg/ml) and αCD28 (1 μg/ml) or other stimuli for the indicated time periods.
To check primary response, depleted cells were primed, and CD4⁺ T cells were purified by positive selection using human CD4 Dynabeads (Invitrogen) at the indicated time points prior to RNA isolation or lysate preparation.

Pancreases embedded in OCT compound (Cellpath, Newtown, UK) were frozen in isopentane (Sigma) cooled with liquid nitrogen. 10 μm sections were fixed in acetone before first staining with rabbit anti-mouse insulin (Ab63820 [Abcam, Cambridge, UK], 2.5 μg/ml) in PBS 0.5% BSA, detected with Vector ImmPress anti-rabbit AP (Vector Labs, Peterborough, UK), and developed with Vector ImmPress Red. Immune cells in the tissues were stained with biotinylated antibodies (all from Biolegend, San Diego, CA, USA) to murine CD4 (clone GK1.5, which also recognises human CD4, 2.5 μg/ml), CD8 (clone 53-6.7, 2.5 μg/ml), CD11b (clone M1/70, 0.25 μg/ml), CD11c (clone N418, 0.625 μg/ml), Ly6G (clone 1A8, 0.5 μg/ml) and B220 (clone RA3-6B2, 0.625 μg/ml) in PBS 0.5% BSA for 2 h. The optimal concentration for each antibody was determined by serial dilution. Staining was detected using VectaStain R.T.U. Elite ABC reagent and DAB peroxidase substrate kit from Vector Labs. Nuclei were stained with Mayer’s haematoxylin (Sigma), before mounting slides with VectaMount (Vector Labs). Images were acquired on a Zeiss Axiovert A1 microscope using the Zen (blue edition) software supplied (Zeiss, Cambridge, UK). No cropping or alteration of the images was made. See electronic supplementary material (ESM) Methods for insulitis scoring.