Multimerized T cell epitopes protect from experimental autoimmune diabetes by inducing dominant tolerance -- Piaggio et al. 104 (22): 9393 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Piaggio et al. 10.1073/pnas.0610423104.

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SI Figure 6
SI Figure 7
SI Methods

SI Figure 6

Fig. 6. Enumeration of Foxp3⁺ CD4 T cells in the spleen or pancreatic lymph nodes of RIP-HA mice injected with HA-specific TH1 cells and treated with either 4-mer or HA peptide. Spleen and pancreatic lymph nodes from 4-mer- or HA_107-119 peptide-treated mice were recovered at days 14 (four mice per group) and 30 (three mice per group) after treatment, and a combination of surface (CD4, CD25) and intranuclear (Foxp3) staining was carried out and analyzed by FACS. The proportion (mean ± SD) of CD25⁺ and CD25^- Foxp3⁺ among CD4 T cells at day 14 is shown. Similar results were obtained at day 30.

SI Figure 7

Fig. 7. Reduced cytokine production in 4-mer-treated mice in response to ex vivo HA stimulation. Spleen and pancreatic lymph nodes from 4-mer- or HA_107-119 peptide-treated mice were recovered at days 14 (four mice per group) and 30 (three mice per group) after treatment and restimulated with or without 10 mg/ml of the HA_107-119 peptide. Cytokine production was evaluated at 72 h by using Luminex technology. Cytokine production by splenocytes after HA peptide stimulation is shown relative to that of unstimulated cultures from the same mouse. The mean production of cytokines by unstimulated splenocytes of HA peptide versus 4-mer-treated mice did not differ significantly (323 vs. 225 pg/ml for IL-6; 214 vs. 262 pg/ml for IL-10; 44 vs. 45 pg/ml for TNF-a; and 2,038 vs. 1,735 pg/ml for IFN-g). In a control experiment in which mice were treated with either PBS, 4-mer, or HA_107-119 peptide (three mice per group) but did not receive the HA-specific TH1 cells, cytokine production was minimal, ranging from <3.2 pg/ml for TNF-a to 18.8 pg/ml for IL-6. Qualitatively similar inhibition of cytokine production by the 4-mer treatment was obtained from cells isolated from pancreatic lymph nodes. The statistical analyses were performed using a two-way ANOVA.

SI Methods

Foxp3 FACS Detection.

For analysis of Foxp3 expression at the single-cell level, cells were stained for the expression of surface molecules and, after fixation and permeabilization, incubated with phycoerythrin-conjugated FJK-16s (anti-mouse Foxp3 mAb; eBioscience, San Diego, CA) according to the manufacturer's instructions. Data were collected on a FACScan (Becton-Dickinson, Mountain View, CA) instrument, and analysis was performed by using the CELLQuest software (Becton Dickinson).

Multiplex Cytokine Detection.

Spleen cells or pancreatic lymph nodes cells from 4-mer- or HA_107-119 peptide-treated RIP-HA mice were stimulated with or without 10 mg/ml of HA_107-119 peptide, and supernatants were harvested at 72 h and kept at -80° until used. Simultaneous quantification of a panel of cytokines was performed in duplicate on a Luminex 100SI (IFR31; Rangueil Hospital, Toulouse, France) with a LINCOplex mouse cytokine panel kit (Linco Research, Saint Charles, MI). In each experiment, a standard curve was run in parallel. The detection limits ranged from 1 to 3.2 pg/ml. TGF-b was detected by ELISA, as previously described (1).

1. Cautain B, Damoiseaux J, Bernard I, van Straaten H, van Breda Vriesman P, Boneu B, Druet P, Saoudi A (2001) Eur J Immunol 31:1132-1140.