Defective Antigen Processing in GILT-Free Mice Maja Maric, Balasubramanian Arunachalam, Uyen T. Phan, Chen Dong, Wendy S. Garrett, Kurt S. Cannon, Christopher Alfonso, Lars Karlsson, Richard A. Flavell, and Peter Cresswell

Supplementary Material

Supplemental Figure 1. Lack of T cell response to HEL by GILT -/- APC is not due to delayed kinetics. Splenocytes were isolated from KO and WT mice and incubated with 1mg/ml of HEL (A and C) or 5 g/ml of 74-88 HEL peptide (B and D) for 30 min. 1 h, 2 h, 4 h, 6 h and 24 h, and in another experiment for 24h and 48h (C and D). Splenocytes were fixed for 5 min at room temperature in 0.2% paraformaldehyde and washed with 0.2 M glycine in RPMI 1640 medium followed by 2 washes with complete medium. Fixed and washed splenocytes were incubated with the BO4 hybridoma (specific for the 74-88 peptide) for an additional 20 h at 37°C. Supernatants were collected and assayed for IL-2 by ELISA.

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Supplemental Figure 2. Knockout mice immunized with HEL lack a recall response against the GILT-dependent epitopes 46-61 and 74-88. Knockout mice and their wild type littermates were immunized with 100 g/mouse of HEL mixed with CFA and after 10 days draining lymph nodes were isolated and incubated for 72 h with various concentrations of either HEL (A) or 4 peptides: 20-35 (B), 30-53 (C), 46-61 (D) and 74-88 (E). The proliferation of draining lymph node cells was assayed by ³H-thymidine incorporation after 72 h incubation with a range of protein or peptide concentrations.

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Supplemental Figure 3. Knockout mice respond to HEL peptide 74-88 when immunized directly. Knockout mice and their wild type littermates were immunized with 50g of HEL peptide 74-88 mixed with CFA (1:1) and after 10 days draining lymph nodes were isolated and cells incubated with various concentrations of peptide 74-88. The proliferation of draining lymph node cells was assayed by ³H-thymidine incorporation after 72 hr.

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