The inhibitory function of B7 costimulators in T cell responses to foreign and self-antigens

Supplementary Fig. 1. (pdf 224KB)
Adoptively transferred DO11 T cell are not reduced in spleens of RIP-mOVA mice compared to BALB/c. 5x10⁶ CFSE-labeled DO11 CD4⁺ T cells were adoptively transferred into irradiated (500cGy) wild-type BALB/c or RIP-mOVA recipients on day 0. Spleens were harvested on d1, d2 and d3, and stained with anti-CD4 and KJ1-26. (a) FACS profile of lymphocytes recovered from spleens of wild-type and RIP-mOVA mice on day 1. The plot on the right is gated on CFSE-labeled CD4⁺ cells only. (b) Recovered CD4⁺ KJ1-26⁺ T cells from spleens of wild-type (open squares) and RIP-mOVA mice (filled diamonds) on day 1, 2 and 3 after adoptive transfer. Indicated numbers show the mean percentage of recovered KJ1-26⁺ CD4⁺ cells of transferred CFSE-labeled CD4⁺ cells from two individual mice.

Supplementary Fig. 2. (pdf 96KB)
Activated antigen-specific CD4⁺CD25⁺ cells from sOVA Tg x DO11 mice mediate suppression in vitro. KJ1-26⁺CD4⁺CD25⁺ cells from sOVA Tg x DO11 mice were purified by cell sorting and activated for 4 days with anti-CD3/anti-CD28 and IL-2. 2.5x10⁴ cells/well were cocultured with KJ1-26^-CD4⁺CD25⁺ cells from DO11.10 mice (2.5x10⁴/well) and 2.5x10⁴/well mitomycin C treated BALB/c splenocytes and 1 µg/ml of OVA_323−339 peptide for 60 h. [³H]thymidine (1 µCi/well) was added during the last 18 h of culture.

Supplementary Fig. 3. (pdf 161KB)
Intravenous administration of anti-CD25 antibody leads to depletion of CD4⁺CD25⁺ cells in RIP-mOVA mice. Wild-type RIP-mOVA mice were injected intravenously with 500 g of anti-CD25 antibody (Clone PC61). 3 days after injection animals were bled and PBMCs analyzed and compared to control mice. By staining with non-crossreactive anti-CD25 (7D4), depletion efficiency was >90% in all experiments.

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