Supporting Figures

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Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Figure 10

Fig. 7. CD4⁺CD25⁺ and CD4⁺CD25^- T cells responded to BLP but not LPS. (A) Purified T cells were cultured with anti-CD3 antibody in the presence of BLP (Pam3CSK, 5 mg/ml) or LPS (Salmonella minnesota, 10 mg/ml) for 3 days. BLP, but not LPS, significantly enhanced CD4⁺CD25^- T cell proliferation and IFN-g and IL-2 production, as well as CD4⁺CD25⁺ T cell proliferation and IFN-g production. (B) Purified CD4⁺CD25⁺ T cells were cultured with mitomycin-C-treated APCs and soluble anti-CD3 antibody for 3 days in the presence of LPS from different strains of bacteria (1, S. minnesota; 2, Salmonella enteritidis; 3, Salmonella typhimurium) or BLP (labeled 4). Only LPS from S. typhimurium gave a modest enhancing effect. Data are mean ± SD (n = 3) and are representative of three experiments. **, P < 0.01 compared with control culture without BLP.

Fig. 8. The effect of BLP did not involve APCs. (A) CD4⁺CD25⁺ and CD4⁺CD25^- T cells were purified from TLR2^-/- or WT (C57BL/6) mice and cultured with plate-bound anti-CD3 antibody for 3 days in the presence or absence of BLP (5 mg/ml). Only cells from WT mice responded to BLP stimulation. (Note the difference in the scale of cpm between the two populations of cells.) (B) Tregs from TLR2^-/- mice were cultured with soluble anti-CD3 antibody and mitomycin-C-treated APCs from TLR2^-/- mice mixed with [APC(KO/WT)] or without [(APC(KO)] 5% live WT CD4-depleted spleen cells. Cell proliferation and cytokine production were determined in 3 days’ culture. Live WT spleen cells did not affect the lack of activation by BLP in T cells from TLR2^-/- mice. Data are mean ± SD (n = 3) and are representative of two experiments. **, P < 0.01 compared with control without BLP.

Fig. 9. IL-2 produced by CD4⁺CD25^- T cells was required for overcoming the suppression by BLP. (A) Purified CD4⁺CD25^- T cells from IL-2^-/- or WT BALB/c mice were cultured with plate-bound anti-CD3 antibody with or without BLP (5 mg/ml) for 3 days. BLP significantly enhanced the proliferation and IFN-g and IL-2 production of CD4⁺CD25^- T cells from WT, but not from IL-2^-/-, mice. (B) CD4⁺CD25^- T cells were purified from IL-2^-/- or WT mice and cocultured with CD4⁺CD25⁺ T cells from WT mice in the presence of mitomycin-C-treated APCs (from IL-2^-/- mice) and soluble anti-CD3 antibody for 3 days. BLP partially reversed the suppressive effect of Tregs in the absence of IL-2 production (CD4⁺CD25^- T cells from IL-2^-/- mice). Data are mean ± SD and are representative of two experiments. **, P < 0.01 compared with control culture without BLP.

Fig. 10. IL-6 did not play a significant role in the BLP-mediated effect on Tregs. (A) CD4⁺CD25⁺ and CD4⁺CD25^- T cells from BALB/c mice were cultured alone or together in the presence of soluble anti-CD3 antibody and APCs for 3 days with or without BLP. Anti-IL-6 antibody (1 mg/ml) or normal IgG control were added in some cultures. Cellular proliferation was determined by [³H]thymidine incorporation. Anti-IL-6 antibody had no influence on the reversal effect of BLP-mediated abrogation of Treg activity. (B and C) Cells were purified from IL-6^-/- or WT (BALB/c) mice and cultured as in A. BLP was able to completely reverse the suppressive effect of Tregs in the absence of IL-6 (produced by WT CD4⁺CD25^- T cells). Data are mean ± SD and are representative of three experiments. **, P < 0.01 compared with control culture without BLP.