TLR4 signaling is necessary for SjHSP60-mediated Treg increase.

<p><b>(A and B)</b> TLR2-/- or TLR4-/- mice or their WT control littermates were immunized with SjHSP60 or PBS. Ten days after the final injection, CD4⁺CD25⁺Foxp3⁺ Tregs in spleen of individual mice were analyzed by FCM. <b>(C)</b> Purified CD4⁺CD25^- T cells were co-cultured with purified APCs (DC, Mφ, TLR2-/- Mφ, TLR4-/- Mφ, or Mφ pre-treated with anti-TLR2, anti-TLR4 or their isotype control antibodies) at a 2:1 ratio of T cell:APC (DC or Mφ) with or without stimulation of SjHSP60 (0.2 μg/ml). After 3 days, Tregs in co-cultures were analyzed by FCM. <b>(D-F)</b> Graphs of CD4⁺CD25⁺Foxp3⁺ Treg frequencies. <b>(G and H)</b> CD4⁺CD25⁺ Tregs purified from mice deficient for TLR2, TLR4 or their control littermates were cultured in medium alone or stimulated with 1 μg/ml anti-CD3 and/or 0.5 μg/ml SjHSP60. On day 3, proliferation of CD4⁺CD25⁺ Tregs was determined by [³H]thymidine incorporation. FCM plots are representative of one of three independent experiments. Cells were gated on CD4⁺ T cells. The bar graph of average percentages ± SD of CD4⁺CD25⁺Foxp3⁺ Tregs are from 5 mice or triplicate cultures representative of one of three independent experiments. **<i>P</i> < 0.01, ***<i>P</i> < 0.001.