Fig 4. The proliferation and cytokine release of naïve splenocytes stimulated by rTs-Hsp70-activated DCs.

(A) The proliferation of naïve CD4⁺ T cells stimulated by rTs-Hsp70-activated DCs from WT and TLR2/4^-/- mice determined by CFSE. The DCs from WT, TLR2^-/- or TLR4^-/- mice were pre-incubated with rTs-Hsp70 or other controls including PBS, BSA, LPS and Pam₃CSK₄, then co-cultured with naïve mouse spleen CD4⁺ T cells. Representative histograms of the gated CD4⁺ T cells are shown in the left panel. The % of proliferation is shown as mean ± SD of three separate experiments in the right panel. n = 3, ** p < 0.01, *** p < 0.001. (B) The cytokine profile secreted by T cells stimulated by rTs-Hsp70-activated DCs from WT TLR2/4^-/- mice was measured by intracellular cytokine staining. (I) gating on viable cells, (II) selection of non-adherent cells, (III) gating on CD3⁺ CD4⁺ T cells, and (IV) selection of IL-2⁺, IL-4⁺, IL-6⁺, IFN-γ⁺ cells from gated CD4⁺ T cells, respectively. Quantified data are shown as mean ± SD of three separate experiments. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001.