A strong TCR stimulus induces the expression of PRELI, and they both inhibit Th2 cell development. (A) PRELI is induced at the mRNA and protein levels by increasing strength of the TCR stimulus. Naive CD4⁺ cells were activated with different concentrations of α-CD3 (125, 250, or 500 ng/well) and α-CD28 for 24 hours and subsequently harvested for Western blotting and Taqman RT-PCR analysis. Representative data from 4 independent experiments are shown. (B) The strength of the TCR stimulus and the amount of PRELI in cells influence Th2 differentiation. CD4⁺ cells nucleofected with scrambled- or PRELI-siRNA oligos (1 or 2) were activated as in panel A and cultured under Th1- or Th2-polarizing conditions for 7 days. The cells were stained with CRTH2-PE and analyzed by flow cytometry. Th1 cells were used as negative controls. Representative data from 2 independent experiments are shown. White bars represent the mean value plus or minus SEM, calculated from PRELI-siRNA1 and PRELI-siRNA2 cultures from the representative experiments. (C) PRELI influences the relative proportion of Th cells under conditions that enable both Th1 and Th2 polarization. CD4⁺ cells nucleofected and activated (as described earlier in this paragraph) were cultured in the presence of both IL-12 (2.5 ng/mL) and IL-4 (10 ng/mL). After 7 days of culture, expression of CRTH2 was analyzed (as described earlier in this paragraph). Production of IFNγ was measured by intracellular cytokine staining. Representative data from 2 biologic replicates with PRELI-siRNA2 are shown.