Adaptive Immunity, Autophagy relatively modest changes in Beclin 1 expression modulate CD40- induced anti-T. gondii activity. CD40 stimulation diminishes p21 expression leading to Beclin 1 upregulation p21 has been reported to decrease Beclin 1 expression through protein degradation [27]. CD40 upregulates Beclin 1 without affecting Beclin 1 mRNA levels as assessed by real time PCR (Figure S4). Thus, we examined the role of p21 in Beclin 1 upregulation caused by CD40. CD40 stimulation of primary microglia and BV-2 cells caused a rapid decrease in p21 levels (Figure 5A). In addition, knockdown of p21 by transfection with siRNA induced Beclin 1 upregulation in BV-2 cells incubated with a sub-optimal concentration of anti-CD40 mAb (Figure 5B). In contrast, p21 overexpression by transfection with a p21-encoding plasmid prevented CD40 from increasing Beclin 1 levels Figure 5. CD40 downregulates p21, increases Beclin 1 expression and autophagic killing of T. gondii. A, Primary brain microglia from B6 mice were incubated with anti-CD40 mAb or control mAb for 1 h followed by assessment of p21 and actin expression. B, BV-2 cells were transfected with control or p21 siRNA. Cells were incubated with a suboptimal concentration of anti-CD40 (1 mg/ml) or control mAb and followed by assessment of Beclin 1 expression. In addition, cells were challenged with RH T. gondii and the numbers of tachyzoites per 100 microglia were determined. C, BV-2 cells were transfected with p21-encoding or empty plasmids. Cells were incubated with an optimal concentration of anti-CD40 (10 mg/ml) or control mAb and followed by assessment of Beclin 1 expression. Cells were challenged with RH T. gondii and the numbers of tachyzoites per 100 microglia were determined. Results are shown as the mean 6 SEM and are representative of 3 independent experiments. D, Proposed model of the role of p21 in modulation of Beclin 1 expression and activation of T. gondii killing induced by CD40. We previously reported that CD40 induces autophagy and killing of T. gondii through the adaptor protein TRAF6 [6,8]. This process would be dependent on down-regulation of p21 that would result in increased levels of Beclin 1 and enhanced autophagy. **p#0.01; ***p#0.001. doi:10.1371/journal.pone.0014472.g005 PLoS ONE | www.plosone.org 7 December 2010 | Volume 5 | Issue 12 | e14472