Treatment with TLR 2, 3, or 9 ligands induces impaired T-cell proliferation and ζ-chain down-regulation. Mice were injected subcutaneously 3 times with MLVs and encapsulated with one of the following TLRLs: Zymosan (TLR 2), poly (I:C) (TLR 3), or CpG (TLR 9), as described in “Antigen, TLRLs, and immunization.” (A) Proliferative response as determined by the percentage of BrdU⁺Thy1.2⁺ of total Thy1.2⁺ cells isolated from control mice (□) relative to TLRL-treated mice (■) after stimulation with anti-CD3 and anti-CD28 antibodies or with PMA and Ca²⁺ ionophore. The results are presented as the mean values of 3 independent experiments, and standard deviations are shown. *P < .005 (Student t test). (B) Equal numbers of splenic T cells from Zymosan, poly (I:C), and CpG-treated and control mice were lysed and subjected to Western blot analysis. Expression of the protein kinase ZAP-70, CD3ε, and ζ-chain was assessed by immunoblotting (IB) using specific antibodies.