(A) RT-PCR for IDO of GEN cells stimulated for 18 hours by HIV or resiquimod (R848). (B) Western blot for IDO of purified primary pDCs stimulated with HIV or resiquimod, in combination with CTLA-4–Ig. HeLa cells treated by IFN-γ were used as a control. (C) Naive CD4⁺ T cells were stimulated by HIV-activated or unactivated pDCs as in Figure 1A, in the presence or absence of 1-methyl-tryptophan (1-MT), and then added to a primary MLR. Proliferation was measured at day 5 by thymidine incorporation. *P < 0.01, Student’s t test, compared with proliferation of naive T cells. Results are representative of at least 5 experiments. (D) GEN cells were electroporated with siRNA for IDO, ICOS-L, or control siRNA (Ct siRNA). After 1 day, they were stimulated by AT-2 HIV or left unstimulated and used as stimulating APCs for naive CD4⁺ T cells. After 7 days, the T cell suppressive activity was measured as in Figure 1B by adding the Tregs to naive CD4⁺ T cells (+Tn, 1:3 ratio). *P < 0.01, Student t test, compared with proliferation of naive T cells alone. (E) GEN cells were electroporated with siRNA for MyD88, TLR7, or TLR9 or control siRNA for 1 day, before stimulation with HIV. IDO transcripts were detected by RT-PCR after 6 hours. Right: Densitometric quantification of the IDO PCR product, normalized to the actin levels. (F) Purified primary pDCs were stimulated for 18 hours by HIV, with or without preincubation with chloroquine (CQ), IRS 661, neutralizing anti-CD4 and anti-gp120 antibodies (CD4/b12), or isotype control (IgG). IDO expression was monitored by Western blot analysis.