Soluble DC-HIL-Fc enhances responses of CD4⁺ T cells by APCs. Effects of soluble DC-HIL-Fc on T-cell activation were examined in MLR (A-B), anti-CD3 response (C), or in OVA-specific antigen presentation (D-E). MLR: C57BL/6 spleen cells (5 × 10⁴) were γ irradiated and mixed with CD4⁺ T cells (2 × 10⁵) purified from BALB/c mouse spleens. (A) Increasing doses of hIgG or DC-HIL-Fc were added to the MLR culture and incubated for 2 days prior to ³H-thymidine pulsing. Proliferative response of T cells was assayed by incorporation of ³H-thymidine. (B) MLR was incubated in the absence/presence of hIgG or DC-HIL-Fc (20 μg/mL) for 1, 2, or 3 days before pulsing. (C) Soluble (Sol) DC-HIL-Fc does not inhibit T-cell activation triggered by immobilized (Im) anti-CD3 Ab. CD4⁺ T cells were cultured in microwells precoated with anti-CD3 Ab (0.3 μg/mL) and 5 μg/mL of DC-HIL-Fc. In some wells, soluble hIgG or DC-HIL-Fc in increasing doses was added to culture in wells coated with the same amount of anti-CD3 Ab. T-cell activation was expressed as proliferative capacity. (D-E) OVA-specific response: CD4⁺ T cells purified from the spleens of BALB/cTac-TgN(DO11.10)-Rag2^tm1 mice were cocultured without (No) or with BM-DCs (from BALB/c mice) previously pulsed with OVA peptide. T-cell activation was assayed by IL-2 production (D) and by FACS for frequency of CD69⁺/CD4⁺ T cells (E). Control staining was performed with FITC-rat IgG (rIgG) and PE-hamster IgG (haIgG). (F) siRNA-mediated knockdown of DC-HIL. At 1 day after transfection of DCs with control (Ctrl; shuffled) siRNA or DC-HIL–targeted siRNA, cells were harvested and assayed by immunoblotting for protein expression of DC-HIL or β-actin. (G) Increasing numbers of transfected DCs were pulsed with OVA peptide and cocultured with a constant number of OVA-specific CD4⁺ T cells. Activation was measured by IL-2 production. (H) At 2 days after coculturing, frequency of CD69⁺ in the CD4⁺ T cells was determined by FACS. *Statistical significance (P < .001) compared with T-cell responses treated with hIgG control. Data shown are representative of at least 3 independent experiments.