CYTIP silencing in DCs induces prolonged contacts with naive T cells and impairs antigen-specific T-cell proliferation. (A) Knockdown of CYTIP expression was monitored by immunofluorescence staining of mature DCs 24 hours after transfection with unrelated control and CYTIP siRNA, respectively. Original magnification ×63. (B) Expression of surface markers known to be up-regulated on maturation of DCs was analyzed by flow cytometry after transfection of wild-type DCs with control siRNA and specific CYTIP siRNA, respectively. Surface staining of CD80 or CD40 on CD11c⁺ DCs is depicted in the 2 bottom rows and was found not to be significantly different in CYTIP-silenced versus control DCs. Specific staining for CD11c, CD80, and CD40 is indicated by filled lines; isotype control is illustrated by unfilled lines. The geometric mean of specific staining was quantified and is shown within each fluorescence-activated cell sorter profile. (C) Mature DCs were transfected with control or CYTIP-specific siRNA and cocultivated in presence of the ovalbumin peptide with naive CD4⁺ OT-II T cells in a 3-D collagen gel. Contact times of individual T-cell/DC pairs were analyzed and were found to be significantly enhanced on silencing of CYTIP expression. (D) Antigen-specific proliferation of CD4⁺ OT-II T cells on cocultivation with DCs that were left untreated or were transfected with control or CYTIP siRNA was assessed by incorporation of [³H]thymidine. Assays were performed in triplicate. One representative assay of 3 is shown. *P < .05. cpm indicates counts per minute.