Active LFA-1 on DCs interferes with T-cell activation and generation of Th1 cells. (A) OT-I T cells were cocultivated for 4 hours with wild-type (WT) or LFA-1^d/d DCs loaded with different doses of the ovalbumin peptide SIINFEKL in a 3-D collagen matrix. On isolation from the matrix, expression of the V_α2-chain of the TCR was measured by flow cytometry. Mean fluorescence intensity (MFI) of V_α2 on CD8⁺ T cells is depicted (n = 3). Amount of TCR down-regulation by wild-type or LFA-1^d/d DCs was found to be insignificant. (B) OT-I T cells were activated by wild-type or LFA-1^d/d DCs pulsed with different doses of SIINFEKL for 3 days in parallel to experiments performed in panel A. Proliferation was measured by incorporation of [³H]thymidine (n = 2). (C) OT-II T cells and ovalbumin peptide–loaded DCs were isolated from the collagen matrix after 24 hours of coculture. Total RNA was extracted, and quantitative real-time PCR was performed. RNA expression levels were normalized to expression of endogenous β-actin mRNA. Relative expression of IL-2 mRNA of wild-type DCs cocultivated with OT-II T cells in the absence of peptide was equalized to 1 (n = 3). *P < .05. (D) Naive CD4⁺ OT-II cells were cocultivated for 24 hours with DCs in the presence or absence of ovalbumin peptide in a 3-D collagen matrix. Expression of intracellular IFN-γ was analyzed by flow cytometry. The number of IFN-γ-positive CD4⁺ T cells was quantified (n = 2). *P < .05. (E) Total RNA from CD4⁺ OT-II T cells cocultivated with ovalbumin-loaded DCs in a 3-D collagen gel for 24 hours was extracted, and quantitative real-time PCR was performed. RNA expression levels were normalized to expression of endogenous β-actin mRNA. Relative expression of Tbet (n = 4), GATA3 (n = 4), and FoxP3 (n = 3) mRNA of wild-type DCs cocultivated with OT-II T cells was equalized to 1. *P < .05. cpm indicates counts per minute.