Naive B cells naive T cells form a mature immunologic synapse. Mature bmDC or naive or preactivated splenic B cells were loaded with pOVA. Cells were mixed with naive DO11.10 T cells and fixed after 120 minutes of interaction. Immunohistologic staining was performed for actin (green) and the clonotypic TCR (red). Individual pairs of T cells and naive B cells (A-D), T cells and preactivated B cells (E-H') or T cells and DC (I-L), respectively, were analyzed by 2-color confocal microscopy making Z-stacks over the entire range of a cell pair. T cells in contact with B cells have the entire TCR signal at the contact plane (A), while T cells attached to DC show TCR staining scattered over the body (I). T cells in contact with preactivated B cells show both accumulation of TCR and distribution over the whole T-cell surface (E). A three-dimensional reconstruction showing the en face view of the contact plane reveals no preferential accumulation of TCR in the contact with DC, see Actin-stain (J), TCR-stain (K), and merged image (L). In contrast, in T-cell–naive B-cell pairs the TCR signal is focused in the center of contact (B-D). Interactions with preactivated B cells show partially the formation of a mature IS (F-H'). Shown is a T-B interface resembling a mature IS, yet with substantially scattered TCR (T cell at 6 o'clock, F-H); and an interface between the same preactivated B cells interacting with a second T cell, showing almost no IS segregation (T cell at 2 o'clock, F'-H'). The scale bar defines 5 μm (A,E) and 10 μm (I), respectively. (M) Quantitative analysis of 300 random individual cell pairs for the distribution of TCR in the contact plane. (N) Mean Fluorescence Intensity (MFI) analysis of the expression of costimulatory molecules on APC. While naive splenic B cells show only a sparse expression of CD80 and CD86, CD86 is clearly up-regulated upon B-cell activation. DCs exhibit a high amount of CD80 as well as CD86 on their surface. (M,N) Bars represent mean plus or minus SD.