Contact duration between FV-infected DCs and naive T cells. DC–T-cell interactions were analyzed within a 3D collagen gel. Transgenic CD4⁺ T cells from DO11.10 mice were isolated and mixed with OVA-antigen loaded DCs in a 10:1 ratio. (A) Contact duration times (minutes) between uninfected DCs (●) or FV-infected DCs (■) and CD4⁺ T cells are shown. The median for both groups (gray line) was statistically significantly different (P < .001). Shown are the cumulative results from 4 independent experiments with similar outcomes. The total number of measured contacts were 330 in uninfected DCs and 329 in FV-infected DCs. (B) In addition, cell contacts between uninfected (□) and FV-infected (■) DCs and CD4⁺ T cells that lasted longer than 38 minutes (3 times the median of the contact times between T cells and uninfected DCs) were counted. Cumulative results from 4 independent experiments are shown. The difference between both groups was statistically significant (P < .001). (C) Representative example of short cell contacts between 1 uninfected DC and 2 naive CD4⁺ T cells. The time point when the picture was taken after initiation of the culture is given. (D) Representative example of a long contact between 1 FV-infected DC and 2 naive CD4⁺ T cells. Arrows indicate cell-to-cell contacts between DC and T cells. See “Analysis of cell-cell interactions within 3D collagen gels” for complete image acquisition information. (E) Contact duration between DCs and natural Treg cells. DC–T-cell interactions were analyzed within a 3D collagen gel. Naive CD25⁺ (Foxp3⁺ but CD69⁻ and CD44⁻) or CD25⁻ transgenic CD4⁺ T cells from DO11.10 mice were isolated and mixed with OVA-antigen–loaded uninfected DCs in a 10:1 ratio. Contact duration times (minutes) between DCs and CD4⁺CD25⁻ (left) or CD4⁺CD25⁺ (right) are shown. The median for both groups (gray line) was statistically significantly different (P < .001). Shown are the cumulative results from 3 independent experiments with similar outcomes. The numbers of measured contacts were 167 for CD4⁺ and 106 for CD4⁺CD25⁺. (F) In addition, cell contacts between CD4⁺CD25⁻ (□) or CD4⁺CD25⁺ (■) T cells and DCs that lasted longer than 3 times the median of the contact times between the total CD4⁺ T cells and uninfected DCs were counted. Cumulative results from 3 independent experiments are shown.