CD28 expression does not affect the contact time and motility of conventional and regulatory T cells. (A) CD4⁺CD25⁻ and CD4⁺ CD25⁺ T cells from DO11.10 x Cd28^+/+ and Cd28^−/− mice show similar values for contact times, velocity, and displacement. Anti-CD3/CD28 activated CD4⁺ CD25⁺ and CD4⁺ CD25⁻ T cells from Cd28^+/+ and Cd28^−/− x DO11.10 Tg mice were labeled with CFSE and tracked for migration on LN slices as described (see supplemental Videos 4-5).^35,36  T cells were seeded with DCs alone or with DCs that had been preincubated with OVA peptide (DC-OVA). Dwell-times were followed on syngeneic LNs in the presence of SNARF-1 labeled DCs. Both Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁻ T cells show comparable contact times (left top panel), velocities (left middle panel), and displacement values (left bottom panel) in the absence (DC) and presence of OVA (DC-OVA). Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁺ T cells show comparable contact times (right top panel), velocities (right middle panel), and displacement values (right bottom panel) in the absence (DC) and presence of OVA (DC-OVA). (B) Top panel: Stable Contact times of Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁻ T cells versus Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁺ T cells with DC-OVA. Contact times were comparable between Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁻ T cells versus Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁺ T cells. Bottom panels: Arrest coefficient values between Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁻ T cells versus Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁺ T cells. Arrest coefficient is comparable between Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁻ T cells (left panel) and between Cd28^+/+ and Cd28^−/− CD4⁺ CD25⁺ T cells (right panel). Differences between means were tested using 2-tailed Student t test (GraphPad Prism 5.0). P < .05 was considered significant. Data are representative of 2 separate experiments.