CD4⁺ CD25⁺ Tregs on LNs slow in response to OVA peptide in a CTLA-4–independent manner. (A) Dwell times of Ctla4^+/+ and Ctla4^−/− CD4⁺ CD25⁻ Tcons and CD4⁺ CD25⁺ Tregs on LNs in response to OVA peptide. Anti-CD3/CD28 activated CD4⁺ CD25⁺ and CD4⁺ CD25⁻ T cells from Ctla4^+/+ and Ctla4^−/− x DO11.10 Tg mice were labeled with CFSE and tracked for migration on LN slices as described (see supplemental Videos 2-3).³⁸  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. Bottom left panel: Ctla4^−/− CD4⁺ CD25⁻ T cells show significantly longer contact times than Ctla4^+/+ CD4⁺ CD25⁻ T cells in the presence of DC-OVA. Bottom right panel: Ctla4^−/− and Ctla4^+/+CD4⁺ CD25⁺ T cells show comparable contact times in the presence of DC-OVA. (B) Velocity and displacement of Ctla4^+/+ and Ctla4^−/− CD4⁺ CD25⁻ Tcons and CD4⁺ CD25⁺ Tregs on LNs in response to OVA peptide. Left panel: Ctla4^−/− CD4⁺ CD25⁻ T cells move significantly slower than Ctla4^+/+ CD4⁺ CD25⁻ T cells in the presence of DC-OVA. Right panel: Ctla4^−/− and Ctla4^+/+ CD4⁺ CD25⁺ T cells show comparable velocities in the presence of DC-OVA. Bottom left panel: Ctla4^−/− CD4⁺ CD25⁻ T cells show significantly reduced displacement compared with Ctla4^+/+ CD4⁺ CD25⁻ T cells in the presence of DC-OVA. Bottom right panel: Ctla4^−/− and Ctla4^+/+ CD4⁺ CD25⁺ T cells show comparable displacement measurements in the presence of DC-OVA. (C) Stable contacts and arrest coefficients of Ctla4^+/+ and Ctla4^−/−CD4⁺CD25⁻ Tcons and CD4⁺CD25⁺ Tregs on LNs in response to OVA peptide. Top panel: Ctla4^−/− CD4⁺ CD25⁻ T cells show significantly more stable contacts with DCs than Ctla4^+/+ CD4⁺ CD25⁻ T cells in the presence of DC-OVA. Ctla4^−/− and Ctla4^+/+ CD4⁺ CD25⁺ T cells show similar behavior for stable T cell/DC interaction. Bottom left panel: Ctla4^−/− CD4⁺ CD25⁻ T cells show a significant higher arrest coefficient than Ctla4^+/+ CD4⁺ CD25⁻ T cells. Bottom right panel: Ctla4^−/− and Ctla4^+/+ CD4⁺ CD25⁺ T cells show similar arrest coefficients. Differences between means were tested using 2-tailed Student t test (GraphPad Prism 5.0). P < .05 was considered significant; *P < .05; **P < .01; ***P < .001. Data are representative of 3 separate experiments.