Figure 1
Figure 1. CTLA-4 reverses TCR stop-signal and limits T cell/DC dwell-times in Ctla4+/+ DO11.10 T cells. Ctla4+/+ T cells fail to stop in response to DC-OVA peptide in contrast to Ctla4−/− T cells. GFP-vector or GFP-CTLA-4, CD4-positive T cells from Ctla4+/+ and Ctla4−/− DO11.10 TCR Tg mice were preactivated with plate-bound anti-CD3/CD28 antibodies for 48 hours, transfected and then rested for 24 hours. Cells were tracked for migration on LN slices as described.35 Ctla4−/− cells plus CTLA-4 represents Ctla4−/− T cells transfected with mouse GFP-CTLA-4. Ctla4−/− cells plus vector represents Ctla4−/− T cells transfected with pEGFP. 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 (see supplemental Video 1). (A) Tracing of the migration of Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− T cells on LN slices. T-cell tracks have been superimposed from their starting positions. (B) CTLA-4 expression in transfected Ctla4−/− T cells. Ctla4−/− CD4-positive T cells were transfected with CTLA-4-GFP and assessed for expression by FACS. (C) Left panel: Unstimulated or anti-CD3/CD28 stimulated Ctla4+/+ and Ctla4−/− DO11.10 CD4-positive T cells for 48 hours followed by resting for 24 hours were stained with anti–LFA-1-PE, anti–CD44-APC or anti–CD69-APC. Right panel: 3H-thymidine incorporation of unstimulated and anti-CD3/CD28 stimulated DO11.10 CD4-positive Ctla4+/+ and Ctla4−/− T cells. (D) Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− DO11.10 CD4-positive T cells were seeded with SNARF-1 labeled DCs alone or with DCs that had been preincubated with OVA peptide (DC-OVA). Migration on LN slices was monitored over a time period of 20 minutes. Left panel: Contact times of Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− T cells with DCs in the presence and absence of OVA peptide on LN slices. Middle panel: Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− T-cell velocities with DCs in the presence and absence of OVA peptide on LN slices. Right panel: Measurements of displacement of Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− T cells with DCs in the presence and absence of OVA peptide on LN slices. Differences between means were tested using 2-tailed Student t test (GraphPad Prism 5.0). P < .05 was considered significant. Data are representative of 6 separate experiments.

CTLA-4 reverses TCR stop-signal and limits T cell/DC dwell-times in Ctla4+/+ DO11.10 T cells.Ctla4+/+ T cells fail to stop in response to DC-OVA peptide in contrast to Ctla4−/− T cells. GFP-vector or GFP-CTLA-4, CD4-positive T cells from Ctla4+/+ and Ctla4−/− DO11.10 TCR Tg mice were preactivated with plate-bound anti-CD3/CD28 antibodies for 48 hours, transfected and then rested for 24 hours. Cells were tracked for migration on LN slices as described.35 Ctla4−/− cells plus CTLA-4 represents Ctla4−/− T cells transfected with mouse GFP-CTLA-4. Ctla4−/− cells plus vector represents Ctla4−/− T cells transfected with pEGFP. 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 (see supplemental Video 1). (A) Tracing of the migration of Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− T cells on LN slices. T-cell tracks have been superimposed from their starting positions. (B) CTLA-4 expression in transfected Ctla4−/− T cells. Ctla4−/− CD4-positive T cells were transfected with CTLA-4-GFP and assessed for expression by FACS. (C) Left panel: Unstimulated or anti-CD3/CD28 stimulated Ctla4+/+ and Ctla4−/− DO11.10 CD4-positive T cells for 48 hours followed by resting for 24 hours were stained with anti–LFA-1-PE, anti–CD44-APC or anti–CD69-APC. Right panel: 3H-thymidine incorporation of unstimulated and anti-CD3/CD28 stimulated DO11.10 CD4-positive Ctla4+/+ and Ctla4−/− T cells. (D) Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− DO11.10 CD4-positive T cells were seeded with SNARF-1 labeled DCs alone or with DCs that had been preincubated with OVA peptide (DC-OVA). Migration on LN slices was monitored over a time period of 20 minutes. Left panel: Contact times of Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− T cells with DCs in the presence and absence of OVA peptide on LN slices. Middle panel: Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− T-cell velocities with DCs in the presence and absence of OVA peptide on LN slices. Right panel: Measurements of displacement of Ctla4+/+, Ctla4−/−, and GFP-CTLA-4 transfected Ctla4−/− T cells with DCs in the presence and absence of OVA peptide on LN slices. Differences between means were tested using 2-tailed Student t test (GraphPad Prism 5.0). P < .05 was considered significant. Data are representative of 6 separate experiments.

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