Figure 3.

Tregs engage immigrant DCs near the lymph node capsule. (a) LPS-activated CMTMR-labeled DCs (red) imaged in the draining inguinal lymph node 24 hr after subcutaneous injection into a Foxp3^EGFP mouse, showing immigrant DCs encountering and making contact with numerous endogenous Tregs (green) directly beneath the collagen capsule (blue) of the lymph node (major tick marks = 20 μm, 50 μm z-stack). See Supplementary Video 6. (b) Close-up image of a DC (red) near the collagen capsule and its interactions with several Tregs (green). Major tick marks = 10 μm. (c) Space-filled rendering of the DC (red) from (b) and an associated Treg (green) with track (grey, 39:27 min:sec duration of imaging), showing the close association between Treg and DC (scale bar = 5 μm). (d) Time sequence (times shown in min:s) showing interactions of Tregs (green with grey tracks) engaging recently immigrated DCs (red). (e) Experimental design to examine the effect of LPS-activated DCs on OTII Tconv cells and Tregs. LPS-activated DCs from a ECFP mouse were injected into a Foxp3^EGFP mouse followed by adoptive transfer of CMTMR-labeled OTII Tconv cells at 24 hr and 2P imaging in the draining lymph node 12 hr later. (f) Snapshot showing adoptively transferred OTII Tconv cells (red), LPS activated DC (blue), and Tregs (green). Scale bar = 30 μm. See Supplementary Video 7. (g) Contact durations of Tregs with LPS-DCs (n = 110 contacts) and Tconvs with LPS-DCs (88 contacts). Data pooled from 3 experiments each. (h) Effect of LPS-activated DCs on Treg velocities. Control velocities in the absence of LPS-DCs: Treg n = 296; OTII Tconv cells n = 213. In the presence of LPS DCs: OTII Tconv cell velocity did not change appreciably (p = 0.7, n = 207, Mann–Whitney U test) whereas Treg velocity was reduced (n = 193) Data pooled from 3 experiments each.