Regulatory T cells inhibit stable contacts between CD4+ T cells and dendritic cells in vivo

Abstract

Regulatory T (T reg) cells exert powerful down-modulatory effects on immune responses, but it is not known how they act in vivo. Using intravital two-photon laser scanning microscopy we determined that, in the absence of T reg cells, the locomotion of autoantigen-specific T cells inside lymph nodes is decreased, and the contacts between T cells and antigen-loaded dendritic cells (DCs) are of longer duration. Thus, T reg cells can exert an early effect on immune responses by attenuating the establishment of stable contacts during priming of naive T cells by DCs.

Figure 1.

In the absence of immunization, T reg cells do not influence the movement of MBP-specific CD4⁺ T cells. (a) Experimental protocol. T/R⁺ or Tg/Tg mice, which have or lack endogenous T reg cells, respectively, received 10⁷ CFSE-labeled MBP Ac1-11–specific CD4⁺ T cells by tail vein injection. 20 h later, PLNs were imaged. (b) Representative tracks of MBP-specific T cells in T/R⁺ and Tg/Tg recipients. (c) Mean speeds of MBP-specific CD4⁺ T cells in both types of recipient mice. (d) Arrest coefficient for CD4⁺ T cells in both types of recipient mice. Results are representative of three independent experiments.

Figure 2.

T reg cells release antigen-specific CD4⁺ T cells in the presence of antigen. (a) Experimental protocol for b–d. T/R⁺ or Tg/Tg mice, which have or do not have endogenous T reg cells, respectively, received 10⁷ CFSE-labeled MBP Ac1-11–specific CD4⁺ T cells via the tail vein. As endogenous control, Tg/Tg animals received, by tail vein injection, 1–2 × 10⁷ CFSE-labeled MBP-specific T cells from Tg/Tg mice together with 10⁷ CD4⁺CD25⁻ T cells from syngeneic CFP-expressing mice. On the same day, mice received 50 μg MBP Ac1-11 peptide emulsified in IFA in the footpad. 20 h after immunization, draining PLNs were imaged. (b) Representative tracks of MBP-specific T cells (green) in T/R⁺ and Tg/Tg immunized recipients. (c) Mean speeds of MBP-specific CD4⁺ T cells in both types of recipient mice. (d) Arrest coefficient for CD4⁺ T cells in both types of recipient mice. Results are representative of three independent experiments. Representative tracks of WT CD4⁺CD25⁻ T cells (blue) are shown in b. Arrest coefficients for WT CD4⁺ T cells (blue) are shown in d.

Figure 3.

The presence of endogenous T reg cells results in shorter contact time between CD4⁺ T cells and peptide-pulsed DCs. (a) Experimental protocol. T/R⁺ or Tg/Tg mice, which have or lack endogenous T reg cells, respectively, received 10⁷ CFSE-labeled MBP Ac1-11–specific CD4⁺ T cells together with 10⁷ CD4⁺CD25⁻ T cells from syngeneic CFP mice by tail vein injection and, 24 h later, received 5–10 × 10⁵ CMTMR-labeled DCs pulsed with MBP Ac1-11[4Y] peptide in the footpad. 20 h after DC transfer, PLNs were imaged. (b) Representative tracks of MBP Ac1-11–specific CD4⁺ T cells in T/R⁺ and Tg/Tg DC-transferred recipients. Trackings of CFP-expressing WT CD4 T cells are represented in blue. (c) Contact time between MBP-specific or WT CD4 T cells and antigen-loaded DCs. (d) Arrest coefficient for transgenic (green or red) or WT (blue) CD4⁺ T cells in T/R⁺ and Tg/Tg DC-transferred recipients. Results in b–d are representative of three independent experiments.

Figure 4.

Reconstitution of Tg/Tg animals with T reg cells diminishes the contact time between CD4⁺ T cells and peptide-pulsed DCs. (a) Experimental protocol. Tg/Tg mice received 10⁷ CFSE-labeled MBP-specific CD4⁺ T cells via tail vein and, on the same day, 5–10 × 10⁵ CD4⁺CD25⁺ T cells (T reg cells) or CD4⁺CD25⁻ T cells (non–T reg cells) from a syngeneic WT animal in the footpad. 24 h later, CMTMR-labeled DCs pulsed with MBP Ac1-11[4Y] peptide were injected in the footpad, and PLNs were imaged 12 h after DC transfer. (b) Representative tracks of CD4 effector T cells in Tg/Tg animals reconstituted with T reg or non–T reg cells. (c) Contact time between CD4 effector T cells and DCs in Tg/Tg mice that received T reg or non– T reg cells. (d) Arrest coefficient of CD4⁺ T cells in Tg/Tg mice that received T reg or non–T reg cells. Results in b–d are representative of three independent experiments.