Figure 6: Suppressive effect of CD4⁺2D2-IEL-T_HIGH cells on T-cell proliferation in vitro.

(a) Sorted spleen CD4⁺CD25⁻ T cells from 2D2 mice were labelled with CellTrace and were used as responder cells. The indicated suppressor cells were sorted and co-cultured. Suppressor cells were not added to control cultures. CD4⁺Vα3.2⁺Vβ11⁺ cells from MLN of 2D2 mice (2D2-MLN), CD4⁺CD25⁺ T cells from WT spleen cells (Treg) and CD4⁺ or CD4⁻ 2D2-IEL-T_HIGH and T_LOW cells sorted from IEL populations of 2D2 mice were used as suppressor cells. Cells were stimulated with APCs (CD3⁻ WT spleen cells) plus anti-CD3 mAb. (b) Per cent suppression was calculated from three to four samples for each condition from three experiments including a. Mean and s.e.m.; *P<0.05, **P<0.01 by unpaired t-test. (c) The responder cells used in a were co-cultured with the indicated suppressor cells and MOG(35-55)-pulsed APCs and average per cent suppression was calculated in d. (e) Blocking mAb or isotype-matched control antibodies were added to the cell cultures. After 4 days of culture in 96-well U-bottom (a–d) or V-bottom (e) plates, intensities of CellTrace in 7AAD⁻CD4⁺ cells were measured by FACS. Bars represent undivided CellTrace-labelled responder cells. (c,e) Data are representative of two experiments. FACS, fluorescence-activated cell sorting.