Figure 8: Suppressive capabilities of CD4⁺ IEL from KBx/N mice and WT mice.

(a) IELs from KBx/N mice were used. Responder CD4⁺CD25⁻ T cells (labelled with CellTrace) and CD3⁻ cells were sorted from the spleens of KBx/N transgene-negative littermates. CD4⁺CD25⁻ T cells sorted from the spleens of transgene-negative littermates were used as responder cells. CD4⁺CD25⁺ T (Treg) cells sorted from the spleen cells of transgene-negative littermates were used as suppressor cells as a positive control. CD4⁺ and CD4⁻ Vβ6⁺ T cells were sorted from KBx/N-IELs and used as suppressor cells. Control data were obtained without adding suppressor cells. Data are representative of three experiments. (b) WT-CD4⁺CD8α⁻ induced IEL and WT-CD4⁺Sp-T cells were analysed for the expression of surface CD25 and intracellular Foxp3. Data represent two experiments. (c) mRNA expression in WT-CD4⁺ induced IELs (CD2⁺CD5⁺CD4⁺CD8α⁻TCRβ⁺IEL), WT natural IELs (CD2⁻CD5⁻CD4⁻TCRβ⁺IEL), and WT-CD4⁺Sp-T cells was analysed by quantitative RT–PCR (n=4) mean and s.e.m. (d) Suppressor assay using IELs from WT mice. Responder T cells, CD3⁻ spleen cells, CD4⁺ induced IELs and CD4⁺CD25⁺ T cells (Treg) in the spleen and MLN-CD4⁺ T cells were sorted from WT mice. After 4 days of culture in 96-well V-bottom (a) or U-bottom (d) plates, intensities of CellTrace in 7-AAD⁻CD4⁺ cells were analysed by FACS. Bars represent undivided CellTrace-labelled responder T cells. Data representative of three experiments are shown. FACS, fluorescence-activated cell sorting; RT–PCR, reverse transcription PCR.