Fig. 5.

Forkhead box protein 3 (FoxP3)^– expression in CD4⁺ CD25⁺ T cells in the reconstituted mice and memory-like phenotype (CD44⁺) in host-derived CD4⁺CD25^-FoxP3^– cells. (a) Frequency of CD4⁺CD25⁺FoxP3⁺ cells in the host (black bar) and donor-derived (white bar) splenocytes of the BDC2·5NOD→B6.g7 chimeras at 12 weeks post-transplant in comparison to B6.g7 mice. Frequency shown as mean ± standard error of the mean (s.e.m.), n = 3; ***P < 0·0004 (t-test). (b) Donor-derived T effectors (CD4⁺CD25^–) and host- or donor-derived regulatory T cells (T_regs) (CD4⁺CD25⁺) sorted from BDC2·5NOD→B6.g7 mice were mixed at a ratio of 1 : 1 and co-cultured for 72 h in the presence of CD3/CD28 co-stimulation. Proliferation (thymidine incorporation) was determined after additional 8 h incubation with [³H]-thymidine. Normalized proliferation of each group was compared to dT_effs alone sample, represented as 100%. Frequency shown as mean ± s.e.m., n = 2; ***P < 0·0001 (t-test). (c) CD44 expression on CD4⁺CD25^–FoxP3^– cells in the spleens of B6.g7 and host-derived CD4⁺CD25^-FoxP3^– cells in the spleens of the BDC2·5NOD→B6.g7 chimeric mice at 12 weeks post-reconstitution. Frequency is shown as mean ± s.e.m. from three BDC2·5NOD→B6.g7 and three B6.g7 mice; **P < 0·0037 (t-test). (d) Expression of FR4 and CD73 on spleen cells in the CD25^–FoxP3^– population. Representative profiles from one of three mice are shown. Average frequencies from all three mice ± s.e.m.; B6.g7 CD73⁺FR4⁺: 26·17 ± 6%; BDC2·5→B6.g7: host CD73⁺FR4⁺: 66 ± 7·5%, donor CD73⁺FR4⁺: 5·3 ± 2%. Statistical significance (t-test) BDC2·5NOD→B6.g7 host versus BDC2·5NOD→B6.g7 donor P < 0·0021; BDC2·5NOD→B6.g7 host versus B6.g7 P < 0·03. (e) CD73 and FR4 mean fluorescence intensity (MFI) from the host (black bar) and donor-derived (white bar) CD4⁺CD25^–FoxP3^– cells in the two chimeras. MFI is shown as mean ± s.e.m. from three mice. **P < 0·004; **P < 0·0194 (t-test), for MFI CD73 and FR4 respectively. Representative data from one of two independent experiments.