Figure 2. CD4⁺ T cells co-cultured with allogeneic DexD3-DCs are hypo-responsive to alloantigen re-challenge in vitro and preferentially expand CD4⁺25⁺ T cells.

(A) CD4⁺ T cells (2 × 10⁵) from CBA mice were co-cultured with BALB/c derived DCs treated or not with DexD3 in the presence or absence of LPS [(LPS-matured DCs (closed circles), immature DCs (open diamonds), DexD3+LPS-DCs (closed triangles) and DexD3-DCs (closed squares)]. On day 6 of culture the CD4⁺ T cells were purified and re-stimulated with immature BALB/c DCs at different ratios. T cell proliferation was measured after 72 hours by addition of ³H thymidine for the last 16 hours. Proliferation is expressed as counts per minute (cpm) +/− SD. One representative experiment is shown out of 5 performed.

(B) BALB/c DCs treated with or without DexD3 in the presence or absence of LPS were co-cultured with total CD4⁺ T cells (1×10⁶), derived from B6 mice, at a ratio of 1:10 in the presence of rIL-2 (200U/ml). On day 5, T cells were harvested and analysed for expression of CD4, CD25 and FoxP3 by flow cytometry. Data plotted is the mean percentage of FoxP3⁺ T cells detected after co-culture with DCs in 4 independent experiments. *** denotes p<0.001.

(C) CFSE labelled total CD4⁺ T cells, or CD4⁺ T cells depleted of CD25⁺ cells (CD4⁺CD25⁻) derived from B6 mice, were cultured with BALB/c DCs preparations as described in (B). On day 5, T cells were stained for expression of CD25 and FoxP3, and also analysed for CFSE dilution by flow cytometry. Data shown is representative of 3 independent experiments and shows the expansion (CD4⁺ T cells, top panels) and induction (CD4⁺CD25⁻ T cells, bottom panels) of Foxp3⁺ Tregs after 5 days of co-culture with DCs.