a, Allostimulated, CFSE-stained CD4⁺CD25⁻ T-cells were phenotyped for FOXP3, CD25, CTLA-4, CD27, CD62L, CCR7 and CD28 expression at day 6 of culture. Dividing CFSE^low cells were positive for most of the markers shown, similar to the phenotype of ex vivo-purified T_regs. b, CD127 (IL7-R) expression was monitored on allostimulated T-cells, revealing CD127 downregulation with the induction of FOXP3. CD127 remained low at a point where FOXP3 was downregulated.

a, Gated CD8⁺ T-cells from an allostimulated CD3⁺CD25⁻ T-cell culture are shown. Transient upregulation of FOXP3 and CD25 on CFSE^low (dividing) cells is depicted. b, CD25⁺ T-cells were sorted from 5-day cultures of anti-CD3-stimulated CD8⁺CD25⁻ T-cells and tested for their ability to suppress autologous CD4⁺CD25⁻ T-cells in ³H-thymidine based suppression assays. Proliferation data was normalized to responder proliferation (100%), which ranged between 50,000 to 80,000 CPM in various experiments. c, Suppressive ability of flow-sorted CFSE^low (dividing) cells from day 4 and day 10 of anti-CD3-activated CD8⁺CD25⁻ T-cell cultures.

Allostimulated CD4⁺CD25⁻ T-cell cultures were flow sorted for CD4⁺, CFSE^high, CD25⁺ fraction (Day 2), CD4⁺, CFSE^low, CD25⁺, dividing fraction (Days 4, 6 and 9) to obtain highly pure induced T_reg populations at different stages of their formation. FOXP3, IFN-γ, TNF-α and IL-10 messages were quantified by real time PCR and normalized to β-actin. Presence of specific product was confirmed by dissociation curve and gel analysis.

Commercially available antibodies were used to quantify total (PCH101) and full-length FOXP3 (FJK16S) in various T_reg subpopulations. Spliced (Δ2) FOXP3 was detected by using a combination of both the antibodies (PCH101⁺/FJK16S⁻). a, Regions of the FOXP3 molecule that are targeted by the two antibodies are diagrammed. b, Using CD25 microbeads, we purified two subsets of CD4⁺ T-cells ex vivo: a subset enriched in CD25^{high/intermediate} T-cells and a CD25⁻ subset. CD4⁺CD25^{high/intermediate} T-cells expressed both isoforms ex vivo, with a larger subset expressing the D2 isoform. c, Bead-sorted CD4⁺CD25⁻ T-cells, CD4⁺CD25^{high/intermediate} T-cells or CD8⁺CD25⁻ T-cells were CFSE-stained and activated with anti-CD3. Following 4 days of anti-CD3 activation, CD4⁺CD25⁻ T-cells induced predominantly the Δ2 isoform with a small subset expressing the full-length isoform. Activated CD4⁺CD25^{high/intermediate} T-cells appeared to harbor a relatively larger subset expressing the full-length isoform. CD8⁺CD25⁻ T-cells also upregulated both the isoforms with the Δ2 isoform predominating.

Bead-sorted, ‘untouched’ CD4⁺CD25⁻ T-cells were activated with either anti-CD3 or allostimulation. a, CD4⁺CD25⁺ T-cells were sorted from PBMC ex vivo (black bars) or at 7 days of culture (as indicated) and tested for the ability to suppress the anti-CD3-mediated proliferation of freshly thawed, autologous responder CD4⁺CD25⁻ T-cells in the presence of irradiated APC. The counts per minute (CPM) from a 5-day ³H-thymidine based assay were obtained and then normalized to the anti-CD3-induced proliferation of responder cells (assigned a value of 100; CPM were in the 50,000 to 80,000 range in most experiments on various donors). The sorted CD4⁺CD25⁺ T-cells were anergic and were able to suppress the responder cells at increasing ratios. In this experiment, the proportion of FOXP3⁺ cells was highest in anti-CD3-activated cells followed by allostimulated cells (data not shown). b, CFSE^low dividing cells were flow sorted on day 5 (black bars) or day 13 (gray bars) from an anti-CD3-activated CD4⁺CD25⁻ T-cell culture and evaluated for suppressive ability. c, CFSE^low dividing cells (black bars) and CFSE^high non-dividing cells (gray bars) were flow sorted on day 5 from an anti-CD3-activated CD4⁺CD25⁻ T-cell culture and evaluated for suppressive ability. CPM values show that the non-dividing cells were not anergic and did not suppress. d, CFSE^high (non-dividing) cells from day 13 of an anti-CD3-activated culture were flow sorted and were re-cultured with fresh autologous APC in media alone or anti-CD3 for a further 6 days. Robust proliferation and upregulation of CD25 and FOXP3 are shown.

Bead sorted ‘untouched’ CD3⁺CD25⁻ T-cells (a, c) or CD4⁺CD25⁻ T-cells (b, e and f) were stained with CFSE and activated with either allogeneic T-cell-depleted PBMC (a) or with anti-CD3 antibody and autologous APC (b,c). CD25 and FOXP3 expression and CFSE dilution were monitored over time. a, Color-coded contour plots of gated CD4⁺ T-cells from allostimulated cultures are shown, with CFSE on the X-axis and CD25 (top row) or FOXP3 (bottom row) on the Y-axis. The vertical lines separate dividing (CFSE^low) from non-dividing (CFSE^high) cells. b, Anti-CD3 stimulated CD4⁺CD25⁻ T-cells at five days of culture. c, Percentage of dividing cells that expressed CD25 (squares) or FOXP3 (circles) are shown at various time points following allogeneic (filled symbols) or anti-CD3 (open symbols) stimulation d, CFSE-stained PBMC stimulated with either CMV lysate (day 7) or tetanus toxoid (day 5) are shown. Data are gated for CD4⁺ T-cells. e, RT-PCR analysis of CD4⁺ T-cells for FOXP3 expression is shown. Primers were picked such that full-length isoform and the shorter alternatively spliced form could be detected as different bands in agarose gels. Cells were obtained from 2-day or 6-day cultures or purified ex vivo, as indicated. f, Total FOXP3 expression by quantitative real time PCR analysis. FOXP3 levels were first normalized to β-actin and then normalized to levels seen in ex vivo-purified CD4⁺CD25⁺ T-cells (assigned a value of 1.0). g, FOXP3 expression in allostimulated CD45RO-depleted CD4⁺CD25⁻ naïve T-cells at 6 days of culture. All results are representative of at least 2 independent replicates performed on different donors (multiple replicates for a and b). Position of the positive gates for FOXP3 and CD25 in all graphs were determined based on the unstimulated control at the same time point and was also confirmed by either FMO (fluorescence minus one) staining (for polychromatic flow cytometry) or isotype control staining.