Determination of optimal length of primary and secondary Treg expansion cycles.

<p>(A) Optimal length of primary Treg expansion cycles. Treg were stimulated with CFSE-labeled alloantigen (solid line, solid circle) or polyclonal (dotted line, open circle) stimulus in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSE^pos allogeneic stimulator cells) and related to Treg numbers at initial set up. (B) Optimal length of secondary Treg expansion cycles. Treg were primed with alloantigen (solid lines) or polyclonal (dotted lines) stimulation as indicated in the presence of IL-2 and IL-15 and rested for 2 days prior to restimulation. Cells were restimulated with CFSE-labeled alloantigen (solid circles) or polyclonal stimulus (open circles) in the presence of IL-2 and IL-15. At indicated time points, Treg numbers were counted by FACS (excluding CFSE^pos allogeneic stimulator cells) and related to Treg numbers at initial set up. Data are representative of three independent experiments.</p

Determination of optimal strength of alloantigen Treg stimulation.

<p>(A) Treg were stimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [³H]Thymidine incorporation at day 5. Data are representative of three independent experiments. (B) Treg primed with alloantigen were restimulated with indicated ratios of irradiated allogeneic PBMC in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [³H]Thymidine incorporation at day 3. Data are representative of three independent experiments. (C) Efficiency of primary alloantigen stimulated expansion is related to the number of HLA-DRB1 mismatches. Freshly isolated Treg were expanded in one cycle in the presence of IL-2 and IL-15 and stimulation by gamma irradiated allogeneic PBMC with one (N = 10) or two (N = 6) mismatches on HLA class II DRB1 genes. Expansion values were calculated by relating the number of cells set up in the initial culture to the number of cells after expansion (after two days rest). Expansion was higher with alloantigen mismatched on two HLA-DRB1 genes as compared to alloantigen with one mismatch. This difference was found to be statistically significant in a Mann Whitney test (P = 0.025).</p

Determination of optimal mode and strength of secondary polyclonal Treg stimulation.

<p>(A) Treg were primed with anti-CD3+ anti-CD28 microbeads and restimulated with indicated ratios of anti-CD3+ anti-CD28 microbeads or with indicated concentrations of platebound anti-CD3+ soluble anti-CD28 in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [³H]Thymidine incorporation at day 3. (B) Indexed cell yield after secondary polyclonal Treg culture using optimal strength of anti-CD3+ anti-CD28 microbeads (set to 100%) or platebound anti-CD3+ soluble anti-CD28 stimulation. (C) Cell division pattern and survival/death signals of Treg after different secondary polyclonal stimulations. Treg were stained with CFSE and stimulated with optimal strength of anti-CD3+ anti-CD28 microbeads or platebound anti-CD3+ soluble anti-CD28. CFSE dilution pattern, 7AAD staining and Bcl-2 expression of Treg population were assessed at day 4. Data are representative of four independent experiments.</p

Treg expansion after primary and secondary cycles with alternated alloantigen or polyclonal stimulation.

<p>Treg and Tconv were expanded according the schedule in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002233#pone-0002233-g001" target="_blank">Figure 1</a> and rested for two days. Treg numbers were determined and related to Treg numbers at initial set up. Data represent average expansion +SD of seven independent experiments.</p

Suppressive capacity of expanded Treg in [³H]Thymidine incorporation suppression assays.

<p>Treg and Tconv were expanded according the schedule in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002233#pone-0002233-g001" target="_blank">Figure 1</a> and rested for two days. (A) Suppressive capacity of Treg in target alloantigen and third party alloantigen responses as determined in a MLR co-culture using [³H]Thymidine incorporation. Autologous naïve CD4^posCD25^neg Tresp cells were stimulated with target alloantigen (same as used for expansion) or third party HLA mismatched gamma irradiated allogeneic PBMC. Expanded Treg (black lines) or Tconv (grey lines) were added into these cultures at indicated Tresp∶Treg/Tconv ratios. Proliferation was determined by measuring [³H]Thymidine incorporation at day 5. Results are expressed as percentage of [³H]Thymidine incorporation +SD, indexed to [³H]Thymidine incorporation of naïve Tresp and antigen only. Data are representative of seven independent experiments. (B) Suppressive capacity of Treg in target alloantigen and third party alloantigen responses as determined in a MLR co-culture using CFSE dilution. CFSE labeled Autologous naïve CD4^posCD25^neg Tresp cells were stimulated with PKH26 labeled target alloantigen (same as used for expansion) or PKH26 labeled third party HLA mismatched gamma irradiated allogeneic PBMC. Expanded Treg (black lines) or Tconv (grey lines) were added into these cultures at indicated Tresp∶Treg/Tconv ratios. Proliferation was determined by measuring CFSE dilution of Tresp at day 5. Results are expressed as percentage of proliferating cells, indexed to percentages of proliferating cells in cultures of naïve Tresp and antigen only. Data are representative of three independent experiments.</p

Phenotypical and functional characteristics of freshly MACS-isolated Treg.

<p>CD4^pos T cells were negatively isolated from PBMC and separated into CD25^pos (Treg) and CD25^neg (Tconv) T cell fractions by magnetic cell sorting. Data from a typical isolation is shown. (A) Cell surface expression of CD25, CD127, CD27, CD70 and CD62L, and intracellular expression of FoxP3 were analyzed on Treg (black filled histograms) or Tconv (grey line histogram). (B) Proliferative capacity of Treg or Tconv upon stimulation with HLA mismatched gamma irradiated allogeneic PBMC was determined in the absence (black bar) or presence (grey bar) of exogenous IL-2. Proliferation was determined by measuring [³H]Thymidine incorporation at day 5. (C) Suppressive capacity of Treg in alloantigen responses was determined in a MLR co-culture using [³H]Thymidine incorporation. Autologous naïve CD4^posCD25^neg Tresp cells were stimulated with HLA mismatched gamma irradiated allogeneic PBMC. Treg (black lines) or Tconv (grey lines) were added into these cultures at indicated Tresp∶Treg/Tconv ratios. Proliferation was determined by measuring [³H]Thymidine incorporation at day 5. Results are expressed as percentage of [³H]Thymidine incorporation +SD, indexed to [³H]Thymidine incorporation of naïve Tresp and antigen only. (D) Suppressive capacity of Treg in alloantigen responses was determined in a MLR co-culture using CFSE dilution. CFSE labeled autologous naïve CD4^posCD25^neg Tresp cells were stimulated with PKH26 labeled HLA mismatched gamma irradiated allogeneic PBMC. Treg (black lines) or Tconv (grey lines) were added into these cultures at indicated Tresp∶Treg/Tconv ratios. Proliferation was determined by measuring CFSE dilution of Tresp at day 5. Results are expressed as percentage of proliferating cells, indexed to percentages of proliferating cells in cultures of naïve Tresp and antigen only. (E) Example CFSE of suppression assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002233#pone-0002233-g002" target="_blank">Figure 2D</a>. Tresp only (grey filled histogram), Tresp+Treg (1∶1, black line) and Tresp+Tconv (1∶1, grey line) are shown, numbers indicate percentage of proliferating cells, indexed to percentages of proliferating cells in cultures of naïve Tresp and antigen only.</p

Determination of optimal mode and strength of primary polyclonal Treg stimulation.

<p>(A) Treg were stimulated with indicated ratios of anti-CD3+ anti-CD28 microbeads or with indicated concentrations of platebound anti-CD3+ soluble anti-CD28 (1 μg/ml) in the presence of IL-2 and IL-15. Proliferation was determined by measurement of [³H]Thymidine incorporation at day 4. (B) Indexed cell yield after primary polyclonal Treg culture using optimal strength of anti-CD3+ anti-CD28 microbeads (set to 100%) or platebound anti-CD3 plus soluble anti-CD28 stimulation. (C) Cell division pattern and survival/death signals of Treg after different primary polyclonal stimulations. Treg were stained with CFSE and stimulated with optimal strength of anti-CD3+ anti-CD28 microbeads or platebound anti-CD3+ soluble anti-CD28. CFSE dilution pattern, 7AAD staining and Bcl-2 expression of Treg population were assessed at day 4. In the lowest row, Bcl-2 expression is depicted gated on dividing Treg only. Data are representative of four independent experiments.</p

Proliferative capacity of expanded Treg.

<p>Treg and Tconv were expanded according the schedule in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002233#pone-0002233-g001" target="_blank">Figure 1</a> and rested for two days. Proliferative capacity of expanded cells upon restimulation with target alloantigen (same as used for expansion) was determined in the absence (black bars) or presence (grey bars) of IL-2. Proliferation was determined by measuring [³H]Thymidine incorporation at day 3. Results are expressed as percentage of [³H]Thymidine incorporation +SD. Data are representative of four independent experiments.</p

Schematic overview of expansion strategies.

<p>Treg were expanded in two cycles, in which alloantigen and polyclonal stimulation was alternated, resulting in four distinct strategies: two subsequent cycles with alloantigen stimulation; primary cycle with alloantigen stimulus and secondary cycle with polyclonal stimulus; primary cycle with polyclonal stimulus and secondary cycle with alloantigen stimulus; and two subsequent cycles with polyclonal stimulation.</p