Method for ex vivo expansion of regulatory T cells

Abstract

The invention relates to a new method for in vitro expansion of CD4+CD25.sup.HighCD127−.sup./LOWfoxP3+Tregs, wherein the process of Treg expansion takes place permanently or temporarily at a temperature below 37° C., optimally at a temperature of 33° C., the isolated Tregs are expanded in SCGM or X-vivo-20 medium supplemented with human serum or with foetal bovine serum, and magnetic beads coated with anti-CD3 and anti-CD28 antibodies at 1:1 (cell:bead) ratio and interleukin-2 are added to the culture.

Claims

1. A method for in vitro expansion of CD4.sup.+CD25.sup.HighCD127.sup.−/lowFoxP3.sup.+ regulatory T cells (Tregs), said method comprising: expanding said Tregs, wherein Treg expansion takes place at 33° C. for at least 14 days in a culture; and adding magnetic beads coated with anti-CD3 and anti-CD28 antibodies at a 1:1 cell:bead ratio and interleukin-2 to the culture.

2. The method of claim 1, wherein the expanded Tregs are used for clinical therapies of adverse immune reactions.

3. The method of claim 2, wherein the adverse immune reactions are selected from the group consisting of autoimmune diseases, transplant rejection, allergic reactions, graft rejection, and graft-versus-host disease.

4. The method of claim 1, wherein the Tregs are polyclonal or antigen-specific cells.

5. The method of claim 1, wherein the Treg expansion provides about 300% more Tregs after a 14-day culture in vitro as compared to an expansion method that takes place at a temperature of 37° C.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1—presents fold increase of the initial Treg number after 14-day culture. The figure depicts a comparison of fold increase of Treg number after 14-days expansion at standard temperature of 37° C. (grey bar; n=8) and at 33° C. (white bar; n=8). This parameter was calculated as a ratio of the final cell number on day 14 of expansion under a given culture condition to the initial cell number. Hence, the values are usually not whole numbers (the results are given with an accuracy to two decimal places). The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(2) FIG. 2—presents the percentage of CD4.sup.+FoxP3.sup.+ cells and the intensity of FoxP3 expression in these cells. The upper panel shows dot plots depicting FoxP3 expression in Tregs from the same donor expanded simultaneously at 37° C. (A) and 33° C. (B) for 14 days. The lower panel depicts frequency of CD4.sup.+FoxP3.sup.+ cells on day 7 and 14 of the culture at two different temperature conditions (37° C. and 33° C., C, n=8) and the intensity of FoxP3 expression in these cells on day 14 (D; n=8). The intensity of FoxP3 expression is expressed as a ratio of median fluorescence intensity (MFI) of the positive signal to median intensity of autofluorescence of unlabeled cells (INDEX MFI FoxP3). The grey and white bars represent the results obtained for the cells expanded at 37° C. and 33° C., respectively. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(3) FIG. 3—presents the stability of FoxP3 expression in the entire Treg population. The figure illustrates the difference between the percentage of CD4.sup.+FoxP3.sup.+ Tregs on day 14 and 7 of the culture at temperature of 37° C. (grey bar; n=8) and 33° C. (white bar; n=8). A significant time-dependent decrease in the percentage of CD4.sup.+FoxP3.sup.+ cells is observed only for Tregs expanded at 37° C. The cells cultured at 33° C. are characterised by a stable expression of FoxP3 over time. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(4) FIG. 4—presents frequency of CD25.sup.High cells within CD4.sup.+FoxP3.sup.+ population and the intensity of CD25 production by these cells on day 7 and 14 of the culture. The upper panel shows dot plots depicting surface expression of CD25 on Teffs after 14 day expansion at 37° C. (A) and on Tregs from the same donor expanded simultaneously at 37° C. (B) and 33° C. (C). The lower panel depicts frequency of CD25.sup.High cells within CD4.sup.+FoxP3.sup.+ Treg population on day 7 and 14 of the culture at two different temperature conditions (37° C. and 33° C., D, n=8) and the intensity of CD25 production by these cells on day 7 and 14 (E; n=8). The intensity of CD25 expression is expressed as a ratio of median fluorescence intensity (MFI) of the positive signal to median intensity of autofluorescence of unlabeled cells (INDEX MFI FoxP3). The grey and white bars represent the results obtained for the cells expanded at 37° C. and 33° C., respectively. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(5) FIG. 5—presents the stability of CD25 expression on CD4.sup.+FoxP3.sup.+ Tregs. The figure illustrates difference in percentage of CD25.sup.High cells within CD4.sup.+FoxP3.sup.+ Treg population between day 14 and 7 of the culture at temperature of 37° C. (grey bar; n=8) and 33° C., (white bar; n=8). A significant time-dependent decrease in the percentage of CD25.sup.High cells is observed only for Tregs expanded at 37° C. The cells cultured at 33° C. are characterised by a high and stable expression of CD25 over time. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(6) FIG. 6—presents the percentage of CD4.sup.+FoxP3.sup.High cells. The upper panel shows dot plots depicting frequency of CD4.sup.+FoxP3.sup.High cells within CD4.sup.+FoxP3.sup.+ Treg population from the same donor expanded simultaneously at 37° C. (A) and 33° C. (B) for 14 days. The lower panel depicts frequency of CD4.sup.+FoxP3.sup.High cells on day 7 and 14 of the culture at two different temperature conditions (37° C. and 33° C., C, n=8). The grey and white bars represent the results obtained for the cells expanded at 37° C. and 33° C., respectively. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(7) FIG. 7—presents frequency of CD25.sup.High cells within CD4.sup.+FoxP3.sup.High population and the intensity of CD25 production by these cells on day 7 and 14 of the culture. The figure depicts frequency of CD25.sup.High cells within CD4.sup.+FoxP3.sup.High Treg population on day 7 and 14 of the culture at two different temperature conditions (37° C. and 33° C., A, n=8) and the intensity of CD25 production by these cells on day 7 and 14 (B; n=8). The intensity of CD25 expression is expressed as a ratio of median fluorescence intensity (MFI) of the positive signal to median autofluorescence intensity of unlabeled cells (INDEX MFI FoxP3). The grey and white bars represent the results obtained for the cells expanded at 37° C. and 33° C., respectively. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(8) FIG. 8—presents the stability of FoxP3 expression in CD4.sup.+FoxP3.sup.High Treg population. The figure illustrates the difference between day 14 and 7 of the culture in percentage of CD4.sup.+FoxP3.sup.High Tregs after expansion at temperature of 37° C. (grey bar; n=8) and 33° C. (white bar; n=8). A significant time-dependent decrease in the percentage of CD4.sup.+FoxP3.sup.High cells is observed only for Tregs expanded at 37° C. The cells cultured at 33° C. are characterised by significantly more stable expression of FoxP3 over time. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(9) FIG. 9—presents % of inhibition of Teff proliferation by Tregs expanded at 37 (black circles, n=5) and 33° C. (red triangles, n=5). Results for various Teff:Treg ratios are shown. All data were processed in the same way and each time proliferation of unstimulated Teffs cultured without Tregs (K) was treated as a background and subtracted from % of dividing Teffs in all tested Teff:Treg proportions. Thus % of inhibition of Teff proliferation for K is always equal to 100% and signifies complete inhibition of Teff proliferation. Adequately, results for stimulated Teffs and cultured without Tregs (1:0) are always equal to 0% and correspond to lack of inhibition of proliferation. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(10) FIG. 10—presents % of inhibition of IFN-γ synthesis in Teffs by Tregs expanded at 37 (gray circles) and 33° C. (red triangles, n=4) for 14 days. Results for various Teff:Treg ratios are shown. All data were processed in the same way and each time IFN-γ production by unstimulated Teffs cultured without Tregs (K) was treated as a background and subtracted from % of IFN-γ.sup.+ Teffs in all tested Teff:Treg proportions. Thus, % of inhibition of IFN-γ production by Teffs for K is always equal to 100% and signifies that 100% of Teffs did not synthesize IFN-γ (100% of inhibition). Adequately, results for stimulated Teffs and cultured without Tregs (1:0) are always equal to 0% and correspond to lack of inhibition of IFN-γ production. The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum.

(11) FIG. 11—presents IFN-γ production by Tregs at day 14 of the culture. The figure depicts percentage of IFN-γ* Tregs after 14-day expansion at temperature of 37° C. (grey bar; n=4) and 33° C. (white bar; n=4). The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(12) FIG. 12—depicts frequency of cells with demethylated TSDRs within Teff (control samples) and Treg cultures expanded at 37 and 33° C. (black, gray and white symbols, respectively) on day 7 and 14 of expansion (n=5). The differences were calculated with Mann-Whitney test for non-parametric data. The values are shown as median, minimum and maximum. An asterisk indicates statistical significance (p<0.05).

(13) FIG. 13—presents representative dot plots depicting percentage of CD25.sup.High cells within CD4.sup.+FoxP3.sup.+ cells derived from the same donor expanded simultaneously for 14 days at 37° C. (A), 33° C. (B) and 29° C. (C).

(14) FIG. 14—presents representative dot plots depicting percentage of CD4.sup.+FoxP3.sup.+ cells within Treg cultures derived from the same donor expanded simultaneously for 14 days at 37° C. (A), 33° C. (B) and 29° C. (C).

(15) The present invention is illustrated by the following example, which is not its limitation

EXAMPLE

Isolation of Tregs and CD4.SUP.+ Teffs

(16) Peripheral blood Tregs and CD4.sup.+ Teffs were isolated from buffy coats derived from female and male volunteer blood donors. First, peripheral blood mononuclear cells (PBMC) were obtained by Ficoll/Uropoline gradient centrifugation. Then, CD4.sup.+ T cells were isolated with negative immunomagnetic selection (isolation purity 90-99%). For this purpose, the EasySep Human CD4+ T Cell Enrichment Kit (Stemcell Technologies) was used. The kit depletes the samples from cells expressing the following antigens: CD8, CD14, CD16, CD19, CD20, CD36, CD56, CD66b, CD123, TCRγ/δ and glycophorin A. Subsequently, the isolated CD4.sup.+ T cells were labeled with monoclonal antibodies (mAb, 5 μl mAB/10.sup.6 cells) specific for the following antigens: CD3, CD4, CD25, CD127, CD8, CD19, CD16 and CD14 (BD Biosciences). The last 4 mAbs were conjugated with the same fluorochrome in aim to minimize the fluorescence overlap and cut-off in one step cytotoxic T cells (Tc), B cells, natural killer (NK) cells and monocytes, respectively. These cells were defined all together in sorting algorithm as lineage.

(17) Then, cells were sorted with FACS sorter into the following phenotype of Tregs: CD3.sup.+CD4.sup.+CD25.sup.highCD127.sup.−/Lowdoublet.sup.−lineage.sup.−dead.sup.− and Teffs: CD3.sup.+CD4.sup.+CD25.sup.−CD127.sup.Highdoublet.sup.−lineage.sup.−dead.sup.−. The post-sort purity of Tregs was ˜100% [median(min−max): 98%(97−99)]. It should be underlined that the cell sorter Influx (BD Biosciences) that was used for Treg isolation is a GMP (Good Manufacturing Practice) adapted machine. Its considerable advantage from the viewpoint of clinical therapy is the replaceable sample line, which eliminates the risk of cross contamination. However, depending on the application (research study/clinical therapy), Tregs can also be obtained with other types of cell sorter. The isolation algorithm described here and the method for Treg expansion according to the present invention, allows to replicate the results of Treg expansion also when different model of cell sorter is used. Therefore, the cell sorter used in this example is not a limitation of the present invention.

(18) The isolated Tregs and Teffs were then seeded into separate plates and expanded at 37° C. and at 33° C. in SCGM culture medium (CellGro) which meets GMP criteria. The medium contained antibiotics (penicillin 100 U/ml and streptomycin 100 mg/ml) and was supplemented with human inactivated serum AB (10%) and interleukin-2 (IL-2; 2000 U/ml; Proleukin; Chiron, San Diego, Calif.). On the day 0 and day 7 of the culture, magnetic beads coated with anti-CD3 and anti-CD28 antibodies (Invitrogen; Carlsbad, Calif.) were added to the culture at a 1:1 cell:bead ratio. The beads mimic stimulation by antigen-presenting cells and thus induce Treg proliferation. In some cultures, a Treg sample was also expanded at 29° C. to determine the lower temperature limit at which Tregs keep their viability, proliferative potential and characteristic phenotype.

Fold Increase of Initial Treg Number after 14-Day Culture

(19) After 14 days, the Tregs were harvested and washed with a PBS, beads were removed and cells were counted. The fold increase of Treg number was calculated as a ratio of the final cell number on day 14 of expansion under a given culture condition to the initial cell count. It was observed that 14-day culture of Tregs at 33° C. resulted in ≈3-fold higher cell counts as compared with Tregs expanded at 37° C. (FIG. 1). It was also found that 29° C. is the lower limit of temperature at which Tregs keep their viability. However, Treg proliferation was suppressed at this temperature. Therefore, a decrease in culture temperature below 33° C. is not recommended, and 33° C. is the optimal temperature for Treg culture.

(20) Prolongation of the culture to 4-5 weeks significantly reduced the percentage of FoxP3.sup.+ cells when they were expanded at 37° C. (≈40-45% of FoxP3.sup.+ cells), while Tregs cultured at 33° C. were constantly characterised by high FoxP3 expression (≈90-95% of FoxP3.sup.+ cells) and intensive proliferation. After 4 and 5 weeks of the culture, Tregs expanded at 33° C. revealed an ≈6000- and 115000-fold increase, respectively. Therefore, prolongation of Treg culture at 33° C. is feasible and results with several-fold higher number of stable cells of high quality. Nevertheless, the decision regarding duration of Treg culture depends on the application. In clinical treatment, a 7-14 day-expansion is sufficient to obtain a therapeutically relevant number of Tregs (Marek-Trzonkowska N, 2012) (Marek-Trzonkowska N, 2014). The cultures at 29° C. had to be ceased after 3 weeks due to the low cell viability and inhibition of their proliferation.

Phenotype Control

(21) On each 7 and 14 day of the culture, samples of Tregs expanded at 33° C., 37° C. and 29° C. were collected and labelled with the following monoclonal antibodies: anti-CD3, anti-CD4, anti-CD25, anti-CD127, anti-CD62L, anti-CD45RA and anti-FoxP3, using the Foxp3 Staining Buffer Set (eBiosciences). Then, the cells were analysed with flow cytometer (Canto II, BD Biosciences).

(22) It was observed that the Tregs cultured at 33° C. were characterized by significantly higher percentage of FoxP3.sup.+ cells on day 7 (p=0.049) and 14 (p=1×10.sup.−4) as compared with those at standard temperature of 37° C. The difference was more pronounced on the last day of the culture. In addition, on day 14 of expansion, FoxP3.sup.+ Tregs expanded at 33° C. showed a higher intensity of FoxP3 expression (p=0.037) measured as a ratio of median fluorescence intensity (MFI) of the positive signal to median autofluorescence intensity of unlabeled cells (INDEX MFI FoxP3; FIG. 2).

(23) When frequency of FoxP3.sup.+ Tregs on day 14 was compared with percentage of these cells on day 7 we found that Tregs cultured at 33° C. were characterised by a significantly higher stability of FoxP3 expression during the culture, than those at 37° C. The median decline in percentage of FoxP3.sup.+ Tregs at day 14 of the culture was equal to 2% and 12.9% (>6-fold greater) in cultures expanded at 33 and 37° C., respectively (FIG. 3; p=0.001).

(24) Similar differences were observed for CD25 molecule expression. A culture at a temperature of 33° C. was associated with a significantly higher percentage of CD25.sup.High cells within CD4.sup.+FoxP3.sup.+ cells on day 7 (p=0.006) and 14 (p=0.003) of expansion. In addition, CD4.sup.+FoxP3.sup.+CD25.sup.High cells cultured at 33° C. were characterized by higher intensity of CD25 expression (INDEX MFI CD25) on day 7 (p=0.014) and 14 (p=0.006) of the culture, than the corresponding cells at of 37° C. (FIG. 4).

(25) Likewise, FoxP3, CD25 expression was also more stable in Tregs expanded at 33° C. The median decrease in frequency of CD4.sup.+FoxP3.sup.+CD25.sup.High Tregs at 33° C. on day 14 of the culture was equal to 1.65%, while at 37° C. it was >6-fold higher (6.2%) (FIG. 5). In addition, it was observed that Tregs expanded at 33° C. were characterised by a significantly higher percentage of cells with the highest intensity of FoxP3 expression (FoxP3.sup.High) on day 7 (p=0.049) and 14 (p=1×10.sup.−4) of the culture as compared with cells at 37° C. (FIG. 6). Furthermore, CD4.sup.+FoxP3.sup.High population at 33° C. contained significantly more CD25.sup.High cells on day 7 (p=0.002) and 14 (p=0.003) of the culture with higher intensity of CD25 expression (INDEX MFI CD25; day 7 p=0.01; day 14 p=0.049), than the corresponding population at 37° C. (FIG. 7). Like CD4.sup.+FoxP3.sup.+ cells, CD4.sup.+FoxP3.sup.High subpopulation was also characterized with higher stability of FoxP3 expression (p=0.037) at 33° C., than at a standard culture temperature (FIG. 8).

(26) As compared to the cells expanded at both 33° C. and 37° C., Tregs cultured at 29° C. were characterised by a rapid loss of characteristic Treg phenotype after the 7.sup.th day of expansion. On day 14 of the culture at 29° C. only 7-20% of Tregs remained CD25.sup.High cells (FIG. 13). Lowering culture temperature to 29° C. also impaired expression of FoxP3. Even though, after the first 7 days of the culture at 29° C. Tregs showed a relatively high expression of this marker (≈80% of FoxP3.sup.+ cells), the percentage of Fox3.sup.+ Tregs on day 14 was merely oscillated around 20% (FIG. 14). Between the first and the second week of the culture at 29° C., approximately 60% of Tregs lost expression of this main marker and regulator of their function. Therefore, our experiments demonstrated that 33° C. is the optimal temperature for Treg culture, and not only intensifies their proliferation but also preserves high quality and stability of Tregs. Further temperature reduction is not recommended, and 29° C. is the lower limit of temperature at which Tregs survive, but at the same time stop to proliferate and rapidly lose their typical phenotypic features.

Test of Inhibition of Teff Proliferation by Tregs

(27) At day 7 of the expansion a proliferation inhibition assay was performed. Teffs were stained with 2 μM Violet Proliferation Dye 450 (VPD-450; BD Horizon) for 15 min. at 37° C. and mixed with unstained Tregs expanded at 37 and 33° C. in the following proportions: 2:1, 1:1, 1:½, 1:¼, and 1:⅛. Cells were cocultured for the next 4 days at 37° C. in SCGM medium supplemented with 10% human inactivated AB serum and expanding beads in a 1:1 Teff:bead ratio. VPD-450 stained Teffs cultured alone in presence or absence of beads were used as controls. After the stimulation cells were labeled with 7-amino-actinomycin D (7-AAD, BD Pharmingen) a compound that binds to DNA of dead cells in aim to exclude them from the analysis. The samples were evaluated with flow cytometry (Canto II, BD Biosciences).

(28) Data were analyzed as % of inhibition of Teff proliferation. Each time proliferation of unstimulated Teffs cultured without Tregs (K) was treated as a background and subtracted from % of dividing Teffs in all tested Teff:Treg proportions. Thus % of inhibition of Teff proliferation for K is always equal to 100% and signifies complete inhibition of Teff proliferation. Adequately, results for stimulated Teffs and cultured without Tregs (1:0) are always equal to 0% and correspond to lack of inhibition.

(29) Tregs cultured at 33° C. were found to suppress proliferation of Teffs more efficiently, than those expanded at 37° C. The effect was more pronounced for lower Treg:Teff ratios: ½:1, ¼:1 and ⅛:1 (Mann-Whitney U test, p=0.03, p=0.03 and p=0.02, respectively; FIG. 9). All tests were performed at 37° C. that is considered a physiological temperature of human body.

(30) Functional tests for Tregs expanded at 29° C. could not be performed because of the complete inhibition of proliferation and thus cell number was insufficient for concomitant phenotype control and assessment of suppressor activity.

IFN-δ Inhibition Assay and Measurement of IFN-γ Production by Tregs

(31) After 11 days, samples of Tregs expanded at 33° C. and 37° C., were collected for assessment of their inhibitory effect on IFN-γ production (a proinflammatory cytokine) by autologous CD4.sup.+ Teffs. Simultaneously, a sample of Teffs expanded, at 37° C. in the same culture medium as the tested Tregs was also harvested. Before the test cells were washed and beads were removed. Then, cells were kept without beads and IL-2 for 48 h in aim to make them resting.

(32) After the next 2 days, i.e. on day 13 of the culture, the cells were washed with PBS and counted. Then, Teffs were stained with a solution of carboxyfluorescein diacetate succinimidyl ester (CFDA; Vybrant® CFDA SE Cell Tracer Kit, Invitrogen; 5 μM; 15 min., 37° C.) in order to discriminate them from unstained Tregs. Subsequently, CFDA-labelled autologous Teffs were mixed in the following proportions with Tregs previously cultured at 33° C. and 37° C.: 1:1, 1:½, 1:¼ and 1:⅛, where number of Teffs was constant and the number of Tregs was variable. The cells were suspended in a fresh culture medium supplemented with human inactivated AB serum (10%), IL-2 (100 U/ml) and monoenzin, a protein transport inhibitor (GolgiStop, BD Biosciences; 2 μl/1000 μl of medium) in aim to inhibit the release of IFN-γ outside the cells. Then, beads coated with anti-CD3 and anti-CD28 antibodies were added in 1:1 Teff:bead ratio. In addition, each time, a positive and negative controls were performed, and IFN-γ production by Tregs cultured without Teffs was also analysed. Positive and negative controls were stimulated and unstimulated Teffs incubated without Tregs, respectively. After 24 h of stimulation at 37° C. (on day 14 day of the culture) the cells were labelled with anti-CD4 and anti-IFN-γ antibodies using a kit for intracellular antigen staining (BD Cytofix/Cytoperm Plus Fixation/Permeabilization Solution Kit with BD GolgiStop; BD Biosciences) and analysed with flow cytometer (Canto II, BD Biosciences).

(33) All the functional tests were conducted at the standard temperature for T cell culture, i.e. 37° C. This temperature was chosen for functional tests because it is a physiological temperature of human body, and ex vivo expanded Tregs will have to function at this temperature after administration to the patient. Data were analyzed as % of inhibition of IFN-γ production by Teffs. Each time IFN-γ production by unstimulated Teffs cultured without Tregs (K) was treated as a background and subtracted from % of IFN-γ.sup.+ Teffs in all tested Teff:Treg proportions. Thus, % of inhibition of IFN-γ production by Teffs for K was always equal to 100% and signified complete lack of IFN-γ synthesis. Adequately, results for stimulated Teffs and cultured without Tregs (1:0) were always equal to 0% (lack of inhibition).

(34) It was observed that the Tregs previously cultured at 33° C. showed a tendency towards stronger inhibition of IFN-γ production by Teffs, as compared with Tregs expanded at 37° C. (FIG. 10). However, these differences did not reach statistical significance.

(35) In addition, it was found that Tregs cultured 33° C. produced only traceable amounts of IFN-γ after 14-day expansion (median 2.5%), while the percentage of IFN-γ.sup.+ cells was >2-fold higher in Treg population expanded at 37° C. (5.3% median, p=0.031; FIG. 11). Because of insufficient number of Tregs cultured at 29° C., these tests could not be performed for these cells.

DNA Methylation of the Treg-Specific Demethylated Region (TSDR)

(36) Genomic DNA from 7-day and 14-day cultures of Teffs, Tregs37 and Tregs33 was extracted with the QIAamp DNA blood mini kit (Qiagen, Hilden, Germany). A minimum of 60 ng bisulfite-treated (EpiTect; Qiagen) genomic DNA was used in a real-time PCR to quantify the Foxp3 Treg-specific demethylated region (TSDR). Real-time PCR was performed in a final reaction volume of 20 μl containing 10 μl FastStart universal probe master (Roche Diagnostics, Mannheim, Germany), 50 ng/μl lamda DNA (New England Biolabs, Frankfurt, Germany), 5 pmol/μl methylation or non-methylation-specific probe, 30 pmol/μl methylation or non-methylation-specific primers and 60 ng bisulfite-treated DNA or a respective amount of plasmid standard. The samples were analyzed in triplicates on a ABI 7500 cycler and reported as % of T cells with demethylated TSDR region. Treg samples with extremely low FoxP3 expression ( 2/13 cultures at 37° C.) were not subjected to this analyses as they were considered not to be the most representative data for this culture condition.

(37) Tregs expanded at 33° C. were characterized by significantly higher frequency of cells with demethylated TSDR after culture in vitro, than those at 37° C. The differences have been escalating with time and reached statistical significance at day 14 (Mann-Whitney U test, p=0.03; FIG. 12). Notably, TSDR demethylation was kept stable over time only in Tregs cultured at 33° C.

REFERENCES

(38) Andrés A (2005). “Cancer incidence after immunosuppressive treatment following kidney transplantation.” Crit Rev Oncol Hematol 56(1): 71-85.

(39) Barbaro M P, Spanevello A, Palladino G P, Salerno F G, Lacedonia D, Carpagnano G E, (2014). “Exhaled matrix metalloproteinase-9 (MMP-9) in different biological phenotypes of asthma.” Eur J Intern Med 25(1): 92-96.

(40) Berney T, Secchi A (2009). “Rapamycin in islet transplantation: friend or foe?” Transpl Int 22(2): 153-161.

(41) Bluestone J A, Buckner J H, Fitch M, Gitelman S E, Gupta S, Hellerstein M K, Herold K C, Lares A, Lee M R, Li K, Liu W, Long S A, Masiello L M, Nguyen V, Putnam A L, Rieck M, Sayre P H, Tang Q, (2015). “Type 1 diabetes immunotherapy using polyclonal regulatory T cells.” Sci Transl Med 7(315): 315ra189.

(42) Bluestone J A, Trotta E, Xu D, (2015). “The therapeutic potential of regulatory T cells for the treatment of autoimmune disease.” Expert Opin Ther Targets 19(8): 1091-1103.

(43) Braza F, Dugast E, Panov I, Paul C, Vogt K, Pallier A, Chesneau M, Baron D, Guerif P, Lei H, Laplaud D A, Volk H D, Degauque N, Giral M, Soulillou J P, Sawitzki B, Brouard S, (2015). “Central role of CD45RA-Foxp3hi memory regulatory T cells in clinical kidney transplantation tolerance.” J Am Soc Nephrol 26(8): 1795-1805.

(44) Di Ianni M, Falzetti F, Carotti A, Terenzi A, Castellino F, Bonifacio E, Del Papa B, Zei T, Ostini R I, Cecchini D, Aloisi T, Perruccio K, Ruggeri L, Balucani C, Pierini A, Sportoletti P, Aristei C, Falini B, Reisner Y, Velardi A, Aversa F, Martelli M F, (2011). “Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation.” Blood 117(14): 3921-398.

(45) Fontenot J D, Gavin M A, Rudensky A Y, (2003). “Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.” Nat Immunol 4(4): 330-336.

(46) Gambineri E, Torgerson T R, Ochs H D, (2003). “Immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance (IPEX), a syndrome of systemic autoimmunity caused by mutations of FOXP3, a critical regulator of T-cell homeostasis.” Curr Opin Rheumatol 15: 430-435.

(47) Gupta S, (2012). “Immunotherapies in diabetes mellitus type 1.” Med Clin North Am 96(3): 621-634.

(48) Hoffmann P, Boeld T J, Eder R, Huehn J, Floess S, Wieczorek G, Olek S, Dietmaier W, Andreesen R, Edinger M, (2009). “Loss of FOXP3 expression in natural human CD4+CD25+ regulatory T cells upon repetitive in vitro stimulation.” Eur J Immunol 39(4): 1088-1097.

(49) Kehrmann J, Tatura R, Zeschnigk M, Probst-Kepper M, Geffers R, Steinmann J, Buer J, (2014). “Impact of 5-aza-2′-deoxycytidine and epigallocatechin-3-gallate for induction of human regulatory T cells.” Immunology 142(3): 384-395.

(50) Lima X T, Cintra M L, Piaza A C, Mamoni R L, Oliveira R T, Magalhães R F, Blotta M H, (2015). “Frequency and characteristics of circulating CD4(+) CD28(null) T cells in patients with psoriasis.” Br J Dermatol 173(4): 998-1005.

(51) Malek T R (2003). “The main function of IL-2 is to promote the development of T regulatory cells.” J Leukoc Biol 74(6): 961-965.

(52) Malek T R, Castro I (2010). “Interleukin-2 receptor signaling: at the interface between tolerance and immunity.” Immunity 33(2): 153-165.

(53) Marek-Trzonkowska N, Mysliwiec M, Dobyszuk A, Grabowska M, Techmanska I, Juscinska J, Wujtewicz M A, Witkowski P, Mlynarski W, Balcerska A, Mysliwska J, Trzonkowski P, (2012). “Administration of CD4+CD25highCD127-regulatory T cells preserves β-cell function in type 1 diabetes in children.” Diabetes Care 35(9): 1817-1820.

(54) Marek-Trzonkowska N, Myśliwec M, Siebert J, Trzonkowski P, (2013). “Clinical application of regulatory T cells in type 1 diabetes.” Pediatr Diabetes 14(5): 322-332.

(55) Marek-Trzonkowska N, Myśliwiec M, Dobyszuk A, Grabowska M, Derkowska I, Juścińska J, Owczuk R, Szadkowska A, Witkowski P, Mynarski W, Jarosz-Chobot P, Bossowski A, Siebert J, Trzonkowski P, (2014). “Therapy of type 1 diabetes with CD4(+) CD25(high)CD127-regulatory T cells prolongs survival of pancreatic islets—results of one year follow-up.” Clin Immunol 153(1): 23-30.

(56) Marek N, Bieniaszewska M, Krzystyniak A, Juścińska J, Myśliwska J, Witkowski P, Hellmann A, Trzonkowski P, (2011). “The time is crucial for ex vivo expansion of T regulatory cells for therapy.” Cell Transplant 20(11-12): 1747-1758.

(57) Martelli M F, Di Ianni M, Ruggeri L, Falzetti F, Carotti A, Terenzi A, Pierini A, Massei M S, Amico L, Urbani E, Del Papa B, Zei T, Iacucci Ostini R, Cecchini D, Tognellini R, Reisner Y, Aversa F, Falini B, Velardi A, (2014). “HLAhaploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents acute leukemia relapse.” Blood 124(4): 638-644.

(58) Mu Q, Zhang H, Luo X M, (2015). “SLE: another autoimmune disorder influenced by microbes and diet?” Front Immunol 6(artyku 608): 1-10.

(59) Orent W, McHenry A R, Rao D A, White C, Klein H U, Bassil R, Srivastava G, Replogle J M, Raj T, Frangieh M, Cimpean M, Cuerdon N, Chibnik L, Khoury S J, Karlson E W, Brenner M B, De Jager P, Bradshaw E M, Elyaman W, (2015). “Rheumatoid arthritis-associated RBPJ polymorphism alters memory CD4+ T cells.” Hum Mol Genet DOI: 10.1093/hmg/ddv474 (in press).

(60) Panettieri R A, Jr Covar R, Grant E, Hillyer E V, Bacharier L, (2008). “Natural history of asthma: persistence versus progression-does the beginning predict the end?” J Allergy Clin Immunol 121(3): 607-613.

(61) Polansky J K, Kretschmer K, Freyer J, Floess S, Garbe A, Baron U, Olek S, Hamann A, von Boehmer H, Huehn J. (2008). “DNA methylation controls Foxp3 gene expression.” Eur J Immunol 38(6): 1654-1663.

(62) Prókai Á, Csohány R, Sziksz E, Pap D, Balicza-Himer L, Boros S, Magda B, Vannay Á, Kis-Petik K, Fekete A, Peti-Peterdi J, Szabó A J, (2015). “Calcineurin-inhibition results in upregulation of local renin and subsequent vascular endothelial growth factor production in renal collecting ducts.” Transplantation DOI: 10.1097/TP.0000000000000961 (in press).

(63) Pujol-Autonell I, Ampudia R M, Monge P, Lucas A M, Carrascal J, Verdaguer J, Vives-Pi M, (2013). “Immunotherapy with Tolerogenic Dendritic Cells Alone or in Combination with Rapamycin Does Not Reverse Diabetes in NOD Mice.” ISRN Endocrinol 2013 (ID 346987): 1-5.

(64) Rama I, Grinyó J M (2010). “Malignancy after renal transplantation: the role of immunosuppression.” Nat Rev Nephrol 6(9): 511-519.

(65) Ryba M, Marek N, Hak , Rybarczyk-Kapturska K, Myśliwiec M, Trzonkowski P, Myśliwska J, (2011). “Anti-TNF rescue CD4+Foxp3+ regulatory T cells in patients with type 1 diabetes from effects mediated by TNF.” Cytokine 55(3): 353-361.

(66) Sénécal V, Deblois G, Beauseigle D, Schneider R, Brandenburg J, Newcombe J, Moore C S, Prat A, Antel J, Arbour N, (2015). “Production of IL-27 in multiple sclerosis lesions by astrocytes and myeloid cells: Modulation of local immune responses.” Glia DOI: 10.1002/glia.22948 (in press).

(67) Tang Q, Bluestone J A (2013). “Regulatory T-cell therapy in transplantation-moving to the clinic.” Cold Spring Harb Perspect Med 3(11): pii: a015552.

(68) Trzonkowski P, Bacchetta R, Battaglia M, Berglund D, Bohnenkamp H R, ten Brinke A, Bushell A, Cools N, Geissler B K, Gregori S, Marieke van Ham S, Hilkens C, Hutchinson J A, Lombardi G, Madrigal J A, Marek-Trzonkowska N, Martinez-Caceres E M, Roncarolo M G, Sanchez-Ramon S, Saudemont A, Sawitzki B, (2015). “Hurdles in therapy with regulatory T cells.” Sci Transl Med 7(304): 304ps18.

(69) Trzonkowski P, Bieniaszewska M, Juścińska J, Dobyszuk A, Krzystyniak A, Marek N, Myśliwska J, Hellmann A, (2009). “First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127-T regulatory cells.” Clin Immunol 133(1): 22-26.

(70) Trzonkowski P, Dukat-Mazurek A, Bieniaszewska M, Marek-Trzonkowska N, Dobyszuk A, Juścińska J, Dutka M, Myśliwska J, Hellmann A, (2013). “Treatment of graft-versus-host disease with naturally occurring T regulatory cells.” BioDrugs 27(6): 605-614.

(71) Trzonkowski P, Szaryńska M, Myśliwska J, Myśliwski A, (2009). “Ex vivo expansion of CD4(+)CD25(+) T regulatory cells for immunosuppressive therapy.” Cytometry A 75(3): 175-188.

(72) Vignali D A, Collison L W, Workman C J, (2008). “How regulatory T cells work.” Nat Rev Immunol 8(7): 523-532.

(73) Wang Y M, Zhang G Y, Wang Y, Hu M, Wu H, Watson D, Hori S, Alexander I E, Harris D C, Alexander S I, (2006). “Foxp3-transduced polyclonal regulatory T cells protect against chronic renal injury from adriamycin.” J Am Soc Nephrol 17(3): 697-706.

(74) Yi S, Ji M, Wu j, Ma X, Phillips P, Hawthorne W J, O'Connell P J, (2012). “Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice.” Diabetes 61(5): 1180-1191.

(75) Zhang N, Su D, Qu S, Tse T, Bottino R, Balamurugan A N, Xu J, Bromberg J S, Dong H H, (2006). “Sirolimus is associated with reduced islet engraftment and impaired beta-cell function.” Diabetes 55(9): 2429-2436.

(76) Zhao K, Ruan S, Yin L, Zhao D, Chen C, Pan B, Zeng L, Li Z, Xu K, (2015). “Dynamic regulation of effector IFN-γ-producing and IL-17-producing T cell subsets in the development of acute graft-versus-host disease.” Mol Med Rep DOI: 10.3892/mmr.2015.4638 (in press).