Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment - PubMed (original) (raw)

Expansion and characteristics of human T regulatory type 1 cells in co-cultures simulating tumor microenvironment

Christoph Bergmann et al. Cancer Immunol Immunother. 2007 Sep.

Abstract

Objective: Chronic inflammation and cancer development are associated with dysregulated immune responses and the presence of regulatory T cells (T(reg)). To study the role of T(reg) in tumor cell escape from immune surveillance, an in vitro model simulating the tumor microenvironment and promoting the induction and expansion of IL-10(+) T(reg )type 1 (Tr1) was established.

Methods: An in vitro co-culture system (IVA) included an irradiated head and neck squamous cell carcinoma cell line, immature dendritic cells (iDC), CD4(+)CD25(- )T cells and cytokines, IL-2 (10 IU/ml), IL-10 (20 IU/ml), IL-15 (20 IU/ml) +/- 1 nM rapamycin. Autologous iDC and CD4(+)CD25(-) T cells were obtained from the peripheral blood of 15 normal donors. Co-cultures were expanded for 10 days. Proliferating lymphocytes were phenotyped by multi-color flow cytometry. Their suppressor function was measured in CFSE inhibition assays +/- neutralizing anti-IL-10 mAb and using transwell cultures. Culture supernatants were tested for IL-4, IL-10, TGF-beta and IFN-gamma in ELISA.

Results: In the IVA, low doses of IL-2, IL-10 and IL-15 promoted induction and expansion of CD3(+)CD4(+)CD25(-)IL2Rbeta(+)IL2Rgamma(+)FoxP3(+)CTLA-4(+)IL-10(+) cells with suppressor activity (mean suppression +/- SD = 58 +/- 12%). These suppressor cells produced IL-10 (mean +/- SD = 535 +/- 12 pg/ml) and TGF-beta (mean +/- SD = 512 +/- 38 pg/ml), but no IL-4 or IFN-gamma. Suppressor function of co-cultures correlated with the percent of expanding IL-10(+) Tr1 cells (r (2 )=( )0.9; P < 0.001). The addition of rapamycin enriched Tr1 cells in all co-cultures. Neutralizing anti-IL-10 mAb abolished suppressive activity. Suppression was cell-contact independent.

Conclusion: The tumor microenvironment promotes generation of Tr1 cells which have the phenotype distinct from that of CD4(+)CD25(high)FoxP3(+) nTreg and mediate IL-10 dependent immune suppression in a cell-contact independent manner. Tr1 cells may play a critical role in cancer progression.

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Figures

Fig. 1

Fig. 1

Components of the model for outgrowth of Tr1 cells. a Numbers of proliferating CD4+CD25− T cells co-cultured with tumor cells, iDC and cytokines used in different combinations. In this HLA-A2-restricted model, T cells and iDCs are autologous. Tumor is an HNSCC cell line, PCI-13. Cells were co-cultured for 10 days. Growth was determined after harvest by viable cell counts of the non-adherent fraction. The data are from three experiments performed with cells of different HLA-A2+ normal donors. b Kinetics of CD4+IL-10+ cell growth in culture and the development of their suppressor activity, analyzed by flow cytometry and in CFSE suppression assays, respectively. The data are mean values ± SD from five experiments performed with cells of two normal donors (solid line). Activated T cells were used as controls (broken line). c Phenotypic analysis of MACS-separated CD4+CD25− T cells (black bar) co-cultured in the IVA under different conditions. c.1 A profile of CD4+CD25− T cells prior to the IVA; c.2 phenotype of CD4+CD25− T cells proliferating under the conditions established and optimized for Tr1 cell expansion. c.3 In absence of tumor cells, the outgrowth of IL-10+CD4+ T cells was significantly decreased (P < 0.01); c.4 outgrowth of Tr1 cells in absence of iDCs; c.5 activated CD4+CD25− T cells (white bar) cultured in presence of IL-2 (1,000 IU/ml) for 10 days were used as reference culture; All data are mean % ± SD of CD4+CD25− cells expressing a given marker were obtained with cells of 15 normal donors. c.6 nTreg generated from MACS-separated CD4+CD25+ T cells (stripped bar) and cultured in the presence of IL-2 (1,000 IU/ml), rapamycin (1 nM), anti-CD3 mAb (1 μg/ml) and anti-CD28 mAb (1 μg/ml) for 10 days, were used as reference. The data obtained with nTreg reference cultures are mean % ± SD of positive cells from experiments with cells of five normal donors

Fig. 1

Fig. 1

Components of the model for outgrowth of Tr1 cells. a Numbers of proliferating CD4+CD25− T cells co-cultured with tumor cells, iDC and cytokines used in different combinations. In this HLA-A2-restricted model, T cells and iDCs are autologous. Tumor is an HNSCC cell line, PCI-13. Cells were co-cultured for 10 days. Growth was determined after harvest by viable cell counts of the non-adherent fraction. The data are from three experiments performed with cells of different HLA-A2+ normal donors. b Kinetics of CD4+IL-10+ cell growth in culture and the development of their suppressor activity, analyzed by flow cytometry and in CFSE suppression assays, respectively. The data are mean values ± SD from five experiments performed with cells of two normal donors (solid line). Activated T cells were used as controls (broken line). c Phenotypic analysis of MACS-separated CD4+CD25− T cells (black bar) co-cultured in the IVA under different conditions. c.1 A profile of CD4+CD25− T cells prior to the IVA; c.2 phenotype of CD4+CD25− T cells proliferating under the conditions established and optimized for Tr1 cell expansion. c.3 In absence of tumor cells, the outgrowth of IL-10+CD4+ T cells was significantly decreased (P < 0.01); c.4 outgrowth of Tr1 cells in absence of iDCs; c.5 activated CD4+CD25− T cells (white bar) cultured in presence of IL-2 (1,000 IU/ml) for 10 days were used as reference culture; All data are mean % ± SD of CD4+CD25− cells expressing a given marker were obtained with cells of 15 normal donors. c.6 nTreg generated from MACS-separated CD4+CD25+ T cells (stripped bar) and cultured in the presence of IL-2 (1,000 IU/ml), rapamycin (1 nM), anti-CD3 mAb (1 μg/ml) and anti-CD28 mAb (1 μg/ml) for 10 days, were used as reference. The data obtained with nTreg reference cultures are mean % ± SD of positive cells from experiments with cells of five normal donors

Fig. 2

Fig. 2

Phenotypic characteristics of in vitro expanded Tr1 cells. a The phenotype of CD3+CD4+ lymphocytes proliferating under conditions established for the Tr1 IVA (black bar). Activated T cells, cultured in presence of IL-2 (1,000 IU/ml) for 10 days served as reference cultures (white bar). Cells were stained with mAb and evaluated by multi-color flow cytometry with gates set on CD3+CD4+. The data are mean % of positive cells ± SD from experiments with cells of 15 normal donors. Asterisks indicate significant differences (*P < 0.05 and **P < 0.01) between % positive cells in the IVA versus reference cultures; b flow cytometry histograms showing relative levels of expression of the selected markers on lymphocytes cultured in the Tr1 IVA for 10 days. Gates were set on CD3+CD4+ cells. The data obtained with T cells of one donor are representative for 15 different normal donors tested

Fig. 3

Fig. 3

Phenotypic characteristics of Tr1 cells expanded in presence of rapamycin. a The phenotype of CD3+CD4+ lymphocytes proliferating in the presence of 1 nM rapamycin and under the conditions established for the Tr1 cell IVA (black bar). Activated T cells, cultured in presence of IL-2 (1,000 IU/ml) for 10 days served as reference cultures (white bar). b Flow cytometry dot blots showing relative expression of selected markers on lymphocytes cultured in the Tr1 IVA and in the presence of rapamycin for 10 days. Gates were set on CD3+CD4+ cells. The data are representative for cells of one donor out of 15 tested

Fig. 4

Fig. 4

Suppression of proliferation by Tr1 cells ± rapamycin. a Freshly MACS-purified CD4+CD25− responder (R) T cells were labeled with CFSE, stimulated with plate-bound anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) mAb and cultured in the presence of 50 IU/ml IL-2 for 5 days. Cell proliferation of CFSE-labeled R cells were evaluated by flow cytometry and subsequent analysis using the ModFit software as described in Materials and methods. The histograms show CFSE intensity (X axis) and the number of events (Y axis). The acquisition gate was restricted to lymphocytes, as determined by characteristic forward and side scatter properties of these cells. Further, analysis gates were restricted to the CD3+CD4+ T-cell and CD4+CFSE+ T-cell subsets; b, c lymphocytes from the Tr1 IVA performed in the absence or presence of rapamycin (R1) were added at the start of the culture (ratio 1:1) and co-incubated with autologous CFSE-labeled R cells; d, e lymphocytes from the reference cultures (activated T cells or nTreg) were added at the start of the culture (ratio 1:1) and co-incubated with autologous CFSE-labeled R cells. The percentages of proliferation inhibition of CFSE labeled R cells alone or after additions of Tr1 cells are indicated in each panel. The results of one representative experiment out of 15 performed with cells of different donors are shown. f A representative experiment shows linearity of suppression depending on the number of Tr1 cells in co-cultures

Fig. 5

Fig. 5

Correlation between IL-10 expression by Tr1 cells and suppressor activity Correlation between suppressor function of Tr1 cells generated in the IVA model and their IL-10 expression was established using a linear regression model. Median percentages of IL-10+CD4+ T cells cultured under various conditions were plotted against their corresponding suppressor activity, determined as % suppression of proliferation of CFSE-labeled autologous CD4+CD25− responder T cells. Medians were calculated from up to 15 unique experiments

Fig. 6

Fig. 6

Tr1 cells mediate suppression by IL-10 secretion. a Tr1 cells generated in IVA were tested for their ability to suppress anti-CD3/CD28 mAbs-induced proliferation of autologous CD4+CD25− T cells in the presence or absence of neutralizing anti-IL-10 mAb (2 μg/ml) (black bar) or control (GAH-IgG: gray bar); b Tr1 cells separated from (R) cells in a transwell system inhibited proliferation of the (R) cells at the levels observed in unseparated co-cultures. Upon the addition of anti-IL-10 mAb to theses cultures almost completely abolished Tr1−mediated suppression (P < 0.001). The results are mean % of suppression ± SD obtained in three experiments with cells of three different normal donors

Fig. 7

Fig. 7

IL-10 and TGF-β1 levels in culture supernatants. Supernatants (SN) from different IVA cultures were collected on day 10 as described in Materials and methods and tested for cytokine levels by EIA; a IL-10 was not detectable in SN from reference cultures. SN from the Tr1 IVA ± rapamycin (R1) were positive for IL-10; b TGF-β1 was undetectable/low in acidified SN from T cells obtained from IL-2 activated or nTreg reference cultures. SN from the Tr1 IVA ± R1 contained high levels of TGF-β1

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