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Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies.

Bonanno G, Procoli A, Mariotti A, Corallo M, Perillo A, Danese S, De Cristofaro R, Scambia G, Rutella S - J Transl Med (2010)

Bottom Line: In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline.Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF.Pegfilgrastim induced significant changes in immune cell number and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology, Catholic University Med, School, Rome, Italy.

ABSTRACT

Background: Pegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the in vivo and ex vivo release of immune modulating cytokines remain unexplored.

Methods: Twelve patients with gynecological cancers received carboplatin/paclitaxel chemotherapy and single-dose pegfilgrastim as prophylaxis of febrile neutropenia. Peripheral blood was collected prior to pegfilgrastim administration (day 0) and on days +7, +11 and +21, to quantify immunoregulatory cytokines and to assess type 1 DC (DC1), type 2 DC (DC2) and Treg cell mobilization. In vitro-differentiated, monocyte-derived DC were used to investigate endocytic activity, expression of DC maturation antigens and ability to activate allogeneic T-cell proliferation.

Results: Pegfilgrastim increased the frequency of circulating DC1 and DC2 precursors. In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline. Serum levels of hepatocyte growth factor and interleukin (IL)-12p40, but not transforming growth factor-β1 or immune suppressive kynurenines, significantly increased after pegfilgrastim administration. Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF. Finally, DC populations differentiated in vitro after clinical provision of pegfilgrastim were phenotypically mature, possessed low endocytic activity, and incited a robust T-cell proliferative response.

Conclusions: Pegfilgrastim induced significant changes in immune cell number and function. The enhancement of monocytic IL-12 secretion portends favorable implications for pegfilgrastim administration to patients with cancer, a clinical context where the induction of immune deviation would be highly undesirable.

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Phenotypic features of DC-like cells from patients receiving pegfilgrastim. Monocytes were purified from the peripheral blood of patients given pegfilgrastim and were cultured in the presence of either pre-G-CSF or post-G-CSF serum (20% v/v) for 6 days, as detailed in Materials and Methods. Control cultures consisted of immunogenic DC preparations that were differentiated with GM-CSF and IL-4 without the provision of additional maturation stimuli (GM4DC). Panel A: Percentage of cells staining positively for a given antigen in a representative experiment out of 12 with similar results. Panel B: Relative expression of informative differentiation antigens. Median values and interquartile range recorded in 12 independent experiments. *denotes a p value < 0.05 compared with the other time-points.
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Figure 5: Phenotypic features of DC-like cells from patients receiving pegfilgrastim. Monocytes were purified from the peripheral blood of patients given pegfilgrastim and were cultured in the presence of either pre-G-CSF or post-G-CSF serum (20% v/v) for 6 days, as detailed in Materials and Methods. Control cultures consisted of immunogenic DC preparations that were differentiated with GM-CSF and IL-4 without the provision of additional maturation stimuli (GM4DC). Panel A: Percentage of cells staining positively for a given antigen in a representative experiment out of 12 with similar results. Panel B: Relative expression of informative differentiation antigens. Median values and interquartile range recorded in 12 independent experiments. *denotes a p value < 0.05 compared with the other time-points.

Mentions: For technical reasons, insufficient quantities of day +7 serum were obtained to be supplemented at 20% v/v to the DC and monocyte cultures. Figure 5 thus illustrates a representative experiment with day +11 and day +21 monocyte-derived DC preparations. Not unexpectedly, monocytes cultured with GM-CSF and IL-4 under serum-free conditions down-regulated CD14, were uniformly CD1a+, and up-regulated costimulatory molecules (CD80 and CD86) and DC maturation antigens such as CD83 and CD209 (Figure 5A). In sharp contrast, monocytes cultured with either pre- or post-pegfilgrastim serum maintained a CD14+CD1a- phenotype, in accordance with previous reports on the phenotype of human serum-supplemented DC cultures [11]. Interestingly, monocyte cultures containing pre- and post-pegfilgrastim serum differed in their expression of costimulatory molecules. CD80 and CD86 were expressed at significantly higher levels after culture with post-pegfilgrastim serum, both in terms of percent positive cells and in terms of MFI (Figure 5A and 5B). In addition, post-pegfilgrastim monocytes up-regulated the DC maturation antigen CD209 compared with cells in pre-G-CSF cultures (Figure 5B). ILT3 was also detected on higher percentages of post-pegfilgrastim monocytic cells, where its expression increased in terms of fluorescence intensity. Finally, CD83, CD11c and CD123 were detected on comparable percentages of pre-G-CSF and post-G-CSF monocytes. Taken together, phenotypic studies revealed that soluble factors contained in post-pegfilgrastim serum promoted the acquisition of a mature DC-like phenotype, with high expression of costimulatory molecules and CD209, and preserved expression of the monocyte/macrophage antigen CD14. In line with this, monocytes nurtured with post-pegfilgrastim serum possessed a diminished ability to endocytose FITC-conjugated dextran, a measure of DC maturation status, compared with monocytes cultured with pre-pegfilgrastim serum and with immature DC differentiated with GM-CSF and IL-4, used as control for optimal incorporation of FITC-dextran (Figure 6A and 6B).


Effects of pegylated G-CSF on immune cell number and function in patients with gynecological malignancies.

Bonanno G, Procoli A, Mariotti A, Corallo M, Perillo A, Danese S, De Cristofaro R, Scambia G, Rutella S - J Transl Med (2010)

Phenotypic features of DC-like cells from patients receiving pegfilgrastim. Monocytes were purified from the peripheral blood of patients given pegfilgrastim and were cultured in the presence of either pre-G-CSF or post-G-CSF serum (20% v/v) for 6 days, as detailed in Materials and Methods. Control cultures consisted of immunogenic DC preparations that were differentiated with GM-CSF and IL-4 without the provision of additional maturation stimuli (GM4DC). Panel A: Percentage of cells staining positively for a given antigen in a representative experiment out of 12 with similar results. Panel B: Relative expression of informative differentiation antigens. Median values and interquartile range recorded in 12 independent experiments. *denotes a p value < 0.05 compared with the other time-points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2992497&req=5

Figure 5: Phenotypic features of DC-like cells from patients receiving pegfilgrastim. Monocytes were purified from the peripheral blood of patients given pegfilgrastim and were cultured in the presence of either pre-G-CSF or post-G-CSF serum (20% v/v) for 6 days, as detailed in Materials and Methods. Control cultures consisted of immunogenic DC preparations that were differentiated with GM-CSF and IL-4 without the provision of additional maturation stimuli (GM4DC). Panel A: Percentage of cells staining positively for a given antigen in a representative experiment out of 12 with similar results. Panel B: Relative expression of informative differentiation antigens. Median values and interquartile range recorded in 12 independent experiments. *denotes a p value < 0.05 compared with the other time-points.
Mentions: For technical reasons, insufficient quantities of day +7 serum were obtained to be supplemented at 20% v/v to the DC and monocyte cultures. Figure 5 thus illustrates a representative experiment with day +11 and day +21 monocyte-derived DC preparations. Not unexpectedly, monocytes cultured with GM-CSF and IL-4 under serum-free conditions down-regulated CD14, were uniformly CD1a+, and up-regulated costimulatory molecules (CD80 and CD86) and DC maturation antigens such as CD83 and CD209 (Figure 5A). In sharp contrast, monocytes cultured with either pre- or post-pegfilgrastim serum maintained a CD14+CD1a- phenotype, in accordance with previous reports on the phenotype of human serum-supplemented DC cultures [11]. Interestingly, monocyte cultures containing pre- and post-pegfilgrastim serum differed in their expression of costimulatory molecules. CD80 and CD86 were expressed at significantly higher levels after culture with post-pegfilgrastim serum, both in terms of percent positive cells and in terms of MFI (Figure 5A and 5B). In addition, post-pegfilgrastim monocytes up-regulated the DC maturation antigen CD209 compared with cells in pre-G-CSF cultures (Figure 5B). ILT3 was also detected on higher percentages of post-pegfilgrastim monocytic cells, where its expression increased in terms of fluorescence intensity. Finally, CD83, CD11c and CD123 were detected on comparable percentages of pre-G-CSF and post-G-CSF monocytes. Taken together, phenotypic studies revealed that soluble factors contained in post-pegfilgrastim serum promoted the acquisition of a mature DC-like phenotype, with high expression of costimulatory molecules and CD209, and preserved expression of the monocyte/macrophage antigen CD14. In line with this, monocytes nurtured with post-pegfilgrastim serum possessed a diminished ability to endocytose FITC-conjugated dextran, a measure of DC maturation status, compared with monocytes cultured with pre-pegfilgrastim serum and with immature DC differentiated with GM-CSF and IL-4, used as control for optimal incorporation of FITC-dextran (Figure 6A and 6B).

Bottom Line: In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline.Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF.Pegfilgrastim induced significant changes in immune cell number and function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Hematology, Catholic University Med, School, Rome, Italy.

ABSTRACT

Background: Pegylated granulocyte colony-stimulating factor (G-CSF; pegfilgrastim) is a longer-acting form of G-CSF, whose effects on dendritic cell (DC) and regulatory T cell (Treg) mobilization, and on the in vivo and ex vivo release of immune modulating cytokines remain unexplored.

Methods: Twelve patients with gynecological cancers received carboplatin/paclitaxel chemotherapy and single-dose pegfilgrastim as prophylaxis of febrile neutropenia. Peripheral blood was collected prior to pegfilgrastim administration (day 0) and on days +7, +11 and +21, to quantify immunoregulatory cytokines and to assess type 1 DC (DC1), type 2 DC (DC2) and Treg cell mobilization. In vitro-differentiated, monocyte-derived DC were used to investigate endocytic activity, expression of DC maturation antigens and ability to activate allogeneic T-cell proliferation.

Results: Pegfilgrastim increased the frequency of circulating DC1 and DC2 precursors. In contrast, CD4+FoxP3+ bona fide Treg cells were unchanged compared with baseline. Serum levels of hepatocyte growth factor and interleukin (IL)-12p40, but not transforming growth factor-β1 or immune suppressive kynurenines, significantly increased after pegfilgrastim administration. Interestingly, pegfilgrastim fostered in vitro monocytic secretion of IL-12p40 and IL-12p70 when compared with unconjugated G-CSF. Finally, DC populations differentiated in vitro after clinical provision of pegfilgrastim were phenotypically mature, possessed low endocytic activity, and incited a robust T-cell proliferative response.

Conclusions: Pegfilgrastim induced significant changes in immune cell number and function. The enhancement of monocytic IL-12 secretion portends favorable implications for pegfilgrastim administration to patients with cancer, a clinical context where the induction of immune deviation would be highly undesirable.

Show MeSH
Related in: MedlinePlus