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Interleukin-21 induces the differentiation of human umbilical cord blood CD34-lineage- cells into pseudomature lytic NK cells.

Bonanno G, Mariotti A, Procoli A, Corallo M, Scambia G, Pierelli L, Rutella S - BMC Immunol. (2009)

Bottom Line: IL-21/IL-15-differentiated cells expressed high levels of mRNA for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRgamma genes.From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-gamma, GM-CSF and CCL3/MIP-1alpha, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules.This study underpins a novel role for IL-21 in the differentiation of pseudo-mature lytic NK cells in a synergistic context with IL-15, and identifies a potential strategy to expand functional NK cells for immunotherapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gynaecology, Catholic University Medical School, Rome, Italy. giuseppina.bonanno@rm.unicatt.it

ABSTRACT

Background: Umbilical cord blood (UCB) is enriched with transplantable CD34+ cells. In addition to CD34-expressing haematopoietic stem cells (HSC), human UCB contains a rare population of CD34-lineage- cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and a stromal cell support. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34-lineage- cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34-lineage- cells.

Results: CD34-lineage- cells were maintained in liquid culture with Flt3-L and SCF, with the addition of IL-15 and IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate cell surface phenotype, expression of molecular determinants of lymphoid/NK cell differentiation, secretion of IFN-gamma, GM-CSF, TNF-alpha and CCL3/MIP-1alpha, and cytolytic activity against NK-sensitive tumour cell targets. CD34-lineage- cells proliferated vigorously in response to IL-15 and IL-21 but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins. CD34-lineage- cells expanded by IL-21 in combination with IL-15 acquired lymphoid morphology and killer-cell immunoglobulin-like receptor (KIR)-CD56+CD16-/+ phenotype, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells expressed high levels of mRNA for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRgamma genes. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-gamma, GM-CSF and CCL3/MIP-1alpha, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules.

Conclusion: This study underpins a novel role for IL-21 in the differentiation of pseudo-mature lytic NK cells in a synergistic context with IL-15, and identifies a potential strategy to expand functional NK cells for immunotherapy.

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Related in: MedlinePlus

Cytokine and chemokine release from CD34-lineage- cells and CD34+ cells differentiated with IL-15 and IL-21. The production of GM-CSF, TNF-α and CCL3/MIP-1α in supernatants of CD34-lineage- cells and CD34+ cells maintained with SCF+Flt3-L either alone or supplemented with IL-15, IL-21 or the combination of both cytokines was monitored weekly with conventional ELISA. Bars depict mean and standard deviation recorded in 3 independent experiments performed in triplicate. FL = Flt3-L; W = week.
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Figure 5: Cytokine and chemokine release from CD34-lineage- cells and CD34+ cells differentiated with IL-15 and IL-21. The production of GM-CSF, TNF-α and CCL3/MIP-1α in supernatants of CD34-lineage- cells and CD34+ cells maintained with SCF+Flt3-L either alone or supplemented with IL-15, IL-21 or the combination of both cytokines was monitored weekly with conventional ELISA. Bars depict mean and standard deviation recorded in 3 independent experiments performed in triplicate. FL = Flt3-L; W = week.

Mentions: We next determined the ability of IL-15+IL-21-differentiated cells to release IFN-γ, GM-CSF, TNF-α and CCL3/MIP-1α in culture supernatants. IFN-γ was undetectable (< 8 pg/ml) after cell culture in the presence of SCF and Flt3-L supplemented with either IL-15 or IL-21 alone. In sharp contrast, exposure to IL-15 and IL-21 induced a highly significant release of IFN-γ in 4-week old cultures, corresponding to a mean of 1210 ± 245 pg/ml in 10 independent experiments. IFN-γ release by CD34+ HSC matured with IL-15 and IL-21 was superimposable (data not shown). GM-CSF was undetectable in Flt3-L+SCF-differentiated cultures but was released at high levels from week +3 onward in CD34-lineage- cultures nurtured with IL-15 alone or with IL-15+IL-21. Conversely, TNF-α was preferentially detected in the supernatants of IL-15-differentiated CD34-lineage- cells compared with cells maintained with IL-15 and IL-21. GM-CSF and TNF-α release by CD34+ HSC followed a similar pattern, although the magnitude of cytokine release was significantly lower when compared with CD34-lineage- cultures. Finally, measurable CCL3/MIP-1α secretion occurred under any culture condition that we established. However, CCL3/MIP-1α release in response to Flt3-L and SCF was further enhanced by IL-15 alone and, to a greater extent, by IL-15 and IL-21 in combination (Figure 5). Notably, NK cells differentiated with IL-15 from CD34+ HSC released the highest levels of CCL3/MIP-1α. Collectively, these experiments suggested that IL-15+IL-21-differentiated CD34-lineage- cells acquired the ability to release soluble factors relevant for NK effector function and NK migration/homing.


Interleukin-21 induces the differentiation of human umbilical cord blood CD34-lineage- cells into pseudomature lytic NK cells.

Bonanno G, Mariotti A, Procoli A, Corallo M, Scambia G, Pierelli L, Rutella S - BMC Immunol. (2009)

Cytokine and chemokine release from CD34-lineage- cells and CD34+ cells differentiated with IL-15 and IL-21. The production of GM-CSF, TNF-α and CCL3/MIP-1α in supernatants of CD34-lineage- cells and CD34+ cells maintained with SCF+Flt3-L either alone or supplemented with IL-15, IL-21 or the combination of both cytokines was monitored weekly with conventional ELISA. Bars depict mean and standard deviation recorded in 3 independent experiments performed in triplicate. FL = Flt3-L; W = week.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743656&req=5

Figure 5: Cytokine and chemokine release from CD34-lineage- cells and CD34+ cells differentiated with IL-15 and IL-21. The production of GM-CSF, TNF-α and CCL3/MIP-1α in supernatants of CD34-lineage- cells and CD34+ cells maintained with SCF+Flt3-L either alone or supplemented with IL-15, IL-21 or the combination of both cytokines was monitored weekly with conventional ELISA. Bars depict mean and standard deviation recorded in 3 independent experiments performed in triplicate. FL = Flt3-L; W = week.
Mentions: We next determined the ability of IL-15+IL-21-differentiated cells to release IFN-γ, GM-CSF, TNF-α and CCL3/MIP-1α in culture supernatants. IFN-γ was undetectable (< 8 pg/ml) after cell culture in the presence of SCF and Flt3-L supplemented with either IL-15 or IL-21 alone. In sharp contrast, exposure to IL-15 and IL-21 induced a highly significant release of IFN-γ in 4-week old cultures, corresponding to a mean of 1210 ± 245 pg/ml in 10 independent experiments. IFN-γ release by CD34+ HSC matured with IL-15 and IL-21 was superimposable (data not shown). GM-CSF was undetectable in Flt3-L+SCF-differentiated cultures but was released at high levels from week +3 onward in CD34-lineage- cultures nurtured with IL-15 alone or with IL-15+IL-21. Conversely, TNF-α was preferentially detected in the supernatants of IL-15-differentiated CD34-lineage- cells compared with cells maintained with IL-15 and IL-21. GM-CSF and TNF-α release by CD34+ HSC followed a similar pattern, although the magnitude of cytokine release was significantly lower when compared with CD34-lineage- cultures. Finally, measurable CCL3/MIP-1α secretion occurred under any culture condition that we established. However, CCL3/MIP-1α release in response to Flt3-L and SCF was further enhanced by IL-15 alone and, to a greater extent, by IL-15 and IL-21 in combination (Figure 5). Notably, NK cells differentiated with IL-15 from CD34+ HSC released the highest levels of CCL3/MIP-1α. Collectively, these experiments suggested that IL-15+IL-21-differentiated CD34-lineage- cells acquired the ability to release soluble factors relevant for NK effector function and NK migration/homing.

Bottom Line: IL-21/IL-15-differentiated cells expressed high levels of mRNA for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRgamma genes.From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-gamma, GM-CSF and CCL3/MIP-1alpha, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules.This study underpins a novel role for IL-21 in the differentiation of pseudo-mature lytic NK cells in a synergistic context with IL-15, and identifies a potential strategy to expand functional NK cells for immunotherapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Gynaecology, Catholic University Medical School, Rome, Italy. giuseppina.bonanno@rm.unicatt.it

ABSTRACT

Background: Umbilical cord blood (UCB) is enriched with transplantable CD34+ cells. In addition to CD34-expressing haematopoietic stem cells (HSC), human UCB contains a rare population of CD34-lineage- cells endowed with the ability to differentiate along the T/NK pathway in response to interleukin (IL)-15 and a stromal cell support. IL-21 is a crucial regulator of NK cell function, whose influence on IL-15-induced differentiation of CD34-lineage- cells has not been investigated previously. The present study was designed and conducted to address whether IL-21 might replace the stromal cell requirements and foster the IL-15-induced NK differentiation of human UCB CD34-lineage- cells.

Results: CD34-lineage- cells were maintained in liquid culture with Flt3-L and SCF, with the addition of IL-15 and IL-21, either alone or in combination. Cultures were established in the absence of feeder cells or serum supplementation. Cytokine-treated cells were used to evaluate cell surface phenotype, expression of molecular determinants of lymphoid/NK cell differentiation, secretion of IFN-gamma, GM-CSF, TNF-alpha and CCL3/MIP-1alpha, and cytolytic activity against NK-sensitive tumour cell targets. CD34-lineage- cells proliferated vigorously in response to IL-15 and IL-21 but not to IL-21 alone, and up-regulated phosphorylated Stat1 and Stat3 proteins. CD34-lineage- cells expanded by IL-21 in combination with IL-15 acquired lymphoid morphology and killer-cell immunoglobulin-like receptor (KIR)-CD56+CD16-/+ phenotype, consistent with pseudo-mature NK cells. IL-21/IL-15-differentiated cells expressed high levels of mRNA for Bcl-2, GATA-3 and Id2, a master switch required for NK-cell development, and harboured un-rearranged TCRgamma genes. From a functional standpoint, IL-21/IL-15-treated cells secreted copious amounts of IFN-gamma, GM-CSF and CCL3/MIP-1alpha, and expressed cell surface CD107a upon contact with NK-sensitive tumour targets, a measure of exocytosis of NK secretory granules.

Conclusion: This study underpins a novel role for IL-21 in the differentiation of pseudo-mature lytic NK cells in a synergistic context with IL-15, and identifies a potential strategy to expand functional NK cells for immunotherapy.

Show MeSH
Related in: MedlinePlus