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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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Molecular profile of CD34-lineage- cells differentiated with IL-15 plus IL-21. Panel A: Molecular features of cytokine-differentiated CD34-lineage- cells. The expression of mRNA specific for the NK-associated transcription factor Id2 and for Bcl-2 and GATA-3 was investigated by quantitative PCR (qPCR) in freshly isolated (day 0) and cytokine-differentiated CD34-lineage- cells (week +4), as previously detailed [5]. CD34+ cells from the same UCB samples were used as control. Panel B: TCRγ rearrangement status after in vitro exposure to IL-15 plus IL-21. The analysis of TCRγ rearrangement status was performed as detailed in Materials and Methods. Peripheral blood CD3+ T cells from healthy donors were used as control for TCRγ rearrangement status. CD34-lineage- cells cultured with IL-15 and IL-21 harboured un-rearranged TCRγ genes, similar to freshly isolated CD34-lineage- cells (day 0) and to cells maintained with SCF and Flt3-L either alone or supplemented with IL-21. M = marker. JP = rearrangement involving the Jγ and Cγ segments. The forward primer mapped to the JγP cassette, whereas the reverse primer mapped to the constant (C) region. JP1 = rearrangement involving the JγP1 region and the Cγ segment. The forward primer mapped to the JγP1 cassette, whereas the reverse primer mapped to the constant (C) region. JP2 = rearrangement involving the JγP2 region and the Cγ segment. The forward primer mapped to the JγP2 cassette, whereas the reverse primer mapped to the constant (C) region. J1/2 = rearrangement involving the Jγ1/2 region and the Cγ segment. The forward primer mapped to the Jγ1–2 cassette, whereas the reverse primer mapped to the constant (C) region. L = linker region (no rearrangement). Forward and reverse primers mapped to the spacer region between Vγ9 and Vγ10 segments.
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Figure 4: Molecular profile of CD34-lineage- cells differentiated with IL-15 plus IL-21. Panel A: Molecular features of cytokine-differentiated CD34-lineage- cells. The expression of mRNA specific for the NK-associated transcription factor Id2 and for Bcl-2 and GATA-3 was investigated by quantitative PCR (qPCR) in freshly isolated (day 0) and cytokine-differentiated CD34-lineage- cells (week +4), as previously detailed [5]. CD34+ cells from the same UCB samples were used as control. Panel B: TCRγ rearrangement status after in vitro exposure to IL-15 plus IL-21. The analysis of TCRγ rearrangement status was performed as detailed in Materials and Methods. Peripheral blood CD3+ T cells from healthy donors were used as control for TCRγ rearrangement status. CD34-lineage- cells cultured with IL-15 and IL-21 harboured un-rearranged TCRγ genes, similar to freshly isolated CD34-lineage- cells (day 0) and to cells maintained with SCF and Flt3-L either alone or supplemented with IL-21. M = marker. JP = rearrangement involving the Jγ and Cγ segments. The forward primer mapped to the JγP cassette, whereas the reverse primer mapped to the constant (C) region. JP1 = rearrangement involving the JγP1 region and the Cγ segment. The forward primer mapped to the JγP1 cassette, whereas the reverse primer mapped to the constant (C) region. JP2 = rearrangement involving the JγP2 region and the Cγ segment. The forward primer mapped to the JγP2 cassette, whereas the reverse primer mapped to the constant (C) region. J1/2 = rearrangement involving the Jγ1/2 region and the Cγ segment. The forward primer mapped to the Jγ1–2 cassette, whereas the reverse primer mapped to the constant (C) region. L = linker region (no rearrangement). Forward and reverse primers mapped to the spacer region between Vγ9 and Vγ10 segments.

Mentions: We next aimed to get insights into the expression levels of mRNA encoding for NK-associated transcription factors. The provision of IL-15 and IL-21 to CD34-lineage- cells was associated with up-regulated mRNA signals for Bcl-2, GATA-3 and, perhaps more importantly, Id2, a master switch implicated in the commitment of bi-potent foetal thymus T/NK progenitors to NK cells (Figure 4A) [22]. Notably, Bcl-2 induction by IL-21 has also been demonstrated in T cells, leading to their enhanced survival through the activation of the PI-3K signalling pathway [23].


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)

Molecular profile of CD34-lineage- cells differentiated with IL-15 plus IL-21. Panel A: Molecular features of cytokine-differentiated CD34-lineage- cells. The expression of mRNA specific for the NK-associated transcription factor Id2 and for Bcl-2 and GATA-3 was investigated by quantitative PCR (qPCR) in freshly isolated (day 0) and cytokine-differentiated CD34-lineage- cells (week +4), as previously detailed [5]. CD34+ cells from the same UCB samples were used as control. Panel B: TCRγ rearrangement status after in vitro exposure to IL-15 plus IL-21. The analysis of TCRγ rearrangement status was performed as detailed in Materials and Methods. Peripheral blood CD3+ T cells from healthy donors were used as control for TCRγ rearrangement status. CD34-lineage- cells cultured with IL-15 and IL-21 harboured un-rearranged TCRγ genes, similar to freshly isolated CD34-lineage- cells (day 0) and to cells maintained with SCF and Flt3-L either alone or supplemented with IL-21. M = marker. JP = rearrangement involving the Jγ and Cγ segments. The forward primer mapped to the JγP cassette, whereas the reverse primer mapped to the constant (C) region. JP1 = rearrangement involving the JγP1 region and the Cγ segment. The forward primer mapped to the JγP1 cassette, whereas the reverse primer mapped to the constant (C) region. JP2 = rearrangement involving the JγP2 region and the Cγ segment. The forward primer mapped to the JγP2 cassette, whereas the reverse primer mapped to the constant (C) region. J1/2 = rearrangement involving the Jγ1/2 region and the Cγ segment. The forward primer mapped to the Jγ1–2 cassette, whereas the reverse primer mapped to the constant (C) region. L = linker region (no rearrangement). Forward and reverse primers mapped to the spacer region between Vγ9 and Vγ10 segments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Molecular profile of CD34-lineage- cells differentiated with IL-15 plus IL-21. Panel A: Molecular features of cytokine-differentiated CD34-lineage- cells. The expression of mRNA specific for the NK-associated transcription factor Id2 and for Bcl-2 and GATA-3 was investigated by quantitative PCR (qPCR) in freshly isolated (day 0) and cytokine-differentiated CD34-lineage- cells (week +4), as previously detailed [5]. CD34+ cells from the same UCB samples were used as control. Panel B: TCRγ rearrangement status after in vitro exposure to IL-15 plus IL-21. The analysis of TCRγ rearrangement status was performed as detailed in Materials and Methods. Peripheral blood CD3+ T cells from healthy donors were used as control for TCRγ rearrangement status. CD34-lineage- cells cultured with IL-15 and IL-21 harboured un-rearranged TCRγ genes, similar to freshly isolated CD34-lineage- cells (day 0) and to cells maintained with SCF and Flt3-L either alone or supplemented with IL-21. M = marker. JP = rearrangement involving the Jγ and Cγ segments. The forward primer mapped to the JγP cassette, whereas the reverse primer mapped to the constant (C) region. JP1 = rearrangement involving the JγP1 region and the Cγ segment. The forward primer mapped to the JγP1 cassette, whereas the reverse primer mapped to the constant (C) region. JP2 = rearrangement involving the JγP2 region and the Cγ segment. The forward primer mapped to the JγP2 cassette, whereas the reverse primer mapped to the constant (C) region. J1/2 = rearrangement involving the Jγ1/2 region and the Cγ segment. The forward primer mapped to the Jγ1–2 cassette, whereas the reverse primer mapped to the constant (C) region. L = linker region (no rearrangement). Forward and reverse primers mapped to the spacer region between Vγ9 and Vγ10 segments.
Mentions: We next aimed to get insights into the expression levels of mRNA encoding for NK-associated transcription factors. The provision of IL-15 and IL-21 to CD34-lineage- cells was associated with up-regulated mRNA signals for Bcl-2, GATA-3 and, perhaps more importantly, Id2, a master switch implicated in the commitment of bi-potent foetal thymus T/NK progenitors to NK cells (Figure 4A) [22]. Notably, Bcl-2 induction by IL-21 has also been demonstrated in T cells, leading to their enhanced survival through the activation of the PI-3K signalling pathway [23].

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