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


CONCLUSIONS
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.
Bonanno
Giuseppina
G
Department of Gynaecology, Catholic University Medical School, Rome, Italy. giuseppina.bonanno@rm.unicatt.it
Mariotti
Andrea
A
Procoli
Annabella
A
Corallo
Maria
M
Scambia
Giovanni
G
Pierelli
Luca
L
Rutella
Sergio
S
D015550:Cell Degranulation / Q000187:drug effects / Q000502:physiology; D002454:Cell Differentiation; D019070:Cell Lineage / Q000276:immunology*; D054405:Chemokine CCL3 / Q000096:biosynthesis / Q000276:immunology; D005312:Fetal Blood / Q000166:cytology / Q000187:drug effects / Q000276:immunology*; D050990:GATA3 Transcription Factor / Q000276:immunology / Q000378:metabolism; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000276:immunology; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2 / Q000276:immunology / Q000378:metabolism; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology; D019409:Interleukin-15 / Q000494:pharmacology*; D007378:Interleukins / Q000276:immunology* / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000276:immunology* / Q000637:transplantation; D052118:Lysosomal-Associated Membrane Protein 1 / Q000276:immunology / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000276:immunology / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000819:agonists / Q000276:immunology / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000819:agonists / Q000276:immunology / Q000378:metabolism; D000098546:Interleukin-21
C515429:CCL3 protein, human; D054405:Chemokine CCL3; D050990:GATA3 Transcription Factor; C497450:ID2 protein, human; D051797:Inhibitor of Differentiation Protein 2; D019409:Interleukin-15; D007378:Interleukins; D052118:Lysosomal-Associated Membrane Protein 1; D019253:Proto-Oncogene Proteins c-bcl-2; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000098546:Interleukin-21