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Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15.

Giuliani M, Giron-Michel J, Negrini S, Vacca P, Durali D, Caignard A, Le Bousse-Kerdiles C, Chouaib S, Devocelle A, Bahri R, Durrbach A, Taoufik Y, Ferrini S, Croce M, Mingari MC, Moretta L, Azzarone B - PLoS ONE (2008)

Bottom Line: Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes.In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors.In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR 542, Université de Paris XI, Hôpital Paul Brousse, Villejuif, France.

ABSTRACT

Background: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated.

Methodology/principal findings: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells.

Conclusions/significance: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.

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Autonomous NK commitment of PB-HP: morphology and expression of cytolytic granules.(A) Ral 555 staining on 3-weeks-old PB-HP cultured in in STEMα Α medium supplemented with 100 ng/ml of FL and SCF. PB-NKP exhibit a lymphoid morphology. (B) Intracytoplasmic expression of perforin and granzyme B in PB-NKP and in PB activated conventional NK cells was analyzed by confocal microscopy. Both cell types were fixed, permeabilized, and stained for perforin and granzyme B. As negative controls, cells were incubated with mouse IgG, and the second reagent. (C) Analysis of the cytolytic activity of freshly purified NK cells from healthy donors (open boxes) and from PB-NKP (grey boxes) against 51Cr-labelled K562 cells. NK cell effectors, treated or not for 18 hours with IL-12 (5 ng/ml), were assayed in a 4-h 51Cr-release assay against K562 cells in a dilution of E/T cell ratios (10∶1) in duplicate wells. Data are representative of three experiments performed.
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pone-0002241-g003: Autonomous NK commitment of PB-HP: morphology and expression of cytolytic granules.(A) Ral 555 staining on 3-weeks-old PB-HP cultured in in STEMα Α medium supplemented with 100 ng/ml of FL and SCF. PB-NKP exhibit a lymphoid morphology. (B) Intracytoplasmic expression of perforin and granzyme B in PB-NKP and in PB activated conventional NK cells was analyzed by confocal microscopy. Both cell types were fixed, permeabilized, and stained for perforin and granzyme B. As negative controls, cells were incubated with mouse IgG, and the second reagent. (C) Analysis of the cytolytic activity of freshly purified NK cells from healthy donors (open boxes) and from PB-NKP (grey boxes) against 51Cr-labelled K562 cells. NK cell effectors, treated or not for 18 hours with IL-12 (5 ng/ml), were assayed in a 4-h 51Cr-release assay against K562 cells in a dilution of E/T cell ratios (10∶1) in duplicate wells. Data are representative of three experiments performed.

Mentions: Phenotypical characterization was strengthened by morphological analysis showing that after three weeks in culture PB-HP autonomously committed into the NK pathway display a typical lymphoid morphology (Fig. 3A). In addition, confocal microcopy demonstrated a spotted intracytoplasmic expression of perforin and granzyme B, suggesting their localization within granule-like structures as observed in conventional activated NK cells (Fig. 3B and 3C).


Generation of a novel regulatory NK cell subset from peripheral blood CD34+ progenitors promoted by membrane-bound IL-15.

Giuliani M, Giron-Michel J, Negrini S, Vacca P, Durali D, Caignard A, Le Bousse-Kerdiles C, Chouaib S, Devocelle A, Bahri R, Durrbach A, Taoufik Y, Ferrini S, Croce M, Mingari MC, Moretta L, Azzarone B - PLoS ONE (2008)

Autonomous NK commitment of PB-HP: morphology and expression of cytolytic granules.(A) Ral 555 staining on 3-weeks-old PB-HP cultured in in STEMα Α medium supplemented with 100 ng/ml of FL and SCF. PB-NKP exhibit a lymphoid morphology. (B) Intracytoplasmic expression of perforin and granzyme B in PB-NKP and in PB activated conventional NK cells was analyzed by confocal microscopy. Both cell types were fixed, permeabilized, and stained for perforin and granzyme B. As negative controls, cells were incubated with mouse IgG, and the second reagent. (C) Analysis of the cytolytic activity of freshly purified NK cells from healthy donors (open boxes) and from PB-NKP (grey boxes) against 51Cr-labelled K562 cells. NK cell effectors, treated or not for 18 hours with IL-12 (5 ng/ml), were assayed in a 4-h 51Cr-release assay against K562 cells in a dilution of E/T cell ratios (10∶1) in duplicate wells. Data are representative of three experiments performed.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376096&req=5

pone-0002241-g003: Autonomous NK commitment of PB-HP: morphology and expression of cytolytic granules.(A) Ral 555 staining on 3-weeks-old PB-HP cultured in in STEMα Α medium supplemented with 100 ng/ml of FL and SCF. PB-NKP exhibit a lymphoid morphology. (B) Intracytoplasmic expression of perforin and granzyme B in PB-NKP and in PB activated conventional NK cells was analyzed by confocal microscopy. Both cell types were fixed, permeabilized, and stained for perforin and granzyme B. As negative controls, cells were incubated with mouse IgG, and the second reagent. (C) Analysis of the cytolytic activity of freshly purified NK cells from healthy donors (open boxes) and from PB-NKP (grey boxes) against 51Cr-labelled K562 cells. NK cell effectors, treated or not for 18 hours with IL-12 (5 ng/ml), were assayed in a 4-h 51Cr-release assay against K562 cells in a dilution of E/T cell ratios (10∶1) in duplicate wells. Data are representative of three experiments performed.
Mentions: Phenotypical characterization was strengthened by morphological analysis showing that after three weeks in culture PB-HP autonomously committed into the NK pathway display a typical lymphoid morphology (Fig. 3A). In addition, confocal microcopy demonstrated a spotted intracytoplasmic expression of perforin and granzyme B, suggesting their localization within granule-like structures as observed in conventional activated NK cells (Fig. 3B and 3C).

Bottom Line: Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes.In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors.In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR 542, Université de Paris XI, Hôpital Paul Brousse, Villejuif, France.

ABSTRACT

Background: NK cells have been long time considered as cytotoxic lymphocytes competent in killing virus-infected cells and tumors. However, NK cells may also play essential immuno-regulatory functions. In this context, the real existence of a defined NK subset with negative regulatory properties has been hypothesized but never clearly demonstrated.

Methodology/principal findings: Herein, we show the in vitro generation from human peripheral blood haematopoietic progenitors (PB-HP), of a novel subset of non-cytolytic NK cells displaying a mature phenotype and remarkable immuno-regulatory functions (NK-ireg). The main functional hallmark of these NK-ireg cells is represented by the surface expression/release of HLA-G, a major immunosuppressive molecule. In addition, NK-ireg cells secrete two powerful immuno-regulatory factors: IL-10 and IL-21. Through these factors, NK-ireg cells act as effectors of the down-regulation of the immune response: reconverting mature myeloid DC (mDC) into immature/tolerogenic DC, blocking cytolytic functions on conventional NK cells and inducing HLA-G membrane expression on PB-derived monocytes. The generation of "NK-ireg" cells is obtained, by default, in culture conditions favouring cell-to-cell contacts, and it is strictly dependent on reciprocal trans-presentation of membrane-bound IL-15 forms constitutively and selectively expressed by human CD34(+) PB-HP. Finally, a small subset of NKp46(+) HLA-G(+) IL-10(+) is detected within freshly isolated decidual NK cells, suggesting that these cells could represent an in vivo counterpart of the NK-ireg cells.

Conclusions/significance: In conclusion, NK-ireg cells represent a novel truly differentiated non-cytolytic NK subset with a self-sustainable phenotype (CD56(+) CD16(+) NKp30(+) NKp44(+) NKp46(+) CD94(+) CD69(+) CCR7(+)) generated from specific pSTAT6(+) GATA3(+) precursors. NK-ireg cells could be employed to develop new immuno-suppressive strategies in autoimmune diseases, transplant rejection or graft versus host diseases. In addition, NK-ireg cells can be easily derived from peripheral blood of the patients and could constitute an autologous biotherapic tool to be used combined or in alternative to other immuno-regulatory cells.

Show MeSH
Related in: MedlinePlus