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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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NKPSG cells secrete immuno-regulatory factors.(A) Detection of IL-10 production by three-weeks-old NKPSG cells using : RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. ELISA assay. Conventional PB-NK cells and BM-MSC were used as negative controls while LPS- activated PB-macrophages were used as positive control. Western blot. recombinant IL-10 was used as positive control. Flow cytometry. Detection of membrane bound IL-10 is represented with continuous black line. Isotype control (shadowed peak). (B) Detection of IL-21 expression on three-weeks-old NKPSG cells using: RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. Western blot. IL-21 expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for IL-21. HL-60 cell line and recombinant IL-21 were used as positive controls. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. (C) Detection of HLA-G expression on three-weeks-old NKPSG cells using: Western blot. HLA-G expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for HLA-G. M8-HLA-G1 cell line was used as positive control. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. Flow cytometry. Detection of surface and intracellular expression of HLA-G on NKPSG cells is represented with continuous black line. Isotype control (shadowed peaks). The data are representative of three separate experiments. Confocal microscopy. As negative controls, NKPSG cells were incubated with Isotype-matched IgG1-FITC.
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pone-0002241-g008: NKPSG cells secrete immuno-regulatory factors.(A) Detection of IL-10 production by three-weeks-old NKPSG cells using : RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. ELISA assay. Conventional PB-NK cells and BM-MSC were used as negative controls while LPS- activated PB-macrophages were used as positive control. Western blot. recombinant IL-10 was used as positive control. Flow cytometry. Detection of membrane bound IL-10 is represented with continuous black line. Isotype control (shadowed peak). (B) Detection of IL-21 expression on three-weeks-old NKPSG cells using: RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. Western blot. IL-21 expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for IL-21. HL-60 cell line and recombinant IL-21 were used as positive controls. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. (C) Detection of HLA-G expression on three-weeks-old NKPSG cells using: Western blot. HLA-G expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for HLA-G. M8-HLA-G1 cell line was used as positive control. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. Flow cytometry. Detection of surface and intracellular expression of HLA-G on NKPSG cells is represented with continuous black line. Isotype control (shadowed peaks). The data are representative of three separate experiments. Confocal microscopy. As negative controls, NKPSG cells were incubated with Isotype-matched IgG1-FITC.

Mentions: Since the supernatant of NKPSG cells displayed immuno-regulatory properties, we attempted to identify factors produced by NKPSG cells that could exert tolerogenic/immuno-suppressive effects. RT-PCR analysis (Fig. 8A) revealed that NKPSG cells expressed the specific transcripts for IL-10 (403 bp). Production of IL-10 was confirmed by the detection of discrete concentrations of IL-10 (30–40 pg/ml) in the cell supernatants of NKPSG cells by ELISA (Fig. 8A). As negative control, we used primary cultures of human BM-derived mesenchymal stem cells [32] and IL-2 stimulated conventional NK cells, while human PB-derived monocytes stimulated with LPS were used as positive controls. In addition, Western blot analysis shows the presence of the specific 36 kDa band (Fig. 8A), and flow cytometry analysis shows that more than 80% of NKPSG cells express IL-10 as a membrane-associated form, similarly to what previously reported in human monocytes releasing IL-10 [23] (Fig. 8A).


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)

NKPSG cells secrete immuno-regulatory factors.(A) Detection of IL-10 production by three-weeks-old NKPSG cells using : RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. ELISA assay. Conventional PB-NK cells and BM-MSC were used as negative controls while LPS- activated PB-macrophages were used as positive control. Western blot. recombinant IL-10 was used as positive control. Flow cytometry. Detection of membrane bound IL-10 is represented with continuous black line. Isotype control (shadowed peak). (B) Detection of IL-21 expression on three-weeks-old NKPSG cells using: RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. Western blot. IL-21 expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for IL-21. HL-60 cell line and recombinant IL-21 were used as positive controls. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. (C) Detection of HLA-G expression on three-weeks-old NKPSG cells using: Western blot. HLA-G expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for HLA-G. M8-HLA-G1 cell line was used as positive control. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. Flow cytometry. Detection of surface and intracellular expression of HLA-G on NKPSG cells is represented with continuous black line. Isotype control (shadowed peaks). The data are representative of three separate experiments. Confocal microscopy. As negative controls, NKPSG cells were incubated with Isotype-matched IgG1-FITC.
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pone-0002241-g008: NKPSG cells secrete immuno-regulatory factors.(A) Detection of IL-10 production by three-weeks-old NKPSG cells using : RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. ELISA assay. Conventional PB-NK cells and BM-MSC were used as negative controls while LPS- activated PB-macrophages were used as positive control. Western blot. recombinant IL-10 was used as positive control. Flow cytometry. Detection of membrane bound IL-10 is represented with continuous black line. Isotype control (shadowed peak). (B) Detection of IL-21 expression on three-weeks-old NKPSG cells using: RT-PCR analysis. The human neuroblastoma cell line Neuro2a was used as negative control, while human PBL activated with PHA and PMA were used as positive control. Amplification of the same cDNAs with β-actin-specific primers is also shown. Western blot. IL-21 expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for IL-21. HL-60 cell line and recombinant IL-21 were used as positive controls. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. (C) Detection of HLA-G expression on three-weeks-old NKPSG cells using: Western blot. HLA-G expression was analyzed in total cell lysates (TL) and cell supernantants (SN) from NKPSG cells using mAbs specific for HLA-G. M8-HLA-G1 cell line was used as positive control. Conventional PB-NK cells were used as negative control. The data are representative of three separate experiments. Flow cytometry. Detection of surface and intracellular expression of HLA-G on NKPSG cells is represented with continuous black line. Isotype control (shadowed peaks). The data are representative of three separate experiments. Confocal microscopy. As negative controls, NKPSG cells were incubated with Isotype-matched IgG1-FITC.
Mentions: Since the supernatant of NKPSG cells displayed immuno-regulatory properties, we attempted to identify factors produced by NKPSG cells that could exert tolerogenic/immuno-suppressive effects. RT-PCR analysis (Fig. 8A) revealed that NKPSG cells expressed the specific transcripts for IL-10 (403 bp). Production of IL-10 was confirmed by the detection of discrete concentrations of IL-10 (30–40 pg/ml) in the cell supernatants of NKPSG cells by ELISA (Fig. 8A). As negative control, we used primary cultures of human BM-derived mesenchymal stem cells [32] and IL-2 stimulated conventional NK cells, while human PB-derived monocytes stimulated with LPS were used as positive controls. In addition, Western blot analysis shows the presence of the specific 36 kDa band (Fig. 8A), and flow cytometry analysis shows that more than 80% of NKPSG cells express IL-10 as a membrane-associated form, similarly to what previously reported in human monocytes releasing IL-10 [23] (Fig. 8A).

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