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Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity

View Article: PubMed Central - PubMed

ABSTRACT

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2−/− mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2−/− HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2−/− mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction.

No MeSH data available.


Increased NK cell differentiation of SOCS2−/− HPCs in vitro.(A,B) NK cells were differentiated in vitro from HPCs of WT and SOCS2−/− mice. After maturation (mNK), CD3 and NK1.1 expression were analyzed by flow cytometry. Data for days 0, 3 and 6 pooled from four experiments. *p < 0.05, **p < 0.01. (C) Differentiated WT and SOCS2−/− NK cells were mixed at the indicated ratio with YAC-1 target cells and NK cytotoxicity was determined by a 51Cr release assay. (D) IFN- γ production of differentiated WT and SOCS2−/− NK cells which are stimulated with IL-12 (20 ng/ml). *p < 0.05, **p < 0.01. (E) Splenic WT and SOCS2−/− NK cells were cultured with IL-2 (30 ng/ml) for 16h. The cytotoxicity of NK cells was determined by a 51Cr release assay against YAC-1 target cells at the indicated E:T ratios. (F) IFN- γ production of splenic WT or SOCS2−/− NK cells was quantified by ELISA. *p < 0.05, **p < 0.01. The data are representative of three independent experiments, and the error bars represent the SD of triplicates.
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f2: Increased NK cell differentiation of SOCS2−/− HPCs in vitro.(A,B) NK cells were differentiated in vitro from HPCs of WT and SOCS2−/− mice. After maturation (mNK), CD3 and NK1.1 expression were analyzed by flow cytometry. Data for days 0, 3 and 6 pooled from four experiments. *p < 0.05, **p < 0.01. (C) Differentiated WT and SOCS2−/− NK cells were mixed at the indicated ratio with YAC-1 target cells and NK cytotoxicity was determined by a 51Cr release assay. (D) IFN- γ production of differentiated WT and SOCS2−/− NK cells which are stimulated with IL-12 (20 ng/ml). *p < 0.05, **p < 0.01. (E) Splenic WT and SOCS2−/− NK cells were cultured with IL-2 (30 ng/ml) for 16h. The cytotoxicity of NK cells was determined by a 51Cr release assay against YAC-1 target cells at the indicated E:T ratios. (F) IFN- γ production of splenic WT or SOCS2−/− NK cells was quantified by ELISA. *p < 0.05, **p < 0.01. The data are representative of three independent experiments, and the error bars represent the SD of triplicates.

Mentions: SOCS2 is a well-known negative regulator of the cytokine-induced signaling pathway. Because IL-15 is an essential cytokine for NK cell development28, we examined whether SOCS2 deficiency influenced IL-15–induced NK cell differentiation of hematopoietic progenitor cells (Lineage−c-Kit+/HPCs). HPCs obtained from WT and SOCS2−/− mice were differentiated into NK cells in vitro and analyzed by flow cytometry. The population of CD3−NK1.1+ cells from HPCs of SOCS2−/− mice was significantly increased (Fig. 2A,B). To investigate whether differentiated NK cells from SOCS2−/− HPCs show NK cell activity, we performed a cytotoxicity assay using NK cells and measured the secretion of cytokines. As shown in Fig. 2C, the differentiated total cells from SOCS2−/− HPCs showed an enhanced capacity to lyse target cells, which may have been due to the high frequency of NK cells among total cells. The differentiated total cells from SOCS2−/− HPCs also showed markedly increased IFN-γ levels in the culture medium (Fig. 2D). To determine the effects of SOCS2−/− deficiency on the activity of primary NK cells, we performed cytolytic activity assays on isolated DX5+ NK cells from WT and SOCS2−/− mice in vitro. The same number of SOCS2−/− NK cells had no significant effect on NK cell cytolytic activity and IFN-γ secretion when compared with WT NK cells in vitro (Fig. 2E,F). Therefore, these results indicate that SOCS2 serves as a negative regulator of NK cell differentiation but does not affect the functional activity of differentiated NK cells.


Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity
Increased NK cell differentiation of SOCS2−/− HPCs in vitro.(A,B) NK cells were differentiated in vitro from HPCs of WT and SOCS2−/− mice. After maturation (mNK), CD3 and NK1.1 expression were analyzed by flow cytometry. Data for days 0, 3 and 6 pooled from four experiments. *p < 0.05, **p < 0.01. (C) Differentiated WT and SOCS2−/− NK cells were mixed at the indicated ratio with YAC-1 target cells and NK cytotoxicity was determined by a 51Cr release assay. (D) IFN- γ production of differentiated WT and SOCS2−/− NK cells which are stimulated with IL-12 (20 ng/ml). *p < 0.05, **p < 0.01. (E) Splenic WT and SOCS2−/− NK cells were cultured with IL-2 (30 ng/ml) for 16h. The cytotoxicity of NK cells was determined by a 51Cr release assay against YAC-1 target cells at the indicated E:T ratios. (F) IFN- γ production of splenic WT or SOCS2−/− NK cells was quantified by ELISA. *p < 0.05, **p < 0.01. The data are representative of three independent experiments, and the error bars represent the SD of triplicates.
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f2: Increased NK cell differentiation of SOCS2−/− HPCs in vitro.(A,B) NK cells were differentiated in vitro from HPCs of WT and SOCS2−/− mice. After maturation (mNK), CD3 and NK1.1 expression were analyzed by flow cytometry. Data for days 0, 3 and 6 pooled from four experiments. *p < 0.05, **p < 0.01. (C) Differentiated WT and SOCS2−/− NK cells were mixed at the indicated ratio with YAC-1 target cells and NK cytotoxicity was determined by a 51Cr release assay. (D) IFN- γ production of differentiated WT and SOCS2−/− NK cells which are stimulated with IL-12 (20 ng/ml). *p < 0.05, **p < 0.01. (E) Splenic WT and SOCS2−/− NK cells were cultured with IL-2 (30 ng/ml) for 16h. The cytotoxicity of NK cells was determined by a 51Cr release assay against YAC-1 target cells at the indicated E:T ratios. (F) IFN- γ production of splenic WT or SOCS2−/− NK cells was quantified by ELISA. *p < 0.05, **p < 0.01. The data are representative of three independent experiments, and the error bars represent the SD of triplicates.
Mentions: SOCS2 is a well-known negative regulator of the cytokine-induced signaling pathway. Because IL-15 is an essential cytokine for NK cell development28, we examined whether SOCS2 deficiency influenced IL-15–induced NK cell differentiation of hematopoietic progenitor cells (Lineage−c-Kit+/HPCs). HPCs obtained from WT and SOCS2−/− mice were differentiated into NK cells in vitro and analyzed by flow cytometry. The population of CD3−NK1.1+ cells from HPCs of SOCS2−/− mice was significantly increased (Fig. 2A,B). To investigate whether differentiated NK cells from SOCS2−/− HPCs show NK cell activity, we performed a cytotoxicity assay using NK cells and measured the secretion of cytokines. As shown in Fig. 2C, the differentiated total cells from SOCS2−/− HPCs showed an enhanced capacity to lyse target cells, which may have been due to the high frequency of NK cells among total cells. The differentiated total cells from SOCS2−/− HPCs also showed markedly increased IFN-γ levels in the culture medium (Fig. 2D). To determine the effects of SOCS2−/− deficiency on the activity of primary NK cells, we performed cytolytic activity assays on isolated DX5+ NK cells from WT and SOCS2−/− mice in vitro. The same number of SOCS2−/− NK cells had no significant effect on NK cell cytolytic activity and IFN-γ secretion when compared with WT NK cells in vitro (Fig. 2E,F). Therefore, these results indicate that SOCS2 serves as a negative regulator of NK cell differentiation but does not affect the functional activity of differentiated NK cells.

View Article: PubMed Central - PubMed

ABSTRACT

Suppressor of cytokine signaling (SOCS) proteins are negative regulators of cytokine responses. Although recent reports have shown regulatory roles for SOCS proteins in innate and adaptive immunity, their roles in natural killer (NK) cell development are largely unknown. Here, we show that SOCS2 is involved in NK cell development. SOCS2&minus;/&minus; mice showed a high frequency of NK cells in the bone marrow and spleen. Knockdown of SOCS2 was associated with enhanced differentiation of NK cells in vitro, and the transplantation of hematopoietic stem cells (HSCs) into congenic mice resulted in enhanced differentiation in SOCS2&minus;/&minus; HSCs. We found that SOCS2 could inhibit Janus kinase 2 (JAK2) activity and JAK2-STAT5 signaling pathways via direct interaction with JAK2. Furthermore, SOCS2&minus;/&minus; mice showed a reduction in lung metastases and an increase in survival following melanoma challenge. Overall, our findings suggest that SOCS2 negatively regulates the development of NK cells by inhibiting JAK2 activity via direct interaction.

No MeSH data available.