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HMGN2, a new anti-tumor effector molecule of CD8⁺ T cells.

Su L, Hu A, Luo Y, Zhou W, Zhang P, Feng Y - Mol. Cancer (2014)

Bottom Line: In this study, we isolated and cultured PBMCs and CD8⁺ T cells to analyze the expression and antitumor effects of HMGN2.This antitumor effect could be significantly blocked by using an anti-HMGN2 antibody.These results suggest that HMGN2 is an anti-tumor effector molecule of CD8⁺ T cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, Chengdu, Sichuan, China. pingzhang68@hotmail.com.

ABSTRACT

Background: Cytolytic T lymphocytes (CTL) and natural killer (NK) cells have been implicated as important cells in antitumor responses. Our previous research has shown that high mobility group nucleosomal-binding domain 2 (HMGN2) could be released by IL-2 and PHA stimulated peripheral blood mononuclear cells (PBMCs) and also induced tumor cells apoptosis at low doses. In this study, we isolated and cultured PBMCs and CD8⁺ T cells to analyze the expression and antitumor effects of HMGN2.

Methods: PBMCs from healthy donors were isolated using Human Lymphocyte Separation tube. CD8⁺ T cells were separated from the PBMCs using MoFlo XDP high-speed flow cytometry sorter. Activation of PBMCs and CD8⁺ T cells were achieved by stimulating with Phytohemagglutinin (PHA) or tumor antigen. In addition, the methods of ELISA, intracellular staining, and fluorescence-labeling assays were used.

Results: PHA induced PBMCs to release high levels of HMGN2, and CD8⁺ T cells was the major cell population in PBMCs that release HMGN2 after PHA activation. Tumor antigen-activated CD8⁺ T cells also released high levels of HMGN2. Supernatants of tumor antigen-activated CD8⁺ T cells were able to kill tumor cells in a dose-dependent manner. This antitumor effect could be significantly blocked by using an anti-HMGN2 antibody. Fluorescence-labeling assays showed that the supernatant proteins of activated CD8⁺ T cells could be transported into tumor cells, and the transport visibly decreased after HMGN2 was depleted by anti-HMGN2 antibody.

Conclusions: These results suggest that HMGN2 is an anti-tumor effector molecule of CD8⁺ T cells.

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PHA induced HMGN2 expression in different cell populations. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 20 μg/ml PHA or 100 IU/ml IL-2 for 72 hours. Normal medium was used as the control. Cells were collected and stained (A) Anti-CD4-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained; (B) Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. Figures are representative of three independent experiments. (C, D) Error bars represent HMGN2 intracellular expression positive rate (%) in CD4+ or CD8+ T cell populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to IL-2 and medium control (p < 0.05), #Significantly higher compared to CD4+ T cell population (p < 0.05). (E) Representative plots of the isotype staining and 2nd-Ab-FITC staining control.
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Figure 2: PHA induced HMGN2 expression in different cell populations. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 20 μg/ml PHA or 100 IU/ml IL-2 for 72 hours. Normal medium was used as the control. Cells were collected and stained (A) Anti-CD4-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained; (B) Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. Figures are representative of three independent experiments. (C, D) Error bars represent HMGN2 intracellular expression positive rate (%) in CD4+ or CD8+ T cell populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to IL-2 and medium control (p < 0.05), #Significantly higher compared to CD4+ T cell population (p < 0.05). (E) Representative plots of the isotype staining and 2nd-Ab-FITC staining control.

Mentions: To determine which T cell population expressed HMGN2, we stained the PHA pre-activated PBMCs with CD4-PE or CD8-PE surface staining. We then stained intracellular HMGN2 with rabbit anti-human HMGN2/goat anti-rabbit IgG-FITC. Results showed that HMGN2 expression significantly increased in both CD4+ and CD8+ T cell populations (Figure 2A, B, C). In addition, HMGN2 expression in CD8+ T cells (50.71 ± 10.34%) was significantly higher than that in CD4+ T cells (16.67 ± 5.61%) after PHA stimulation (Figure 2D). Finally, we used a MoFlo XDP high-speed flow cytometry sorter to purify PHA pre-activated CD3+CD8+ T cells, CD3+CD8- T cells and CD3- mix cells, followed by culturing in complete medium with 2000 IU/ml IL-2 for 5 days and analyzed the expression of HMGN2 by ELISA and intracellular staining. Results showed that HMGN2 was still expressed at high levels in CD8+ T cells (539.00 ± 118 ng/ml; 68.37 ± 15.21%). On the other hand, the expression of HMGN2 in the CD3+CD8- T cells (mainly CD4+ T cells) (307.67 ± 97.34 ng/ml; 36.23 ± 11.52%) and the CD3- mixed PBMCs population (386.67 ± 105.46 ng/ml; 35.73 ± 13.71%) was significantly lower in both supernatants and intercellular (Figure 3A, B and C). These results confirmed that activated CD8 + T cells expressed high levels of HMGN2.


HMGN2, a new anti-tumor effector molecule of CD8⁺ T cells.

Su L, Hu A, Luo Y, Zhou W, Zhang P, Feng Y - Mol. Cancer (2014)

PHA induced HMGN2 expression in different cell populations. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 20 μg/ml PHA or 100 IU/ml IL-2 for 72 hours. Normal medium was used as the control. Cells were collected and stained (A) Anti-CD4-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained; (B) Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. Figures are representative of three independent experiments. (C, D) Error bars represent HMGN2 intracellular expression positive rate (%) in CD4+ or CD8+ T cell populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to IL-2 and medium control (p < 0.05), #Significantly higher compared to CD4+ T cell population (p < 0.05). (E) Representative plots of the isotype staining and 2nd-Ab-FITC staining control.
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Figure 2: PHA induced HMGN2 expression in different cell populations. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 20 μg/ml PHA or 100 IU/ml IL-2 for 72 hours. Normal medium was used as the control. Cells were collected and stained (A) Anti-CD4-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained; (B) Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. Figures are representative of three independent experiments. (C, D) Error bars represent HMGN2 intracellular expression positive rate (%) in CD4+ or CD8+ T cell populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to IL-2 and medium control (p < 0.05), #Significantly higher compared to CD4+ T cell population (p < 0.05). (E) Representative plots of the isotype staining and 2nd-Ab-FITC staining control.
Mentions: To determine which T cell population expressed HMGN2, we stained the PHA pre-activated PBMCs with CD4-PE or CD8-PE surface staining. We then stained intracellular HMGN2 with rabbit anti-human HMGN2/goat anti-rabbit IgG-FITC. Results showed that HMGN2 expression significantly increased in both CD4+ and CD8+ T cell populations (Figure 2A, B, C). In addition, HMGN2 expression in CD8+ T cells (50.71 ± 10.34%) was significantly higher than that in CD4+ T cells (16.67 ± 5.61%) after PHA stimulation (Figure 2D). Finally, we used a MoFlo XDP high-speed flow cytometry sorter to purify PHA pre-activated CD3+CD8+ T cells, CD3+CD8- T cells and CD3- mix cells, followed by culturing in complete medium with 2000 IU/ml IL-2 for 5 days and analyzed the expression of HMGN2 by ELISA and intracellular staining. Results showed that HMGN2 was still expressed at high levels in CD8+ T cells (539.00 ± 118 ng/ml; 68.37 ± 15.21%). On the other hand, the expression of HMGN2 in the CD3+CD8- T cells (mainly CD4+ T cells) (307.67 ± 97.34 ng/ml; 36.23 ± 11.52%) and the CD3- mixed PBMCs population (386.67 ± 105.46 ng/ml; 35.73 ± 13.71%) was significantly lower in both supernatants and intercellular (Figure 3A, B and C). These results confirmed that activated CD8 + T cells expressed high levels of HMGN2.

Bottom Line: In this study, we isolated and cultured PBMCs and CD8⁺ T cells to analyze the expression and antitumor effects of HMGN2.This antitumor effect could be significantly blocked by using an anti-HMGN2 antibody.These results suggest that HMGN2 is an anti-tumor effector molecule of CD8⁺ T cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, Chengdu, Sichuan, China. pingzhang68@hotmail.com.

ABSTRACT

Background: Cytolytic T lymphocytes (CTL) and natural killer (NK) cells have been implicated as important cells in antitumor responses. Our previous research has shown that high mobility group nucleosomal-binding domain 2 (HMGN2) could be released by IL-2 and PHA stimulated peripheral blood mononuclear cells (PBMCs) and also induced tumor cells apoptosis at low doses. In this study, we isolated and cultured PBMCs and CD8⁺ T cells to analyze the expression and antitumor effects of HMGN2.

Methods: PBMCs from healthy donors were isolated using Human Lymphocyte Separation tube. CD8⁺ T cells were separated from the PBMCs using MoFlo XDP high-speed flow cytometry sorter. Activation of PBMCs and CD8⁺ T cells were achieved by stimulating with Phytohemagglutinin (PHA) or tumor antigen. In addition, the methods of ELISA, intracellular staining, and fluorescence-labeling assays were used.

Results: PHA induced PBMCs to release high levels of HMGN2, and CD8⁺ T cells was the major cell population in PBMCs that release HMGN2 after PHA activation. Tumor antigen-activated CD8⁺ T cells also released high levels of HMGN2. Supernatants of tumor antigen-activated CD8⁺ T cells were able to kill tumor cells in a dose-dependent manner. This antitumor effect could be significantly blocked by using an anti-HMGN2 antibody. Fluorescence-labeling assays showed that the supernatant proteins of activated CD8⁺ T cells could be transported into tumor cells, and the transport visibly decreased after HMGN2 was depleted by anti-HMGN2 antibody.

Conclusions: These results suggest that HMGN2 is an anti-tumor effector molecule of CD8⁺ T cells.

Show MeSH
Related in: MedlinePlus