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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 was mainly expressed in CD8+ T cell population. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 20 μg/ml PHA for 72 hours. Cells were collected and stained with CD3-FITC and CD8-PE. CD3+CD8+T cells (Gate R1), CD3+CD8-T cells (Gate R2) and CD3- mixed cells (Gate R3) were purified with MoFlo XDP sorter. 4 × 106 CD3+CD8+T cells, CD3+CD8-T cells and CD3- mixed cells were incubated in 24-well plates respectively for 5 days. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD8+ T cells populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to CD8-CD3+ and MIX cells (p < 0.05).
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Figure 3: PHA-induced HMGN2 was mainly expressed in CD8+ T cell population. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 20 μg/ml PHA for 72 hours. Cells were collected and stained with CD3-FITC and CD8-PE. CD3+CD8+T cells (Gate R1), CD3+CD8-T cells (Gate R2) and CD3- mixed cells (Gate R3) were purified with MoFlo XDP sorter. 4 × 106 CD3+CD8+T cells, CD3+CD8-T cells and CD3- mixed cells were incubated in 24-well plates respectively for 5 days. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD8+ T cells populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to CD8-CD3+ and MIX cells (p < 0.05).

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 was mainly expressed in CD8+ T cell population. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 20 μg/ml PHA for 72 hours. Cells were collected and stained with CD3-FITC and CD8-PE. CD3+CD8+T cells (Gate R1), CD3+CD8-T cells (Gate R2) and CD3- mixed cells (Gate R3) were purified with MoFlo XDP sorter. 4 × 106 CD3+CD8+T cells, CD3+CD8-T cells and CD3- mixed cells were incubated in 24-well plates respectively for 5 days. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD8+ T cells populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to CD8-CD3+ and MIX cells (p < 0.05).
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Figure 3: PHA-induced HMGN2 was mainly expressed in CD8+ T cell population. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 20 μg/ml PHA for 72 hours. Cells were collected and stained with CD3-FITC and CD8-PE. CD3+CD8+T cells (Gate R1), CD3+CD8-T cells (Gate R2) and CD3- mixed cells (Gate R3) were purified with MoFlo XDP sorter. 4 × 106 CD3+CD8+T cells, CD3+CD8-T cells and CD3- mixed cells were incubated in 24-well plates respectively for 5 days. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD8+ T cells populations. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to CD8-CD3+ and MIX cells (p < 0.05).
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