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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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Related in: MedlinePlus

PHA induced HMGN2 expression in PBMCs. 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. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and stained with anti-CD3-PE and anti-HMGN2-IgG-FITC, followed to run on a Beckman coulter FC500 Flow cytometry. Data were analyzed by using Submit 5.2 software after gate lymphocytes (R5) group on dot plot graph. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD3+ T cells. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to medium control (p < 0.05). (D) Representative plots of the isotype staining and 2nd-Ab-FITC staining control.
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Figure 1: PHA induced HMGN2 expression in PBMCs. 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. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and stained with anti-CD3-PE and anti-HMGN2-IgG-FITC, followed to run on a Beckman coulter FC500 Flow cytometry. Data were analyzed by using Submit 5.2 software after gate lymphocytes (R5) group on dot plot graph. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD3+ T cells. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to medium control (p < 0.05). (D) Representative plots of the isotype staining and 2nd-Ab-FITC staining control.

Mentions: PHA is a lectin found in plants, especially legumes, and is a mitogen that triggers T-lymphocyte cell division and activation. Supernatants of PBMCs that have been stimulated by PHA contain a series of effector proteins. In our experiments, PBMCs were isolated from healthy donors and stimulated with 20 μg/ml PHA or 100 IU/ml IL-2 for 72 hours. Supernatants were collected and HMGN2 concentrations were analyzed by ELISA. The results showed that PBMCs release high levels of HMGN2 (771.33 ± 123.12 ng/ml) following 20 μg/ml PHA stimulation for 72 hours. Compared with the medium control (289.67 ± 98.5 ng/ml) and IL-2 stimulated (397.67 ± 134.15 ng/ml), HMGN2 concentration significantly increased (Figure 1A). In order to make sure that HMGN2 was released by activated T cells, we used surface and indirect intracellular staining to analyze HMGN2 expression in CD3+ T cells. Consistent with the ELISA results (Figure 1A), compared with the medium control (9.06 ± 3.8%) and IL-2 stimulated (18.85 ± 8.71%), the percentage of HMGN2+CD3+ cell population significantly increased after PHA stimulation (47.79 ± 11.87%) (Figure 1B and C).


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 PBMCs. 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. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and stained with anti-CD3-PE and anti-HMGN2-IgG-FITC, followed to run on a Beckman coulter FC500 Flow cytometry. Data were analyzed by using Submit 5.2 software after gate lymphocytes (R5) group on dot plot graph. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD3+ T cells. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to medium control (p < 0.05). (D) Representative plots of the isotype staining and 2nd-Ab-FITC staining control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4126642&req=5

Figure 1: PHA induced HMGN2 expression in PBMCs. 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. (A) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. (B) The cells were removed and stained with anti-CD3-PE and anti-HMGN2-IgG-FITC, followed to run on a Beckman coulter FC500 Flow cytometry. Data were analyzed by using Submit 5.2 software after gate lymphocytes (R5) group on dot plot graph. Figures are representative of three independent experiments. (C) Error bars represent HMGN2 intracellular expression positive rate (%) in CD3+ T cells. Data are represented as means ± SD of three independent experiments. *Significantly higher compared to medium control (p < 0.05). (D) Representative plots of the isotype staining and 2nd-Ab-FITC staining control.
Mentions: PHA is a lectin found in plants, especially legumes, and is a mitogen that triggers T-lymphocyte cell division and activation. Supernatants of PBMCs that have been stimulated by PHA contain a series of effector proteins. In our experiments, PBMCs were isolated from healthy donors and stimulated with 20 μg/ml PHA or 100 IU/ml IL-2 for 72 hours. Supernatants were collected and HMGN2 concentrations were analyzed by ELISA. The results showed that PBMCs release high levels of HMGN2 (771.33 ± 123.12 ng/ml) following 20 μg/ml PHA stimulation for 72 hours. Compared with the medium control (289.67 ± 98.5 ng/ml) and IL-2 stimulated (397.67 ± 134.15 ng/ml), HMGN2 concentration significantly increased (Figure 1A). In order to make sure that HMGN2 was released by activated T cells, we used surface and indirect intracellular staining to analyze HMGN2 expression in CD3+ T cells. Consistent with the ELISA results (Figure 1A), compared with the medium control (9.06 ± 3.8%) and IL-2 stimulated (18.85 ± 8.71%), the percentage of HMGN2+CD3+ cell population significantly increased after PHA stimulation (47.79 ± 11.87%) (Figure 1B and C).

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