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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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HMGN2 expression in Tumor antigen stimulated PBMCs. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 150 μg/ml T-Ag for 7 days. Normal medium was used as the control. (A) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (B) Error bars represent HMGN2 intracellular expression positive rate (%) in PBMCs after stimulation with T-Ag. (C) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. Data are represented as means ± SD of three independent experiments.
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Figure 4: HMGN2 expression in Tumor antigen stimulated PBMCs. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 150 μg/ml T-Ag for 7 days. Normal medium was used as the control. (A) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (B) Error bars represent HMGN2 intracellular expression positive rate (%) in PBMCs after stimulation with T-Ag. (C) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. Data are represented as means ± SD of three independent experiments.

Mentions: In order to ensure that HMGN2 was an effector protein of tumor antigen activated T cells, we used tumor full protein as tumor antigen (T-Ag) to stimulate PBMCs for 7 days. Supernatants were collected for assaying HMGN2 levels by ELISA and cells were collected for HMGN2 intracellular staining. Results showed that there was no significant change of HMGN2 expression after stimulation with T-Ag compared with the medium control (Figure 4). In order to ensure that T cells were activated by T-Ag, we used CD44high as the activated marker of T cells. T-Ag stimulated PBMCs were stained with CD8-PE/CD44-APC surface and HMGN2 indirect intracellular staining. CD44high was used as the activated T cell population (Figure 5Aa, gate R2) and CD44low was used as the naive T cell population (Figure 5Aa, gate R6). T-Ag induced only 18.35 ± 6.20% PBMCs activated (Figure 5Aa, gate R2 and5B). There was about 43.69 ± 12.51% of activated CD8+ T cells expressed HMGN2 (Figure 5Ab and C), where only 18.71 ± 6.34% naive CD8+ T cells expressed HMGN2 (Figure 5Ac 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)

HMGN2 expression in Tumor antigen stimulated PBMCs. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 150 μg/ml T-Ag for 7 days. Normal medium was used as the control. (A) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (B) Error bars represent HMGN2 intracellular expression positive rate (%) in PBMCs after stimulation with T-Ag. (C) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. Data are represented as means ± SD of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: HMGN2 expression in Tumor antigen stimulated PBMCs. PBMCs were seeded at a density of 1 × 107 per well in 6 well plates and stimulated with 150 μg/ml T-Ag for 7 days. Normal medium was used as the control. (A) The cells were removed and intracellularly stained for HMGN2. Figures are representative of three independent experiments. (B) Error bars represent HMGN2 intracellular expression positive rate (%) in PBMCs after stimulation with T-Ag. (C) The supernatants were collected and HMGN2 concentrations were analyzed by ELISA. Data are represented as means ± SD of three independent experiments.
Mentions: In order to ensure that HMGN2 was an effector protein of tumor antigen activated T cells, we used tumor full protein as tumor antigen (T-Ag) to stimulate PBMCs for 7 days. Supernatants were collected for assaying HMGN2 levels by ELISA and cells were collected for HMGN2 intracellular staining. Results showed that there was no significant change of HMGN2 expression after stimulation with T-Ag compared with the medium control (Figure 4). In order to ensure that T cells were activated by T-Ag, we used CD44high as the activated marker of T cells. T-Ag stimulated PBMCs were stained with CD8-PE/CD44-APC surface and HMGN2 indirect intracellular staining. CD44high was used as the activated T cell population (Figure 5Aa, gate R2) and CD44low was used as the naive T cell population (Figure 5Aa, gate R6). T-Ag induced only 18.35 ± 6.20% PBMCs activated (Figure 5Aa, gate R2 and5B). There was about 43.69 ± 12.51% of activated CD8+ T cells expressed HMGN2 (Figure 5Ab and C), where only 18.71 ± 6.34% naive CD8+ T cells expressed HMGN2 (Figure 5Ac 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