Limits...
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.

Show MeSH

Related in: MedlinePlus

T-Ag activated CD8+T cells released HMGN2 to kill tumor cells. 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. (A) Cells were removed and stained with CD44-APC/CD8-FITC. CD44high/CD8+T activated T cells (Gate R4) were purified with MoFlo XDP sorter. The purified CD44high/CD8+T cells were cultured in complete medium with 2000 IU/ml IL-2 for 5 days. (B) The cells were removed and stained with Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. (C) The antitumor effects of the supernatants at different concentration. (D) The antitumor effects of the 20% (v/v) supernatants after blocking HMHN2 using anti-HMGN2 antibody. Figures are representative of three independent experiments. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to medium control (p < 0.05). #Significantly decreased after with anti-HMGN2 antibody (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: T-Ag activated CD8+T cells released HMGN2 to kill tumor cells. 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. (A) Cells were removed and stained with CD44-APC/CD8-FITC. CD44high/CD8+T activated T cells (Gate R4) were purified with MoFlo XDP sorter. The purified CD44high/CD8+T cells were cultured in complete medium with 2000 IU/ml IL-2 for 5 days. (B) The cells were removed and stained with Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. (C) The antitumor effects of the supernatants at different concentration. (D) The antitumor effects of the 20% (v/v) supernatants after blocking HMHN2 using anti-HMGN2 antibody. Figures are representative of three independent experiments. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to medium control (p < 0.05). #Significantly decreased after with anti-HMGN2 antibody (p < 0.05).

Mentions: We used a Beckman Coulter MoFlo XDP high-speed flow cytometry sorter to isolate T-Ag-activated the CD44highCD8+ T cell population (Figure 6A, Gate R4). The purified cells were cultured with 2000 IU/ml IL-2 for 5 days and then the cells were identified with CD44-APC and CD8-FITC staining (Figure 6A). Cells were collected and stained with CD8-PE surface and HMGN2 intracellular staining. A total of 69.35 ± 12.13% of CD8+ T cells still expressed HMGN2 (Figure 6B).


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)

T-Ag activated CD8+T cells released HMGN2 to kill tumor cells. 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. (A) Cells were removed and stained with CD44-APC/CD8-FITC. CD44high/CD8+T activated T cells (Gate R4) were purified with MoFlo XDP sorter. The purified CD44high/CD8+T cells were cultured in complete medium with 2000 IU/ml IL-2 for 5 days. (B) The cells were removed and stained with Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. (C) The antitumor effects of the supernatants at different concentration. (D) The antitumor effects of the 20% (v/v) supernatants after blocking HMHN2 using anti-HMGN2 antibody. Figures are representative of three independent experiments. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to medium control (p < 0.05). #Significantly decreased after with anti-HMGN2 antibody (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: T-Ag activated CD8+T cells released HMGN2 to kill tumor cells. 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. (A) Cells were removed and stained with CD44-APC/CD8-FITC. CD44high/CD8+T activated T cells (Gate R4) were purified with MoFlo XDP sorter. The purified CD44high/CD8+T cells were cultured in complete medium with 2000 IU/ml IL-2 for 5 days. (B) The cells were removed and stained with Anti-CD8-PE surface stained and anti-HMGN2/IgG-FITC intracellular stained. (C) The antitumor effects of the supernatants at different concentration. (D) The antitumor effects of the 20% (v/v) supernatants after blocking HMHN2 using anti-HMGN2 antibody. Figures are representative of three independent experiments. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to medium control (p < 0.05). #Significantly decreased after with anti-HMGN2 antibody (p < 0.05).
Mentions: We used a Beckman Coulter MoFlo XDP high-speed flow cytometry sorter to isolate T-Ag-activated the CD44highCD8+ T cell population (Figure 6A, Gate R4). The purified cells were cultured with 2000 IU/ml IL-2 for 5 days and then the cells were identified with CD44-APC and CD8-FITC staining (Figure 6A). Cells were collected and stained with CD8-PE surface and HMGN2 intracellular staining. A total of 69.35 ± 12.13% of CD8+ T cells still expressed HMGN2 (Figure 6B).

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