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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

HMGN2, released by T-Ag activated CD8+ T cells, transmembrane transported into tumor cells. HMGN2 protein and the supernatant of T-Ag activated CD8+ T cells were pre-labeled with FITC. Tca8113 cells were seeded at a density of 3 × 104 per well in 24-well plates. After overnight growth, the cells were cultured in medium with FITC pre-labeled samples. (A) HMGN2 transport into tumor cells analyzed with fluorescence microscope. The three figures are the same area. (a) Light micrographs of Tca8113 cells. (b) Fluorescent micrographs of Tca8113 cells of Hoechst 33258 nuclear staining. (c) Fluorescent micrographs of FITC labeled HMGN2 protein distribution in Tca8113 cells. (B) The Tca8113 cells were analyzed with fluorescent microscope. (a, b, c) FITC pre-labeled HMGN2 as the positive control. (d, e, f) FITC pre-labeled CD8+ T cells supernatant. (a, d) Cells under a light microscope. (b, e) Cells under a fluorescent microscope. (c, f) Cells under a fluorescent microscope after cultured in medium with HMGN2 depleted samples. (C) The Tca8113 cells were analyzed with Flow Cytometry. (a) Untreated Tca8113 control. (b, d) Tca8113 cultured in medium with FITC labeled samples. (c, e) Tca8113 cells cultured in medium with HMGN2 depleted samples. Figures are representative of three independent experiments. (f) Error bars represent FITC positive rate (%) of Tca8113 cells after cultured in medium with FITC labeled or HMGN2 depleted sample for 1 hour. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to HMGN2 undepleted (p < 0.05).
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Figure 7: HMGN2, released by T-Ag activated CD8+ T cells, transmembrane transported into tumor cells. HMGN2 protein and the supernatant of T-Ag activated CD8+ T cells were pre-labeled with FITC. Tca8113 cells were seeded at a density of 3 × 104 per well in 24-well plates. After overnight growth, the cells were cultured in medium with FITC pre-labeled samples. (A) HMGN2 transport into tumor cells analyzed with fluorescence microscope. The three figures are the same area. (a) Light micrographs of Tca8113 cells. (b) Fluorescent micrographs of Tca8113 cells of Hoechst 33258 nuclear staining. (c) Fluorescent micrographs of FITC labeled HMGN2 protein distribution in Tca8113 cells. (B) The Tca8113 cells were analyzed with fluorescent microscope. (a, b, c) FITC pre-labeled HMGN2 as the positive control. (d, e, f) FITC pre-labeled CD8+ T cells supernatant. (a, d) Cells under a light microscope. (b, e) Cells under a fluorescent microscope. (c, f) Cells under a fluorescent microscope after cultured in medium with HMGN2 depleted samples. (C) The Tca8113 cells were analyzed with Flow Cytometry. (a) Untreated Tca8113 control. (b, d) Tca8113 cultured in medium with FITC labeled samples. (c, e) Tca8113 cells cultured in medium with HMGN2 depleted samples. Figures are representative of three independent experiments. (f) Error bars represent FITC positive rate (%) of Tca8113 cells after cultured in medium with FITC labeled or HMGN2 depleted sample for 1 hour. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to HMGN2 undepleted (p < 0.05).

Mentions: To confirm that the HMGN2 in the supernatant of T-Ag activated CD8+ T cells could be transmembrane transported into tumor cells, we used fluorescence FITC to label the proteins in the supernatant, before adding to the medium. Human FITC labeled HMGN2 protein was used as the positive control. Results showed that the protein in the supernatant could effectively be transported into the tumor cells, as could the human HMGN2 protein control (Figure 7A, Figure 7B b&e, Figure 7C b&d). After HMGN2-depleted by anti-HMGN2, the number of FITC-positive cells visibly decreased comparing with HMGN2 undepleted samples analyzed by both fluorescent microscope (Figure 7B b vs c, e vs f) and Flow Cytometry (Figure 7C b vs c, d vs e).


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, released by T-Ag activated CD8+ T cells, transmembrane transported into tumor cells. HMGN2 protein and the supernatant of T-Ag activated CD8+ T cells were pre-labeled with FITC. Tca8113 cells were seeded at a density of 3 × 104 per well in 24-well plates. After overnight growth, the cells were cultured in medium with FITC pre-labeled samples. (A) HMGN2 transport into tumor cells analyzed with fluorescence microscope. The three figures are the same area. (a) Light micrographs of Tca8113 cells. (b) Fluorescent micrographs of Tca8113 cells of Hoechst 33258 nuclear staining. (c) Fluorescent micrographs of FITC labeled HMGN2 protein distribution in Tca8113 cells. (B) The Tca8113 cells were analyzed with fluorescent microscope. (a, b, c) FITC pre-labeled HMGN2 as the positive control. (d, e, f) FITC pre-labeled CD8+ T cells supernatant. (a, d) Cells under a light microscope. (b, e) Cells under a fluorescent microscope. (c, f) Cells under a fluorescent microscope after cultured in medium with HMGN2 depleted samples. (C) The Tca8113 cells were analyzed with Flow Cytometry. (a) Untreated Tca8113 control. (b, d) Tca8113 cultured in medium with FITC labeled samples. (c, e) Tca8113 cells cultured in medium with HMGN2 depleted samples. Figures are representative of three independent experiments. (f) Error bars represent FITC positive rate (%) of Tca8113 cells after cultured in medium with FITC labeled or HMGN2 depleted sample for 1 hour. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to HMGN2 undepleted (p < 0.05).
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Figure 7: HMGN2, released by T-Ag activated CD8+ T cells, transmembrane transported into tumor cells. HMGN2 protein and the supernatant of T-Ag activated CD8+ T cells were pre-labeled with FITC. Tca8113 cells were seeded at a density of 3 × 104 per well in 24-well plates. After overnight growth, the cells were cultured in medium with FITC pre-labeled samples. (A) HMGN2 transport into tumor cells analyzed with fluorescence microscope. The three figures are the same area. (a) Light micrographs of Tca8113 cells. (b) Fluorescent micrographs of Tca8113 cells of Hoechst 33258 nuclear staining. (c) Fluorescent micrographs of FITC labeled HMGN2 protein distribution in Tca8113 cells. (B) The Tca8113 cells were analyzed with fluorescent microscope. (a, b, c) FITC pre-labeled HMGN2 as the positive control. (d, e, f) FITC pre-labeled CD8+ T cells supernatant. (a, d) Cells under a light microscope. (b, e) Cells under a fluorescent microscope. (c, f) Cells under a fluorescent microscope after cultured in medium with HMGN2 depleted samples. (C) The Tca8113 cells were analyzed with Flow Cytometry. (a) Untreated Tca8113 control. (b, d) Tca8113 cultured in medium with FITC labeled samples. (c, e) Tca8113 cells cultured in medium with HMGN2 depleted samples. Figures are representative of three independent experiments. (f) Error bars represent FITC positive rate (%) of Tca8113 cells after cultured in medium with FITC labeled or HMGN2 depleted sample for 1 hour. Data are represented as means ± SD of three independent experiments. *Significantly decreased compared to HMGN2 undepleted (p < 0.05).
Mentions: To confirm that the HMGN2 in the supernatant of T-Ag activated CD8+ T cells could be transmembrane transported into tumor cells, we used fluorescence FITC to label the proteins in the supernatant, before adding to the medium. Human FITC labeled HMGN2 protein was used as the positive control. Results showed that the protein in the supernatant could effectively be transported into the tumor cells, as could the human HMGN2 protein control (Figure 7A, Figure 7B b&e, Figure 7C b&d). After HMGN2-depleted by anti-HMGN2, the number of FITC-positive cells visibly decreased comparing with HMGN2 undepleted samples analyzed by both fluorescent microscope (Figure 7B b vs c, e vs f) and Flow Cytometry (Figure 7C b vs c, d vs e).

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