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MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

Xu H, Yan Y, Williams MS, Carey GB, Yang J, Li H, Zhang GX, Rostami A - PLoS ONE (2010)

Bottom Line: CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively.In contrast, knockdown of MS4a4B accelerated T cell proliferation.MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America. hui.xu@jefferson.edu

ABSTRACT
MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

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Expression of MS4a4B in T cells modulates cell cycle progression.A, MS4a4B-LV or mock LV-infected EL4 thymoma cells were synchronized by treatments with 2.5 mM thymidine and serum starvation. The synchronized cells were adjusted into 5 × 105/ml and were stimulated with complete medium containing 15% FCS. For cell cycle analysis, cells were harvested from culture at the time indicated. DNA content in cells was determined by propidium iodide staining and flow cytometric analysis. Results are shown as histogram from each sample with numbers indicating percentage of S-G2/M phase cells. Data presented are representative of three repeat experiments. B, Infection of T32 cells by shMS4a4B-expressing LV vector decreased MS4a4B protein expression. T32 cells stably infected with shMS4a4B-LV or shLuc-LV control were sorted by flow cytometry. MS4a4B expression in the sorted T32 cells was assessed by flow cytometry with anti-MS4a4B staining. Data are shown as representative histograms from analysis with Flowjo. C, shMS4a4B-LV or shLuc-LV control-infected T32 were co-stained with DAPI and anti-MS4a4B antibody, followed by labeling with anti-rabbit IgG-Cy3 conjugate. Knockdown of MS4a4B protein in T32 cells by shMS4a4B-LV was examined by confocal microscopy (Magnification, ×40). D, Knockdown of MS4a4B expression in T32 cells by shMS4a4B-LV inhibited cell cycle progression. shMS4a4B-LV or shLuc-LV control-infected T32 cells were synchronized as described in “A”. Synchronized cells were stimulated by 15% FCS RPMI medium containing 20 U/ml IL-2. Cell samples were harvested from culture at the time indicated. Cell cycle was analyzed by propidium iodide staining. Data are shown as the representative histogram of three repeat experiments. The numbers in histogram are the percentage of cells in S-G2/M phase.
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pone-0013780-g004: Expression of MS4a4B in T cells modulates cell cycle progression.A, MS4a4B-LV or mock LV-infected EL4 thymoma cells were synchronized by treatments with 2.5 mM thymidine and serum starvation. The synchronized cells were adjusted into 5 × 105/ml and were stimulated with complete medium containing 15% FCS. For cell cycle analysis, cells were harvested from culture at the time indicated. DNA content in cells was determined by propidium iodide staining and flow cytometric analysis. Results are shown as histogram from each sample with numbers indicating percentage of S-G2/M phase cells. Data presented are representative of three repeat experiments. B, Infection of T32 cells by shMS4a4B-expressing LV vector decreased MS4a4B protein expression. T32 cells stably infected with shMS4a4B-LV or shLuc-LV control were sorted by flow cytometry. MS4a4B expression in the sorted T32 cells was assessed by flow cytometry with anti-MS4a4B staining. Data are shown as representative histograms from analysis with Flowjo. C, shMS4a4B-LV or shLuc-LV control-infected T32 were co-stained with DAPI and anti-MS4a4B antibody, followed by labeling with anti-rabbit IgG-Cy3 conjugate. Knockdown of MS4a4B protein in T32 cells by shMS4a4B-LV was examined by confocal microscopy (Magnification, ×40). D, Knockdown of MS4a4B expression in T32 cells by shMS4a4B-LV inhibited cell cycle progression. shMS4a4B-LV or shLuc-LV control-infected T32 cells were synchronized as described in “A”. Synchronized cells were stimulated by 15% FCS RPMI medium containing 20 U/ml IL-2. Cell samples were harvested from culture at the time indicated. Cell cycle was analyzed by propidium iodide staining. Data are shown as the representative histogram of three repeat experiments. The numbers in histogram are the percentage of cells in S-G2/M phase.

Mentions: It has been documented that CD20 regulates B cell proliferation by impacting cell cycle progression in these cells [22], [33]. HTm4 was found to prevent cell cycle progression from G0/G1 phase into S-G2/M phase in hematopoeotic cells [15], [24]. To get some insight into how MS4a4B modulates T cell proliferation, we tested if MS4a4B protein regulates cell cycle progression by using MS4a4B-lentivirus-infected EL4 cells. EL4 cells stably infected by MS4a4B-lentivirus (or mock lentiviral vector as control) were synchronized by two cycle treatment with 2.5 mM thymidine and serum starvation. Synchronized cells then were stimulated with complete medium containing 15% FCS. Cell samples were collected at serial time points after stimulation and cell cycle was analyzed by flow cytometry with propidium iodide staining. After 8 hours of stimulation, 68.5% of control cells entered S-G2/M phase. In contrast, the percentage of cells that entered S-G2/M phase was markedly reduced in MS4a4B-expressing vector-infected EL4 cells (Fig. 4A). These results suggest that MS4a4B protein inhibits cell proliferation by preventing cell cycle progression from G0/G1 phase into S-G2/M phases.


MS4a4B, a CD20 homologue in T cells, inhibits T cell propagation by modulation of cell cycle.

Xu H, Yan Y, Williams MS, Carey GB, Yang J, Li H, Zhang GX, Rostami A - PLoS ONE (2010)

Expression of MS4a4B in T cells modulates cell cycle progression.A, MS4a4B-LV or mock LV-infected EL4 thymoma cells were synchronized by treatments with 2.5 mM thymidine and serum starvation. The synchronized cells were adjusted into 5 × 105/ml and were stimulated with complete medium containing 15% FCS. For cell cycle analysis, cells were harvested from culture at the time indicated. DNA content in cells was determined by propidium iodide staining and flow cytometric analysis. Results are shown as histogram from each sample with numbers indicating percentage of S-G2/M phase cells. Data presented are representative of three repeat experiments. B, Infection of T32 cells by shMS4a4B-expressing LV vector decreased MS4a4B protein expression. T32 cells stably infected with shMS4a4B-LV or shLuc-LV control were sorted by flow cytometry. MS4a4B expression in the sorted T32 cells was assessed by flow cytometry with anti-MS4a4B staining. Data are shown as representative histograms from analysis with Flowjo. C, shMS4a4B-LV or shLuc-LV control-infected T32 were co-stained with DAPI and anti-MS4a4B antibody, followed by labeling with anti-rabbit IgG-Cy3 conjugate. Knockdown of MS4a4B protein in T32 cells by shMS4a4B-LV was examined by confocal microscopy (Magnification, ×40). D, Knockdown of MS4a4B expression in T32 cells by shMS4a4B-LV inhibited cell cycle progression. shMS4a4B-LV or shLuc-LV control-infected T32 cells were synchronized as described in “A”. Synchronized cells were stimulated by 15% FCS RPMI medium containing 20 U/ml IL-2. Cell samples were harvested from culture at the time indicated. Cell cycle was analyzed by propidium iodide staining. Data are shown as the representative histogram of three repeat experiments. The numbers in histogram are the percentage of cells in S-G2/M phase.
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Related In: Results  -  Collection

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pone-0013780-g004: Expression of MS4a4B in T cells modulates cell cycle progression.A, MS4a4B-LV or mock LV-infected EL4 thymoma cells were synchronized by treatments with 2.5 mM thymidine and serum starvation. The synchronized cells were adjusted into 5 × 105/ml and were stimulated with complete medium containing 15% FCS. For cell cycle analysis, cells were harvested from culture at the time indicated. DNA content in cells was determined by propidium iodide staining and flow cytometric analysis. Results are shown as histogram from each sample with numbers indicating percentage of S-G2/M phase cells. Data presented are representative of three repeat experiments. B, Infection of T32 cells by shMS4a4B-expressing LV vector decreased MS4a4B protein expression. T32 cells stably infected with shMS4a4B-LV or shLuc-LV control were sorted by flow cytometry. MS4a4B expression in the sorted T32 cells was assessed by flow cytometry with anti-MS4a4B staining. Data are shown as representative histograms from analysis with Flowjo. C, shMS4a4B-LV or shLuc-LV control-infected T32 were co-stained with DAPI and anti-MS4a4B antibody, followed by labeling with anti-rabbit IgG-Cy3 conjugate. Knockdown of MS4a4B protein in T32 cells by shMS4a4B-LV was examined by confocal microscopy (Magnification, ×40). D, Knockdown of MS4a4B expression in T32 cells by shMS4a4B-LV inhibited cell cycle progression. shMS4a4B-LV or shLuc-LV control-infected T32 cells were synchronized as described in “A”. Synchronized cells were stimulated by 15% FCS RPMI medium containing 20 U/ml IL-2. Cell samples were harvested from culture at the time indicated. Cell cycle was analyzed by propidium iodide staining. Data are shown as the representative histogram of three repeat experiments. The numbers in histogram are the percentage of cells in S-G2/M phase.
Mentions: It has been documented that CD20 regulates B cell proliferation by impacting cell cycle progression in these cells [22], [33]. HTm4 was found to prevent cell cycle progression from G0/G1 phase into S-G2/M phase in hematopoeotic cells [15], [24]. To get some insight into how MS4a4B modulates T cell proliferation, we tested if MS4a4B protein regulates cell cycle progression by using MS4a4B-lentivirus-infected EL4 cells. EL4 cells stably infected by MS4a4B-lentivirus (or mock lentiviral vector as control) were synchronized by two cycle treatment with 2.5 mM thymidine and serum starvation. Synchronized cells then were stimulated with complete medium containing 15% FCS. Cell samples were collected at serial time points after stimulation and cell cycle was analyzed by flow cytometry with propidium iodide staining. After 8 hours of stimulation, 68.5% of control cells entered S-G2/M phase. In contrast, the percentage of cells that entered S-G2/M phase was markedly reduced in MS4a4B-expressing vector-infected EL4 cells (Fig. 4A). These results suggest that MS4a4B protein inhibits cell proliferation by preventing cell cycle progression from G0/G1 phase into S-G2/M phases.

Bottom Line: CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively.In contrast, knockdown of MS4a4B accelerated T cell proliferation.MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America. hui.xu@jefferson.edu

ABSTRACT
MS4a4B, a CD20 homologue in T cells, is a novel member of the MS4A gene family in mice. The MS4A family includes CD20, FcεRIβ, HTm4 and at least 26 novel members that are characterized by their structural features: with four membrane-spanning domains, two extracellular domains and two cytoplasmic regions. CD20, FcεRIβ and HTm4 have been found to function in B cells, mast cells and hematopoietic cells respectively. However, little is known about the function of MS4a4B in T cell regulation. We demonstrate here that MS4a4B negatively regulates mouse T cell proliferation. MS4a4B is highly expressed in primary T cells, natural killer cells (NK) and some T cell lines. But its expression in all malignant T cells, including thymoma and T hybridoma tested, was silenced. Interestingly, its expression was regulated during T cell activation. Viral vector-driven overexpression of MS4a4B in primary T cells and EL4 thymoma cells reduced cell proliferation. In contrast, knockdown of MS4a4B accelerated T cell proliferation. Cell cycle analysis showed that MS4a4B regulated T cell proliferation by inhibiting entry of the cells into S-G2/M phase. MS4a4B-mediated inhibition of cell cycle was correlated with upregulation of Cdk inhibitory proteins and decreased levels of Cdk2 activity, subsequently leading to inhibition of cell cycle progression. Our data indicate that MS4a4B negatively regulates T cell proliferation. MS4a4B, therefore, may serve as a modulator in the negative-feedback regulatory loop of activated T cells.

Show MeSH
Related in: MedlinePlus