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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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MS4a4B is expressed on cell surface and is potentially associated with activation of T cells.A, To determine whether MS4a4B is expressed on cell surface, mouse spleen cells were surface-labeled with EZ-Link Sulfo-NHS-SS-Biotin according to the manufacturer's protocol (Pierce). Unlabeled spleen cells were used as control. Cells were lysed by lysis buffer containing 1% NP-40. Cell lysate was immunoprecipitated by anti-MS4a4B-coupled protein A-beads and was separated on 12% SDS-PAGE, followed by blotting with streptavidin-HRP. MS4a4B was confirmed by re-blotting with anti-MS4a4B antibody. B, EL4 cells, infected with either MS4a4B-expressing lentivirus (LV) vector or mock LV vector, were stained with rabbit anti-MS4a4B antibody followed by labeling with anti-rabbit-IgG-Cy3 conjugate. Expression and localization of MS4a4B were observed by confocal microscopy. Magnification, ×40. C, Spleen cells were cultured for 24, 48 and 72 hrs in the presence or absence of Con A (5 µg/ml). Spleen cells before culture were used as control (0 hr). Cells were first stained with anti-CD3-PE, and then intracellular stained with biotinylated anti-MS4a4B antibody (blue line) or biotinylated Ig control (red line), followed by labeling with streptavidin-Red 670. For flow cytometric analysis, cells were first gated on CD3, and then were analyzed for MS4a4B expression. D, Spleen cells pre- or 48 hr post Con A stimulation were co-immunostained with anti-CD3 and anti-MS4a4B antibody. Cells were analyzed by flow cytometry. Note the CD3low/MS4a4Blow population. E, Spleen cells were stimulated with Con A for 48 hr and were co-stained with Annexin V, anti-CD3 and anti-MS4a4B antibodies. CD3low/MS4a4Blow and CD3high/MS4a4Bhigh populations were gated for assessment of apoptosis indicated by Annexin V binding.
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pone-0013780-g001: MS4a4B is expressed on cell surface and is potentially associated with activation of T cells.A, To determine whether MS4a4B is expressed on cell surface, mouse spleen cells were surface-labeled with EZ-Link Sulfo-NHS-SS-Biotin according to the manufacturer's protocol (Pierce). Unlabeled spleen cells were used as control. Cells were lysed by lysis buffer containing 1% NP-40. Cell lysate was immunoprecipitated by anti-MS4a4B-coupled protein A-beads and was separated on 12% SDS-PAGE, followed by blotting with streptavidin-HRP. MS4a4B was confirmed by re-blotting with anti-MS4a4B antibody. B, EL4 cells, infected with either MS4a4B-expressing lentivirus (LV) vector or mock LV vector, were stained with rabbit anti-MS4a4B antibody followed by labeling with anti-rabbit-IgG-Cy3 conjugate. Expression and localization of MS4a4B were observed by confocal microscopy. Magnification, ×40. C, Spleen cells were cultured for 24, 48 and 72 hrs in the presence or absence of Con A (5 µg/ml). Spleen cells before culture were used as control (0 hr). Cells were first stained with anti-CD3-PE, and then intracellular stained with biotinylated anti-MS4a4B antibody (blue line) or biotinylated Ig control (red line), followed by labeling with streptavidin-Red 670. For flow cytometric analysis, cells were first gated on CD3, and then were analyzed for MS4a4B expression. D, Spleen cells pre- or 48 hr post Con A stimulation were co-immunostained with anti-CD3 and anti-MS4a4B antibody. Cells were analyzed by flow cytometry. Note the CD3low/MS4a4Blow population. E, Spleen cells were stimulated with Con A for 48 hr and were co-stained with Annexin V, anti-CD3 and anti-MS4a4B antibodies. CD3low/MS4a4Blow and CD3high/MS4a4Bhigh populations were gated for assessment of apoptosis indicated by Annexin V binding.

Mentions: To define the expression of MS4a4B, we detected MS4a4B protein in mouse cells, including normal T cells, non-T cells, and malignant T cells, by using anti-MS4a4B antibodies and flow cytometric analysis. Consistent with our previous findings [1], MS4a4B was strongly expressed in naïve T cells but was not expressed in B cells. In addition to T cells, we also examined expression of MS4a4B in other cells. The results showed that MS4a4B was also expressed at high levels in NK cells (marked by NK1.1) and moderately expressed in macrophages (marked by Mac1) (supplementary Fig. S1A). In bone marrow cells, only the Mac1+ population expressed low levels of MS4a4B, suggesting MS4a4B expression in early hematopoietic progenitors (supplementary Fig. S1A). Surprisingly, all malignant T cells, including thymoma and T hybridoma cell lines that we have examined so far, lose expression of MS4a4B (Table 1 and supplementary Fig. S2). Lack of MS4a4B protein in thymoma cells led us to postulate that MS4a4B may be required for appropriate functioning of mature T cells and the absence of this protein may be, at least in part, responsible for the uncontrollable growth of thymoma. Western blotting with surface-biotin labeling of primary T cells and histological studies with confocal microscopy of MS4a4B-expressing retrovirus-infected EL4 thymoma cells showed that MS4a4B was indeed expressed on cell surface (Fig. 1A and B), suggesting that MS4a4B may potentially interact with other cell membrane proteins in T cell regulation. Interestingly, expression of MS4a4B is regulated not only during thymocyte development [1] but also during primary T cell activation (Fig. 1C). Although MS4a4B was constitutively expressed in primary naïve T cells, levels of its expression were markedly increased upon stimulation by mitogen concanavalin A (Con A). However, expression of MS4a4B was gradiently decreased by 72 hour of activation. It is of note that the down-regulation of MS4a4B was closely associated with reduction of surface CD3 expression in T cells (Fig. 1D), which led us to speculate whether this CD3low/MS4a4Blow population might represent the cells undergoing apoptosis. We further examined apoptosis of this population by Annexin V assay. The CD3low/MS4a4Blow population showed markedly high levels of apoptotic cells (Fig. 1E). Thus, temporal expression of MS4a4B in activated T cells and silence of the MS4a4B gene in malignant T cells led us to hypothesize that MS4a4B may play a regulatory role in propagation of T cells.


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)

MS4a4B is expressed on cell surface and is potentially associated with activation of T cells.A, To determine whether MS4a4B is expressed on cell surface, mouse spleen cells were surface-labeled with EZ-Link Sulfo-NHS-SS-Biotin according to the manufacturer's protocol (Pierce). Unlabeled spleen cells were used as control. Cells were lysed by lysis buffer containing 1% NP-40. Cell lysate was immunoprecipitated by anti-MS4a4B-coupled protein A-beads and was separated on 12% SDS-PAGE, followed by blotting with streptavidin-HRP. MS4a4B was confirmed by re-blotting with anti-MS4a4B antibody. B, EL4 cells, infected with either MS4a4B-expressing lentivirus (LV) vector or mock LV vector, were stained with rabbit anti-MS4a4B antibody followed by labeling with anti-rabbit-IgG-Cy3 conjugate. Expression and localization of MS4a4B were observed by confocal microscopy. Magnification, ×40. C, Spleen cells were cultured for 24, 48 and 72 hrs in the presence or absence of Con A (5 µg/ml). Spleen cells before culture were used as control (0 hr). Cells were first stained with anti-CD3-PE, and then intracellular stained with biotinylated anti-MS4a4B antibody (blue line) or biotinylated Ig control (red line), followed by labeling with streptavidin-Red 670. For flow cytometric analysis, cells were first gated on CD3, and then were analyzed for MS4a4B expression. D, Spleen cells pre- or 48 hr post Con A stimulation were co-immunostained with anti-CD3 and anti-MS4a4B antibody. Cells were analyzed by flow cytometry. Note the CD3low/MS4a4Blow population. E, Spleen cells were stimulated with Con A for 48 hr and were co-stained with Annexin V, anti-CD3 and anti-MS4a4B antibodies. CD3low/MS4a4Blow and CD3high/MS4a4Bhigh populations were gated for assessment of apoptosis indicated by Annexin V binding.
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getmorefigures.php?uid=PMC2967469&req=5

pone-0013780-g001: MS4a4B is expressed on cell surface and is potentially associated with activation of T cells.A, To determine whether MS4a4B is expressed on cell surface, mouse spleen cells were surface-labeled with EZ-Link Sulfo-NHS-SS-Biotin according to the manufacturer's protocol (Pierce). Unlabeled spleen cells were used as control. Cells were lysed by lysis buffer containing 1% NP-40. Cell lysate was immunoprecipitated by anti-MS4a4B-coupled protein A-beads and was separated on 12% SDS-PAGE, followed by blotting with streptavidin-HRP. MS4a4B was confirmed by re-blotting with anti-MS4a4B antibody. B, EL4 cells, infected with either MS4a4B-expressing lentivirus (LV) vector or mock LV vector, were stained with rabbit anti-MS4a4B antibody followed by labeling with anti-rabbit-IgG-Cy3 conjugate. Expression and localization of MS4a4B were observed by confocal microscopy. Magnification, ×40. C, Spleen cells were cultured for 24, 48 and 72 hrs in the presence or absence of Con A (5 µg/ml). Spleen cells before culture were used as control (0 hr). Cells were first stained with anti-CD3-PE, and then intracellular stained with biotinylated anti-MS4a4B antibody (blue line) or biotinylated Ig control (red line), followed by labeling with streptavidin-Red 670. For flow cytometric analysis, cells were first gated on CD3, and then were analyzed for MS4a4B expression. D, Spleen cells pre- or 48 hr post Con A stimulation were co-immunostained with anti-CD3 and anti-MS4a4B antibody. Cells were analyzed by flow cytometry. Note the CD3low/MS4a4Blow population. E, Spleen cells were stimulated with Con A for 48 hr and were co-stained with Annexin V, anti-CD3 and anti-MS4a4B antibodies. CD3low/MS4a4Blow and CD3high/MS4a4Bhigh populations were gated for assessment of apoptosis indicated by Annexin V binding.
Mentions: To define the expression of MS4a4B, we detected MS4a4B protein in mouse cells, including normal T cells, non-T cells, and malignant T cells, by using anti-MS4a4B antibodies and flow cytometric analysis. Consistent with our previous findings [1], MS4a4B was strongly expressed in naïve T cells but was not expressed in B cells. In addition to T cells, we also examined expression of MS4a4B in other cells. The results showed that MS4a4B was also expressed at high levels in NK cells (marked by NK1.1) and moderately expressed in macrophages (marked by Mac1) (supplementary Fig. S1A). In bone marrow cells, only the Mac1+ population expressed low levels of MS4a4B, suggesting MS4a4B expression in early hematopoietic progenitors (supplementary Fig. S1A). Surprisingly, all malignant T cells, including thymoma and T hybridoma cell lines that we have examined so far, lose expression of MS4a4B (Table 1 and supplementary Fig. S2). Lack of MS4a4B protein in thymoma cells led us to postulate that MS4a4B may be required for appropriate functioning of mature T cells and the absence of this protein may be, at least in part, responsible for the uncontrollable growth of thymoma. Western blotting with surface-biotin labeling of primary T cells and histological studies with confocal microscopy of MS4a4B-expressing retrovirus-infected EL4 thymoma cells showed that MS4a4B was indeed expressed on cell surface (Fig. 1A and B), suggesting that MS4a4B may potentially interact with other cell membrane proteins in T cell regulation. Interestingly, expression of MS4a4B is regulated not only during thymocyte development [1] but also during primary T cell activation (Fig. 1C). Although MS4a4B was constitutively expressed in primary naïve T cells, levels of its expression were markedly increased upon stimulation by mitogen concanavalin A (Con A). However, expression of MS4a4B was gradiently decreased by 72 hour of activation. It is of note that the down-regulation of MS4a4B was closely associated with reduction of surface CD3 expression in T cells (Fig. 1D), which led us to speculate whether this CD3low/MS4a4Blow population might represent the cells undergoing apoptosis. We further examined apoptosis of this population by Annexin V assay. The CD3low/MS4a4Blow population showed markedly high levels of apoptotic cells (Fig. 1E). Thus, temporal expression of MS4a4B in activated T cells and silence of the MS4a4B gene in malignant T cells led us to hypothesize that MS4a4B may play a regulatory role in propagation of T cells.

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