Limits...
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.

Show MeSH

Related in: MedlinePlus

MS4a4B negatively regulates T cell proliferation.A, Retrovirus-driven over-expression of MS4a4B inhibits proliferation of primary T cells. Primary CD4+ T cells isolated from spleen with anti-CD4 magnetic beads were stimulated by anti-CD3/ anti-CD28 antibodies and then were infected with MS4a4B-retroviral vector or mock vector. After infection, cells were labeled with PKH-26 Red Fluorescent linker kit. Proliferation of the tested cells was analyzed by flow cytometry (FL2). Data shown are representative of three repeat experiments. B, Forced expression of MS4a4B by LV-vector reduced proliferation of EL4 cells. Purified EL4 cells with stable lentiviral infection were labeled with PKH-26 Red and then cultured in complete RPMI 1640 medium. Cells were collected on the days indicated. Cell proliferation was assessed by flow cytometry. Data shown are representative of three repeat experiments. The numbers in histograms are mean fluorescence intensity (MFI) ± SD. ***, p<0.001. C, Knockdown of MS4a4B by siRNA. T32 cells were transfected with either FAM-labeled negative control siRNA or FAM-labeled MS4a4B-specific siRNAs (siMS4a4B1 and siMS4a4B2). Cells were collected on day 3 after transfection. Transcription of MS4a4B mRNA was analyzed by RT-PCR. D, Cell samples collected from culture on day 4 were subjected to MS4a4B protein detection by flow cytometry with anti-MS4a4B antibody. Data are presented as fluorescence intensity of FL-2 in FAM-positive population. Blue line: siMS4a4B2-transfected cells; red line: control siRNA-transfected cells. E and F, Knockdown of MS4a4B expression accelerated proliferation of T32 cells (E) and primary T cells (F). T32 cells or anti-CD3/anti-CD28-stimulated CD4+ primary T cells were labeled with PKH-26 Red and were transfected with FAM-labeled negative control siRNA or FAM-labeled siMS4a4B2. The labeled cells were stimulated with complete medium containing 20 U/ml IL-2. Cells were collected from culture at the time indicated for determination of proliferation rate by flow cytometric analysis. Data are presented as representative of three repeat experiments. Tinted grey peak: unlabeled cells; blue line: siMS4a4B2-transfected cells; red line: control siRNA-transfected cells. The numbers in histograms are MFI ± SD. *, p<0.05; **, p<0.01; ***, p<0.001. G, knockdown of MS4a4B accelerated IL-2-induced proliferation of T32 cells. T32 cells were infected with either shMS4a4B- or shLuc-lentiviral vector. Infected cells (1×105) were cultured in 96 well plate for 24 hr in the presence of serially diluted IL-2, followed by incubation with 1μCi 3H-thymidine for an additional 16 hr. Cell proliferation was assayed by 3H-thymidine incorporation. A representative of two independent experiments is shown. *, p<0.05; **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2967469&req=5

pone-0013780-g002: MS4a4B negatively regulates T cell proliferation.A, Retrovirus-driven over-expression of MS4a4B inhibits proliferation of primary T cells. Primary CD4+ T cells isolated from spleen with anti-CD4 magnetic beads were stimulated by anti-CD3/ anti-CD28 antibodies and then were infected with MS4a4B-retroviral vector or mock vector. After infection, cells were labeled with PKH-26 Red Fluorescent linker kit. Proliferation of the tested cells was analyzed by flow cytometry (FL2). Data shown are representative of three repeat experiments. B, Forced expression of MS4a4B by LV-vector reduced proliferation of EL4 cells. Purified EL4 cells with stable lentiviral infection were labeled with PKH-26 Red and then cultured in complete RPMI 1640 medium. Cells were collected on the days indicated. Cell proliferation was assessed by flow cytometry. Data shown are representative of three repeat experiments. The numbers in histograms are mean fluorescence intensity (MFI) ± SD. ***, p<0.001. C, Knockdown of MS4a4B by siRNA. T32 cells were transfected with either FAM-labeled negative control siRNA or FAM-labeled MS4a4B-specific siRNAs (siMS4a4B1 and siMS4a4B2). Cells were collected on day 3 after transfection. Transcription of MS4a4B mRNA was analyzed by RT-PCR. D, Cell samples collected from culture on day 4 were subjected to MS4a4B protein detection by flow cytometry with anti-MS4a4B antibody. Data are presented as fluorescence intensity of FL-2 in FAM-positive population. Blue line: siMS4a4B2-transfected cells; red line: control siRNA-transfected cells. E and F, Knockdown of MS4a4B expression accelerated proliferation of T32 cells (E) and primary T cells (F). T32 cells or anti-CD3/anti-CD28-stimulated CD4+ primary T cells were labeled with PKH-26 Red and were transfected with FAM-labeled negative control siRNA or FAM-labeled siMS4a4B2. The labeled cells were stimulated with complete medium containing 20 U/ml IL-2. Cells were collected from culture at the time indicated for determination of proliferation rate by flow cytometric analysis. Data are presented as representative of three repeat experiments. Tinted grey peak: unlabeled cells; blue line: siMS4a4B2-transfected cells; red line: control siRNA-transfected cells. The numbers in histograms are MFI ± SD. *, p<0.05; **, p<0.01; ***, p<0.001. G, knockdown of MS4a4B accelerated IL-2-induced proliferation of T32 cells. T32 cells were infected with either shMS4a4B- or shLuc-lentiviral vector. Infected cells (1×105) were cultured in 96 well plate for 24 hr in the presence of serially diluted IL-2, followed by incubation with 1μCi 3H-thymidine for an additional 16 hr. Cell proliferation was assayed by 3H-thymidine incorporation. A representative of two independent experiments is shown. *, p<0.05; **, p<0.01.

Mentions: T cell proliferation is a critical process for T cell-mediated immunity, which is reciprocally regulated at multiple levels by numerous activators and inhibitors [26], [27], [28]. Its regulation, however, is still not clearly understood. Studies on CD20 suggest that MS4A proteins may regulate cell proliferation [13], [23]. To determine the role of MS4a4B in T cell proliferation, we constructed a MS4a4B-expressing retroviral vector, which co-expresses GFP as a selection marker. We used the MS4a4B-retroviral vector to manipulate expression of MS4a4B in primary T cells, which were subsequently labeled with PHK-26, a red fluorescent dye, and analyzed T cell proliferation upon CD3/CD28 stimulation by flow cytometry. Over-expression of MS4a4B by retroviral vector indeed inhibited proliferation of primary T cells (Fig. 2A). Since primary T cells constitutively expressed high levels of MS4a4B, we next used EL4 cells (MS4a4B−), a widely used thymoma cell line [29], [30], [31], to confirm the inhibitory effect of MS4a4B on T cell proliferation. We constructed a lentiviral vector (LV) to express MS4a4B and a GFP marker in EL4 cells. We sorted GFP+ cells and generated a stable MS4a4B-expressing EL4 cell line. To test proliferation of EL4 cells, the infected EL4 cells were labeled with PHK-26 and cell proliferation was analyzed by flow cytometry. As predicted, forced expression of MS4a4B inhibited EL4 thymoma cell proliferation (Fig. 2B).


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 negatively regulates T cell proliferation.A, Retrovirus-driven over-expression of MS4a4B inhibits proliferation of primary T cells. Primary CD4+ T cells isolated from spleen with anti-CD4 magnetic beads were stimulated by anti-CD3/ anti-CD28 antibodies and then were infected with MS4a4B-retroviral vector or mock vector. After infection, cells were labeled with PKH-26 Red Fluorescent linker kit. Proliferation of the tested cells was analyzed by flow cytometry (FL2). Data shown are representative of three repeat experiments. B, Forced expression of MS4a4B by LV-vector reduced proliferation of EL4 cells. Purified EL4 cells with stable lentiviral infection were labeled with PKH-26 Red and then cultured in complete RPMI 1640 medium. Cells were collected on the days indicated. Cell proliferation was assessed by flow cytometry. Data shown are representative of three repeat experiments. The numbers in histograms are mean fluorescence intensity (MFI) ± SD. ***, p<0.001. C, Knockdown of MS4a4B by siRNA. T32 cells were transfected with either FAM-labeled negative control siRNA or FAM-labeled MS4a4B-specific siRNAs (siMS4a4B1 and siMS4a4B2). Cells were collected on day 3 after transfection. Transcription of MS4a4B mRNA was analyzed by RT-PCR. D, Cell samples collected from culture on day 4 were subjected to MS4a4B protein detection by flow cytometry with anti-MS4a4B antibody. Data are presented as fluorescence intensity of FL-2 in FAM-positive population. Blue line: siMS4a4B2-transfected cells; red line: control siRNA-transfected cells. E and F, Knockdown of MS4a4B expression accelerated proliferation of T32 cells (E) and primary T cells (F). T32 cells or anti-CD3/anti-CD28-stimulated CD4+ primary T cells were labeled with PKH-26 Red and were transfected with FAM-labeled negative control siRNA or FAM-labeled siMS4a4B2. The labeled cells were stimulated with complete medium containing 20 U/ml IL-2. Cells were collected from culture at the time indicated for determination of proliferation rate by flow cytometric analysis. Data are presented as representative of three repeat experiments. Tinted grey peak: unlabeled cells; blue line: siMS4a4B2-transfected cells; red line: control siRNA-transfected cells. The numbers in histograms are MFI ± SD. *, p<0.05; **, p<0.01; ***, p<0.001. G, knockdown of MS4a4B accelerated IL-2-induced proliferation of T32 cells. T32 cells were infected with either shMS4a4B- or shLuc-lentiviral vector. Infected cells (1×105) were cultured in 96 well plate for 24 hr in the presence of serially diluted IL-2, followed by incubation with 1μCi 3H-thymidine for an additional 16 hr. Cell proliferation was assayed by 3H-thymidine incorporation. A representative of two independent experiments is shown. *, p<0.05; **, p<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2967469&req=5

pone-0013780-g002: MS4a4B negatively regulates T cell proliferation.A, Retrovirus-driven over-expression of MS4a4B inhibits proliferation of primary T cells. Primary CD4+ T cells isolated from spleen with anti-CD4 magnetic beads were stimulated by anti-CD3/ anti-CD28 antibodies and then were infected with MS4a4B-retroviral vector or mock vector. After infection, cells were labeled with PKH-26 Red Fluorescent linker kit. Proliferation of the tested cells was analyzed by flow cytometry (FL2). Data shown are representative of three repeat experiments. B, Forced expression of MS4a4B by LV-vector reduced proliferation of EL4 cells. Purified EL4 cells with stable lentiviral infection were labeled with PKH-26 Red and then cultured in complete RPMI 1640 medium. Cells were collected on the days indicated. Cell proliferation was assessed by flow cytometry. Data shown are representative of three repeat experiments. The numbers in histograms are mean fluorescence intensity (MFI) ± SD. ***, p<0.001. C, Knockdown of MS4a4B by siRNA. T32 cells were transfected with either FAM-labeled negative control siRNA or FAM-labeled MS4a4B-specific siRNAs (siMS4a4B1 and siMS4a4B2). Cells were collected on day 3 after transfection. Transcription of MS4a4B mRNA was analyzed by RT-PCR. D, Cell samples collected from culture on day 4 were subjected to MS4a4B protein detection by flow cytometry with anti-MS4a4B antibody. Data are presented as fluorescence intensity of FL-2 in FAM-positive population. Blue line: siMS4a4B2-transfected cells; red line: control siRNA-transfected cells. E and F, Knockdown of MS4a4B expression accelerated proliferation of T32 cells (E) and primary T cells (F). T32 cells or anti-CD3/anti-CD28-stimulated CD4+ primary T cells were labeled with PKH-26 Red and were transfected with FAM-labeled negative control siRNA or FAM-labeled siMS4a4B2. The labeled cells were stimulated with complete medium containing 20 U/ml IL-2. Cells were collected from culture at the time indicated for determination of proliferation rate by flow cytometric analysis. Data are presented as representative of three repeat experiments. Tinted grey peak: unlabeled cells; blue line: siMS4a4B2-transfected cells; red line: control siRNA-transfected cells. The numbers in histograms are MFI ± SD. *, p<0.05; **, p<0.01; ***, p<0.001. G, knockdown of MS4a4B accelerated IL-2-induced proliferation of T32 cells. T32 cells were infected with either shMS4a4B- or shLuc-lentiviral vector. Infected cells (1×105) were cultured in 96 well plate for 24 hr in the presence of serially diluted IL-2, followed by incubation with 1μCi 3H-thymidine for an additional 16 hr. Cell proliferation was assayed by 3H-thymidine incorporation. A representative of two independent experiments is shown. *, p<0.05; **, p<0.01.
Mentions: T cell proliferation is a critical process for T cell-mediated immunity, which is reciprocally regulated at multiple levels by numerous activators and inhibitors [26], [27], [28]. Its regulation, however, is still not clearly understood. Studies on CD20 suggest that MS4A proteins may regulate cell proliferation [13], [23]. To determine the role of MS4a4B in T cell proliferation, we constructed a MS4a4B-expressing retroviral vector, which co-expresses GFP as a selection marker. We used the MS4a4B-retroviral vector to manipulate expression of MS4a4B in primary T cells, which were subsequently labeled with PHK-26, a red fluorescent dye, and analyzed T cell proliferation upon CD3/CD28 stimulation by flow cytometry. Over-expression of MS4a4B by retroviral vector indeed inhibited proliferation of primary T cells (Fig. 2A). Since primary T cells constitutively expressed high levels of MS4a4B, we next used EL4 cells (MS4a4B−), a widely used thymoma cell line [29], [30], [31], to confirm the inhibitory effect of MS4a4B on T cell proliferation. We constructed a lentiviral vector (LV) to express MS4a4B and a GFP marker in EL4 cells. We sorted GFP+ cells and generated a stable MS4a4B-expressing EL4 cell line. To test proliferation of EL4 cells, the infected EL4 cells were labeled with PHK-26 and cell proliferation was analyzed by flow cytometry. As predicted, forced expression of MS4a4B inhibited EL4 thymoma cell proliferation (Fig. 2B).

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