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CD6 attenuates early and late signaling events, setting thresholds for T-cell activation.

Oliveira MI, Gonçalves CM, Pinto M, Fabre S, Santos AM, Lee SF, Castro MA, Nunes RJ, Barbosa RR, Parnes JR, Yu C, Davis SJ, Moreira A, Bismuth G, Carmo AM - Eur. J. Immunol. (2011)

Bottom Line: Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor.Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6.Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.

View Article: PubMed Central - PubMed

Affiliation: Group of Cell Activation and Gene Expression, IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal.

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CD6 decreases the amplitude of Ca2+ responses. (A) Cytometric analysis of ex vivo human T cells transfected with an rCD6-YFP vector. Live cells were gated based on the Forward Scatter (FSC) and Side Scatter (SSC) profiles. The expression of rCD6 is indicated by a thin line (upper panel) and the negative control is shown (in all panels) as a thick line. Expression of hCD6, (middle panel), and human CD3 (hCD3, lower panel) is shown before (thin line) and after transfection (dashed line) of T cells with the vector. (B) Raji cells preincubated with sAg interacted with Fura-2 loaded T lymphocytes transfected with a cDNA construct encoding rCD6-YFP. Time-lapse video imaging with 20× magnification showing superimposed differential interference contrast (DIC) and calcium signals (upper panel) and rCD6 expression (lower panel) on one rCD6− T cells (black arrow) and one rCD6+ T cell (white arrow), interacting with APCs in the same field. Scale bar=10 μm. (C) Percentage of rCD6− or rCD6+ T cells that increase (ratio >1.5) or not their calcium after conjugate formation with sAg-pulsed Raji cells. Bars represent the mean (±SD) values of three separate experiments with at least 30 individual cells analyzed in each. (D) Intracellular calcium was measured sequentially every 10 s in T cells interacting with sAg-pulsed Raji cells. Calcium was measured for T cells expressing (dashed line) or not (solid line) rCD6-YFP. Calcium signals were averaged from individual responses of all the cells in each set. Results are from one of the three experiments showing similar results. (E) Dot plots showing maximal calcium signals obtained in individual cells following stimulation by sAg-loaded Raji cells. Each dot represents one cell. Horizontal lines indicate mean values [rCD6− (n=12); rCD6+ (n=10)]. The probability that the Ca2+ signals of rCD6+ T cells in response to sAg stimulation were similar to those of rCD6− T cells was assessed using Students t-test (p=0.038).
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fig01: CD6 decreases the amplitude of Ca2+ responses. (A) Cytometric analysis of ex vivo human T cells transfected with an rCD6-YFP vector. Live cells were gated based on the Forward Scatter (FSC) and Side Scatter (SSC) profiles. The expression of rCD6 is indicated by a thin line (upper panel) and the negative control is shown (in all panels) as a thick line. Expression of hCD6, (middle panel), and human CD3 (hCD3, lower panel) is shown before (thin line) and after transfection (dashed line) of T cells with the vector. (B) Raji cells preincubated with sAg interacted with Fura-2 loaded T lymphocytes transfected with a cDNA construct encoding rCD6-YFP. Time-lapse video imaging with 20× magnification showing superimposed differential interference contrast (DIC) and calcium signals (upper panel) and rCD6 expression (lower panel) on one rCD6− T cells (black arrow) and one rCD6+ T cell (white arrow), interacting with APCs in the same field. Scale bar=10 μm. (C) Percentage of rCD6− or rCD6+ T cells that increase (ratio >1.5) or not their calcium after conjugate formation with sAg-pulsed Raji cells. Bars represent the mean (±SD) values of three separate experiments with at least 30 individual cells analyzed in each. (D) Intracellular calcium was measured sequentially every 10 s in T cells interacting with sAg-pulsed Raji cells. Calcium was measured for T cells expressing (dashed line) or not (solid line) rCD6-YFP. Calcium signals were averaged from individual responses of all the cells in each set. Results are from one of the three experiments showing similar results. (E) Dot plots showing maximal calcium signals obtained in individual cells following stimulation by sAg-loaded Raji cells. Each dot represents one cell. Horizontal lines indicate mean values [rCD6− (n=12); rCD6+ (n=10)]. The probability that the Ca2+ signals of rCD6+ T cells in response to sAg stimulation were similar to those of rCD6− T cells was assessed using Students t-test (p=0.038).

Mentions: Following transfection with a cDNA encoding rCD6 fused to YFP, the human T cells expressed variable levels of the chimeric protein (Fig. 1A, top panel). Endogenous human CD6 (hCD6) as well as human CD3 (hCD3) were highly expressed and their expression was unaffected by the coexpression of rCD6 (Fig. 1A, middle and bottom panels, respectively). Similarly, Raji cells (used as APCs) express human CD166 (hCD166), and so presumably in T cell–APC conjugates, T-cell surface hCD6 interacts with hCD166 expressed in Raji cells. We have considered untransfected T cells interacting with untransfected Raji the standard experimental setting, and changes that may occur following the expression of rCD6 and rCD166 should be due to specific functions of these heterologous rat molecules.


CD6 attenuates early and late signaling events, setting thresholds for T-cell activation.

Oliveira MI, Gonçalves CM, Pinto M, Fabre S, Santos AM, Lee SF, Castro MA, Nunes RJ, Barbosa RR, Parnes JR, Yu C, Davis SJ, Moreira A, Bismuth G, Carmo AM - Eur. J. Immunol. (2011)

CD6 decreases the amplitude of Ca2+ responses. (A) Cytometric analysis of ex vivo human T cells transfected with an rCD6-YFP vector. Live cells were gated based on the Forward Scatter (FSC) and Side Scatter (SSC) profiles. The expression of rCD6 is indicated by a thin line (upper panel) and the negative control is shown (in all panels) as a thick line. Expression of hCD6, (middle panel), and human CD3 (hCD3, lower panel) is shown before (thin line) and after transfection (dashed line) of T cells with the vector. (B) Raji cells preincubated with sAg interacted with Fura-2 loaded T lymphocytes transfected with a cDNA construct encoding rCD6-YFP. Time-lapse video imaging with 20× magnification showing superimposed differential interference contrast (DIC) and calcium signals (upper panel) and rCD6 expression (lower panel) on one rCD6− T cells (black arrow) and one rCD6+ T cell (white arrow), interacting with APCs in the same field. Scale bar=10 μm. (C) Percentage of rCD6− or rCD6+ T cells that increase (ratio >1.5) or not their calcium after conjugate formation with sAg-pulsed Raji cells. Bars represent the mean (±SD) values of three separate experiments with at least 30 individual cells analyzed in each. (D) Intracellular calcium was measured sequentially every 10 s in T cells interacting with sAg-pulsed Raji cells. Calcium was measured for T cells expressing (dashed line) or not (solid line) rCD6-YFP. Calcium signals were averaged from individual responses of all the cells in each set. Results are from one of the three experiments showing similar results. (E) Dot plots showing maximal calcium signals obtained in individual cells following stimulation by sAg-loaded Raji cells. Each dot represents one cell. Horizontal lines indicate mean values [rCD6− (n=12); rCD6+ (n=10)]. The probability that the Ca2+ signals of rCD6+ T cells in response to sAg stimulation were similar to those of rCD6− T cells was assessed using Students t-test (p=0.038).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig01: CD6 decreases the amplitude of Ca2+ responses. (A) Cytometric analysis of ex vivo human T cells transfected with an rCD6-YFP vector. Live cells were gated based on the Forward Scatter (FSC) and Side Scatter (SSC) profiles. The expression of rCD6 is indicated by a thin line (upper panel) and the negative control is shown (in all panels) as a thick line. Expression of hCD6, (middle panel), and human CD3 (hCD3, lower panel) is shown before (thin line) and after transfection (dashed line) of T cells with the vector. (B) Raji cells preincubated with sAg interacted with Fura-2 loaded T lymphocytes transfected with a cDNA construct encoding rCD6-YFP. Time-lapse video imaging with 20× magnification showing superimposed differential interference contrast (DIC) and calcium signals (upper panel) and rCD6 expression (lower panel) on one rCD6− T cells (black arrow) and one rCD6+ T cell (white arrow), interacting with APCs in the same field. Scale bar=10 μm. (C) Percentage of rCD6− or rCD6+ T cells that increase (ratio >1.5) or not their calcium after conjugate formation with sAg-pulsed Raji cells. Bars represent the mean (±SD) values of three separate experiments with at least 30 individual cells analyzed in each. (D) Intracellular calcium was measured sequentially every 10 s in T cells interacting with sAg-pulsed Raji cells. Calcium was measured for T cells expressing (dashed line) or not (solid line) rCD6-YFP. Calcium signals were averaged from individual responses of all the cells in each set. Results are from one of the three experiments showing similar results. (E) Dot plots showing maximal calcium signals obtained in individual cells following stimulation by sAg-loaded Raji cells. Each dot represents one cell. Horizontal lines indicate mean values [rCD6− (n=12); rCD6+ (n=10)]. The probability that the Ca2+ signals of rCD6+ T cells in response to sAg stimulation were similar to those of rCD6− T cells was assessed using Students t-test (p=0.038).
Mentions: Following transfection with a cDNA encoding rCD6 fused to YFP, the human T cells expressed variable levels of the chimeric protein (Fig. 1A, top panel). Endogenous human CD6 (hCD6) as well as human CD3 (hCD3) were highly expressed and their expression was unaffected by the coexpression of rCD6 (Fig. 1A, middle and bottom panels, respectively). Similarly, Raji cells (used as APCs) express human CD166 (hCD166), and so presumably in T cell–APC conjugates, T-cell surface hCD6 interacts with hCD166 expressed in Raji cells. We have considered untransfected T cells interacting with untransfected Raji the standard experimental setting, and changes that may occur following the expression of rCD6 and rCD166 should be due to specific functions of these heterologous rat molecules.

Bottom Line: Measuring calcium mobilization in single cells responding to superantigen, we found that human T cells expressing rat CD6 react significantly less well compared with T cells not expressing the exogenous receptor.Calcium responses, and also late indicators of T-cell activation such as IL-2 release, were also diminished in TCR-activated Jurkat cells expressing human CD6, compared with CD6-deficient cells or cells expressing a cytoplasmic deletion mutant of human CD6.Our data suggest that CD6 is a signaling attenuator whose expression alone, i.e. in the absence of ligand engagement, is sufficient to restrain signaling in T cells.

View Article: PubMed Central - PubMed

Affiliation: Group of Cell Activation and Gene Expression, IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal.

Show MeSH
Related in: MedlinePlus