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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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The inhibitory role of CD6 is dependent on its cytoplasmic domain. (A) Percentage of calcium responsive (ratio >1.5) or unresponsive (ratio <1.5) T cells expressing a rCD6 cytoplasmic deletion mutant (rCD6CY5-YFP), 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. (B) Calcium signals were acquired sequentially every 10 s from rCD6CY5-YFP T cells (solid line) versus rCD6+ T cells (expressing full-length rCD6, dashed line), interacting with sAg-pulsed Raji cells. Averaged responses were determined from 15 rCD6+ T cells and 10 rCD6CY5-expressing T cells interacting with sAg-loaded Raji cells. Results are from one of two experiments, both gave similar results. (C) Surface expression of hCD6 in untreated E6.1 Jurkat cells (top), in Jurkat cells following transfection with a cDNA construct encoding full-length hCD6 (hCD6-FL) (middle), and in Jurkat cells following transfection with a cDNA construct encoding a cytoplasmatic deletion mutant of hCD6 (hCD6CY5) (bottom). Live cells were gated based on the FSC and SSC profiles. (D) E6.1-hCD6-FL and E6.1-hCD6CY5 cells were cell-surface biotinylated, lysed in detergent, and CD6 species were immunoprecipitated using MEM-98 mAb and run on SDS-PAGE. (E) E6.1 Jurkat cells expressing hCD6-FL (black lines) or hCD6CY5 (gray lines), and CD6− cells (dashed lines) were preincubated with the calcium indicators Fluo-3 and Fura-red and, upon anti-CD3 stimulation, total intracellular calcium levels were monitored by cytometry during 5 min. Results are from one of three experiments, all gave similar results.
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fig04: The inhibitory role of CD6 is dependent on its cytoplasmic domain. (A) Percentage of calcium responsive (ratio >1.5) or unresponsive (ratio <1.5) T cells expressing a rCD6 cytoplasmic deletion mutant (rCD6CY5-YFP), 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. (B) Calcium signals were acquired sequentially every 10 s from rCD6CY5-YFP T cells (solid line) versus rCD6+ T cells (expressing full-length rCD6, dashed line), interacting with sAg-pulsed Raji cells. Averaged responses were determined from 15 rCD6+ T cells and 10 rCD6CY5-expressing T cells interacting with sAg-loaded Raji cells. Results are from one of two experiments, both gave similar results. (C) Surface expression of hCD6 in untreated E6.1 Jurkat cells (top), in Jurkat cells following transfection with a cDNA construct encoding full-length hCD6 (hCD6-FL) (middle), and in Jurkat cells following transfection with a cDNA construct encoding a cytoplasmatic deletion mutant of hCD6 (hCD6CY5) (bottom). Live cells were gated based on the FSC and SSC profiles. (D) E6.1-hCD6-FL and E6.1-hCD6CY5 cells were cell-surface biotinylated, lysed in detergent, and CD6 species were immunoprecipitated using MEM-98 mAb and run on SDS-PAGE. (E) E6.1 Jurkat cells expressing hCD6-FL (black lines) or hCD6CY5 (gray lines), and CD6− cells (dashed lines) were preincubated with the calcium indicators Fluo-3 and Fura-red and, upon anti-CD3 stimulation, total intracellular calcium levels were monitored by cytometry during 5 min. Results are from one of three experiments, all gave similar results.

Mentions: We addressed the role of the cytoplasmic domain of CD6 using an engineered isoform of rCD6 lacking its cytoplasmic domain, rCD6CY5 fused to YFP, which was expressed in ex vivo human T cells. rCD6CY5 moved to the synapse when the T cells were allowed to form conjugates with rCD166-expressing Raji cells (data not shown). Calcium signals were measured in individual cells conjugated with rCD166+ Raji cells, and we found that the fraction of T cells expressing rCD6CY5 that increased their calcium levels following conjugate formation (i.e. ∼66%; Fig. 4A) closely matched that of rCD6− cells (Fig. 1C). For the responding T cells expressing rCD6CY5 (n=10), we measured calcium signals and compared these with signals from responding T cells expressing full-length rCD6 (n=15; Fig. 4B and Supporting Information Fig. 2). The results indicate that rCD6-expressing cells displayed lower and more transient calcium signals than cells expressing rCD6CY5, and thus the inhibitory effects of CD6 can be assigned to its cytoplasmic tail.


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)

The inhibitory role of CD6 is dependent on its cytoplasmic domain. (A) Percentage of calcium responsive (ratio >1.5) or unresponsive (ratio <1.5) T cells expressing a rCD6 cytoplasmic deletion mutant (rCD6CY5-YFP), 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. (B) Calcium signals were acquired sequentially every 10 s from rCD6CY5-YFP T cells (solid line) versus rCD6+ T cells (expressing full-length rCD6, dashed line), interacting with sAg-pulsed Raji cells. Averaged responses were determined from 15 rCD6+ T cells and 10 rCD6CY5-expressing T cells interacting with sAg-loaded Raji cells. Results are from one of two experiments, both gave similar results. (C) Surface expression of hCD6 in untreated E6.1 Jurkat cells (top), in Jurkat cells following transfection with a cDNA construct encoding full-length hCD6 (hCD6-FL) (middle), and in Jurkat cells following transfection with a cDNA construct encoding a cytoplasmatic deletion mutant of hCD6 (hCD6CY5) (bottom). Live cells were gated based on the FSC and SSC profiles. (D) E6.1-hCD6-FL and E6.1-hCD6CY5 cells were cell-surface biotinylated, lysed in detergent, and CD6 species were immunoprecipitated using MEM-98 mAb and run on SDS-PAGE. (E) E6.1 Jurkat cells expressing hCD6-FL (black lines) or hCD6CY5 (gray lines), and CD6− cells (dashed lines) were preincubated with the calcium indicators Fluo-3 and Fura-red and, upon anti-CD3 stimulation, total intracellular calcium levels were monitored by cytometry during 5 min. Results are from one of three experiments, all gave similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3298641&req=5

fig04: The inhibitory role of CD6 is dependent on its cytoplasmic domain. (A) Percentage of calcium responsive (ratio >1.5) or unresponsive (ratio <1.5) T cells expressing a rCD6 cytoplasmic deletion mutant (rCD6CY5-YFP), 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. (B) Calcium signals were acquired sequentially every 10 s from rCD6CY5-YFP T cells (solid line) versus rCD6+ T cells (expressing full-length rCD6, dashed line), interacting with sAg-pulsed Raji cells. Averaged responses were determined from 15 rCD6+ T cells and 10 rCD6CY5-expressing T cells interacting with sAg-loaded Raji cells. Results are from one of two experiments, both gave similar results. (C) Surface expression of hCD6 in untreated E6.1 Jurkat cells (top), in Jurkat cells following transfection with a cDNA construct encoding full-length hCD6 (hCD6-FL) (middle), and in Jurkat cells following transfection with a cDNA construct encoding a cytoplasmatic deletion mutant of hCD6 (hCD6CY5) (bottom). Live cells were gated based on the FSC and SSC profiles. (D) E6.1-hCD6-FL and E6.1-hCD6CY5 cells were cell-surface biotinylated, lysed in detergent, and CD6 species were immunoprecipitated using MEM-98 mAb and run on SDS-PAGE. (E) E6.1 Jurkat cells expressing hCD6-FL (black lines) or hCD6CY5 (gray lines), and CD6− cells (dashed lines) were preincubated with the calcium indicators Fluo-3 and Fura-red and, upon anti-CD3 stimulation, total intracellular calcium levels were monitored by cytometry during 5 min. Results are from one of three experiments, all gave similar results.
Mentions: We addressed the role of the cytoplasmic domain of CD6 using an engineered isoform of rCD6 lacking its cytoplasmic domain, rCD6CY5 fused to YFP, which was expressed in ex vivo human T cells. rCD6CY5 moved to the synapse when the T cells were allowed to form conjugates with rCD166-expressing Raji cells (data not shown). Calcium signals were measured in individual cells conjugated with rCD166+ Raji cells, and we found that the fraction of T cells expressing rCD6CY5 that increased their calcium levels following conjugate formation (i.e. ∼66%; Fig. 4A) closely matched that of rCD6− cells (Fig. 1C). For the responding T cells expressing rCD6CY5 (n=10), we measured calcium signals and compared these with signals from responding T cells expressing full-length rCD6 (n=15; Fig. 4B and Supporting Information Fig. 2). The results indicate that rCD6-expressing cells displayed lower and more transient calcium signals than cells expressing rCD6CY5, and thus the inhibitory effects of CD6 can be assigned to its cytoplasmic tail.

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