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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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Expression of hCD6 downregulates calcium signals. (A) Purified human primary T lymphocytes were treated with specific hCD6 MOs to suppress endogenous hCD6 expression (hCD6 kd), or with control scramble morpholinos (Control). Knockdown of hCD6 was evaluated by Western blot of cell lysates, using β-actin as a loading control. Numbers on the left indicate molecular mass markers (MM) in kilodaltons (kDa). (B) Cells were incubated with calcium indicators and after stimulation with CD3 mAb (OKT3), cells expressing hCD6+ (grey line) and cells where hCD6 was knocked down (hCD6kd, black line) were evaluated for calcium mobilization by cytometry. Data are shown from one out of the three experiments with similar results.
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fig02: Expression of hCD6 downregulates calcium signals. (A) Purified human primary T lymphocytes were treated with specific hCD6 MOs to suppress endogenous hCD6 expression (hCD6 kd), or with control scramble morpholinos (Control). Knockdown of hCD6 was evaluated by Western blot of cell lysates, using β-actin as a loading control. Numbers on the left indicate molecular mass markers (MM) in kilodaltons (kDa). (B) Cells were incubated with calcium indicators and after stimulation with CD3 mAb (OKT3), cells expressing hCD6+ (grey line) and cells where hCD6 was knocked down (hCD6kd, black line) were evaluated for calcium mobilization by cytometry. Data are shown from one out of the three experiments with similar results.

Mentions: To determine whether endogenous hCD6 would exhibit comparable inhibitory behavior in these cells, we suppressed its expression using hCD6-specific morpholino oligomers (MOs); we were successful in reducing expression by over 95% as assessed by western blotting (Fig. 2A). Using CD3 mAb (OKT3), we activated the untransfected T cells as well as T cells treated with the MOs, and measured calcium mobilization. At 1 μg/mL to activate 3×106 cells/mL, OKT3 induced a very modest increase in calcium in hCD6-expressing T cells, whereas a pronounced increase in calcium signaling was observed in the hCD6-depleted T cells (Fig. 2B), confirming the observations made for the rCD6 transfections.


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)

Expression of hCD6 downregulates calcium signals. (A) Purified human primary T lymphocytes were treated with specific hCD6 MOs to suppress endogenous hCD6 expression (hCD6 kd), or with control scramble morpholinos (Control). Knockdown of hCD6 was evaluated by Western blot of cell lysates, using β-actin as a loading control. Numbers on the left indicate molecular mass markers (MM) in kilodaltons (kDa). (B) Cells were incubated with calcium indicators and after stimulation with CD3 mAb (OKT3), cells expressing hCD6+ (grey line) and cells where hCD6 was knocked down (hCD6kd, black line) were evaluated for calcium mobilization by cytometry. Data are shown from one out of the three experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: Expression of hCD6 downregulates calcium signals. (A) Purified human primary T lymphocytes were treated with specific hCD6 MOs to suppress endogenous hCD6 expression (hCD6 kd), or with control scramble morpholinos (Control). Knockdown of hCD6 was evaluated by Western blot of cell lysates, using β-actin as a loading control. Numbers on the left indicate molecular mass markers (MM) in kilodaltons (kDa). (B) Cells were incubated with calcium indicators and after stimulation with CD3 mAb (OKT3), cells expressing hCD6+ (grey line) and cells where hCD6 was knocked down (hCD6kd, black line) were evaluated for calcium mobilization by cytometry. Data are shown from one out of the three experiments with similar results.
Mentions: To determine whether endogenous hCD6 would exhibit comparable inhibitory behavior in these cells, we suppressed its expression using hCD6-specific morpholino oligomers (MOs); we were successful in reducing expression by over 95% as assessed by western blotting (Fig. 2A). Using CD3 mAb (OKT3), we activated the untransfected T cells as well as T cells treated with the MOs, and measured calcium mobilization. At 1 μg/mL to activate 3×106 cells/mL, OKT3 induced a very modest increase in calcium in hCD6-expressing T cells, whereas a pronounced increase in calcium signaling was observed in the hCD6-depleted T cells (Fig. 2B), confirming the observations made for the rCD6 transfections.

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