CD6 attenuates early and late signaling events, setting thresholds for T-cell activation.
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.
Affiliation: Group of Cell Activation and Gene Expression, IBMC-Instituto de Biologia Molecular e Celular, Porto, Portugal.
- Antigens, CD/immunology*
- Antigens, Differentiation, T-Lymphocyte/immunology*
- Signal Transduction/immunology*
- Activated-Leukocyte Cell Adhesion Molecule/immunology
- Antigens, CD3/immunology
- Flow Cytometry
- Jurkat Cells
- Lymphocyte Activation
- Receptors, Antigen, T-Cell/immunology
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fig03: Ligand-binding interactions are required for CD6 accumulation at the immunological synapse but not for downmodulation of Ca2+ signaling. (A) Conjugates formed between sAg-pulsed Raji cells and rCD6-YFP-expressing T cells. Left panels: differential interference contrast (DIC) images. Upper right panel: localization of rCD6 (visualized in red) when rCD166 is not expressed on the surface of Raji cells; lower right panel: localization of rCD6 (red) and rCD166 (green) when rCD166 is expressed on the surface of Raji cells. Fluorescence overlays (yellow) of rCD6-YFP and rCD166-CFP localization are shown. Magnification is 40×, scale bar=10 μm. (B) Intracellular calcium was measured in rCD6+ T cells establishing contacts with rCD166+ Raji cells (thin line, n=16), rCD6+ T cells establishing contacts with rCD166− Raji cells (dashed line, n=11), or rCD6− T cells establishing contacts with rCD166+ Raji cells (thick line, n=15). Raji cells were previously pulsed with sAg. Averaged responses shown are from the calcium profiles of individual cells from each set, from two independent experiments. (C) Expression of human CD166 in untransfected (left) or hCD166-cDNA-transfected cells (middle), and of rat CD166 in rCD166-cDNA-transfected cells (right). Cells were gated based on the expression of hCD166 or rCD166, respectively, and these gates were used for the analysis in (D). The negative control (Neg) is also shown. (D) Binding of hCD6 tetramers (hCD6-tet, gray lines) to untransfected K562 cells (left), K562-hCD166 cells (middle), and K562-rCD166 cells (right). Binding of hCD58 tetramers (hCD58-tet, thick lines) to the cells was used as negative control, as K562 cells do not express the hCD58 ligand, hCD2. Profiles of hCD58-tet overlapped with the profiles of unlabeled cells (data not shown). (E) Interactions between one T cell expressing rCD6-YFP (T in red) and three sAg-loaded Raji cells, one of which expresses rCD166-CFP (B in green) and two that do not express rCD166 (B in white). Fluorescence overlays (yellow) of rCD6 and rCD166 localization are shown. Magnification is 40×, scale bar=10 μm. (F) Raji cells transfected with a rCD166-CFP vector were pulsed with a sAg mix before incubation with ex vivo T cells expressing rCD6Δd3, a naturally occurring isoform devoid of the rCD166 binding domain. Cells were fixed after 45 min of interaction. Localization of rCD166-CFP (green) and rCD6Δd3-YFP (red) as well as fluorescence overlays (yellow) are shown with a magnification of 40×. Scale bar=10 μm. (G) Intracellular calcium was measured sequentially every 10 s in T cells interacting with sAg-pulsed Raji cells. Calcium mobilization was measured for T cells lacking rCD6 (rCD6−, solid line) or T cells expressing rCD6Δd3 (dashed line), interacting with rCD166-expressing Raji cells. Averaged responses were determined from 12 rCD6− T cells and 16 rCD6Δd3-expressing T cells interacting with sAg-loaded Raji cells from two independent experiments.
In order to promote rCD6 localization to the immune synapse (IS), we transfected Raji cells with a cDNA-encoding rat CD166 (rCD166) fused to CFP. As expected, when Raji cells lacked expression of rCD166, rCD6 in T cells was distributed homogeneously over the cell surface and there was no particular enrichment at the IS (Fig. 3A, upper panels). In contrast, for Raji cells expressing rCD166, both the rCD6 expressed by the T cells and the rCD166 expressed by the Raji cells accumulated in the contact area (Fig. 3A, bottom panels). Measurements of calcium mobilization in rCD6+ cells establishing contacts with Raji cells expressing rCD166 (rCD166+; n=16) or not expressing rCD166 (rCD166−; n=11) revealed no differences in signaling for T cells interacting with the two sets of target cells (Fig. 3B and Supporting Information Fig. 1A). This clearly indicates that the localization of rCD6 at the IS per se is not required in order for it to suppress calcium signaling.