T Cell Costimulation by CD6 Is Dependent on Bivalent Binding of a GADS/SLP-76 Complex

Abstract

The cell surface receptor CD6 regulates T cell activation in both activating and inhibitory manners. The adaptor protein SLP-76 is recruited to the phosphorylated CD6 cytoplasmic Y662 residue during T cell activation, providing an activating signal to T cells. In this study, a biochemical approach identified the SH2 domain-containing adaptor protein GADS as the dominant interaction partner for the CD6 cytoplasmic Y629 residue. Functional experiments in human Jurkat and primary T cells showed that both mutations Y629F and Y662F abolished costimulation by CD6. In addition, a restraint on T cell activation by CD6 was revealed in primary T cells expressing CD6 mutated at Y629 and Y662. These data are consistent with a model in which bivalent recruitment of a GADS/SLP-76 complex is required for costimulation by CD6.

Keywords: CD6; GADS; SLP-76; T cells; signal transduction.

FIG 1

CD6 Y629 binds directly to the SH2 domains of GADS, GRB2, and TSAd. Equilibrium binding fitted curves and K_Ds (left) were derived from the sensorgram data (right) for 2-fold serial dilutions of GADS (35 μM) (A), GRB2 (106 μM) (B), and TSAd (314 μM) (C) SH2 domains over immobilized peptides. Background signals for proteins over streptavidin were subtracted.

FIG 2

The GADS/SLP-76 complex is recruited to CD6 Y629 and Y662. (A, left) Human CD6, Y629F and Y662F single mutant, and Y629F Y662F double mutant proteins fused to EGFP and stained with a CD6 MAb (T12.1) were expressed at similar levels on Jurkat cells. (Right) CD6 surface staining is correlated with the EGFP signal. (B and C) CD6 was immunoprecipitated from Jurkat cells (3 × 10⁶ cells per sample). Western blots of lysates and CD6 immunoprecipitates (IP) were probed for SLP-76, GADS, TSAd, GRB2, and EGFP to detect the CD6-EGFP fusion protein. A representative blot (B) and combined data from densitometric analyses for three experiments (C) are shown. The bars (means ± standard errors of the means) represent the ratios of coimmunoprecipitated CD6/CD6 in the lysate normalized to the ratio of immunoprecipitated CD6/CD6 in the lysate to measure the relative abundance, in arbitrary units (AU), of intracellular proteins in CD6 immunoprecipitates. The unpaired Student t test was used to compare values for the mutants with those for CD6. *, P < 0.05; **, P < 0.01; ns, not significant.

FIG 3

GADS SH2 and SLP-76 SH2 are specific for CD6 Y629 and Y662, respectively. Equilibrium binding fitted curves and K_Ds (left) were derived from SPR sensorgram data (right) for 2-fold serial dilutions of GADS (10 μM) (A) and SLP-76 (20 μM) (B) SH2 domains over the long CD6 peptides. There was no binding of the GADS SH2 domain to pY662 or of the SLP-76 SH2 domain to pY629 (data not shown). Background signals for proteins over streptavidin were subtracted.

FIG 4

Costimulation by CD6 is dependent on CD6 Y629 and Y662 in Jurkat cells. Flow cytometry analysis of Jurkat cells transduced with human CD6, the Y629F or Y662F single mutant, or the Y629F Y662F double mutant fused with EGFP and stained with CD3 MAb UCHT1, which showed that CD3 levels were unchanged by transduction (A), or stimulated with different concentrations of CD3 MAb with (B) or without (C) CD6 MAb (T12.1) (2.5 μg/ml) and measured for CD69 MFI on EGFP-positive cells. Compared with untransduced cells, the CD69 MFI was increased in cells transduced with CD6, but the effect of CD6 was reduced by the Y629F or Y662F single mutation (B) or the Y629F Y662F double mutation (B and C). *, P < 0.05; **, P < 0.01. Combined data from three experiments (means ± standard errors of the means) are shown. The unpaired Student t test was used to compare the maximum effect (E_max) values of each dose-response curve.

FIG 5

Costimulation by CD6 is dependent on CD6 Y629 and Y662 in primary CD4⁺ T cells. Flow cytometry analysis of primary CD4⁺ T cells transduced with rat domain 1-containing human CD6 or the Y629F Y662F double mutant protein fused to EGFP. (A) Cells stained with a rat domain 1-containing CD6 MAb (OX52) (left), a human domain 3-containing CD6 MAb (OX124) (middle), and a CD3 MAb (UCHT1) (right) show that there are similar levels of CD6 expression on transduced T cell blasts with ∼40-fold-higher MFIs and that CD3 levels in T cells were unchanged compared with those in untransduced cells. Negative isotype controls are shown for each MAb. (B and C) Cells were stimulated with different concentrations of CD3 MAb with (B) and without (C) CD6 MAb (OX52) (5 μg/ml) and measured for the percentage of CD69⁺ EGFP-positive cells. Compared with those for untransduced cells, the percentages of CD69⁺ cells were increased in cells transduced with CD6, but the effect of CD6 was reduced by the Y629F Y662F double mutation. *, P < 0.05; **, P < 0.01. Combined data from three experiments (means ± standard errors of the means) are shown. The unpaired Student t test was used to compare the E_max values of each dose-response curve.

FIG 6

CD6-mediated IL-2 production is dependent on CD6 Y629 and Y662 in primary CD4⁺ T cells. T cell blasts transduced with rat domain 1-containing human CD6 and the Y629F Y662F mutant fused to EGFP were stimulated with CD3 MAb (2 μg/ml) and/or CD6 MAb (OX52) (5 μg/ml), and the percentage of IL-2-positive, EGFP-positive cells was measured by flow cytometry. Combined data from three experiments (means ± standard errors of the means) are shown. The unpaired Student t test was used to compare values (*, P < 0.05; **, P < 0.01).

FIG 7

Model for bivalent recruitment of the GADS/SLP-76 complex by CD6. The GADS and SLP-76 SH2 domains bind to the phosphorylated tyrosine residues Y629 and Y662, respectively. The C-terminal SH3 domain of GADS binds to a proline-rich region of SLP-76, and we provide evidence that the GADS/SLP-76 complex binds bivalently to the CD6 C terminus.