CD3δ Establishes a Functional Link between the T Cell Receptor and CD8

Marie-Agnès Doucey,Laurence Goffin,Dieter Naeher,Olivier Michielin,Petra Baumgärtner,Philippe Guillaume,Ed Palmer,Immanuel F Luescher

doi:10.1074/jbc.m208119200

Marie-Agnès Doucey, Laurence Goffin + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m208119200

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Abstract

T cells expressing T cell receptor (TCR) complexes that lack CD3 delta, either due to deletion of the CD3 delta gene, or by replacement of the connecting peptide of the TCR alpha chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Because the same defects have been observed in mice expressing no CD8 beta or tailless CD8 beta, we examined whether CD3 delta serves to couple TCR.CD3 with CD8. To this end we used T cell hybridomas and transgenic mice expressing the T1 TCR, which recognizes a photoreactive derivative of the PbCS 252-260 peptide in the context of H-2K(d). We report that, in thymocytes and hybridomas expressing the T1 TCR.CD3 complex, CD8 alpha beta associates with the TCR. This association was not observed on T1 hybridomas expressing only CD8 alpha alpha or a CD3 delta(-) variant of the T1 TCR. CD3 delta was selectively co-immunoprecipitated with anti-CD8 antibodies, indicating an avid association of CD8 with CD3 delta. Because CD8 alpha beta is a raft constituent, due to this association a fraction of TCR.CD3 is raft-associated. Cross-linking of these TCR-CD8 adducts results in extensive TCR aggregate formation and intracellular calcium mobilization. Thus, CD3 delta couples TCR.CD3 with raft-associated CD8, which is required for effective activation and positive selection of CD8(+) T cells.

Highlights

T cells expressing T cell receptor (TCR) complexes that lack CD3␦, either due to deletion of the CD3␦ gene, or by replacement of the connecting peptide of the TCR␣ chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells
CD8␣␤ and Wild Type TCR ␣CPM Are Required for Efficient Intracellular Calcium Mobilization—To examine the role of CD8 and the TCR ␣CPM for T cell activation we first assessed intracellular calcium mobilization in T cell hybridomas expressing CD8 and wild type T1 TCR or T1 TCR in which the ␣CPM was replaced with the corresponding sequence of TCR␦ (T1 TCR ␣IV/␤III)
In the presence of mAb H35, no marked calcium mobilization was observed, indicating that CD8 was required for this response (Fig. 1C). Taken together these results indicate that, for efficient calcium mobilization, CD8␣␤ and CD3␦ϩ TCR are required, which is in accordance with previously studies showing that CD8 and the ␣CPM are crucial for efficient TCR signaling and positive selection of CD8 SP T cells (16 –22)

Summary

Introduction

T cells expressing T cell receptor (TCR) complexes that lack CD3␦, either due to deletion of the CD3␦ gene, or by replacement of the connecting peptide of the TCR␣ chain, exhibit severely impaired positive selection and TCR-mediated activation of CD8 single-positive T cells. Using TCR photoaffinity labeling, FRET, co-immunoprecipitation, and confocal microscopy, we find that CD8␣␤, but not CD8␣␣, associates with the TCR via CD3␦ and that this is required for efficient T cell activation. CD8 Increases MHC-peptide Binding on Cells Expressing Wild Type but Not ␣CPM Variant TCR—TCR photoaffinity labeling with soluble monomeric Kd-125“IASA”-YIPSAEK(ABA)I complexes allows direct assessment of TCR-ligand binding and its dependence on CD8 (30 –32).

Results

Conclusion