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Class I MHC alpha 3 domain can function as an independent structural unit to bind CD8 alpha
J Fayen1, J H Huang, H Meyerson
1Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.
Insights
This study reveals that CD8 alpha directly binds to the alpha 3 domain of class I MHC molecules. This interaction is specific and does not require other cellular components for T cell recognition.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- Cytotoxic T cell function relies on CD8-mediated interactions with target cells.
- CD8 interacts with a specific region on the alpha 3 domain of class I MHC molecules.
Purpose of the Study:
- To develop a cell-free assay to directly measure the molecular interaction between CD8 and class I MHC.
- To characterize the binding kinetics and specificity of this interaction.
Main Methods:
- A cell-free system using soluble CD8 alpha and a plate-bound HLA-A2.1 alpha 3/MalE fusion protein.
- Measurement of binding affinity (Kd) and inhibition studies using monoclonal antibodies.
Main Results:
- Demonstrated specific and saturable binding between soluble CD8 alpha and the alpha 3 domain.
- Determined the dissociation constant (Kd) for this interaction to be 4.5 x 10(-7) M.
- Monoclonal antibodies targeting CD8 or the alpha 3 domain inhibited binding, while others did not.
Conclusions:
- The interaction between CD8 alpha and the class I MHC alpha 3 domain is direct and specific.
- This interaction does not necessitate neighboring class I MHC sequences or beta 2-microglobulin.
Abstract:
Functional interactions between CD8-dependent cytotoxic T cells and their targets require physical contact between CD8 and a non-polymorphic determinant on the alpha 3 domain of the class I MHC molecule. We developed a cell-free assay to directly monitor this molecular interaction, specifically excluding the participation of other cellular proteins and lipids. This assay employed a soluble CD8 derivative and a plate-bound HLA-A2.1 derivative, alpha 3/MalE, in which the alpha 3 domain has been expressed independently of its neighboring polypeptide domains on the native class I MHC molecule and beta 2-microglobulin (beta 2-m). These proteins were produced using eukaryotic and prokaryotic expression systems, respectively. Our data demonstrated specific, saturable binding between soluble CD8 alpha (sCD8 alpha) and alpha 3/MalE, and the Kd of this interaction was determined to be 4.5 x 10(-7) M. Monoclonal antibodies (mAb) directed against either CD8 or the alpha 3 domain of class I MHC inhibited binding; mAb directed against other sites on class I MHC and beta 2-m did not. Our data suggest that the interaction between CD8 alpha and the alpha 3 domain of class I MHC does not require the participation of neighboring class I sequences or beta 2-m.