MHC class I tetramers are widely used, usually in combination with an antibody to CD8, to detect antigen specific T cells. Some anti-CD8α antibodies block the interaction of murine MHC class I tetramers with CD8 T cells, while others such as 53.6.7, enhance. To understand the molecular basis for this effect, we mapped the epitope for the enhancing antibody 53.6.7 and three other blocking antibodies using a panel of murine CD8α (Lyt-2) mutants expressed on COS-7 transfectants. Mutations in residues that contact MHC class I affected binding of the blocking antibodies. In contrast, antibody 53.6.7 was affected by a mutation in the residue T81A located on the D-E loop. In the cocrystal of CD8αα with MHC class I, two different complexes (A and B) were observed, indicating the existence of different CD8 conformations. The T81 residue does not make contact with MHC class I in either complex, however, neighboring residues in the D-E loop make very different contacts in the two different complexes. The most likely explanation for antibody enhancement of tetramer bindings is that binding of 53.6.7 to CD8αβ stabilizes a conformation with a higher affinity for interaction with MHC class I and suggests that the CD8 binding site is flexible.
Bibliographical noteFunding Information:
We wish to thank Dr. Linda Rogozinski for her input and reading of the manuscript and Ms. Usha Soundararajan for excellent technical assistance. This work was funded by a grant from the National Institute of Health: RO1 #CA5411 (P.K.) and RO1 AI52163 (S.C.J.). M.A.D. was supported by an NIH Immunology Training Grant (T32 AI07313).
- Cell surface molecules
- Immunoglobulin domain
- MHC class I tetramers
- T lymphocytes