RESUMO
Cytotoxic T lymphocytes recognize short peptides presented in association with MHC class I (MHCI) molecules on the surface of target cells. The Ag specificity of T lymphocytes is conferred by the TCR, but invariable regions of the peptide-MHCI (pMHCI) molecule also interact with the cell surface glycoprotein CD8. The distinct binding sites for CD8 and the TCR allow pMHCI to be bound simultaneously by both molecules. Even before it was established that the TCR recognized pMHCI, it was shown that CTL exhibit clonal heterogeneity in their ability to activate in the presence of anti-CD8 Abs. These Ab-based studies have since been interpreted in the context of the interaction between pMHCI and CD8 and have recently been extended to show that anti-CD8 Ab can affect the cell surface binding of multimerized pMHCI Ags. In this study, we examine the role of CD8 further using point-mutated pMHCI Ag and show that anti-CD8 Abs can either enhance or inhibit the activation of CTL and the stable cell surface binding of multimerized pMHCI, regardless of whether there is a pMHCI/CD8 interaction. We further demonstrate that multimerized pMHCI Ag can recruit CD8 in the absence of a pMHCI/CD8 interaction and that anti-CD8 Abs can generate an intracellular activation signal resulting in CTL effector function. These results question many previous assumptions as to how anti-CD8 Abs must function and indicate that CD8 has multiple roles in CTL activation that are not necessarily dependent on an interaction with pMHCI.
Assuntos
Adjuvantes Imunológicos/farmacologia , Anticorpos Bloqueadores/farmacologia , Sítios de Ligação de Anticorpos , Antígenos CD8/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunossupressores/farmacologia , Ativação Linfocitária/imunologia , Peptídeos/metabolismo , Animais , Ligação Competitiva/imunologia , Antígenos CD8/metabolismo , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Clonais , Reagentes de Ligações Cruzadas/metabolismo , Citotoxicidade Imunológica/imunologia , Antígenos H-2/genética , Antígenos H-2/metabolismo , Antígeno HLA-A2/metabolismo , Humanos , Camundongos , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismoRESUMO
Tetrameric MHC/peptide complexes are important tools for enumerating, phenotyping, and rapidly cloning Ag-specific T cells. It remains however unclear whether they can reliably distinguish between high and low avidity T cell clones. In this report, tetramers with mutated CD8 binding site selectively stain higher avidity human and murine CTL capable of recognizing physiological levels of Ag. Furthermore, we demonstrate that CD8 binding significantly enhances the avidity as well as the stability of interactions between CTL and cognate tetramers. The use of CD8-null tetramers to identify high avidity CTL provides a tool to compare vaccination strategies for their ability to enhance the frequency of high avidity CTL. Using this technique, we show that DNA priming and vaccinia boosting of HHD A2 transgenic mice fail to selectively expand large numbers of high avidity NY-ESO-1(157-165)-specific CTL, possibly due to the large amounts of antigenic peptide delivered by the vaccinia virus. Furthermore, development of a protocol for rapid identification of high avidity human and murine T cells using tetramers with impaired CD8 binding provides an opportunity not only to monitor expansion of high avidity T cell responses ex vivo, but also to sort high avidity CTL clones for adoptive T cell transfer therapy.