Increased signalling efficiency is mediated by optimal co-localization of TCR and CD8 within lipid rafts: A novel mechanism for the control of functional avidity

The functional avidity of cytotoxic T cells (CTL) is a measure of the amount of peptide antigen that is required in order to elicit effector function. High avidity CTL are capable of responding to very low concentrations of peptide antigen, while low avidity CTL require substantially more peptide in order to become activated. The ability to respond to lower concentrations of peptide antigen allows for the increased efficacy of high avidity CTL in reducing viral burden in vivo, underscoring the importance of understanding the mechanisms which confer enhanced sensitivity exhibited by high avidity CTL.; The full range of parameters that can influence functional avidity are not clear. Although T cell receptor (TCR) affinity is one parameter that can contribute to the sensitivity to peptide antigen, we investigated additional mechanisms that may also modulate functional avidity. By developing an assay system utilizing TCR transgenic mice we have eliminated TCR affinity as a variable. These studies have demonstrated that CTL of high or low functional avidity can be generated independent of TCR affinity. We showed that low avidity CTL require more TCR engagement events in order to become activated compared to high avidity CTL. Furthermore, we found that high avidity CTL expressed CD8 predominantly as an αβ heterodimer, whereas low avidity CTL express a substantial amount of CD8 in the αα homodimeric form. Through the use of confocal microscopy, we have examined the membrane organization of CD8 and TCR in high and low avidity CTL generated from TCR transgenic mice, demonstrating that high and low avidity CTL differ dramatically in the membrane organization of TCR and CD8. Co-capping studies revealed that high avidity CTL form highly polarized caps of co-localized CD8 and TCR. In contrast, low avidity CTL fail to co-localize a substantial amount of TCR with CD8 and display patches of co-localization which fail to fully cap. The ability to co-cap CD8 and TCR is dependent on the integrity of lipid rafts, as treatment with methyl-β cylcodextrin (MBCD) completely abrogates TCR/CD8 co-capping. In addition to abolishing TCR/CD8 co-capping, raft disruption results in a lower functional avidity phenotype.; These data support a mechanism in which the increased sensitivity to TCR engagement demonstrated by high avidity CTL is the result of optimal compartmentalization of CD8 and TCR within membrane microdomains. Additionally, these studies have furthered our understanding of how CD8 can influence functional avidity by identifying a differential use of CD8 as a co-receptor by high and low avidity CTL.