Viral antigen density and confinement time regulate the reactivity pattern of CD4 T-cell responses to vaccinia virus infection

T-cell recognition of ligands is polyspecific. This translates into antiviral T-cell responses having a range of potency and specificity for viral ligands. How these ligand recognition patterns are established is not fully understood. Here, we show that an activation threshold regulates whether robust CD4 T-cell activation occurs following viral infection. The activation threshold was variable because of its dependence on the density of the viral peptide (p)MHC displayed on infected cells. Furthermore, the activation threshold was not observed to be a specific equilibrium affinity (K_D) or half-life (t_1/2) of the TCR–viral pMHC interaction, rather it correlated with the confinement time of TCR–pMHC interactions, i.e., the half-life (t_1/2) of the interaction accounting for the effects of TCR–pMHC rebinding. One effect of a variable activation threshold is to allow high-density viral pMHC ligands to expand CD4 T cells with a variety of potency and peptide cross-reactivity patterns for the viral pMHC ligand, some of which are only poorly activated by infections that produce a lower density of the viral pMHC ligand. These results argue that antigen concentration is a key component in determining the pattern of K_D, t_1/2 and peptide cross-reactivity of the TCRs expressed on CD4 T cells responding to infection.