Clonotypic Analysis of CMV-Specific CD4+ T Cells in Human and Nonhuman Primates

Abstract

Cytomegalovirus (CMV) is a complex pathogen with the ability to persist in a host via mechanisms of immune evasion. CD4+ T cells are known to play a role in maintaining life-long immunity against CMV; however, the cellular requirements for establishing and maintaining protection against CMV disease have not been characterized. The objective of this work was to understand the nature of a protective CD4+ T cell memory response in primates using CMV as a model viral pathogen. First, we characterized the clonotypic hierarchy of an established CMV-specific CD4+ memory T cell response in human subjects and Rhesus macaques (RM). In both we found that long-term CD4+ memory responses to CMV are characterized by highly skewed clonotypic hierarchies, and these hierarchies remained stable over the months examined. We then used the RM model to elucidate the evolution of the CMV-specific CD4+ clonotypic hierarchy during and after primary infection. The clonotypic composition of an emerging CMV-specific response during a primary infection was strikingly diverse. Third, we reinfected the RM and found that reinfection with CMV recruited new clonotypes into the response, further increasing clonotypic complexity. Taken together, these data indicate the CMV-specific CD4+ T cell response undergoes an evolution during primary and secondary infection and includes the generation of a large initial repertoire, followed by selection of a few dominant clonotypes. In chronic infection, stable oligoclonal hierarchies predominate, suggesting that long-term surveillance for CMV reactivation or reinfection is mediated by a small number of clones, which are maintained at higher frequency. Having found these large single clonotype responses to CMV, we examined T cell receptor (TCR) activation requirements at the level of single clonotypes. We found that single clonotypes have heterogeneous activation thresholds and the activation thresholds for elaborating IL-2 and IFN-gamma differed. Finally, we found that the threshold heterogeneity within a clonotype was independent of CD27 expression.