233: Multiparametric high dimensional analysis of normal & VZV infected human tonsil T cells at a single cell resolution by mass cytometry

Abstract

Varicella Zoster Virus (VZV), an alpha-herpesvirus, is a host and cell type restricted pathogen that infects humans to cause “Varicella” (chicken pox) and “Zoster” (shingles). VZV infects tonsil CD4+ T lymphocytes that carry the virus to the skin cells and sensory ganglion neurons; how VZV hijacks immune T cells to replicate and be transported is poorly understood. Mass Cytometry (Cytof) has recently been successfully applied to study complex biological systems at a single cell resolution by using isotope labeled antibodies in a multiplex system. A dual panel comprising of (a) “phenotypic panel” (to detect 20 different surface proteins) and (b) “functional panel” (to detect 11 surface +15 phospho-proteins) were used for this study. VZV-infected & uninfected primary T cells were subjected to the dual panel Cytof analysis; infected cells were detected by an antibody to glycoprotein E tagged to isotope Dy164. Data was analyzed by the SPADE software and other novel statistical algorithms that were designed to detect phenotypic and signaling changes within and between the infected and uninfected populations. Reproducibility of detected changes was addressed through the new notion of “stable assimilation” of single cell level information. This multiplexed study provided an extensive insight into the presence of phenotypically and functionally different sub-populations of T cells in human tonsils. In context of infection, there was decreased expression of CD3 and increased expression of PD-1 and CD69 on infected cells correlating with increased phosphorylation of proteins in the TCR pathway similar to changes observed during T cell activation. In addition, infected naive T cells appeared to be transitioning to the memory-like phenotype – a phenomenon also associated with T cell activation. VZV-infected cells demonstrated decreased CD7, a feature characteristic of cutaneous T cell lymphoma. Several other interesting findings along with these observations will be discussed in context of herpesvirus pathogenesis.