Mapping Influenza-Induced Posttranslational Modifications on Histones from CD8+ T Cells.

Svetlana Rezinciuc,Shawna Hengel,Zhixin Tian,Heather S. Smallwood,Ljiljana Pasa-Tolic,Si Wu

doi:10.3390/v12121409

Abstract

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection.

Highlights

Clearance of viral infection depends upon a well-orchestrated immune response and requires precise control of the immediate effector T cell response as well as the formation and maintenance of the memory T cell population
Using our established methods [3,5,52], female C57BL/6 mice were intranasally infected with influenza strain X31, spleens harvested nine days later, and T cells isolated by Fluorescence-activated cell sorting (FACS) (Figure 1a)
All core histones were loaded onto the columnand separated into individual families H4, H2B, H2A, and H3 that eluted in increasing order of molecular weight, which roughly correlates with increasing hydrophobicity (i.e., 11,352.5, 13,757.1, 14,019.9, and 15,350.8 Da, respectively) (Figure 1a–c)

Summary

Introduction

Clearance of viral infection depends upon a well-orchestrated immune response and requires precise control of the immediate effector T cell response as well as the formation and maintenance of the memory T cell population. Activation of naïve T cells (Tn) initiates an autonomous program of differentiation and the acquisition of effector functions, including pro-inflammatory cytokine and cytolytic effector molecule production [1,2,3,4,5]. Effector CD8 T cells (Teff) modulate their transcriptional programs as they adapt to activation stimuli and immune resolution, which influences their differentiation status and function. In response to dynamic environmental conditions, naïve cells alter their signaling cascades and pathways, leading to the induction of cytokine production and robust cytolytic activity. After the resolution of viral infection, Teff populations contract, and a small population of pathogen-specific long-lived memory cells remain. Memory CD8 T cells (Tm) are Viruses 2020, 12, 1409; doi:10.3390/v12121409 www.mdpi.com/journal/viruses

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Discussion

Conclusion