In Situ Peptide-MHC-II Tetramer Staining of Antigen-Specific CD4+ T Cells in Tissues.

Thamotharampillai Dileepan,Hyeon O Kim,P Patrick Cleary,Pamela J Skinner

doi:10.1371/journal.pone.0128862

Abstract

The invention of peptide-MHC-tetramer technology to label antigen-specific T cells has led to an enhanced understanding of T lymphocyte biology. Here we describe the development of an in situ pMHC-II tetramer staining method to visualize antigen-specific CD4+ T cells in tissues. This method complements other methods developed that similarly use MHC class II reagents to stain antigen-specific CD4+ T cells in situ. In this study, we used group A streptococcus (GAS) expressing a surrogate peptide (2W) to inoculate C57BL/6 mice, and used fresh nasal-associated lymphoid tissues (NALT) in optimizing the in situ staining of 2W:I-Ab specific CD4+ T cells. The results showed 2W:I-Ab tetramer-binding CD4+ T cells in GAS-2W but not GAS infected mice. This method holds promise to be broadly applicable to study the localization, abundance, and phenotype of antigen-specific CD4+ T cells in undisrupted tissues.

Highlights

The development of peptide-MHC-II tetramer staining has revolutionized our ability to study antigen specific CD4+ T cells [1,2,3]
We describe an in situ MHC-class II tetramer staining technique that should be generally applicable to visualizing antigen-specific CD4 T cells in tissues
Intranasal group A streptococcus (GAS) infections induce a strong antigen specific Th17 response in nasal-associated lymphoid tissue (NALT) of mice and multiple infections increase the number of these cells in NALT [11,13]

Summary

Introduction

The development of peptide-MHC-II (pMHC-II) tetramer staining has revolutionized our ability to study antigen specific CD4+ T cells [1,2,3]. Several groups including ours have developed methods using MHC class II reagents to visualize antigen-specific CD4+ T cells in tissues with their spatial relationship to other cells intact. Bischof et al, used mouse class II I-As tetramers to label self-reactive CD4 T cells in fresh PBS perfused lymph node and central nervous tissues from experimental autoimmune encephalomyelitis (EAE) mice [8]. We describe the successful development of an additional in situ staining technique using pMHC-II tetramers to visualize antigen specific CD4+ T cells in tissues with their spatial relationship to other cells intact. We describe an in situ MHC-class II tetramer staining technique that should be generally applicable to visualizing antigen-specific CD4 T cells in tissues

Materials and Methods

Ethics Statement

Background levels

Results and Discussion