In situ peptide-MHC-II tetramer staining of antigen-specific CD4+ T cells in tissues (65.37)

Abstract

Abstract The invention of peptide-MHC-tetramer (pMHC) technology to label antigen-specific T cells has lead to a greatly enhanced understanding of T lymphocyte biology. Although in situ staining of antigen specific CD8+ T cells with pMHCI tetramers has been well characterized and permits visualization and characterization of antigen-specific CD8+ T cells relative to other cell types in stained undisrupted tissue sections, similar staining of antigen specific CD4+ T cells in situ with pMHCII tetramers has been elusive. Thus far, detection of antigen specific CD4+ T cells with pMHCII tetramers has been limited to cells isolated from blood or disrupted tissues that are analyzed by flow cytometry. In this report, we describe our development of an in situ pMHCII tetramer staining method to directly visualize antigen-specific CD4+ T cells in tissues. We used an infectious model system, where Group A streptococcus (GAS) expressing a surrogate peptide (2W) fused to the M1 protein was used to inoculate C57BL/6 mice. NALT from these mice were used in optimizing the in situ staining of 2W:I-Ab specific CD4+ T cells. Parallel flow cytometric analysis was performed to validate the findings of in situ staining. The results showed pMHCII tetramer stained cells in GAS-2W but not GAS infected mice. Thus, we have developed a pMHCII in situ staining method, and this novel methodology can be used to study the localization, abundance and phenotype of antigen specific CD4+ T cells in tissues.