Understanding immune phenotypes in human gastric disease tissues by multiplexed immunohistochemistry

Le Ying,Yiqun Hu,David W Chan,Feng Yan,Dakang Xu,Liang Yu,Xuefeng Wang,Peihua Ni,Liyun Shi,Qiaohong Meng,Bryan R G Williams,Yugang Tu,Xiangliang Yuan

doi:10.1186/s12967-017-1311-8

Abstract

BackgroundUnderstanding immune phenotypes and human gastric disease in situ requires an approach that leverages multiplexed immunohistochemistry (mIHC) with multispectral imaging to facilitate precise image analyses.MethodsWe developed a novel 4-color mIHC assay based on tyramide signal amplification that allowed us to reliably interrogate immunologic checkpoints, including programmed death-ligand 1 (PD-L1), cytotoxic T cells (CD8+T) and regulatory T cells (Foxp3), in formalin-fixed, paraffin-embedded tissues of various human gastric diseases. By observing cell phenotypes within the disease tissue microenvironment, we were able to determine specific co-localized staining combinations and various measures of cell density.ResultsWe found that PD-L1 was expressed in gastric ulcer and in tumor cells (TCs), as well as in tumor-infiltrating immune cells (TIICs), but not in normal gastric mucosa or other gastric intraepithelial neoplastic tissues. Furthermore, we found no significant reduction in CD8+T cells, whereas the ratio of CD8+T:Foxp3 cells and CD8+T:PD-L1 cells was suppressed in tumor tissues and elevated in adjacent normal tissues. An unsupervised hierarchical analysis also identified correlations between CD8+T and Foxp3+ tumor-infiltrating lymphocyte (TIL) densities and average PD-L1 levels. Three main groups were identified based on the results of CD8+T:PD-L1 ratios in gastric tumor tissues. Furthermore, integrating CD8+T:Foxp3 ratios, which increased the complexity for immune phenotype status, revealed 6–7 clusters that enabled the separation of gastric cancer patients at the same clinical stage into different risk-group subsets.ConclusionsCharacterizing immune phenotypes in human gastric disease tissues via multiplexed immunohistochemistry may help guide PD-L1 clinical therapy. Observing unique disease tissue microenvironments can improve our understanding of immune phenotypes and cell interactions within these microenvironments, providing the ability to predict safe responses to immunotherapies.

Highlights

Understanding immune phenotypes and human gastric disease in situ requires an approach that leverages multiplexed immunohistochemistry with multispectral imaging to facilitate precise image analyses
Among all regulatory T cells (Tregs), Foxp3-expressing Tregs are well known to play a critical role in tumor immune evasion [7], which has been reported in a wide array of human malignancies including our study in gastric cancer [7, 8]
Comparison of immunohistochemistry and multiplexed immunohistochemistry (mIHC) to understand immune phenotypes in human gastric cancer We hypothesized that analyzing cell–cell relationships via mIHC will provide a more complete view of tumor microenvironments than traditional IHC

Summary

Introduction

Understanding immune phenotypes and human gastric disease in situ requires an approach that leverages multiplexed immunohistochemistry (mIHC) with multispectral imaging to facilitate precise image analyses. Many recent studies have reported that the response to immunotherapy primarily depends on the expression of PD-L1 in the tumor microenvironment. These findings suggest that the response to anti-PD-1 hinges on pre-existing anti-tumor immune response and that anti-PD-1 acts to free the CD8 T cells from inhibition to exert their anti-tumor activities [3]. Studies using quantitative IHC have identified CD8+ T cell infiltration as an important prognostic factor in predicting outcomes in patients with gastric cancer [6]. Upregulation of Tregs is associated with significantly reduced CD8+ T cell infiltration of tumors and with worse outcomes for cancer patients [9]. The clinical implications of immunosuppressive processes related to immunologic checkpoints (PD-L1, CD8, and Foxp3) in tumors or immune cells in the tumor microenvironment remain controversial, and the potential use of these checkpoints as prognostic markers requires further study

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