CD4-Transgenic Zebrafish Reveal Tissue-Resident Th2- and Regulatory T Cell-like Populations and Diverse Mononuclear Phagocytes.

Christopher T Dee,Ana Cvejic,Adam F L Hurlstone,Christopher J Secombes,Caroline Gray,Raghavendar T Nagaraju,Simon A Johnston,Laura Fernandez Del Ama,Emmanouil I Athanasiadis

doi:10.4049/jimmunol.1600959

Abstract

CD4+ T cells are at the nexus of the innate and adaptive arms of the immune system. However, little is known about the evolutionary history of CD4+ T cells, and it is unclear whether their differentiation into specialized subsets is conserved in early vertebrates. In this study, we have created transgenic zebrafish with vibrantly labeled CD4+ cells allowing us to scrutinize the development and specialization of teleost CD4+ leukocytes in vivo. We provide further evidence that CD4+ macrophages have an ancient origin and had already emerged in bony fish. We demonstrate the utility of this zebrafish resource for interrogating the complex behavior of immune cells at cellular resolution by the imaging of intimate contacts between teleost CD4+ T cells and mononuclear phagocytes. Most importantly, we reveal the conserved subspecialization of teleost CD4+ T cells in vivo. We demonstrate that the ancient and specialized tissues of the gills contain a resident population of il-4/13b–expressing Th2-like cells, which do not coexpress il-4/13a. Additionally, we identify a contrasting population of regulatory T cell–like cells resident in the zebrafish gut mucosa, in marked similarity to that found in the intestine of mammals. Finally, we show that, as in mammals, zebrafish CD4+ T cells will infiltrate melanoma tumors and obtain a phenotype consistent with a type 2 immune microenvironment. We anticipate that this unique resource will prove invaluable for future investigation of T cell function in biomedical research, the development of vaccination and health management in aquaculture, and for further research into the evolution of adaptive immunity.

Highlights

We described specific populations of CD4-1+ mononuclear phagocyte (MNP) suggesting that this cell type had already emerged in early vertebrates
Recent studies employing antisera to CD4-1 in zebrafish and rainbow trout have shown that stimulation with Ag or infection results in the expression of relevant cytokines and transcription factor (TF), but they were unable to identify cell populations skewed toward a particular Th phenotype [14,15,16]
To look for Th2-like cells we examined gene expression in CD4-1+ T cells from the gills and compared them to those isolated from the gut and kidney and spleen

Summary

Introduction

To provide further detail on the specificity of the reporter, we isolated single mCherry+ cells from the spleen and gills of Tg(cd4-1:mCherry) transgenic animals and subjected them to RNA sequencing (Supplemental Fig. 1D–G). Hierarchical clustering independently confirmed that the cells labeled by the reporter identify as either T cells or MNPs, and it indicated that cd4-1 expression is considerably higher in the T cell population (Supplemental Fig. 1D–G).

Results

Conclusion