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

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.

Single-cell RNA-Sequencing uncovers transcriptional states and fate decisions in haematopoiesis

“…The maintenance of wild-type (Tubingen Long Fin) and transgenic zebrafish lines [29][30][31][42][43][44][45][46] (Supplementary Table 1) was performed in accordance with EU regulations on laboratory animals, as previously described 47 .…”

Single-cell transcriptional analysis reveals naïve helper ILC-like cells in zebrafish

“…These cells had a high expression of macrophage receptor with collagenous structure (marco) and macrophage expressed gene 1 (mpeg1.1), ( Figure 1B) strongly indicative of their macrophage identity. This was not surprising, as cd4-1:mCherry has been found to label both CD4 T cells and macrophages (Dee et al, 2016).…”

“…To capture the diversity of lymphoid cell types, we purified and sequenced the RNA from single cells collected from primary lymphoid organs (kidney and thymus), secondary lymphoid organs (spleen) as well as barrier tissues (gut and gills) of healthy, unstimulated adult zebrafish. We used three different transgenic lines: Tg(lck:EGFP) (Langenau et al, 2004), which labels T cells as well as NK cells (Carmona et al, 2017); Tg(cd4-1:mCherry) (Dee et al, 2016), which labels CD4 T cells and macrophages, and Tg(mhc2dab:GFP, cd45:dsRed) (Wittamer et al, 2011) ( Figure 1A) that is expected to label B cells (when sorted as GFP+/DsRed-).…”

“…The maintenance of zebrafish wild-type line (AB), transgenic lines Tg(lck:EGFP) (Langenau et al, 2004), Tg(cd4-1:mcherry) (Dee et al, 2016), Tg(mhc2dab:GFP, cd45:dsRed) (Wittamer et al, 2011) and rag1 hu1999 mutants (also known as rag1 t26683/26683 (Wienholds et al, 2002)) was performed in accordance with EU regulations on laboratory animals. Hu1999 mutation results in a premature stop codon in the middle of the catalytic domain of the Rag1 protein and is considered a null allele (Wienholds et al, 2002).…”

High cholesterol diet modulates macrophage polarization and liver inflammation during early hepatocellular carcinoma progression in zebrafish

“…To quantify leukocyte recruitment we outcrossed double transgenic HCC lines carrying the EGFP liver marker, Tg(fabp10a:ptβ-catenin_cryaa:venus)/(fabp10a:egfp), with a doubletransgenic line with labelled macrophages and neutrophils, Tg(mfap4:tdTomato-CAAX/lyzc:bfp). For T cell recruitment quantification, we outcrossed HCC line, Tg(fabp10a:ptβ-catenin_cryaa:venus), with transgenic line with labelled T cells, Tg(CD4.1-mCherry/lck:egfp) (24). After live imaging, Z series images were reconstructed in 2D maximum intensity projections (MIP) on ZEN pro 2012 software (Zeiss).…”