#Photoconvert linage tracking skin#

Note that Icos, Il1rl1, Klrg1, and Il7r are markers of ILC2s.

( c) Single-gene expression t-SNE plots of the single-cell RNA-seq data. ( b) Heatmaps of the representative ILC2-related genes and chemokine receptor‒related genes from each cluster. Hierarchical clustering based on gene expression profiles was performed, and the ILC2s split into two clusters: cluster 1 (circulating ILC2s) and cluster 2 (skin-resident ILC2s). t-SNE plot of the single-cell RNA-seq data of 1,052 ILC2s combined from three mice. ( a) Single-cell RNA-seq identifies two clusters.

#Photoconvert linage tracking skin#

However, both cell populations demonstrated minimal expression of chemokine receptor‒related genes ( Figure 1b, right panel), consistent with the findings ofįigure 1 Single-cell RNA-seq analysis of sorted ILC2s from the skin and the dLNs of transgenic mice expressing IL-33 (I元3tg). Both the circulating ILC2s (cluster 1) and the skin-resident ILC2s (cluster 2) expressed Icos and Cd69. Gene-generated heatmaps revealed differences in the gene signatures expressed in each cluster ( Figure 1b).

We named cluster 1, found in both the skin and lymph nodes, circulating ILC2s and named cluster 2, found only in the skin, skin-resident ILC2s. Therefore, we hypothesized that among the ILC2s in the skin, only cluster 1 migrated to the dLNs, and cluster 2 remained in the skin.

Conversely, only one cluster of lymph node ILC2s was found (cluster 1). Using hierarchical clustering, a cluster analysis method that attempts to build a hierarchy of clusters, we found that the skin ILC2s had split into two clusters: clusters 1 and 2 ( Figure 1a). To resolve the molecular signatures of the skin ILC2s, we sorted the ILC2s from the skin lesions and dLNs of I元3tg mice and analyzed their transcriptomes using the BD Rhapsody Single-Cell Analysis System (BD Biosciences, San Jose, CA) for single-cell RNA sequencing (RNA-seq) analysis ( Figure 1). Abbreviations: AD ( atopic dermatitis), DC ( dendritic cell), dLN ( draining lymph node), ILC2 ( group 2 innate lymphoid cell), KikGR ( Kikume Green-Red), MHC ( major histocompatibility complex), RNA-seq ( RNA sequencing) Thus, the skin ILC2s turned out to be a heterogeneous cell population. Conversely, the skin-resident ILC2s produced a large number of cytokines. Cutaneous local innate responses could transition to systemic type 2 responses by migrating the activated ILC2s from the skin into the draining lymph node. Exposing the I元3tg–Kikume Green-Red mice’s inflamed skin to violet light allowed us to label the circulating ILC2s in their skin and track the ILC2 migration from the skin to the draining lymph nodes. Next, we tracked ILC2 migration using I元3tg–Kikume Green-Red mice. Conversely, the skin-resident ILC2s demonstrated increased mRNA expression of the ICOS, IL-5, and IL-13. The circulating ILC2s expressed H2-related major histocompatibility complex class II genes. We sorted ILC2s from the skin and draining lymph nodes of I元3tg mice and analyzed their transcriptomes using the single-cell RNA sequencing technique, which revealed that the skin ILC2s had split into two clusters: circulating ILC2 and skin-resident ILC2. ILC2s are believed to be tissue-resident cells under steady-state conditions, but the dynamics of ILC2 migration are not fully understood. We previously generated a transgenic mouse line expressing skin-specific IL-33 (I元3tg mice) and showed that IL-33 elicits group 2 innate lymphoid cell (ILC2)–dependent atopic dermatitis–like skin inflammation.