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Skin-derived dendritic cells (DCs) play a crucial role in the maintenance of immune homeostasis due to their role in antigen trafficking from the skin to the draining lymph nodes (dLNs). To quantify the spatiotemporal regulation of skin-derived DCs in vivo, we generated knock-in mice expressing the photoconvertible fluorescent protein KikGR. By exposing the skin or dLN of these mice to violet light, we were able to label and track the migration and turnover of endogenous skin-derived DCs. Langerhans cells and CD103+DCs, including Langerin+CD103+dermal DCs (DDCs), remained in the dLN for 4–4.5 days after migration from the skin, while CD103−DDCs persisted for only two days. Application of a skin irritant (chemical stress) induced a transient >10-fold increase in CD103−DDC migration from the skin to the dLN. Tape stripping (mechanical injury) induced a long-lasting four-fold increase in CD103−DDC migration to the dLN and accelerated the trafficking of exogenous protein antigens by these cells. Both stresses increased the turnover of CD103−DDCs within the dLN, causing these cells to die within one day of arrival. Therefore, CD103−DDCs act as sentinels against skin invasion that respond with increased cellular migration and antigen trafficking from the skin to the dLNs.

Flow cytometric analysis with multicolor fluoroprobes is an essential method for detecting biological signatures of cells. Here, we present a new full‐spectral flow cytometer (spectral‐FCM). Unlike conventional flow cytometer, this spectral‐FCM acquires the emitted fluorescence for all probes across the full‐spectrum from each cell with 32 channels sequential PMT unit after dispersion with prism, and extracts the signals of each fluoroprobe based on the spectral shape of each fluoroprobe using unique algorithm in high speed, high sensitive, accurate, automatic and real‐time. The spectral‐FCM detects the continuous changes in emission spectra from green to red of the photoconvertible protein, KikGR with high‐spectral resolution and separates spectrally‐adjacent fluoroprobes, such as FITC (Emission peak (Em) 519 nm) and EGFP (Em 507 nm). Moreover, the spectral‐FCM can measure and subtract autofluorescence of each cell providing increased signal‐to‐noise ratios and improved resolution of dim samples, which leads to a transformative technology for investigation of single cell state and function. These advances make it possible to perform 11‐color fluorescence analysis to visualize movement of multilinage immune cells by using KikGR‐expressing mice. Thus, the novel spectral flow cytometry improves the combinational use of spectrally‐adjacent various FPs and multicolor fluorochromes in metabolically active cell for the investigation of not only the immune system but also other research and clinical fields of use. © 2015 The Authors. Cytometry Part A Published by Wiley Periodicals, Inc. on behalf of ISAC

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Foxp3 Regulatory T cells (Tregs) play a critical role in the maintenance of colon homeostasis. Here we utilized photoconvertible KikGR mice to track immune cells from the caecum and ascending (proximal) colon in the steady state and DSS-induced colitis. We found that Tregs from the proximal colon (colonic migratory Tregs) migrated exclusively to the distal part of mesenteric lymph nodes (dMLN) in an S1PR1-dependent process. In the steady state, colonic migratory CD25 Tregs expressed higher levels of CD103, ICOS, LAG3 and CTLA-4 in comparison with pre-existing LN Tregs. Intestinal inflammation led to accelerated Treg replacement in the colon, bidirectional Treg migration from the colon to dMLN and vice versa, as well as increases in Treg number, proliferation and expression of immunosuppressive molecules. This was especially apparent for CD25 very high Tregs induced in colitis. Furthermore, colonic migratory Tregs from the inflamed colon included more interleukin (IL)-10 producing cells, and demonstrated greater inhibition of T-cell proliferation in comparison with pre-existing LN Tregs. Thus, our results suggest that Tregs with superior immunosuppressive capacity are increased both in the colon and dMLN upon inflammation. These Tregs recirculate between the colon and dMLN, and are likely to contribute to the downregulation of intestinal inflammation.