Highlights d Two precursor stages of ST2 + nonlymphoid-tissue Tregs are evident in the spleen and LNs d Precursor stages are defined by differential expression of Nfil3, PD1, and Klrg1 d Chromatin accessibility and scRNA-seq suggests a stepwise precursor reprogramming d Batf drives the molecular tissue program in the precursors Authors

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers.

Highlights d Comparison of chromatin accessibility between murine und human tissue Treg cells d TCR tracking revealed clonal relationship between tissue and blood BATF + CCR8 + Tregs d Treg cells from healthy tissues were similar to CCR8 + Treg cells from tumors d Tfh-like differentiation program induces tissue Treg cell repair characteristics

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