Summary We identify the interfollicular (IF) zone as the site where germinal center B cell and T follicular helper (Tfh) cell differentiation initiates. For the first two days post-immunization, antigen-specific T and B cells remained confined within the IF zone, formed long-lived interactions, and upregulated the transcriptional repressor Bcl6. T cells also acquired the Tfh cell markers CXCR5, PD-1 and GL7. Responding B and T cells migrated to the follicle interior directly from the IF zone, T cell immigration preceding B cells by one day. Notably, in the absence of cognate B cells, Tfh cells still formed and migrated to the follicle. However, without such B cells, PD-1, ICOS and GL7 were no longer expressed on follicular Bcl6hi T cells that nevertheless persisted in the follicle. Thus, Ag-specific B cells are required for the maintenance of the PD-1hi ICOShi GL7hi Tfh cell phenotype within the follicle, but not for their initial differentiation in the IF zone.

Tissue development and regeneration depend on cell–cell interactions and signals that target stem cells and their immediate progeny1. However, the cellular behaviours that lead to a properly regenerated tissue are not well understood. Using a new, non-invasive, two-photon (intravital instead of two-photon?) imaging approach we study physiological hair-follicle regeneration in real time in live mice. By these means we have monitored the behaviour of epithelial stem cells and their progeny2–4 during physiological hair regeneration and addressed how the mesenchyme5 influences their behaviour. Consistent with earlier studies6, stem cells are quiescent during the initial stages of hair regeneration, whereas the progeny is more actively dividing. Moreover, stem cell progeny divisions are spatially organized within follicles. In addition to cell divisions, coordinated cell movements of the progeny allow the rapid expansion of the hair follicle. Finally, we show the requirement of the mesenchyme for hair regeneration through targeted cell ablation and long-term tracking of live hair follicles. Thus, we have established an in vivo approach that has led to the direct observation of cellular mechanisms of growth regulation within the hair follicle and that has enabled us to precisely investigate functional requirements of hair-follicle components during the process of physiological regeneration.

The tumour microenvironment thwarts conventional immunotherapy through multiple immunologic mechanisms, such as the secretion of the transforming growth factor-β (TGF-β), which stunts local tumour immune responses. Therefore, high doses of interleukin-2 (IL-2), a conventional cytokine for metastatic melanoma, induces only limited responses. To overcome the immunoinhibitory nature of the tumour microenvironment, we developed nanoscale liposomal polymeric gels (nanolipogels; nLGs) of drug-complexed cyclodextrins and cytokine-encapsulating biodegradable polymers that can deliver small hydrophobic molecular inhibitors and water-soluble protein cytokines in a sustained fashion to the tumour microenvironment. nLGs releasing TGF-β inhibitor and IL-2 significantly delayed tumour growth, increased survival of tumour-bearing mice, and increased the activity of natural killer cells and of intratumoral-activated CD8+ T-cell infiltration. We demonstrate that the efficacy of nLGs in tumour immunotherapy results from a crucial mechanism involving activation of both innate and adaptive immune responses.

Adult tissues replace lost cells via pools of stem cells. However, the mechanisms of cell self-renewal, commitment, and functional integration into the tissue remain unsolved. Using imaging techniques in live mice, we captured the lifetime of individual cells in the ear and paw epidermis. Our data suggest that epidermal stem cells have equal potential to either divide or directly differentiate. Tracking stem cells over multiple generations reveals that cell behavior is not coordinated between generations. However, sibling cell fate and lifetimes are coupled. We did not observe regulated asymmetric cell divisions. Lastly, we demonstrated that differentiating stem cells integrate into preexisting ordered spatial units of the epidermis. This study elucidates how a tissue is maintained by both temporal and spatial coordination of stem cell behaviors.

Summary: Maintenance of adult tissues depends on sustained activity of resident stem cell populations, yet the mechanisms that regulate stem cell self-renewal during homeostasis remain largely unknown. Using an imaging and tracking approach that captures all epidermal stem cell activity in large regions of living mice, we show that self-renewal is locally coordinated with epidermal differentiation, with a lag time of one to two days. In both homeostasis and upon experimental perturbation, we find that differentiation of a single stem cell is followed by division of a direct neighbor, but not vice versa. Finally, we show that exit from the stem cell compartment is sufficient to drive neighboring stem cell self-renewal. Together, these findings establish that epidermal stem cell self-renewal is not the constitutive driver of homeostasis. Instead it is precisely tuned to tissue demand and responds directly to neighbor cell differentiation.