Jaring Schreuder

Microfold (M) cells transcytose luminal antigens to initiate mucosal adaptive immunity, but their role in organizing innate responses in Peyer’s patches is unclear. Here we showed that Peyer’s patch M cells organized an epithelial–group 3 innate lymphoid cell (ILC3) axis, establishing a spatial niche within the dome epithelium that drove ILC3 localization, proliferation and IL-22 production. We found that epithelial, but not hematopoietic, SPI-B was required for intestinal IgA responses to establish this niche. Single-cell profiling of intestinal SPI-B ⁺ epithelial cells revealed that M cells were highly heterogeneous, displaying tissue- and pathogen-specific transcriptional programs. Using subset-specific genetic perturbation and whole-mount imaging, we found that this circuit relied on CCR6-dependent positioning cues and RANK–RANKL signaling to ILC3 to regulate Peyer’s patch ILC3 homeostasis. Together, these findings identified Peyer’s patch M cells as organizers that spatially coordinated innate cell localization, proliferation and cytokine production to maintain mucosal barrier defense.

Natural killer cells defend against malignancies and viral infections through a tightly controlled program of differentiation and maturation. However, the transcriptional mechanisms guiding this process remain incompletely defined. Using paired single-cell multiomic profiling, we identify GFI1 as an epigenetic regulator of NK cell differentiation, coordinating EOMES and T-BET transcriptional balance to promote NK cell proliferation and the transition from immature to terminally differentiated NK cell states. GFI1 represses FOXO1 chromatin accessibility in mature NK cells, which normally limits NK cell proliferation and maturation. Co-deletion of both GFI1 and FOXO1 largely rescues NK cell differentiation, identifying a critical GFI1–FOXO1 axis required for protection against tumour metastasis. These findings position GFI1 as a key transcriptional node integrating NK cell differentiation, activation and effector programs.

Long-lived memory CD8⁺ T cells are essential for the control of persistent viral infections. The mechanisms that preserve memory cells are poorly understood. Fate mapping of the transcriptional repressor GFI1 identified that GFI1 was differentially regulated in virus-specific CD8⁺ T cells and was selectively expressed in stem cell memory and central memory cells. Deletion of GFI1 led to reduced proliferation and progressive loss of memory T cells, which in turn resulted in failure to maintain antigen-specific CD8⁺ T cell populations following infection with chronic lymphocytic choriomeningitis virus or murine cytomegalovirus. Ablation of GFI1 resulted in downregulation of the transcription factors EOMES and BCL-2 in memory CD8⁺ T cells. Ectopic expression of EOMES rescued the expression of BCL-2, but the persistence of memory CD8⁺ T cells was only partially rescued. These findings highlight the critical role of GFI1 in the long-term maintenance of memory CD8⁺ T cells in persistent infections by sustaining their proliferative potential.

Tissue-resident innate lymphoid cells (ILCs) play a vital role in the frontline defense of various tissues, including the lung. The development of type 2 ILCs (ILC2s) depends on transcription factors such as GATA3, RORα, GFI1, and Bcl11b; however, the factors regulating lung-resident ILC2s remain unclear. Through fate mapping analysis of the paralog transcription factors GFI1 and GFI1B, we show that GFI1 is consistently expressed during the transition from progenitor to mature ILC2s. In contrast, GFI1B expression is limited to specific subsets of bone marrow progenitors and lung-resident ILC progenitors. We found that GFI1B ⁺ lung ILC progenitors represent a multi-lineage subset with tissue-resident characteristics and the potential to form lung-derived ILC subsets and liver-resident ILC1s. Loss of GFI1B in bone marrow progenitors led to the selective loss of lung-resident IL-18R ⁺ ILCs and mature ILC2, subsequently preventing the emergence of effector ILCs that could protect the lung against inflammatory or tumor challenge.

In classic ‘ball-and-stick’ models of haematopoiesis the implicit assumption is that all cells within each defined stem or progenitor cell population are equivalent in their fate. Instead, more recent models suggest a haematopoietic stem and progenitor cell (HSPC) ‘continuum’ of lineage bias and commitment, which is largely inferred through ‘snapshot’ analysis of single cell gene expression or clonal fate. However, the dynamic assessment of lineage commitment of specific HSPC populations and their clonal output over time in vivo is still lacking but is essential to fully inform accurate models of haematopoiesis. Here, using cellular barcoding we compare the single cell output of long-term haematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (LMPPs), common myeloid progenitors (CMPs), common lymphoid progenitors (CLPs), and macrophage/dendritic cell progenitors (MDPs). Each population was assessed for their output to multiple haematopoietic cell types spanning a subset of time points from 9 to 112 days of haematopoiesis after transplantation. These analyses revealed a wide range of clonal fate patterns that were inconsistent with their eponymous labels, i.e. stem and multipotent progenitors were rarely multi- or equipotent, and ‘common’ progenitors were often highly restricted in their fate. To better describe how these clonal patterns integrate into a revised landscape, a novel agent-based mathematical modelling approach that explicitly accounts for haematopoiesis at a clonal level was developed to allow the simulation of growth, timing and branching of clonal trajectories that underlie the process. Rather than a continuum, the proposed model is suggestive of multiple tracks down which clonal trajectories progress, and where fate can branch to a track of lower potency at multiple points down the entire cascade of haematopoiesis. Graphical In a multi-track model, while some HSPCs are multipotent and branch into daughters with committed fate (left trajectory), other HSPCs may already committed or biased to a certain lineage such that their daughters inherit and maintain it in subsequent divisions during haematopoiesis. However, this commitment/fate bias is not evident through current phenotypic definitions of HSPC subsets (background colour) but occurs through putative expression of transcription factors, epigenetic programming or other factor that is currently unresolved (as indicated by the coloured fate potential triangles inside cells).

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Regulation of haematopoietic stem and progenitor cell (HSPC) fate is crucial during homeostasis and under stress conditions. Here we examine the aetiology of the Flt3 ligand (Flt3L)-mediated increase of type 1 conventional dendritic cells (cDC1s). Using cellular barcoding we demonstrate this occurs through selective clonal expansion of HSPCs that are primed to produce cDC1s and not through activation of cDC1 fate by other HSPCs. In particular, multi/oligo-potent clones selectively amplify their cDC1 output, without compromising the production of other lineages, via a process we term tuning. We then develop Divi-Seq to simultaneously profile the division history, surface phenotype and transcriptome of individual HSPCs. We discover that Flt3L-responsive HSPCs maintain a proliferative ‘early progenitor’-like state, leading to the selective expansion of multiple transitional cDC1-primed progenitor stages that are marked by Irf8 expression. These findings define the mechanistic action of Flt3L through clonal tuning, which has important implications for other models of ‘emergency’ haematopoiesis.

A classical view of blood cell development is that multipotent hematopoietic stem and progenitor cells (HSPCs) become lineage-restricted at defined stages. Lin⁻c-Kit⁺Sca-1⁺Flt3⁺ cells, termed lymphoid-primed multipotent progenitors (LMPPs), have lost megakaryocyte and erythroid potential but are heterogeneous in their fate. Here, through single-cell RNA sequencing, we identify the expression of Dach1 and associated genes in this fraction as being coexpressed with myeloid/stem genes but inversely correlated with lymphoid genes. Through generation of Dach1–GFP reporter mice, we identify a transcriptionally and functionally unique Dach1–GFP⁻ subpopulation within LMPPs with lymphoid potential with low to negligible classic myeloid potential. We term these ‘lymphoid-primed progenitors’ (LPPs). These findings define an early definitive branch point of lymphoid development in hematopoiesis and a means for prospective isolation of LPPs.