Blockade of the co-inhibitory molecule PD-1 unleashes ILC2-dependent antitumor immunity in melanoma.

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.

A new lymphoid-primed progenitor marked by Dach1 downregulation identified with single cell multi-omics.

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.

Clonal multi-omics reveals Bcor as a negative regulator of emergency dendritic cell development.

Despite advances in single-cell multi-omics, a single stem or progenitor cell can only be tested once. We developed clonal multi-omics, in which daughters of a clone act as surrogates of the founder, thereby allowing multiple independent assays per clone. With SIS-seq, clonal siblings in parallel "sister" assays are examined either for gene expression by RNA sequencing (RNA-seq) or for fate in culture. We identified, and then validated using CRISPR, genes that controlled fate bias for different dendritic cell (DC) subtypes. This included Bcor as a suppressor of plasmacytoid DC (pDC) and conventional DC type 2 (cDC2) numbers during Flt3 ligand-mediated emergency DC development. We then developed SIS-skew to examine development of wild-type and Bcor-deficient siblings of the same clone in parallel. We found Bcor restricted clonal expansion, especially for cDC2s, and suppressed clonal fate potential, especially for pDCs. Therefore, SIS-seq and SIS-skew can reveal the molecular and cellular mechanisms governing clonal fate.

Transcription Factor PU.1 Promotes Conventional Dendritic Cell Identity and Function via Induction of Transcriptional Regulator DC-SCRIPT.

Dendritic cells (DCs) are can be broadly divided into conventional (cDC) and plasmacytoid (pDC) subsets. Despite the importance of this lineage diversity, its genetic basis is not fully understood. We found that conditional ablation of the Ets-family transcription factor PU.1 in DC-restricted progenitors led to increased pDC production at the expense of cDCs. PU.1 controlled many of the cardinal functions of DCs, such as antigen presentation by cDCs and type I interferon production by pDCs. Conditional ablation of PU.1 de-repressed the pDC transcriptional signature in cDCs. The combination of genome-wide mapping of PU.1 binding and gene expression analysis revealed a key role for PU.1 in maintaining cDC identity through the induction of the transcriptional regulator DC-SCRIPT. PU.1 activated DC-SCRIPT expression, which in turn promoted cDC formation, particularly of cDC1s, and repressed pDC development. Thus, cDC identity is regulated by a transcriptional node requiring PU.1 and DC-SCRIPT.

Identification of cDC1- and cDC2-committed DC progenitors reveals early lineage priming at the common DC progenitor stage in the bone marrow.

Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α(+) (CD103(+)) cDC1 lineage, and the CD11b(+) cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H(-)Ly6C(-) pre-DCs) or cDC2 lineage (Siglec-H(-)Ly6C(+) pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.

CIS and TGF-ß regulatory pathways influence immunity to bacterial infection.

Immunotherapy has revolutionized cancer therapy by reactivating tumour-resident cytotoxic lymphocytes. More recently, immunotherapy has emerged to restore immunity against infectious agents, including bacterial infections. Immunotherapy primarily targets inhibitory pathways in T cells, however interest in other effector populations, such as natural killer (NK) cells, is growing. We have previously discovered that NK cell metabolism, proliferation and activation can be neutralized through the immunosuppressive transforming growth factor (TGF)-ß pathway by inducing plasticity of NK cells and differentiation into innate lymphoid cell (ILC)1-like subsets. NK cells are also regulated through cytokine-inducible SH2-containing protein (CIS), which is induced by interleukin (IL)-15 and is a potent intracellular checkpoint suppressing NK cell survival and function. Targeting these two distinct pathways to restore NK cell function has shown promise in cancer models, but their application in bacterial infection remains unknown. Here, we investigate whether enhancement of NK cell function can improve anti-bacterial immunity, using Salmonella Typhimurium as a model. We identified conversion of NK cells to ILC1-like for the first time in the context of bacterial infection, where TGF-ß signalling contributed to this plasticity. Future study should focus on identifying further drivers of ILC1 plasticity and its functional implication in bacterial infection model. We further describe that CIS-deficient mice displayed enhanced pro-inflammatory function and dramatically enhanced anti-bacterial immunity. Inhibition of CIS may present as a viable therapeutic option to enhance immunity towards bacterial infection.

RelB suppresses type I Interferon signaling in dendritic cells.

Type I Interferon (IFN) signaling plays a critical role in dendritic cell (DC) development and functions. Inhibition of hyper type I IFN signaling promotes cDC2 subtype development. Relb is essential to development of cDC2 subtype and here we analyzed its effect on type I IFN signaling in DCs. We show that Relb suppresses the homeostatic type I IFN signaling in cDC2 cultures. TLR stimulation of FL-DCs led to RelB induction coinciding with fall in IFN signatures; conforming with the observation Relb expression reduced TLR stimulated IFN induction along with decrease in ISGs. Towards understanding mechanism, we show that effects of RelB are mediated by increased levels of IκBα. We demonstrate that RelB dampened antiviral responses by lowering ISG levels and the defect in cDC2 development in RelB null mice can be rescued in Ifnar1-/- background. Overall, we propose a novel role of RelB as a negative regulator of the type I IFN signaling pathway; fine tuning development of cDC2 subtype.

MOZ (KAT6A) is essential for the maintenance of classically defined adult hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are conventionally thought to be at the apex of a hierarchy that produces all mature cells of the blood. The quintessential property of these cells is their ability to reconstitute the entire hematopoietic system of hemoablated recipients. This characteristic has enabled HSCs to be used to replenish the hematopoietic system of patients after chemotherapy or radiotherapy. Here, we use deletion of the monocytic leukemia zinc finger gene (Moz/Kat6a/Myst3) to examine the effects of removing HSCs. Loss of MOZ in adult mice leads to the rapid loss of HSCs as defined by transplantation. This is accompanied by a reduction of the LSK-CD48-CD150+ and LSK-CD34-Flt3- populations in the bone marrow and a reduction in quiescent cells in G0 Surprisingly, the loss of classically defined HSCs did not affect mouse viability, and there was no recovery of the LSK-CD48-CD150+ and LSK-CD34-Flt3- populations 15 to 18 months after Moz deletion. Clonal analysis of myeloid progenitors, which produce short-lived granulocytes, demonstrate that these are derived from cells that had undergone recombination at the Moz locus up to 2 years earlier, suggesting that early progenitors have acquired extended self-renewal. Our results establish that there are essential differences in HSC requirement for steady-state blood cell production compared with the artificial situation of reconstitution after transplantation into a hemoablated host. A better understanding of steady-state hematopoiesis may facilitate the development of novel therapies engaging hematopoietic cell populations with previously unrecognized traits, as well as characterizing potential vulnerability to oncogenic transformation.

GFI1B specifies developmental potential of innate lymphoid cell progenitors in the lungs.

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.

Single-cell analyses reveal the clonal and molecular aetiology of Flt3L-induced emergency dendritic cell development.

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.

Clonal analysis reveals multiple functional defects of aged murine hematopoietic stem cells.

Hematopoietic stem cell (HSC) populations change with aging, but the extent to which this is caused by qualitative versus quantitative alterations in HSC subtypes is unclear. Using clonal assays, in this study we show that the aging HSC compartment undergoes both quantitative and qualitative changes. We observed a variable increase of HSC pool size with age, accompanied by the accumulation of predominantly myeloid-biased HSCs that regenerate substantially fewer mature progeny than young myeloid-biased HSCs and exhibit reduced self-renewal activity as measured by long-term secondary transplantation. Old HSCs had a twofold reduction in marrow-homing efficiency and a similar decrease in functional frequency as measured using long-term transplantation assays. Similarly, old HSCs had a twofold reduced seeding efficiency and a significantly delayed proliferative response compared with young HSCs in long-term stromal cell co-cultures but were indistinguishable in suspension cultures. We show that these functional defects are characteristics of most or all old HSCs and are not indicative of a nonfunctional subset of cells that express HSC markers. Furthermore, we demonstrate that cells with functional properties of old HSCs can be generated directly from young HSCs by extended serial transplantation, which is consistent with the possibility that they arise through a process of cellular aging.