Large numbers of melanoma lesions develop resistance to targeted inhibition of mutant BRAF or fail to respond to checkpoint blockade. We explored whether modulation of intratumoral antigen presenting cells (APCs) could increase responses to these therapies. Using mouse melanoma models, we found that CD103+ dendritic cells (DCs) were the only APCs transporting intact antigens to the lymph nodes and priming tumor-specific CD8+ T cells. CD103+ DCs were required to promote anti-tumoral effects upon blockade of the checkpoint ligand PDL1; however, PD-L1 inhibition only led to partial responses. Systemic administration of the cytokine Flt3L followed by intratumoral poly I:C injections expanded and activated CD103+ DC progenitors in the tumor, enhancing responses to BRAF and PD-L1 blockade and protecting mice from tumor rechallenge. Thus, the paucity of activated CD103+ DCs in tumors limits checkpoint blockade efficacy and Flt3L-poly I:C therapy can enhance tumor responses to checkpoint and BRAF blockade.

Macrophages are a heterogeneous cell population involved in tissue homeostasis, inflammation, and various pathologies. Although the major tissue-resident macrophage populations have been extensively studied, interstitial macrophages (IMs) residing within the tissue parenchyma remain poorly defined. Here we studied IMs from murine lung, fat, heart, and dermis. We identified two independent IM subpopulations that are conserved across tissues: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) monocyte-derived IMs, with distinct gene expression profiles, phenotypes, functions, and localizations. Using a new mouse model of inducible macrophage depletion (Slco2b1flox/DTR), we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung fibrosis. Thus, we demonstrate that two independent populations of IMs coexist across tissues and exhibit conserved niche-dependent functional programming.

SummaryDendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.

Highlights d Identification of fetal-associated endothelial cells and macrophages in HCC d Embryonic-like reprogramming in a subset of FOLR2 + TAM1s d Conserved GRN in mouse embryonically seeded, human fetal-liver, and TAM1 macrophages d Shared onco-fetal ecosystem between human fetal liver and HCC

Most tissue-resident macrophage (RTM) populations are seeded by waves of embryonic hematopoiesis and are self-maintained independently of a bone marrow contribution during adulthood. A proportion of RTMs, however, is constantly replaced by blood monocytes, and their functions compared to embryonic RTMs remain unclear. The kinetics and extent of the contribution of circulating monocytes to RTM replacement during homeostasis, inflammation, and disease are highly debated. Here, we identified Ms4a3 as a specific gene expressed by granulocyte-monocyte progenitors (GMPs) and subsequently generated Ms4a3 TdT reporter, Ms4a3 Cre , and Ms4a3 CreERT2 fate-mapping models. These models traced efficiently monocytes and granulocytes, but no lymphocytes or tissue dendritic cells. Using these models, we precisely quantified the contribution of monocytes to the RTM pool during homeostasis and inflammation. The unambiguous identification of monocyte-derived cells will permit future studies of their function under any condition.

Resident tissue macrophages (RTMs) have a broad spectrum of immune-and non-immune-related tissuesupporting activities. The roots of this heterogeneity and versatility are only beginning to be understood. Here, we propose a conceptual framework for considering the RTM heterogeneity that organizes the factors shaping RTM identity within four cardinal points: (1) ontogeny and the view that adult RTM populations comprise a defined mixture of cells that arise from either embryonic precursors or adult monocytes; (2) local factors unique to the niche of residence, evolving during development and aging; (3) inflammation status; and (4) the cumulative effect of time spent in a specific tissue that contributes to the resilient adaptation of macrophages to their dynamic environment. We review recent findings within this context and discuss the technological advances that are revolutionizing the study of macrophage biology.

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies.

Resident tissue macrophages (RTM) can fulfill various tasks during development, homeostasis, inflammation and repair. In the lung, non-alveolar RTM, called interstitial macrophages (IM), importantly contribute to tissue homeostasis but remain little characterized. Here we show, using single-cell RNA-sequencing (scRNA-seq), two phenotypically distinct subpopulations of long-lived monocyte-derived IM, i.e. CD206 + and CD206 − IM, as well as a discrete population of extravasating CD64 + CD16.2 + monocytes. CD206 + IM are peribronchial self-maintaining RTM that constitutively produce high levels of chemokines and immunosuppressive cytokines. Conversely, CD206 − IM preferentially populate the alveolar interstitium and exhibit features of antigen-presenting cells. In addition, our data support that CD64 + CD16.2 + monocytes arise from intravascular Ly-6C lo patrolling monocytes that enter the tissue at steady-state to become putative precursors of CD206 − IM. This study expands our knowledge about the complexity of lung IM and reveals an ontogenic pathway for one IM subset, an important step for elaborating future macrophage-targeted therapies.