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Building an immune system is a monumental task critical to the survival of the fetus and newborn. A functional fetal immune system must complement the maternal immune system in handling in utero infection; abstain from damaging non-self-reactions that would compromise the materno-fetal interface; mobilize in response to infection and equip mucosal tissues for pathogen exposure at birth. There is growing appreciation that immune cells also have noncanonical roles in development and specifically may contribute to tissue morphogenesis. In this review we detail how hematopoietic and lymphoid organs jointly establish cellular constituents of the immune system; how these constituents are organized in 2 mucosal sites-gut and lung-where early life immune function has long-term consequences for health; and how exemplar diseases of prematurity and inborn errors of immunity reveal dominant pathways in prenatal immunity.

The repertoire of dendritic cells (DCs), monocytes and macrophages in adult humans is diverse and we are appreciating this to a greater extent as high throughput methods, such a single-cell RNA sequencing, become widely adopted and scalable. This powerful lens of analysis is also beginning to shed light on prenatal immunology, allowing us to chart the emergence, tissue distribution and developmental regulation of DCs, monocytes and macrophages during early human life. In this review, we will integrate recent insights from studies of the developing immune system into our understanding of adult DC, monocyte and macrophage organization, illustrating where insights from early life both affirm and challenge current understanding.

The blood and immune systems develop in parallel during early prenatal life. Waves of hematopoiesis separated in anatomical space and time give rise to circulating and tissue-resident immune cells. Previous observations have relied on animal models, which differ from humans in both their developmental timeline and exposure to microorganisms. Decoding the composition of the human immune system is now tractable using single-cell multi-omics approaches. Large-scale single-cell genomics, imaging technologies, and the Human Cell Atlas initiative have together enabled a systems-level mapping of the developing human immune system and its emergent properties. Although the precise roles of specific immune cells during development require further investigation, the system as a whole displays malleable and responsive properties according to developmental need and environmental challenge.

Funder: Lister Institute of Preventive Medicine; doi: https://doi.org/10.13039/501100001255, Funder: Newcastle NIHR-Biomedical Research Centre, KMT2A-rearranged infant ALL is an aggressive childhood leukemia with poor prognosis. Here, we investigated the developmental state of KMT2A-rearranged infant B-cell acute lymphoblastic leukemia (B-ALL) using bulk messenger RNA (mRNA) meta-analysis and examination of single lymphoblast transcriptomes against a developing bone marrow reference. KMT2A-rearranged infant B-ALL was uniquely dominated by an early lymphocyte precursor (ELP) state, whereas less adverse NUTM1-rearranged infant ALL demonstrated signals of later developing B cells, in line with most other childhood B-ALLs. We compared infant lymphoblasts with ELP cells and revealed that the cancer harbored hybrid myeloid-lymphoid features, including nonphysiological antigen combinations potentially targetable to achieve cancer specificity. We validated surface coexpression of exemplar combinations by flow cytometry. Through analysis of shared mutations in separate leukemias from a child with infant KMT2A-rearranged B-ALL relapsing as AML, we established that KMT2A rearrangement occurred in very early development, before hematopoietic specification, emphasizing that cell of origin cannot be inferred from the transcriptional state.

Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic–skewing of PB is perturbed with age in essential thrombocythemia and β-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.

1.AbstractThis paper reviews efforts across 16 international consortia to construct human anatomical structures, cell types, and biomarkers (ASCT+B) tables and three-dimensional reference organs in support of a Human Reference Atlas. We detail the ontological descriptions and spatial three-dimensional anatomical representations together with user interfaces that support the registration and exploration of human tissue data. Four use cases are presented to demonstrate the utility of ASCT+B tables for advancing biomedical research and improving health.

Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11–12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6–7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21). A single-cell atlas of human fetal bone marrow in healthy fetuses and fetuses with Down syndrome provides insight into developmental haematopoiesis in humans and the transcription and functional differences that occur in Down syndrome.

Mononuclear phagocytes (MPs) including monocytes, macrophages and dendritic cells (DCs) are critical innate immune effectors and initiators of the adaptive immune response. MPs are present in the alveolar airspace at steady state, however little is known about DC recruitment in acute pulmonary inflammation. Here we use lipopolysaccharide inhalation to induce acute inflammation in healthy volunteers and examine the impact on bronchoalveolar lavage fluid and blood MP repertoire. Classical monocytes and two DC subsets (DC2/3 and DC5) are expanded in bronchoalveolar lavage fluid 8 h after lipopolysaccharide inhalation. Surface phenotyping, gene expression profiling and parallel analysis of blood indicate recruited DCs are blood-derived. Recruited monocytes and DCs rapidly adopt typical airspace-resident MP gene expression profiles. Following lipopolysaccharide inhalation, alveolar macrophages strongly up-regulate cytokines for MP recruitment. Our study defines the characteristics of human DCs and monocytes recruited into bronchoalveolar space immediately following localised acute inflammatory stimulus in vivo., The diversity of human mononuclear phagocyte subsets remains to be characterized in many tissue-specific and functional contexts, including pulmonary inflammation. Here the authors characterize dendritic cell and monocyte subset recruitment to the bronchoalveolar space in a human LPS inhalation model.

T cell pathology in the skin leads to monocyte influx, but we have little understanding of the fate of recruited cells within the diseased niche, or the long-term impact on cutaneous immune homeostasis. By combining a murine model of acute graft-versus-host disease (aGVHD) with analysis of patient samples, we demonstrate that pathology initiates dermis-specific macrophage differentiation and show that aGVHD-primed macrophages continue to dominate the dermal compartment at the relative expense of quiescent MHCIIint cells. Exposure of the altered dermal niche to topical haptens after disease resolution results in hyper-activation of regulatory T cells (Treg), but local breakdown in tolerance. Disease-imprinted macrophages express increased IL-1β and are predicted to elicit altered TNF superfamily interactions with cutaneous Treg, and we demonstrate the direct loss of T cell regulation within the resolved skin. Thus, T cell pathology leaves an immunological scar in the skin marked by failure to re-set immune homeostasis.

The human lung harbors diverse macrophages that provide barrier immunity and maintain homeostasis, but their precursors are unclear. In this issue of Immunity, Evren et al. use a humanized mouse model to discern that classical monocytes give rise to alveolar and interstitial macrophages, whereas non-classical monocytes contribute to pulmonary intravascular macrophages.

Cellular beauty is skin deep Human skin works as barrier, preventing the entry of pathogens, among other functions. Reynolds et al. used single-cell sequencing to generate an atlas of the human skin from both developing and adult sources, identifying differences and similarities across heterogeneous populations of skin cells. In this atlas, gene expression in the two disease states studied—atopic dermatitis and psoriasis—varied from that in a healthy adult, suggesting that a fetal skin signature is expressed in adult inflamed skin. Furthermore, differences in immune cell composition between healthy fetal and adult skin and that of individuals suffering from disease were observed. Science , this issue p. eaba6500

The global COVID-19 pandemic has caused substantial morbidity and mortality to humanity. Remarkable progress has been made in understanding both the innate and adaptive mechanisms involved in the host response to the causative SARS-CoV-2 virus, but much remains to be discovered. Robust upper airway defenses are critical in restricting SARS-CoV-2 replication and propagation. Further, the nasal abundance of viral uptake receptor, ACE2, and the host epithelial transcriptional landscape, are associated with differential disease outcomes across different patient cohorts. The adaptive host response to systemic COVID-19 is heterogeneous and complex. Blunted responses to interferon and robust cytokine generation are hallmarks of the disease, particularly at the advanced stages. Excessive immune cell influx into tissues can lead to substantial collateral damage to the host akin to sepsis. This review offers a contemporary summary of these mechanisms of disease and highlights potential avenues for diagnostic and therapeutic development. These include improved disease stratification, targeting effectors of immune-mediated tissue damage, and blunting of immune cell-mediated tissue damage.

International audience; Definitive haematopoiesis in the fetal liver supports self-renewal anddifferentiation of haematopoietic stem cells and multipotent progenitors(HSC/MPPs) but remains poorly defined in humans. Here, using single-celltranscriptome profiling of approximately 140,000 liver and 74,000 skin, kidneyand yolk sac cells, we identify the repertoire of human blood and immune cellsduring development. We infer differentiation trajectories from HSC/MPPs andevaluate the influence of the tissue microenvironment on blood and immune celldevelopment. We reveal physiological erythropoiesis in fetal skin and thepresence of mast cells, natural killer and innate lymphoid cell precursors in theyolk sac. We demonstrate a shift in the haemopoietic composition of fetal liverduring gestation away from being predominantly erythroid, accompanied by aparallel change in differentiation potential of HSC/MPPs, which we functionallyvalidate. Our integrated map of fetal liver haematopoiesis provides a blueprintfor the study of paediatric blood and immune disorders, and a reference forharnessing the therapeutic potential of HSC/MPPs.

Summary Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4+ regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation., Graphical Abstract, Highlights • scRNA-seq reveals Treg cell tissue-specific signatures • The phenotype of Treg cells shows progressive adaptation to barrier tissues • Treg cells share transcriptional dynamics of adaptation to colon and skin • Modules of peripheral Treg cell identity are conserved between mouse and human, Treg cells are known to display tissue-specific heterogeneity. Miragaia and colleagues probe Treg cells in lymphoid and barrier tissues by single-cell transcriptomics. They show that Treg cells segregate into subpopulations along a continuum of tissue adaptation and present conserved expression programs between homeostasis and disease and mouse and human.

Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic stem cell transplantation induced by the influx of donor-derived effector T cells (TE) into peripheral tissues. Current treatment strategies rely on targeting systemic T cells; however, the precise location and nature of instructions that program TE to become pathogenic and trigger injury are unknown. We therefore used weighted gene coexpression network analysis to construct an unbiased spatial map of TE differentiation during the evolution of GVHD and identified wide variation in effector programs in mice and humans according to location. Idiosyncrasy of effector programming in affected organs did not result from variation in T cell receptor repertoire or the selection of optimally activated TE. Instead, TE were reprogrammed by tissue-autonomous mechanisms in target organs for site-specific proinflammatory functions that were highly divergent from those primed in lymph nodes. In the skin, we combined the correlation-based network with a module-based differential expression analysis and showed that Langerhans cells provided in situ instructions for a Notch-dependent T cell gene cluster critical for triggering local injury. Thus, the principal determinant of TE pathogenicity in GVHD is the final destination, highlighting the need for target organ–specific approaches to block immunopathology while avoiding global immune suppression., During the evolution of graft-versus-host disease, effector CD8+ T cells are re-programmed by tissue-autonomous mechanisms to develop idiosyncratic pathogenic functions in target organs.

Alemtuzumab conditioning is highly effective at reducing the incidence of acute and chronic graft-versus-host disease (GVHD) in reduced-intensity fludarabine and melphalan transplantation with cyclosporine monotherapy. Less frequent and lower dose scheduling may be used with sibling donors, but an optimal regimen for matched unrelated donors has not been defined. In this retrospective observational study of 313 patients, the incidence and severity of GVHD was compared in patients receiving 3 different dose schedules: the standard 100-mg regimen (20 mg on days –7 to –3), 60 mg (30 mg on days –4 and –2), or 50 mg (10 mg on days –7 to –3). Patients treated with 100 mg, 60 mg, or 50 mg developed acute GVHD grades I to IV with an incidence of 74%, 65%, and 64%, respectively, whereas 36%, 32%, and 41% developed chronic GHVD. An excess of severe acute grades III/IV GVHD was observed in the 50-mg cohort (15% versus 2% to 6%; P = .016). The relative risk of severe acute grade GVHD remained more than 3-fold higher in the 50-mg cohort compared with the 100-mg cohort after adjustment for differences in HLA match, age, gender mismatch, cytomegalovirus risk, and diagnosis (P = .030). The findings indicate that the 60-mg alemtuzumab schedule was comparable with the 100-mg schedule, but more attenuated schedules may increase the risk of severe grade GVHD.

INTRODUCTION Dendritic cells (DCs) and monocytes consist of multiple specialized subtypes that play a central role in pathogen sensing, phagocytosis, and antigen presentation. However, their identities and interrelationships are not fully understood, as these populations have historically been defined by a combination of morphology, physical properties, localization, functions, developmental origins, and expression of a restricted set of surface markers. RATIONALE To overcome this inherently biased strategy for cell identification, we performed single-cell RNA sequencing of ~2400 cells isolated from healthy blood donors and enriched for HLA-DR + lineage − cells. This single-cell profiling strategy and unbiased genomic classification, together with follow-up profiling and functional and phenotypic characterization of prospectively isolated subsets, led us to identify and validate six DC subtypes and four monocyte subtypes, and thus revise the taxonomy of these cells. RESULTS Our study reveals: 1) A new DC subset, representing 2 to 3% of the DC populations across all 10 donors tested, characterized by the expression of AXL , SIGLEC1 , and SIGLEC6 antigens, named AS DCs. The AS DC population further divides into two populations captured in the traditionally defined plasmacytoid DC (pDC) and CD1C + conventional DC (cDC) gates. This split is further reflected through AS DC gene expression signatures spanning a spectrum between cDC-like and pDC-like gene sets. Although AS DCs share properties with pDCs, they more potently activate T cells. This discovery led us to reclassify pDCs as the originally described “natural interferon-producing cells (IPCs)” with weaker T cell proliferation induction ability. 2) A new subdivision within the CD1C + DC subset: one defined by a major histocompatibility complex class II–like gene set and one by a CD14 + monocyte–like prominent gene set. These CD1C + DC subsets, which can be enriched by combining CD1C with CD32B, CD36, and CD163 antigens, can both potently induce T cell proliferation. 3) The existence of a circulating and dividing cDC progenitor giving rise to CD1C + and CLEC9A + DCs through in vitro differentiation assays. This blood precursor is defined by the expression of CD100 + CD34 int and observed at a frequency of ~0.02% of the LIN – HLA-DR + fraction. 4) Two additional monocyte populations: one expressing classical monocyte genes and cytotoxic genes, and the other with unknown functions. 5) Evidence for a relationship between blastic plasmacytoid DC neoplasia (BPDCN) cells and healthy DCs. CONCLUSION Our revised taxonomy will enable more accurate functional and developmental analyses as well as immune monitoring in health and disease. The discovery of AS DCs within the traditionally defined pDC population explains many of the cDC properties previously assigned to pDCs, highlighting the need to revisit the definition of pDCs. Furthermore, the discovery of blood cDC progenitors represents a new therapeutic target readily accessible in the bloodstream for manipulation, as well as a new source for better in vitro DC generation. Although the current results focus on DCs and monocytes, a similar strategy can be applied to build a comprehensive human immune cell atlas.

Dendritic cells (DCs) are specialized leukocytes with antigen-processing and antigen-presenting functions. DCs can be divided into distinct subsets by anatomical location, phenotype and function. In human, the two most accessible tissues to study leukocytes are peripheral blood and skin. DCs are rare in human peripheral blood (1 % of mononuclear cells) and have a less mature phenotype than their tissue counterparts (MacDonald et al., Blood. 100:4512-4520, 2002; Haniffa et al., Immunity 37:60-73, 2012). In contrast, the skin covering an average total surface area of 1.8 m(2) has approximately tenfold more DCs than the average 5 L of total blood volume (Wang et al., J Invest Dermatol 134:965-974, 2014). DCs migrate spontaneously from skin explants cultured ex vivo, which provide an easy method of cell isolation (Larsen et al., J Exp Med 172:1483-1493, 1990; Lenz et al., J Clin Invest 92:2587-2596, 1993; Nestle et al., J Immunol 151:6535-6545, 1993). These factors led to the extensive use of skin DCs as the "prototype" migratory DCs in human studies. In this chapter, we detail the protocols to isolate DCs and resident macrophages from human skin. We also provide a multiparameter flow cytometry gating strategy to identify human skin DCs and to distinguish them from macrophages.

Human immunodeficiency syndrome with loss of DCs, monocytes, and T reg cells; preservation of Langerhans cells; associated loss of BM multilymphoid progenitors; and overproduction of Flt3 ligand., Congenital or acquired cellular deficiencies in humans have the potential to reveal much about normal hematopoiesis and immune function. We show that a recently described syndrome of monocytopenia, B and NK lymphoid deficiency additionally includes the near absence of dendritic cells. Four subjects showed severe depletion of the peripheral blood HLA-DR+ lineage− compartment, with virtually no CD123+ or CD11c+ dendritic cells (DCs) and very few CD14+ or CD16+ monocytes. The only remaining HLA-DR+ lineage− cells were circulating CD34+ progenitor cells. Dermal CD14+ and CD1a+ DC were also absent, consistent with their dependence on blood-derived precursors. In contrast, epidermal Langerhans cells and tissue macrophages were largely preserved. Combined loss of peripheral DCs, monocytes, and B and NK lymphocytes was mirrored in the bone marrow by complete absence of multilymphoid progenitors and depletion of granulocyte-macrophage progenitors. Depletion of the HLA-DR+ peripheral blood compartment was associated with elevated serum fms-like tyrosine kinase ligand and reduced circulating CD4+CD25hiFoxP3+ T cells, supporting a role for DC in T reg cell homeostasis.

Highlights • Human skin dendritic cells (DCs) are heterogenous and functionally specialised. • Factor XIIIa+ dermal dendrocytes are resident dermal macrophages. • Dermal CD14+ cells, previously defined as DCs, are monocyte-derived macrophages. • Dynamic changes occur in the composition of recruited ‘inflammatory’ DCs and resident DCs in inflamed skin., Summary Dendritic cells (DCs) are specialized antigen presenting cells abundant in peripheral tissues such as skin where they function as immune sentinels. Skin DCs migrate to draining lymph node where they interact with naïve T cells to induce immune responses to microorganisms, vaccines, tumours and self-antigens. In this review, we present the key historical developments and recent advances in human skin DC research. We also integrate the current understanding on the origin and functional specializations of DC subsets in healthy skin with findings in inflammatory skin diseases focusing on psoriasis and atopic eczema. A comprehensive understanding of the dynamic changes in DC subsets in health and disease will form a strong foundation to facilitate the clinical translation of DC-based therapeutic and vaccination strategies.

Langerhans cells (LCs) have been scrutinized many times in studies of the pathogenesis of graft-versus-host disease (GVHD). As migratory dendritic cells, LCs are capable of direct antigen presentation to cytotoxic T cells. Their self-renewal capacity has led to speculation that persistent recipient LCs could provide a continuous source of host antigen to donor T cells infused during hematopoietic stem cell transplantation (HSCT). In this issue of Experimental Dermatology, a new study examines at the relationship between recipient LCs and chronic GVHD.

Summary Mouse and human dendritic cells (DCs) are composed of functionally specialized subsets, but precise interspecies correlation is currently incomplete. Here, we showed that murine lung and gut lamina propria CD11b+ DC populations were comprised of two subsets: FLT3- and IRF4-dependent CD24+CD64− DCs and contaminating CSF-1R-dependent CD24−CD64+ macrophages. Functionally, loss of CD24+CD11b+ DCs abrogated CD4+ T cell-mediated interleukin-17 (IL-17) production in steady state and after Aspergillus fumigatus challenge. Human CD1c+ DCs, the equivalent of murine CD24+CD11b+ DCs, also expressed IRF4, secreted IL-23, and promoted T helper 17 cell responses. Our data revealed heterogeneity in the mouse CD11b+ DC compartment and identifed mucosal tissues IRF4-expressing DCs specialized in instructing IL-17 responses in both mouse and human. The demonstration of mouse and human DC subsets specialized in driving IL-17 responses highlights the conservation of key immune functions across species and will facilitate the translation of mouse in vivo findings to advance DC-based clinical therapies., Highlights • Mucosal CD11b+ DCs consist of CD24+CD64− DCs and CD24−CD64+ macrophages • Mucosal CD24+CD11b+ DCs are IRF4-dependent • IRF4-dependent CD24+CD11b+ DCs secrete IL-23α and control mucosal IL-17 responses • Human CD1c+CD11b+ DCs are functional homologs of murine CD24+CD11b+ DCs

Natural killer (NK) cell immunosurveillance may be impaired by malignant disease, resulting in tumour escape and disease progression. Therapies that enhance NK cytotoxicity may therefore prove valuable in remission-induction and maintenance treatment regimes. Acute lymphoblastic leukaemia (ALL) has previously been considered resistant to NK cell lysis and not tractable to this approach. Our study demonstrates that Bortezomib, Valproate and Troglitzaone can upregulate NK activating ligands on a B-ALL cell line and on a proportion but not all adult primary B-ALL samples. Drug-treated ALL cells trigger higher levels of NK degranulation, as measured by CD107a expression, and this effect is dependent on signalling through the NK activating receptor NKG2D. These results suggest that Bortezomib, Valproate and Troglitzaone may have clinical utility in sensitizing ALL to NK mediated lysis in vivo.