Microglia maintain structural integrity during fetal brain morphogenesis.

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development.

Breast cancer remotely imposes a myeloid bias on haematopoietic stem cells by reprogramming the bone marrow niche.

Myeloid cell infiltration of solid tumours generally associates with poor patient prognosis and disease severity1-13. Therefore, understanding the regulation of myeloid cell differentiation during cancer is crucial to counteract their pro-tumourigenic role. Bone marrow (BM) haematopoiesis is a tightly regulated process for the production of all immune cells in accordance to tissue needs14. Myeloid cells differentiate during haematopoiesis from multipotent haematopoietic stem and progenitor cells (HSPCs)15-17. HSPCs can sense inflammatory signals from the periphery during infections18-21 or inflammatory disorders22-27. In these settings, HSPC expansion is associated with increased myeloid differentiation28,29. During carcinogenesis, the elevation of haematopoietic growth factors supports the expansion and differentiation of committed myeloid progenitors5,30. However, it is unclear whether cancer-related inflammation also triggers demand-adapted haematopoiesis at the level of multipotent HSPCs. In the BM, HSPCs reside within the haematopoietic niche which delivers HSC maintenance and differentiation cues31-35. Mesenchymal stem cells (MSCs) are a major cellular component of the BM niche and contribute to HSC homeostasis36-41. Modifications of MSCs in systemic disorders have been associated with HSC differentiation towards myeloid cells22,42. It is unknown if MSCs are regulated in the context of solid tumours and if their myeloid supportive activity is impacted by cancer-induced systemic changes. Here, using unbiased transcriptomic analysis and in situ imaging of HSCs and the BM niche during breast cancer, we show that both HSCs and MSCs are transcriptionally and spatially modified. We demonstrate that breast tumour can distantly remodel the cellular cross-talks in the BM niche leading to increased myelopoiesis.

In Vitro Generation of Murine Bone Marrow-Derived Dendritic Cells.

Dendritic cells (DCs) are mononuclear phagocytes of hematopoietic origin residing in lymphoid and nonlymphoid tissues. DCs are often referred as the sentinels of the immune system as they can sense pathogens and danger signals. Upon activation, DCs migrate to the draining lymph nodes and present antigens to naïve T cells to trigger adaptive immunity. Hematopoietic progenitors for DCs reside in the adult bone marrow (BM). Therefore, BM cell culture systems have been developed to generate large amounts of primary DCs in vitro conveniently enabling to analyze their developmental and functional features. Here, we review various protocols enabling to generate DCs in vitro from murine BM cells and discuss the cellular heterogeneity of each culture system.

In Vitro Generation of Human Dendritic Cell Subsets from CD34+ Cord Blood Progenitors.

Dendritic cells (DCs) are professional antigen-presenting cells controlling the activation of T cells and thus regulating adaptive immune response against pathogens or tumors. Modeling human DC differentiation and function is crucial for our understanding of immune response and the development of new therapies. Considering DC rarity in human blood, in vitro systems allowing their faithful generation are needed. This chapter will describe a DC differentiation method based on the co-culture of CD34+ cord blood progenitors together with mesenchymal stromal cells (eMSCs) engineered to deliver growth factors and chemokines.

A Population of TIM4+FOLR2+ Macrophages Localized in Tertiary Lymphoid Structures Correlates to an Active Immune Infiltrate Across Several Cancer Types.

TIM4 has previously been associated with antitumor immunity, yet the pattern of expression and the function of this receptor across human cancer tissues remain poorly explored. Here we combined extensive immunolabeling of human tissues with in silico analysis of pan-cancer transcriptomic data sets to explore the clinical significance of TIM4 expression. Our results unveil that TIM4 is expressed on a fraction of cavity macrophages (CATIM4+MΦ) of carcinoma patients. Moreover, we uncover a high expression of TIM4 on macrophages of the T-cell zone of the carcinoma-associated tertiary lymphoid structures (TLSTIM4+MΦ). In silico analysis of a pan-cancer data set revealed a positive correlation between TIM4 expression and markers of B cells, effector CD8+ T cells, and a 12-chemokine signature defining tertiary lymphoid structure. In addition, TLSTIM4+MΦ were enriched in cancers displaying microsatellite instability and high CD8+ T-cell infiltration, confirming their association with immune-reactive tumors. Both CATIM4+MΦ and TLSTIM4+MΦ express FOLR2, a marker of tissue-resident MΦ. However, CATIM4+MΦ had a higher expression of the immunosuppressive molecules TREM2, IL10, and TGFß as compared with TLSTIM4+MΦ. By analyzing a scRNA sequence data set of tumor-associated myeloid cells, we identified two TIM4+FOLR2+ clusters coherent with CATIM4+MΦ and TLSTIM4+MΦ. We defined specific gene signatures for each subset and found that the CATIM4+ MΦ signature was associated with worse patient survival. In contrast, TLSTIM4+MΦ gene signature positively correlates with a better prognosis. Together, these data illustrate that TIM4 marks two distinct macrophage populations with distinct phenotypes and tissue localization and that may have opposing roles in tumor immunity.

Tissue-resident FOLR2+ macrophages associate with CD8+ T cell infiltration in human breast cancer.

Macrophage infiltration is a hallmark of solid cancers, and overall macrophage infiltration correlates with lower patient survival and resistance to therapy. Tumor-associated macrophages, however, are phenotypically and functionally heterogeneous. Specific subsets of tumor-associated macrophage might be endowed with distinct roles on cancer progression and antitumor immunity. Here, we identify a discrete population of FOLR2+ tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors. FOLR2+ macrophages localize in perivascular areas in the tumor stroma, where they interact with CD8+ T cells. FOLR2+ macrophages efficiently prime effector CD8+ T cells ex vivo. The density of FOLR2+ macrophages in tumors positively correlates with better patient survival. This study highlights specific roles for tumor-associated macrophage subsets and paves the way for subset-targeted therapeutic interventions in macrophages-based cancer therapies.

Epithelial colonization by gut dendritic cells promotes their functional diversification.

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.

In vivo genome-wide CRISPR screens identify SOCS1 as intrinsic checkpoint of CD4+ TH1 cell response.

Although CD8+ T cells undergo autonomous clonal proliferation after antigen stimulation in vivo, the expansion of activated CD4+ T cells is limited by intrinsic factors that are poorly characterized. Using genome-wide CRISPR-Cas9 screens and an in vivo system modeling of antigen-experienced CD4+ T cell recruitment and proliferation during a localized immune response, we identified suppressor of cytokine signaling 1 (SOCS1) as a major nonredundant checkpoint imposing a brake on CD4+ T cell proliferation. Using anti­interleukin-2 receptor (IL-2R) blocking antibodies, interferon-γ receptor (IFN-γR) knockout mice, and transcriptomic analysis, we show that SOCS1 is a critical node integrating both IL-2 and IFN-γ signals to block multiple downstream signaling pathways abrogating CD4+ T helper 1 (TH1) cell response. Inactivation of SOCS1 in both murine and human CD4+ T cell antitumor adoptive therapies restored intratumor accumulation, proliferation/survival, persistence, and polyfunctionality and promoted rejection of established tumors. However, in CD8+ T cells, SOCS1 deletion did not affect the proliferation but rather improved survival and effector functions, which allowed for optimal therapeutic outcome when associated with SOCS1 inactivation in CD4+ T cells. Together, these findings identify SOCS1 as a major intracellular negative checkpoint of adoptive T cell response, opening new possibilities to optimize CAR-T cell therapy composition and efficacy.

Infiltration by CXCL10 Secreting Macrophages Is Associated With Antitumor Immunity and Response to Therapy in Ovarian Cancer Subtypes.

Ovarian carcinomas (OCs) are poorly immunogenic and immune checkpoint inhibitors (ICIs) have offered a modest benefit. In this study, high CD3+ T-cells and CD163+ tumor-associated macrophages (TAMs) densities identify a subgroup of immune infiltrated high-grade serous carcinomas (HGSCs) with better outcomes and superior response to platinum-based therapies. On the contrary, in most clear cell carcinomas (CCCs) showing poor prognosis and refractory to platinum, a high TAM density is associated with low T cell frequency. Immune infiltrated HGSC are characterized by the 30-genes signature (OC-IS30) covering immune activation and IFNγ polarization and predicting good prognosis (n = 312, TCGA). Immune infiltrated HGSC contain CXCL10 producing M1-type TAM (IRF1+pSTAT1Y701+) in close proximity to T-cells. A fraction of these M1-type TAM also co-expresses TREM2. M1-polarized TAM were barely detectable in T-cell poor CCC, but identifiable across various immunogenic human cancers. Single cell RNA sequencing data confirm the existence of a tumor-infiltrating CXCL10+IRF1+STAT1+ M1-type TAM overexpressing antigen processing and presentation gene programs. Overall, this study highlights the clinical relevance of the CXCL10+IRF1+STAT1+ macrophage subset as biomarker for intratumoral T-cell activation and therefore offers a new tool to select patients more likely to respond to T-cell or macrophage-targeted immunotherapies.

Development and function of human dendritic cells in humanized mice models.

Dendritic cells (DCs) are sentinel cells of the immune system arising from hematopoietic stem cells. DCs play a key role in the regulation of both adaptive and innate lymphocyte responses. As such, experimental models enabling a thorough analysis of human DCs development and function are needed. Humanized mice models (termed collectively as HIS mice, or human immune system mice models) provide unique opportunities to model human hematopoiesis and tackle the function of human immune cell types in vivo. Here, we review experimental approaches enabling to recapitulate the ontogeny of DC subsets in HIS mice and discuss studies addressing the biology of human DC subsets implementing HIS mice models.

Transcriptional and Functional Analysis of CD1c+ Human Dendritic Cells Identifies a CD163+ Subset Priming CD8+CD103+ T Cells.

Dendritic cells (DCs) are antigen-presenting cells controlling T cell activation. In humans, the diversity, ontogeny, and functional capabilities of DC subsets are not fully understood. Here, we identified circulating CD88-CD1c+CD163+ DCs (called DC3s) as immediate precursors of inflammatory CD88-CD14+CD1c+CD163+FcεRI+ DCs. DC3s develop via a specific pathway activated by GM-CSF, independent of cDC-restricted (CDP) and monocyte-restricted (cMoP) progenitors. Like classical DCs but unlike monocytes, DC3s drove activation of naive T cells. In vitro, DC3s displayed a distinctive ability to prime CD8+ T cells expressing a tissue homing signature and the epithelial homing alpha-E integrin (CD103) through transforming growth factor ß (TGF-ß) signaling. In vivo, DC3s infiltrated luminal breast cancer primary tumors, and DC3 infiltration correlated positively with CD8+CD103+CD69+ tissue-resident memory T cells. Together, these findings define DC3s as a lineage of inflammatory DCs endowed with a strong potential to regulate tumor immunity.

Engineered niches support the development of human dendritic cells in humanized mice.

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34+ hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34+ HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123+AXL+CD327+ pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.

Origin and development of classical dendritic cells.

Classical dendritic cells (cDCs) are mononuclear phagocytes of hematopoietic origin specialized in the induction and regulation of adaptive immunity. Initially defined by their unique T cell activation potential, it became quickly apparent that cDCs would be difficult to distinguish from other phagocyte lineages, by solely relying on marker-based approaches. Today, cDCs definition increasingly embed their unique ontogenetic features. A growing consensus defines cDCs on multiple criteria including: (1) dependency on the fms-like tyrosine kinase 3 ligand hematopoietic growth factor, (2) development from the common DC bone marrow progenitor, (3) constitutive expression of the transcription factor ZBTB46 and (4) the ability to induce, after adequate stimulation, the activation of naïve T lymphocytes. cDCs are a heterogeneous cell population that contains two main subsets, named type 1 and type 2 cDCs, arising from divergent ontogenetic pathways and populating multiple lymphoid and non-lymphoid tissues. Here, we present recent knowledge on the cellular and molecular pathways controlling the specification and commitment of cDC subsets from murine and human hematopoietic stem cells.

Clonally Expanded T Cells Reveal Immunogenicity of Rhabdoid Tumors.

Rhabdoid tumors (RTs) are genomically simple pediatric cancers driven by the biallelic inactivation of SMARCB1, leading to SWI/SNF chromatin remodeler complex deficiency. Comprehensive evaluation of the immune infiltrates of human and mice RTs, including immunohistochemistry, bulk RNA sequencing and DNA methylation profiling studies showed a high rate of tumors infiltrated by T and myeloid cells. Single-cell RNA (scRNA) and T cell receptor sequencing highlighted the heterogeneity of these cells and revealed therapeutically targetable exhausted effector and clonally expanded tissue resident memory CD8+ T subpopulations, likely representing tumor-specific cells. Checkpoint blockade therapy in an experimental RT model induced the regression of established tumors and durable immune responses. Finally, we show that one mechanism mediating RTs immunogenicity involves SMARCB1-dependent re-expression of endogenous retroviruses and interferon-signaling activation.

Lysosome signaling controls the migration of dendritic cells.

Dendritic cells (DCs) patrol their environment by linking antigen acquisition by macropinocytosis to cell locomotion. DC activation upon bacterial sensing inhibits macropinocytosis and increases DC migration, thus promoting the arrival of DCs to lymph nodes for antigen presentation to T cells. The signaling events that trigger such changes are not fully understood. We show that lysosome signaling plays a critical role in this process. Upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1), which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. We found that the TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration.

Dendritic Cell Lineage Potential in Human Early Hematopoietic Progenitors.

Conventional dendritic cells (cDCs) are thought to descend from a DC precursor downstream of the common myeloid progenitor (CMP). However, a mouse lymphoid-primed multipotent progenitor has been shown to generate cDCs following a DC-specific developmental pathway independent of monocyte and granulocyte poiesis. Similarly, here we show that, in humans, a large fraction of multipotent lymphoid early progenitors (MLPs) gives rise to cDCs, in particular the subset known as cDC1, identified by co-expression of DNGR-1 (CLEC9A) and CD141 (BDCA-3). Single-cell analysis indicates that over one-third of MLPs have the potential to efficiently generate cDCs. cDC1s generated from CMPs or MLPs do not exhibit differences in transcriptome or phenotype. These results demonstrate an early imprinting of the cDC lineage in human hematopoiesis and highlight the plasticity of developmental pathways giving rise to human DCs.

Oncogenic Transformation of Dendritic Cells and Their Precursors Leads to Rapid Cancer Development in Mice.

Dendritic cells (DCs) are powerful APCs that can induce Ag-specific adaptive immune responses and are increasingly recognized as important players in innate immunity to both infection and malignancy. Interestingly, although there are multiple described hematological malignancies, DC cancers are rarely observed in humans. Whether this is linked to the immunogenic potential of DCs, which might render them uniquely susceptible to immune control upon neoplastic transformation, has not been fully investigated. To address the issue, we generated a genetically engineered mouse model in which expression of Cre recombinase driven by the C-type lectin domain family 9, member a (Clec9a) locus causes expression of the Kirsten rat sarcoma viral oncogene homolog (Kras)(G12D) oncogenic driver and deletion of the tumor suppressor p53 within developing and differentiated DCs. We show that these Clec9a(Kras-G12D) mice rapidly succumb from disease and display massive accumulation of transformed DCs in multiple organs. In bone marrow chimeras, the development of DC cancer could be induced by a small number of transformed cells and was not prevented by the presence of untransformed DCs. Notably, activation of transformed DCs did not happen spontaneously but could be induced upon stimulation. Although Clec9a(Kras-G12D) mice showed altered thymic T cell development, peripheral T cells were largely unaffected during DC cancer development. Interestingly, transformed DCs were rejected upon adoptive transfer into wild-type but not lymphocyte-deficient mice, indicating that immunological control of DC cancer is in principle possible but does not occur during spontaneous generation in Clec9a(Kras-G12D) mice. Our findings suggest that neoplastic transformation of DCs does not by default induce anti-cancer immunity and can develop unhindered by immunological barriers.

GM-CSF Mouse Bone Marrow Cultures Comprise a Heterogeneous Population of CD11c(+)MHCII(+) Macrophages and Dendritic Cells.

Dendritic cells (DCs) are key players in the immune system. Much of their biology has been elucidated via culture systems in which hematopoietic precursors differentiate into DCs under the aegis of cytokines. A widely used protocol involves the culture of murine bone marrow (BM) cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) to generate BM-derived DCs (BMDCs). BMDCs express CD11c and MHC class II (MHCII) molecules and share with DCs isolated from tissues the ability to present exogenous antigens to T cells and to respond to microbial stimuli by undergoing maturation. We demonstrate that CD11c(+)MHCII(+) BMDCs are in fact a heterogeneous group of cells that comprises conventional DCs and monocyte-derived macrophages. DCs and macrophages in GM-CSF cultures both undergo maturation upon stimulation with lipopolysaccharide but respond differentially to the stimulus and remain separable entities. These results have important implications for the interpretation of a vast array of data obtained with DC culture systems.