Development and Plasticity of murine plasmacytoid dendritic cells

The data presented in this thesis identifies a subpopulation of CCR9- MHC class IIlow BST2+ Siglec H+ plasmacytoid dendritic cells (pDCs) within the bone marrow (BM) pDC population. CCR9- MHC class IIlow BST2+ Siglec H+ pDCs express the essential pDC transcription factor E2-2 and produce high levels of interferon-α (IFN-α) and proinflammatory cytokines upon toll-like receptor 9 (TLR9) stimulation. This phenotypically immature pDC population is an immediate precursor for fully differentiated CCR9+ pDCs in vitro as well as in vivo, but does not harbour a pDC-specific gene rearrangement in the Ig gene locus. CCR9- pDCs retain plasticity to downregulate pDC specific surface molecules and upregulate CD11b and MHC class II, acquiring phenotype and function of CD8α- CD11b+ conventional dendritic cell (cDC) - like cells after conditioning with supernatant derived from colonic epithelial cells or exposure to recombinant granulocyte macrophage colony stimulating factor (GM-CSF) in vitro. Functionally cDC-like cells generated from CCR9- pDCs acquire properties of cDCs such as efficient T cell activation and the production of high levels of proinflammatory cytokines comparable to those of splenic CD8α- DCs. This phenotypic and functional change is also reflected on the level of transcription factor expression by downregulation of E2-2, Spi-b and IRF8 but upregulation of ID2, PU.1 and BATF3. CCR9- pDCs can give rise to fully differentiated CCR9+ pDCs and CD11b+ MHC class IIhigh cDC-like cells locally in the tissue in vivo in the steady state. However the plasticity and lineage commitment of CCR9- pDCs is regulated in a tissue specific manner. In BM and liver CCR9- pDCs primarily give rise to CCR9+ pDCs, whereas in spleen, lymph nodes, lung and small intestine a substantial fraction deviates from the pDC lineage to the cDC lineage. Furthermore, this study shows that GM-CSF is necessary for the appearance of CCR9- pDCs and CCR9+ pDCs in lung and small intestine but is dispensable for the generation of CD11b+ MHC class IIhigh cDCs from CCR9- pDCs in vivo. Moreover, GM-CSF controls the proliferation of CCR9- and CCR9+ pDCs in BM and spleen upon adoptive transfer. In conclusion these results show that CCR9- pDCs are tissue resident precursors of pDCs and cDCs and that the generation of DC subsets is regulated by tissue derived factors, thereby allowing adaptation to local microenvironments. This increases the flexibility of the DC compartment under circumstances of infection or inflammation

Cellular Differentiation of Human Monocytes Is Regulated by Time-Dependent Interleukin-4 Signaling and the Transcriptional Regulator NCOR2

Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies

Recent advances in understanding dendritic cell development, classification, and phenotype [version 1; referees: 2 approved]

Dendritic cells (DCs) play an essential role in the induction of adaptive immune responses against infectious agents and in the generation of tolerance to self-antigens. In this mini-review, we summarize new evidence suggesting that the tissue of residence significantly shapes the last developmental steps of DCs into locally adapted cellular entities, enabling them to perform tissue-specific tasks while maintaining the core DC properties. We also discuss recent advances that have highlighted DCs’ rather complex phenotypic and functional heterogeneity in the tumor microenvironment, based on their physical characteristics, such as activation status, maturity, and polarization, illustrating a key role for DCs in the induction of anti-tumor immunity

Modulation of Myelopoiesis Progenitors Is an Integral Component of Trained Immunity

Trained innate immunity fosters a sustained favorable response of myeloid cells to a secondary challenge, despite their short lifespan in circulation. We thus hypothesized that trained immunity acts via modulation of hematopoietic stem and progenitor cells (HSPCs). Administration of β-glucan (prototypical trained-immunity-inducing agonist) to mice induced expansion of progenitors of the myeloid lineage, which was associated with elevated signaling by innate immune mediators, such as IL-1β and granulocyte-macrophage colony-stimulating factor (GM-CSF), and with adaptations in glucose metabolism and cholesterol biosynthesis. The trained-immunity-related increase in myelopoiesis resulted in a beneficial response to secondary LPS challenge and protection from chemotherapy-induced myelosuppression in mice. Therefore, modulation of myeloid progenitors in the bone marrow is an integral component of trained immunity, which to date, was considered to involve functional changes of mature myeloid cells in the periphery

Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow

Blood neutrophil homeostasis is essential for successful host defense against invading pathogens. Circulating neutrophil counts are positively regulated by CXCR2 signaling and negatively regulated by the CXCR4-CXCL12 axis. In particular, G-CSF, a known CXCR2 signaler, and plerixafor, a CXCR4 antagonist, have both been shown to correct neutropenia in human patients. G-CSF directly induces neutrophil mobilization from the bone marrow (BM) into the blood, but the mechanisms underlying plerixafor-induced neutrophilia remain poorly defined. Using a combination of intravital multiphoton microscopy, genetically modified mice and novel in vivo homing assays, we demonstrate that G-CSF and plerixafor work through distinct mechanisms. In contrast to G-CSF, CXCR4 inhibition via plerixafor does not result in neutrophil mobilization from the BM. Instead, plerixafor augments the frequency of circulating neutrophils through their release from the marginated pool present in the lung, while simultaneously preventing neutrophil return to the BM. Our study demonstrates for the first time that drastic changes in blood neutrophils can originate from alternative reservoirs other than the BM, while implicating a role for CXCR4-CXCL12 interactions in regulating lung neutrophil margination. Collectively, our data provides valuable insights into the fundamental regulation of neutrophil homeostasis, which may lead to the development of improved treatment regimens for neutropenic patients.This research was funded by SIgN, A*STAR, Singapore. C.N.Z. Mattar and J.K.Y. Chan received salary support from the National Medical Research Council of Singapore (NMRC/TA/003/2012 and NMRC/CSA/012/2009, respectively).S

Human dermal CD14⁺ cells are a transient population of monocyte-derived macrophages.

Dendritic cells (DCs), monocytes, and macrophages are leukocytes with critical roles in immunity and tolerance. The DC network is evolutionarily conserved; the homologs of human tissue CD141(hi)XCR1⁺ CLEC9A⁺ DCs and CD1c⁺ DCs are murine CD103⁺ DCs and CD64⁻ CD11b⁺ DCs. In addition, human tissues also contain CD14⁺ cells, currently designated as DCs, with an as-yet unknown murine counterpart. Here we have demonstrated that human dermal CD14⁺ cells are a tissue-resident population of monocyte-derived macrophages with a short half-life of <6 days. The decline and reconstitution kinetics of human blood CD14⁺ monocytes and dermal CD14⁺ cells in vivo supported their precursor-progeny relationship. The murine homologs of human dermal CD14⁺ cells are CD11b⁺ CD64⁺ monocyte-derived macrophages. Human and mouse monocytes and macrophages were defined by highly conserved gene transcripts, which were distinct from DCs. The demonstration of monocyte-derived macrophages in the steady state in human tissue supports a conserved organization of human and mouse mononuclear phagocyte system

IRF4 transcription factor-dependent CD11b+ dendritic cells in human and mouse control mucosal IL-17 cytokine responses.

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

Human fetal dendritic cells promote prenatal T-cell immune suppression through arginase-2.

During gestation the developing human fetus is exposed to a diverse range of potentially immune-stimulatory molecules including semi-allogeneic antigens from maternal cells, substances from ingested amniotic fluid, food antigens, and microbes. Yet the capacity of the fetal immune system, including antigen-presenting cells, to detect and respond to such stimuli remains unclear. In particular, dendritic cells, which are crucial for effective immunity and tolerance, remain poorly characterized in the developing fetus. Here we show that subsets of antigen-presenting cells can be identified in fetal tissues and are related to adult populations of antigen-presenting cells. Similar to adult dendritic cells, fetal dendritic cells migrate to lymph nodes and respond to toll-like receptor ligation; however, they differ markedly in their response to allogeneic antigens, strongly promoting regulatory T-cell induction and inhibiting T-cell tumour-necrosis factor-α production through arginase-2 activity. Our results reveal a previously unappreciated role of dendritic cells within the developing fetus and indicate that they mediate homeostatic immune-suppressive responses during gestation