T Cell Zone Resident Macrophages Silently Dispose of Apoptotic Cells in the Lymph Node.

In lymph nodes (LNs), dendritic cells (DCs) are thought to dispose of apoptotic cells, a function pertaining to macrophages in other tissues. We found that a population of CX3CR1+ MERTK+ cells located in the T cell zone of LNs, previously identified as DCs, are efferocytic macrophages. Lineage-tracing experiments and shield chimeras indicated that these T zone macrophages (TZM) are long-lived macrophages seeded in utero and slowly replaced by blood monocytes after birth. Imaging the LNs of mice in which TZM and DCs express different fluorescent proteins revealed that TZM-and not DCs-act as the only professional scavengers, clearing apoptotic cells in the LN T cell zone in a CX3CR1-dependent manner. Furthermore, similar to other macrophages, TZM appear inefficient in priming CD4 T cells. Thus, efferocytosis and T cell activation in the LN are uncoupled processes designated to macrophages and DCs, respectively, with implications to the maintenance of immune homeostasis.

Clonal Proliferation and Stochastic Pruning Orchestrate Lymph Node Vasculature Remodeling.

Lymph node (LN) expansion during an immune response relies on the transient remodeling of its vasculature. Although the mechanisms driving LN endothelial cell division are beginning to be understood, a comprehensive view of LN endothelial cell dynamics at the single-cell level is lacking. Here, we used multicolored fluorescent fate-mapping models to track the behavior of blood endothelial cells during LN expansion upon inflammation and subsequent return to homeostasis. We found that expansion of the LN vasculature relied on the sequential assembly of endothelial cell proliferative units. This segmented growth was sustained by the clonal proliferation of high endothelial venule (HEV) cells, which act as local progenitors to create capillaries and HEV neo-vessels at the periphery of the LN. Return to homeostasis was accompanied by the stochastic death of pre-existing and neo-synthesized LN endothelial cells. Thus, our fate-mapping studies unravel-at a single-cell level-the complex dynamics of vascular-tree remodeling during LN expansion and contraction.

Broad and Largely Concordant Molecular Changes Characterize Tolerogenic and Immunogenic Dendritic Cell Maturation in Thymus and Periphery.

Dendritic cells (DCs) are instrumental in the initiation of T cell responses, but how thymic and peripheral tolerogenic DCs differ globally from Toll-like receptor (TLR)-induced immunogenic DCs remains unclear. Here, we show that thymic XCR1(+) DCs undergo a high rate of maturation, accompanied by profound gene-expression changes that are essential for central tolerance and also happen in germ-free mice. Those changes largely overlap those occurring during tolerogenic and, more unexpectedly, TLR-induced maturation of peripheral XCR1(+) DCs, arguing against the commonly held view that tolerogenic DCs undergo incomplete maturation. Interferon-stimulated gene (ISG) expression was among the few discriminators of immunogenic and tolerogenic XCR1(+) DCs. Tolerogenic XCR1(+) thymic DCs were, however, unique in expressing ISGs known to restrain virus replication. Therefore, a broad functional convergence characterizes tolerogenic and immunogenic XCR1(+) DC maturation in the thymus and periphery, maximizing antigen presentation and signal delivery to developing and to conventional and regulatory mature T cells.

Dynamics and Transcriptomics of Skin Dendritic Cells and Macrophages in an Imiquimod-Induced, Biphasic Mouse Model of Psoriasis.

Psoriasis is a chronic inflammatory skin disease of unknown etiology. Previous studies showed that short-term, 5-7 d-long application of imiquimod (IMQ), a TLR7 agonist, to the skin of mice triggers a psoriasis-like inflammation. In the current study, by applying IMQ for 14 consecutive d, we established an improved mouse psoriasis-like model in that it recapitulated many of the clinical and cellular hallmarks observed in human patients during both the early-onset and the late-stable phase of psoriasis. Although macrophages and dendritic cells (DCs) have been proposed to drive the psoriatic cascade, their largely overlapping phenotype hampered studying their respective role. Based on our ability to discriminate Langerhans cells (LCs), conventional DCs, monocytes, monocyte-derived DCs, macrophages, and plasmacytoid DCs in the skin, we addressed their dynamics during both phases of our biphasic psoriasis-like model. Plasmacytoid DCs were not detectable during the whole course of IMQ treatment. During the early phase, neutrophils infiltrated the epidermis, whereas monocytes and monocyte-derived DCs were predominant in the dermis. During the late phase, LCs and macrophage numbers transiently increased in the epidermis and dermis, respectively. LC expansion resulted from local proliferation, a conclusion supported by global transcriptional analysis. Genetic depletion of LCs permitted to evaluate their function during both phases of the biphasic psoriasis-like model and demonstrated that their absence resulted in a late phase that is associated with enhanced neutrophil infiltration. Therefore, our data support an anti-inflammatory role of LCs during the course of psoriasis-like inflammation.

Identification of a new stromal cell type involved in the regulation of inflamed B cell follicles.

Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-ß deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.

Galectin-1-expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow.

In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7-producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1(+) reticular cells. We also determined that IL-7(+) and GAL1(+) cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7(+) to GAL1(+) supportive BM niches during their development.

High endothelial venules as traffic control points maintaining lymphocyte population homeostasis in lymph nodes.

Millions of lymphocytes enter and exit mammal lymph nodes (LNs) each day, accessing the parenchyma via high endothelial venules (HEVs) and egressing via lymphatics. Despite this high rate of cellular flux and the many entry and exit sites within a given LN, the number of lymphocytes present in a resting LN is extraordinary stable over time, raising the question of how this steady-state is maintained. Here we have examined the anatomic details of lymphocyte movement in HEVs, finding that HEVs create pockets within which lymphocytes reside for several minutes before entering the LN proper. The function of these pockets was revealed in experiments performed under conditions in which lymphocyte egress from the LN was compromised by any of several approaches. Under such conditions, the HEVs pockets behaved as "waiting areas" in which lymphocytes were held until space was made available to them for entry into the parenchyma. Thus, rather than being simple entry ports, HEVs act as gatekeepers able to stack, hold and grant lymphocytes access to LN parenchyma in proportion to the rate of lymphocyte egress from the LN, enabling the LN to maintain a constant steady-state cellularity while supporting the extensive cellular trafficking necessary for repertoire scanning.

Epidermal γδ T cells originate from yolk sac hematopoiesis and clonally self-renew in the adult.

The murine epidermis harbors two immune cell lineages, Langerhans cells (LCs) and γδ T cells known as dendritic epidermal T cells (DETCs). LCs develop from both early yolk sac (YS) progenitors and fetal liver monocytes before locally self-renewing in the adult. For DETCs, the mechanisms of homeostatic maintenance and their hematopoietic origin are largely unknown. Here, we exploited multicolor fate mapping systems to reveal that DETCs slowly turn over at steady state. Like for LCs, homeostatic maintenance of DETCs is achieved by clonal expansion of tissue-resident cells assembled in proliferative units. The same mechanism, albeit accelerated, facilitates DETC replenishment upon injury. Hematopoietic lineage tracing uncovered that DETCs are established independently of definitive hematopoietic stem cells and instead originate from YS hematopoiesis, again reminiscent of LCs. DETCs thus resemble LCs concerning their maintenance, replenishment mechanisms, and hematopoietic development, suggesting that the epidermal microenvironment exerts a lineage-independent influence on the initial seeding and homeostatic maintenance of its resident immune cells.

Fate mapping reveals origin and dynamics of lymph node follicular dendritic cells.

Follicular dendritic cells (FDCs) regulate B cell function and development of high affinity antibody responses but little is known about their biology. FDCs associate in intricate cellular networks within secondary lymphoid organs. In vitro and ex vivo methods, therefore, allow only limited understanding of the genuine immunobiology of FDCs in their native habitat. Herein, we used various multicolor fate mapping systems to investigate the ontogeny and dynamics of lymph node (LN) FDCs in situ. We show that LN FDC networks arise from the clonal expansion and differentiation of marginal reticular cells (MRCs), a population of lymphoid stromal cells lining the LN subcapsular sinus. We further demonstrate that during an immune response, FDCs accumulate in germinal centers and that neither the recruitment of circulating progenitors nor the division of local mature FDCs significantly contributes to this accumulation. Rather, we provide evidence that newly generated FDCs also arise from the proliferation and differentiation of MRCs, thus unraveling a critical function of this poorly defined stromal cell population.

Multicolor fate mapping of Langerhans cell homeostasis.

Langerhans cells (LCs) constitute a network of immune sentinels in the skin epidermis that is seeded during embryogenesis. Whereas the development of LCs has been extensively studied, much less is known about the homeostatic renewal of adult LCs in "nonmanipulated" animals. Here, we present a new multicolor fluorescent fate mapping system and quantification approach to investigate adult LC homeostasis. This novel approach enables us to propose and provide evidence for a model in which the adult epidermal LC network is not formed by mature coequal LCs endowed with proliferative capabilities, but rather constituted by adjacent proliferative units composed of "dividing" LCs and their terminally differentiated daughter cells. Altogether, our results demonstrate the general utility of our novel fate-mapping system to follow cell population dynamics in vivo and to establish an alternative model for LC homeostasis.