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The IRF8-dependent subset of classical dendritic cells (cDCs), termed cDC1, is important for cross-priming cytotoxic T cell responses against pathogens and tumors. Culture of hematopoietic progenitors with DC growth factor FLT3 ligand (FLT3L) yields very few cDC1s (in humans) or only immature “cDC1-like” cells (in the mouse). We report that OP9 stromal cells expressing the Notch ligand Delta-like 1 (OP9-DL1) optimize FLT3L-driven development of cDC1s from murine immortalized progenitors and primary bone marrow cells. Co-culture with OP9-DL1 induced IRF8-dependent cDC1s with a phenotype (CD103+ Dec205+ CD8α+) and expression profile resembling primary splenic cDC1s. OP9-DL1-induced cDC1s showed preferential migration toward CCR7 ligands in vitro and superior T cell cross-priming and antitumor vaccination in vivo. Co-culture with OP9-DL1 also greatly increased the yield of IRF8-dependent CD141+ cDC1s from human bone marrow progenitors cultured with FLT3L. Thus, Notch signaling optimizes cDC generation in vitro and yields authentic cDC1s for functional studies and translational applications. Dendritic cells (DCs) are critical inducers of immune responses, but current methods to generate them in vitro are suboptimal. Kirkling et al. report that Notch signaling facilitates the generation of DCs that closely resemble their in vivo counterparts and show superior capacity to vaccinate against tumors in vivo.

Antigen presentation is a critical step in the activation of naïve T lymphocytes. In mammals, dendritic cells (DCs), macrophages, and B lymphocytes can all function as antigen presenting cells (APCs). Although APCs have been identified in zebrafish, it is unclear if they fulfill similar roles in the initiation of adaptive immunity. Here we review the characterization of zebrafish macrophages, DCs, and B cells and evidence of their function as true APCs. Finally, we discuss the conservation of APC activity in vertebrates and the use of zebrafish to provide a new perspective on the evolution of these functions.

Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b(+) DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esam(hi) DC subset required lymphotoxin beta receptor signaling, proliferated in situ, and facilitated CD4(+) T cell priming. The Notch-independent Esam(lo) DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b(+)CD103(+) DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4(+) T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b(+) DC subsets in the spleen and intestine.

Plasmacytoid dendritic cells (pDCs) are specialized in rapid and massive secretion of type I interferon (IFN-α/β) in response to foreign nucleic acids. Combined with their antigen presentation capacity, this powerful functionality enables pDCs to orchestrate innate and adaptive immune responses. pDCs combine features of both lymphocytes and classical dendritic cells and display unique molecular adaptations to nucleic acid sensing and IFN production. In the decade since the identification of the pDC as a distinct immune cell type, our understanding of its molecular underpinnings and role in immunity has progressed rapidly. Here we review select aspects of pDC biology including cell fate establishment and plasticity, specific molecular mechanisms of pDC function, and the role of pDCs in T cell responses, antiviral immunity, and autoimmune diseases. Important unresolved questions remain in these areas, promising exciting times in pDC research for years to come.

Lymphoid organs are characterized by a complex network of phenotypically distinct dendritic cells (DC) with potentially unique roles in pathogen recognition and immunostimulation. Classical DC (cDC) include two major subsets distinguished in the mouse by the expression of CD8α. Here we describe a subset of CD8α + DC in lymphoid organs of naïve mice characterized by expression of the CX 3 CR1 chemokine receptor. CX 3 CR1 + CD8α + DC lack hallmarks of classical CD8α + DC, including IL-12 secretion, the capacity to cross-present antigen, and their developmental dependence on the transcriptional factor BatF3. Gene-expression profiling showed that CX 3 CR1 + CD8α + DC resemble CD8α − cDC. The microarray analysis further revealed a unique plasmacytoid DC (PDC) gene signature of CX 3 CR1 + CD8α + DC. A PDC relationship of the cells is supported further by the fact that they harbor characteristic D–J Ig gene rearrangements and that development of CX 3 CR1 + CD8α + DC requires E2-2, the critical transcriptional regulator of PDC. Thus, CX 3 CR1 + CD8α + DC represent a unique DC subset, related to but distinct from PDC. Collectively, the expression-profiling data of this study refine the resolution of previous DC definitions, sharpen the border of classical CD8α + and CD8α − DC, and should assist the identification of human counterparts of murine DC subsets.

The IL-2 receptor γ common (IL-2Rγc) chain is the shared subunit of the receptors for the IL-2 family of cytokines, which mediate signaling through JAK3 and various downstream pathways to regulate lymphopoiesis. Inactivating mutations in human IL-2Rγc result in SCID, a primary immunodeficiency characterized by greatly reduced numbers of lymphocytes. This study used bioinformatics, expression analysis, gene ablation, and specific pharmacologic inhibitors to investigate the function of two putative zebrafish IL-2Rγc paralogs, il-2rγc.a and il-2rγc.b, and downstream signaling components during early lymphopoiesis. Expression of il-2rγc.a commenced at 16 h post fertilization (hpf) and rose steadily from 4–6 d postfertilization (dpf) in the developing thymus, with il-2rγc.a expression also confirmed in adult T and B lymphocytes. Transcripts of il-2rγc.b were first observed from 8 hpf, but waned from 16 hpf before reaching maximal expression at 6 dpf, but this was not evident in the thymus. Knockdown of il-2rγc.a, but not il-2rγc.b, substantially reduced embryonic lymphopoiesis without affecting other aspects of hematopoiesis. Specific targeting of zebrafish Jak3 exerted a similar effect on lymphopoiesis, whereas ablation of zebrafish Stat5.1 and pharmacologic inhibition of PI3K and MEK also produced significant but smaller effects. Ablation of il-2rγc.a was further demonstrated to lead to an absence of mature T cells, but not B cells in juvenile fish. These results indicate that conserved IL-2Rγc signaling via JAK3 plays a key role during early zebrafish lymphopoiesis, which can be potentially targeted to generate a zebrafish model of human SCID.

Teleost fish are among the most ancient vertebrates possessing an adaptive immune system with B and T lymphocytes that produce memory responses to pathogens. Most bony fish, however, have only 2 types of B lymphocytes, in contrast to the 4 types available to mammals. To better understand the evolution of adaptive immunity, we generated transgenic zebrafish in which the major immunoglobulin M (IgM(+)) B-cell subset expresses green fluorescence protein (GFP) (IgM1:eGFP). We discovered that the earliest IgM(+) B cells appear between the dorsal aorta and posterior cardinal vein and also in the kidney around 20 days postfertilization. We also examined B-cell ontogeny in adult IgM1:eGFP;rag2:DsRed animals, where we defined pro-B, pre-B, and immature/mature B cells in the adult kidney. Sites of B-cell development that shift between the embryo and adult have previously been described in birds and mammals. Our results suggest that this developmental shift occurs in all jawed vertebrates. Finally, we used IgM1:eGFP and cd45DsRed;blimp1:eGFP zebrafish to characterize plasma B cells and investigate B-cell function. The IgM1:eGFP reporter fish are the first nonmammalian B-cell reporter animals to be described. They will be important for further investigation of immune cell evolution and development and host-pathogen interactions in zebrafish.

Infections with persistent viruses are a frequent cause of immunosuppression, autoimmune sequelae, and/or neoplastic disease. Plasmacytoid dendritic cells (pDCs) are innate immune cells that produce type I interferon (IFN-I) and other cytokines in response to virus-derived nucleic acids. Persistent viruses often cause depletion or functional impairment of pDCs, but the role of pDCs in the control of these viruses remains unclear. We used conditional targeting of pDC-specific transcription factor E2-2 to generate mice that constitutively lack pDCs in peripheral lymphoid organs and tissues. The profound impact of pDC deficiency on innate antiviral responses was revealed by the failure to control acute infection with the cytopathic mouse hepatitis virus. Furthermore, pDC-deficient animals failed to clear lymphocytic choriomeningitis virus (LCMV) from hematopoietic organs during persistent LCMV infection. This failure was associated with reduced numbers and functionality of LCMV-specific CD4 + helper T cells and impaired antiviral CD8 + T-cell responses. Adoptive transfer of LCMV-specific T cells revealed that both CD4 + and CD8 + T cells required IFN-I for expansion, but only CD4 + T cells required the presence of pDCs. In contrast, mice with pDC-specific loss of MHC class II expression supported normal CD4 + T-cell response to LCMV. These data suggest that pDCs facilitate CD4 + helper T-cell responses to persistent viruses independently of direct antigen presentation. Thus pDCs provide an essential link between innate and adaptive immunity to chronic viral infection, likely through the secretion of IFN-I and other cytokines.

Pore-forming toxins are essential to the virulence of a wide variety of pathogenic bacteria.Gardnerella vaginalisis a bacterial species associated with bacterial vaginosis (BV) and its significant adverse sequelae, including preterm birth and acquisition of human immunodeficiency virus.G. vaginalismakes a protein toxin that generates host immune responses and has been hypothesized to be involved in the pathogenesis of BV. We demonstrate thatG. vaginalisproduces a toxin (vaginolysin [VLY]) that is a member of the cholesterol-dependent cytolysin (CDC) family, most closely related to intermedilysin fromStreptococcus intermedius. Consistent with this predicted relationship, VLY lyses target cells in a species-specific manner, dependent upon the complement regulatory molecule CD59. In addition to causing erythrocyte lysis, VLY activates the conserved epithelial p38 mitogen-activated protein kinase pathway and induces interleukin-8 production by human epithelial cells. Transfection of human CD59 into nonsusceptible cells renders them sensitive to VLY-mediated lysis. In addition, a single amino acid substitution in the VLY undecapeptide [VLY(P480W)] generates a toxoid that does not form pores, and introduction of the analogous proline residue into another CDC, pneumolysin, significantly decreases its cytolytic activity. Further investigation of the mechanism of action of VLY may improve understanding of the functions of the CDC family as well as diagnosis and therapy for BV.

Dendritic cells (DCs) link innate immune sensing of the environment to the initiation of adaptive immune responses. Given their supreme capacity to interact with and present antigen to T cells, DCs have been proposed as key mediators of immunological tolerance in the steady state. However, recent evidence suggests that the role of DCs in central and peripheral T-cell tolerance is neither obligate nor dominant. Instead, DCs appear to regulate multiple aspects of T-cell physiology including tonic antigen receptor signaling, priming of effector T-cell response, and the maintenance of regulatory T cells. These diverse contributions of DCs may reflect the significant heterogeneity and “division of labor” observed between and within distinct DC subsets. The emerging complex role of different DC subsets should form the conceptual basis of DC-based therapeutic approaches toward induction of tolerance or immunization.