Clare L. Bennett

Clearance of influenza virus from the lung depends on migratory langerin+CD11b− but not plasmacytoid dendritic cells

Although dendritic cells (DCs) play an important role in mediating protection against influenza virus, the precise role of lung DC subsets, such as CD11b− and CD11b+ conventional DCs or plasmacytoid DCs (pDCs), in different lung compartments is currently unknown. Early after intranasal infection, tracheal CD11b−CD11chi DCs migrated to the mediastinal lymph nodes (MLNs), acquiring co-stimulatory molecules in the process. This emigration from the lung was followed by an accumulation of CD11b+CD11chi DCs in the trachea and lung interstitium. In the MLNs, the CD11b+ DCs contained abundant viral nucleoprotein (NP), but these cells failed to present antigen to CD4 or CD8 T cells, whereas resident CD11b−CD8α+ DCs presented to CD8 cells, and migratory CD11b−CD8α− DCs presented to CD4 and CD8 T cells. When lung CD11chi DCs and macrophages or langerin+CD11b−CD11chi DCs were depleted using either CD11c–diphtheria toxin receptor (DTR) or langerin-DTR mice, the development of virus-specific CD8+ T cells was severely delayed, which correlated with increased clinical severity and a delayed viral clearance. 120G8+ CD11cint pDCs also accumulated in the lung and LNs carrying viral NP, but in their absence, there was no effect on viral clearance or clinical severity. Rather, in pDC-depleted mice, there was a reduction in antiviral antibody production after lung clearance of the virus. This suggests that multiple DCs are endowed with different tasks in mediating protection against influenza virus.

Murine epidermal Langerhans cells and langerin-expressing dermal dendritic cells are unrelated and exhibit distinct functions

A new langerin+ DC subset has recently been identified in murine dermis (langerin+ dDC), but the lineage and functional relationships between these cells and langerin+ epidermal Langerhans cells (LC) are incompletely characterized. Selective expression of the cell adhesion molecule EpCAM by LC allowed viable LC to be easily distinguished from langerin+ dDC in skin and lymphoid tissue and ex vivo as well. Differential expression of EpCAM and langerin revealed the presence of at least 3 distinct skin DC subsets. We determined that LC and langerin+ dDC exhibit different migratory capabilities in vitro and repopulate distinct anatomic compartments in skin at different rates after conditional depletion in vivo. Langerin+ dDC, in contrast to LC, did not require TGFβ1 for development. Carefully timed gene gun immunization studies designed to take advantage of the distinct repopulation kinetics of langerin+ dDC and LC revealed that langerin+ dDC were required for optimal production of β-galactosidase-specific IgG2a/c and IgG2b in the acute phase. In contrast, immunization via LC-deficient skin resulted in persistent and strikingly reduced IgG1 and enhanced IgG2a Ab production. Our data support the concepts that LC and langerin+ dDC represent distinct DC subsets that have specialized functions and that LC are important immunoregulatory cells. The presence of at least 3 functionally distinct skin DC subsets may have particular relevance for vaccines that are administered epicutaneously.

Nicotinic Acid-Induced Flushing Is Mediated by Activation of Epidermal Langerhans Cells

The antidyslipidemic drug nicotinic acid (niacin) has been used for decades. One of the major problems of the therapeutical use of nicotinic acid is a strong cutaneous vasodilation called flushing, which develops in almost every patient taking nicotinic acid. Nicotinic acid-induced flushing has been shown to be mediated by the nicotinic acid receptor GPR109A and to involve the formation of vasodilatory prostanoids. However, the cellular mechanisms underlying this short-term effect are unknown. Here, we show that epidermal Langerhans cells are essential for the cutaneous flushing response induced by nicotinic acid. Langerhans cells respond with an increase in [Ca2+]i to nicotinic acid and express prostanoid synthases required for the formation of the vasodilatory prostanoids prostaglandin E2 and prostaglandin D2. Depletion of epidermal Langerhans cells but not of macrophages or dendritic cells abrogates nicotinic acid-induced flushing. These data unexpectedly identify epidermal Langerhans cells as essential mediators of nicotinic acid-induced flushing and may help to generate new strategies to suppress the unwanted effects of nicotinic acid. In addition, our results suggest that Langerhans cells besides their immunological roles are also involved in the local regulation of dermal blood flow.

Langerhans cells are negative regulators of the anti-Leishmania response

Langerhans cells suppress the immune response to low-dose Leishmania major infection in part by inducing regulatory T cells.

Langerhans Cells Are Required for Efficient Presentation of Topically Applied Hapten to T Cells

Dendritic cells (DC) play a pivotal role in the control of T cell immunity due to their ability to stimulate naive T cells and direct effector function. Murine and human DC are composed of a number of phenotypically, and probably developmentally, distinct subsets, which may play unique roles in the initiation and regulation of T cell responses. The skin is populated by at least two subsets of DC: Langerhans cells (LC), which form a contiguous network throughout the epidermis, and dermal DC. LC have classically been thought vital to initiate T cell responses to cutaneous Ags. However, recent data have highlighted the importance of dermal DC in cutaneous immunity, and the requirement for LC has become unclear. To define the relative roles of LC and dermal DC, we and others generated mouse models in which LC were specifically depleted in vivo. Unexpectedly, these studies yielded conflicting data as to the role of LC in cutaneous contact hypersensitivity (CHS). Extending our initial finding, we demonstrate that topical Ag is inefficiently transported to draining lymph nodes in the absence of LC, resulting in suboptimal priming of T cells and reduced CHS. However, dermal DC may also prime cutaneous T cell responses, suggesting redundancy between the two different skin DC subsets in this model.

Deletional Self-Tolerance to a Melanocyte/Melanoma Antigen Derived from Tyrosinase Is Mediated by a Radio-Resistant Cell in Peripheral and Mesenteric Lymph Nodes

Self-tolerance to melanocyte differentiation Ags limits the ability to generate therapeutic antimelanoma responses. However, the mechanisms responsible for CD8 T cell tolerance to these Ags are unknown. We have used a newly generated TCR-transgenic mouse to establish the basis of tolerance to one such Ag from tyrosinase. Despite expression of tyrosinase transcripts in the thymus, central deletion does not shape the tyrosinase-specific CD8 T cell repertoire. We demonstrate that this endogenously expressed melanocyte Ag is constitutively presented in both peripheral and mesenteric lymph nodes, leading to abortive activation and deletion of tyrosinase-specific CD8 T cells. Importantly, this Ag is not presented by either radio-sensitive dendritic cells, or by radio-resistant Langerhans cells. Thus, for this endogenous Ag, cross-tolerization does not appear to be an operative mechanism. Instead, we find radioresistant tyrosinase mRNA expression in lymphoid compartments where CD8 T cell deletion occurs. This suggests that direct presentation of tyrosinase by radio-resistant lymph node resident cells is entirely responsible for tolerance to this endogenous melanocyte differentiation Ag.

Lipopolysaccharide or Whole Bacteria Block the Conversion of Inflammatory Monocytes into Dendritic Cells In Vivo

Monocytes can develop into dendritic cells (DCs) that migrate to lymph nodes (LNs) and present antigens to T cells. However, we find that this differentiation is blocked when monocytes accumulate subcutaneously in response to bacteria or lipopolysaccharide (LPS). The inhibition of DC differentiation is mediated by the bacteria and in conjunction with inflammatory cells recruited at the site of injection. Inhibition of migratory DC development was reversed in Toll-like receptor (TLR)4-mutated mice when LPS, but not whole bacteria, was injected, suggesting that TLR4 is one but not the only mediator of the inhibition. The block imposed by bacteria was partly relieved by the absence of interleukin (IL)-12 p40, but not by individual absence of several cytokines involved in DC differentiation or in inflammation, i.e., IL-6, IL-10, IL-12 p35, and interferon γ. Consistent with the inability of monocytes to yield migrating DCs, and the finding that other DCs had limited access to particulate or bacterial antigens, these antigens were weakly presented to T cells in the draining LN. These results illustrate that bacteria-associated signals can have a negative regulatory role on adaptive immunity and that local innate responses for containment of infectious bacteria can at least initially supersede development of adaptive responses.

PD-L1 co-stimulation contributes to ligand-induced T cell receptor down-modulation on CD8(+) T cells

T cell receptor (TCR) down‐modulation after antigen presentation is a fundamental process that regulates TCR signal transduction. Current understanding of this process is that intrinsic TCR/CD28 signal transduction leads to TCR down‐modulation. Here, we show that the interaction between programmed cell death 1 ligand 1 (PD‐L1) on dendritic cells (DCs) and programmed death 1 (PD‐1) on CD8 T cells contributes to ligand‐induced TCR down‐modulation. We provide evidence that this occurs via Casitas B‐lymphoma (Cbl)‐b E3 ubiquitin ligase up‐regulation in CD8 T cells. Interference with PD‐L1/PD‐1 signalling markedly inhibits TCR down‐modulation leading to hyper‐activated, proliferative CD8 T cells as assessed in vitro and in vivo in an arthritis model. PD‐L1 silencing accelerates anti‐tumour immune responses and strongly potentiates DC anti‐tumour capacities, when combined with mitogen‐activated kinase (MAPK) modulators that promote DC activation.

Silent infection of bone marrow-derived dendritic cells by Leishmania mexicana amastigotes

Resolution of infection by Leishmania sp. is critically dependent on activation of CD4+ T helper cells. Naive CD4+ T helper cells are primed by dendritic cells which have responded to an activation signal in the periphery. However, the role of Leishmania‐infected dendritic cells in the activation of an anti‐Leishmania immune response has not been comprehensively addressed. Using the highly controlled model system of bone marrow‐derived dendritic cell infection by Leishmania mexicana cultured in vitro, we show that uptake of L. mexicana parasites does not result in activation of immature dendritic cells or secretion of IL‐12. Incubation with L. mexicana promastigotes results in the activation of a small percentage of dendritic cells which do not appear to contain whole parasites. Activation of dendritic cells is not suppressed by infection, since infected cells can be fully activated on addition of activating stimuli. Therefore, uptake of intact Leishmania mexicana parasites is not sufficient to activate dendritic cells in vitro. We propose that these data provide a basis for interpreting the interactions between dendritic cells and all Leishmania sp.

Nonhematopoietic antigen blocks memory programming of alloreactive CD8 + T cells and drives their eventual exhaustion in mouse models of bone marrow transplantation

Allogeneic blood or BM transplantation (BMT) is the most commonly applied form of adoptive cellular therapy for cancer. In this context, the ability of donor T cells to respond to recipient antigens is coopted to generate graft-versus-tumor (GVT) responses. The major reason for treatment failure is tumor recurrence, which is linked to the eventual loss of functional, host-specific CTLs. In this study, we have explored the role of recipient antigen expression by nonhematopoietic cells in the failure to sustain effective CTL immunity. Using clinically relevant models, we found that nonhematopoietic antigen severely disrupts the formation of donor CD8+ T cell memory at 2 distinct levels that operate in the early and late phases of the response. First, initial and direct encounters between donor CD8+ T cells and nonhematopoietic cells blocked the programming of memory precursors essential for establishing recall immunity. Second, surviving CD8+ T cells became functionally exhausted with heightened expression of the coinhibitory receptor programmed death-1 (PD-1). These 2 factors acted together to induce even more profound failure in long-term immunosurveillance. Crucially, the functions of exhausted CD8+ T cells could be partially restored by late in vivo blockade of the interaction between PD-1 and its ligand, PD-L1, without induction of graft-versus-host disease, suggestive of a potential clinical strategy to prevent or treat relapse following allogeneic BMT.