Anna I Proietto

Cutting Edge: Generation of Splenic CD8+ and CD8− Dendritic Cell Equivalents in Fms-Like Tyrosine Kinase 3 Ligand Bone Marrow Cultures

We demonstrate that functional and phenotypic equivalents of mouse splenic CD8+ and CD8− conventional dendritic cell (cDC) subsets can be generated in vitro when bone marrow is cultured with fms-like tyrosine kinase 3 (flt3) ligand. In addition to CD45RAhigh plasmacytoid DC, two distinct CD24high and CD11bhigh cDC subsets were present, and these subsets showed equivalent properties to splenic CD8+ and CD8− cDC, respectively, in the following: 1) surface expression of CD11b, CD24, and signal regulatory protein-α; 2) developmental dependence on, and mRNA expression of, IFN regulatory factor-8; 3) mRNA expression of TLRs and chemokine receptors; 4) production of IL-12 p40/70, IFN-α, MIP-1α, and RANTES in response to TLR ligands; 5) expression of cystatin C; and 6) cross-presentation of exogenous Ag to CD8 T cells. Furthermore, despite lacking surface CD8 expression, the CD24high subset contained CD8 mRNA and up-regulated surface expression when transferred into mice. This culture system allows access to bona fide counterparts of the splenic DC subsets.

The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement.

A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.

Dendritic cells in the thymus contribute to T-regulatory cell induction

Central tolerance is established through negative selection of self-reactive thymocytes and the induction of T-regulatory cells (TRs). The role of thymic dendritic cells (TDCs) in these processes has not been clearly determined. In this study, we demonstrate that in vivo, TDCs not only play a role in negative selection but in the induction of TRs. TDCs include two conventional dendritic cell (DC) subtypes, CD8loSirpαhi/+ (CD8loSirpα+) and CD8hiSirpαlo/− (CD8loSirpα−), which have different origins. We found that the CD8hiSirpα+ DCs represent a conventional DC subset that originates from the blood and migrates into the thymus. Moreover, we show that the CD8loSirpα+ DCs demonstrate a superior capacity to induce TRs in vitro. Finally, using a thymic transplantation system, we demonstrate that the DCs in the periphery can migrate into the thymus, where they efficiently induce TR generation and negative selection.

Differential MHC class II synthesis and ubiquitination confers distinct antigen-presenting properties on conventional and plasmacytoid dendritic cells

The importance of conventional dendritic cells (cDCs) in the processing and presentation of antigen is well established, but the contribution of plasmacytoid dendritic cells (pDCs) to these processes, and hence to T cell immunity, remains unclear. Here we showed that unlike cDCs, pDCs continued to synthesize major histocompatibility complex (MHC) class II molecules and the MHC class II ubiquitin ligase MARCH1 long after activation. Sustained MHC class II–peptide complex formation, ubiquitination and turnover rendered pDCs inefficient in the presentation of exogenous antigens but enabled pDCs to continuously present endogenous viral antigens in their activated state. As the antigen-presenting abilities of cDCs and pDCs are fundamentally distinct, these two cell types may activate largely nonoverlapping repertoires of CD4+ T cells.

c-Rel is required for the development of thymic Foxp3+ CD4 regulatory T cells

During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-κB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel−/− mice, thymic T reg cell numbers are markedly reduced as a result of a T cell–intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-β conversion of peripheral CD4+CD25− T cells into CD4+Foxp3+ cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel−/− mice, the residual peripheral c-rel−/− T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.

Human Dendritic Cell Subsets from Spleen and Blood Are Similar in Phenotype and Function but Modified by Donor Health Status

Mouse dendritic cells (DC) have been extensively studied in various tissues, especially spleen, and they comprise subsets with distinct developmental origins, surface phenotypes, and functions. Considerably less is known about human DC due to their rarity in blood and inaccessibility of other human tissues. The study of DC in human blood has revealed four subsets distinct in phenotype and function. In this study, we describe four equivalent DC subsets in human spleen obtained from deceased organ donors. We identify three conventional DC subsets characterized by surface expression of CD1b/c, CD141, and CD16, and one plasmacytoid DC subset characterized by CD304 expression. Human DC subsets in spleen were very similar to those in human blood with respect to surface phenotype, TLR and transcription factor expression, capacity to stimulate T cells, cytokine secretion, and cross-presentation of exogenous Ag. However, organ donor health status, in particular treatment with corticosteroid methylprednisolone and brain death, may affect DC phenotype and function. DC T cell stimulatory capacity was reduced but DC were qualitatively unchanged in methylprednisolone-treated deceased organ donor spleen compared with healthy donor blood. Overall, our findings indicate that human blood DC closely resemble human spleen DC. Furthermore, we confirm parallels between human and mouse DC subsets in phenotype and function, but also identify differences in transcription factor and TLR expression as well as functional properties. In particular, the hallmark functions of mouse CD8α+ DC subsets, that is, IL-12p70 secretion and cross-presentation, are not confined to the equivalent human CD141+ DC but are shared by CD1b/c+ and CD16+ DC subsets.

Aire regulates the transfer of antigen from mTECs to dendritic cells for induction of thymic tolerance

To investigate the role of Aire in thymic selection, we examined the cellular requirements for generation of ovalbumin (OVA)-specific CD4 and CD8 T cells in mice expressing OVA under the control of the rat insulin promoter. Aire deficiency reduced the number of mature single-positive OVA-specific CD4(+) or CD8(+) T cells in the thymus, independent of OVA expression. Importantly, it also contributed in 2 ways to OVA-dependent negative selection depending on the T-cell type. Aire-dependent negative selection of OVA-specific CD8 T cells correlated with Aire-regulated expression of OVA. By contrast, for OVA-specific CD4 T cells, Aire affected tolerance induction by a mechanism that operated independent of the level of OVA expression, controlling access of antigen presenting cells to medullary thymic epithelial cell (mTEC)-expressed OVA. This study supports the view that one mechanism by which Aire controls thymic negative selection is by regulating the indirect presentation of mTEC-derived antigens by thymic dendritic cells. It also indicates that mTECs can mediate tolerance by direct presentation of Aire-regulated antigens to both CD4 and CD8 T cells.

Selective suicide of cross-presenting CD8+ dendritic cells by cytochrome c injection shows functional heterogeneity within this subset.

Cross-presentation as a fundamental pathway of activating CD8+ T cells has been well established. So far the application of this concept in vivo is limited, and the mechanisms that specialize CD8+ dendritic cells (DCs) for this task are not fully understood. Here we take advantage of the specific cytosolic export feature of cross-presenting DCs together with the property of cytosolic cytochrome c (cyt c) in initiating Apaf-1-dependent apoptosis selectively in cross-presenting DCs. A single i.v. injection of cyt c in B6 mice produced a 2- to 3-fold reduction in splenic CD8+ DCs but not in Apaf-1-deficient mice. Functional studies both in vivo and in vitro showed that cyt c profoundly abrogated OVA-specific CD8+ T cell proliferation through its apoptosis-inducing effect on cross-presenting DCs. More importantly, in vivo injection of cyt c abolished the induction of cytotoxic T lymphocytes to exogenous antigen and reduced subsequent immunity to tumor challenge. In addition, only a proportion of CD8+ DCs that express abundant IL-12 and Toll-like receptor 3 were efficient cross-presenters. Our data support the hypothesis that cross-presentation in vivo requires cytosolic diversion of endocytosed proteins, conferring cross-presentation specialization to a proportion of CD8+ DCs. We propose that DCs incapable of such transfer, even within the CD8+ DC subset, are unable to cross-present. Our model opens an avenue to specifically target cross-presenting DCs in vivo for manipulating cytotoxic T lymphocyte responses toward infections, tumors, and transplants.

Signal Regulatory Protein Molecules Are Differentially Expressed by CD8− Dendritic Cells

A normalized subtracted gene expression library was generated from freshly isolated mouse dendritic cells (DC) of all subtypes, then used to construct cDNA microarrays. The gene expression profiles of the three splenic conventional DC (cDC) subsets were compared by microarray hybridization and two genes encoding signal regulatory protein β (Sirpβ1 and Sirpβ4) molecules were identified as differentially expressed in CD8− cDC. Genomic sequence analysis revealed a third Sirpβ member localized in the same gene cluster. These Sirpβ genes encode cell surface molecules containing extracellular Ig domains and short intracytoplasmic domains that have a charged amino acid in the transmembrane region which can potentially interact with ITAM-bearing molecules to mediate signaling. Indeed, we demonstrated interactions between Sirpβ1 and β2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpβ genes were expressed by CD8− cDC, but not by CD8+ cDC or plasmacytoid pre-DC. The related Sirpα gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpα and Sirpβ1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpβ1. Cross-linking of Sirpβ1 on DC resulted in a reduction in phagocytosis of Leishmania major parasites, but did not affect phagocytosis of latex beads, perhaps indicating that the regulation of phagocytosis by Sirpβ1 is a ligand-dependent interaction. Thus, we postulate that the differential expression of these molecules may confer the ability to regulate the phagocytosis of particular ligands to CD8− cDC.

The C-Type Lectin Clec12A Present on Mouse and Human Dendritic Cells Can Serve as a Target for Antigen Delivery and Enhancement of Antibody Responses

We have cloned the mouse and human C-type lectin Clec12A, expressed both, and produced mAb recognizing both. Mouse Clec12A is highly expressed on splenic CD8+ dendritic cells (DC) and plasmacytoid DC. A proportion of CD8−DC also expresses lower levels of Clec12A, as do monocytes, macrophages, and B cells. Human CLEC12A, like the mouse counterpart, is expressed on blood monocytes and DC, including pDC and BDCA-3+DC, the proposed equivalent of mouse CD8+DC. To determine whether Ag targeted to Clec12A could induce immune responses, mice were injected with a rat mAb recognizing Clec12A, or a control rat mAb, then production of anti-rat Ig was measured. Anti-Clec12A mAb alone produced only moderate responses, but these were amplified by coinjecting only small amounts of LPS as a DC activation agent. Furthermore, when OVA was conjugated to anti-Clec12A mAb, OVA-specific T cells were induced to proliferate. This Ag presentation to naive T cells was due to targeting conventional DC, because their ablation eliminated T cell activation. The potent Ab responses induced using microgram amounts of anti-Clec12A and minimal amounts of adjuvant demonstrate that this molecule can be used as an Ag-delivery target to enhance Ab responses to vaccines.