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.

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.

Cutting edge: priming of CD8 T cell immunity to herpes simplex virus type 1 requires cognate TLR3 expression in vivo.

Despite its potential for involvement in viral immunity, little evidence links TLR3 to adaptive antiviral responses. Here we show that TLR3 is required for the generation of CD8 T cell immunity to HSV-1. The magnitude of the gB-specific CD8 T cell response after flank infection by HSV-1 was significantly reduced in mice lacking TIR domain-containing adaptor-inducing IFN-beta or TLR3, but not MyD88. Impaired CTL induction was evident in chimeric mice lacking TLR3 in bone marrow (BM)-derived cells. Among the dendritic cell subsets, TLR3 was expressed by CD8alpha(+) dendritic cells, known to be involved in priming HSV-1-specific CD8 T cells. Use of mixed BM chimeras revealed that TLR3 and the MHC class I-restriction element must be expressed by the same BM-derived cell for effective priming. These data imply that a cognate linkage between TLR3 and MHC class I is required for efficient CTL priming to HSV-1.

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.

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.

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 (T(R)s). 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 T(R)s. TDCs include two conventional dendritic cell (DC) subtypes, CD8(lo)Sirpalpha(hi/+) (CD8(lo)Sirpalpha(+)) and CD8(hi)Sirpalpha(lo/-) (CD8(hi)Sirpalpha(-)) [corrected] which have different origins. We found that the CD8(hi)Sirpalpha(+) DCs represent a conventional DC subset that originates from the blood and migrates into the thymus. Moreover, we show that the CD8(lo)Sirpalpha(+) DCs demonstrate a superior capacity to induce T(R)s 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 T(R) generation and negative selection.

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.

Differential production of inflammatory chemokines by murine dendritic cell subsets.

Dendritic cells (DC) are efficient antigen presenting cells with the ability to activate naïve T cells. Murine DC represent a heterogeneous population that can be subdivided into distinct subsets, including the conventional DC (cDC) which are either CD4(-)CD8(-) (DN), CD4(+)CD8(-) (CD4+) or CD4(-)CD8(+) (CD8+) subsets and the plasmacytoid DC (pDC), which have different immune regulatory functions. In this study, we investigated the differential expression of genes encoding the inflammatory chemokines Mip-1alpha, Mip-1beta and Rantes, and the secretion of these chemokines, among splenic DC subsets. These chemokine genes were expressed at higher levels by the splenic CD4+ and DN cDC subsets compared with the CD8+ cDC, in both the resting and activated states in vivo. Both the pDC and cDC subsets displayed increases in chemokine secretion in response to a range of toll-like receptor (TLR) stimuli in vitro. Whilst the pDC were the highest producers of Mip-1alpha and Mip-1beta in response to some TLR stimuli, the DN and CD4+ cDC subsets were the superior producers of Rantes. Overall, of the cDC, the CD4+ cDC produced all chemokines most efficiently, both at a basal level, and in response to most TLR stimuli. Thus, we report a new functional difference between the murine splenic cDC subsets, with the CD4+ cDC demonstrating the most efficient production of the inflammatory chemokines Mip-1alpha, Mip-1beta and Rantes.

The impact of circulating dendritic cells on the development and differentiation of thymocytes.

Central tolerance is established through the negative selection of self-reactive thymocytes and the induction of T-regulatory cells (T-regs). A role for thymic epithelial cells in mediating both negative selection and T-reg induction has been clearly shown. The role of thymic dendritic cells (DCs) in these processes has not been clearly determined but has been the focus of recent studies. Thymic DCs include two conventional DC (cDC) subtypes, CD8(lo)Sirpalpha(hi/+) (CD8(lo)Sirpalpha(+) herein) and CD8(hi)Sirpalpha(lo/-) (CD8(hi)Sirpalpha(-) herein). It has been shown that these DC subsets have distinct developmental origins, the CD8(hi)Sirpalpha(-) cDCs developing intra-thymically and the CD8(lo)Sirpalpha(+) migrating into the thymus from the periphery. Furthermore, an important role for thymic DCs in the induction of T-regs has been shown. In this review, the role of DCs in the development and education of T cells in the thymus will be reviewed, with emphasis on the role of circulatory DCs in mediating these processes.

Monoclonal antibodies generated by DNA immunization recognize CD2 from a broad range of primates.

Using heterologous prime-boost (DNA immunization followed by immunization with transfected cells), we have generated depleting mouse anti-baboon CD2 monoclonal antibodies (mAb). These anti-CD2 mAb recognized a diverse range of primate CD2 from New World monkeys and Old World monkeys to humans and have potent immunosuppressive activity for human allo-MLR responses and anti-tetanus-toxoid recall responses. There was no upregulation of activation markers or release of cytokines when the mAb were incubated with human peripheral blood mononuclear cells. Using chimeric NOD-SCID IL2rgamma(null) mice, the mAb were shown to deplete human and cynomolgus monkey T cells in vivo. These anti-CD2 mAb may therefore be important immunological tools in allo- and xenotransplantation.

Distinct functional capacities of mouse thymic and splenic dendritic cell populations.

Dendritic cells (DC) are antigen-presenting cells that activate naive T cells. Murine DC are a heterogeneous population and can be subdivided into distinct subsets with different immune regulatory functions, namely the conventional DC (cDC), which include the CD8(+)Sirpalpha(-) and CD8(-)Sirpalpha(+) DC, and the plasmacytoid DC (pDC). In this study, the phenotype and function of DC subsets in both the thymus and spleen were compared. Significant differences between the thymic and splenic DC were observed in the expression of genes encoding chemokine receptors (CCRs), toll-like receptors (TLRs) and chemokines. Thymic DC expressed high levels of genes encoding a unique set of chemokines (CCL17 and CCL22) known to be important for T-cell development. Moreover, the capacity of the DC from the two organs to produce IL-6, IFN-alpha and IL-12p70 in response to the TLR 9 agonist CpG differed markedly, indicating intrinsic functional differences between subsets with similar surface phenotype. These results indicate that the microenvironment is an important factor that contributes to the functional specification of DC subsets in different lymphoid tissues.

Development of plasmacytoid and conventional dendritic cell subtypes from single precursor cells derived in vitro and in vivo.

The development of functionally specialized subtypes of dendritic cells (DCs) can be modeled through the culture of bone marrow with the ligand for the cytokine receptor Flt3. Such cultures produce DCs resembling spleen plasmacytoid DCs (pDCs), CD8(+) conventional DCs (cDCs) and CD8(-) cDCs. Here we isolated two sequential DC-committed precursor cells from such cultures: dividing 'pro-DCs', which gave rise to transitional 'pre-DCs' en route to differentiating into the three distinct DC subtypes (pDCs, CD8(+) cDCs and CD8(-) cDCs). We also isolated an in vivo equivalent of the DC-committed pro-DC precursor cell, which also gave rise to the three DC subtypes. Clonal analysis of the progeny of individual pro-DC precursors demonstrated that some pro-DC precursors gave rise to all three DC subtypes, some produced cDCs but not pDCs, and some were fully committed to a single DC subtype. Thus, commitment to particular DC subtypes begins mainly at this pro-DC stage.

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 beta (Sirpbeta1 and Sirpbeta4) molecules were identified as differentially expressed in CD8(-) cDC. Genomic sequence analysis revealed a third Sirpbeta member localized in the same gene cluster. These Sirpbeta 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 Sirpbeta1 and beta2 with the ITAM-bearing signaling molecule Dap12. Real-time PCR analysis showed that all three Sirpbeta genes were expressed by CD8(-) cDC, but not by CD8(+) cDC or plasmacytoid pre-DC. The related Sirpalpha gene showed a similar expression profile on cDC subtypes but was also expressed by plasmacytoid pre-DC. The differential expression of Sirpalpha and Sirpbeta1 molecules on DC was confirmed by staining with mAbs, including a new mAb recognizing Sirpbeta1. Cross-linking of Sirpbeta1 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 Sirpbeta1 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.

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 CD45RA(high) plasmacytoid DC, two distinct CD24(high) and CD11b(high) 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-alpha; 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-alpha, MIP-1alpha, 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 CD24(high) 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.