CD300f signalling induces inhibitory human monocytes/macrophages.

The CD300 glycoproteins are a family of related leucocyte surface molecules that regulate the immune response via their paired triggering and inhibitory receptors. Here we studied CD300f, an apoptotic cell receptor, and how it modulates the function of human monocytes and macrophages. We showed that CD300f signalling by crosslinking with anti-CD300f mAb (DCR-2) suppressed monocytes causing upregulation of the inhibitory molecule, CD274 (PD-L1) and their inhibition of T cell proliferation. Furthermore, CD300f signalling drove macrophages preferentially towards M2-type with upregulation of CD274, which was further enhanced by IL-4. CD300f signalling activates the PI3K/Akt pathway in monocytes. Inhibition of PI3K/Akt signalling resulting from CD300f crosslinking leads to downregulation of CD274 expression on monocytes. These findings highlight the potential use of CD300f blockade in cancer immune therapy to target immune suppressive macrophages in the tumour microenvironment, a known resistance mechanism to PD-1/PD-L1 checkpoint inhibitors.

Anti-Mouse CD83 Monoclonal Antibody Targeting Mature Dendritic Cells Provides Protection Against Collagen Induced Arthritis.

Antibodies targeting the activation marker CD83 can achieve immune suppression by targeting antigen-presenting mature dendritic cells (DC). This study investigated the immunosuppressive mechanisms of anti-CD83 antibody treatment in mice and tested its efficacy in a model of autoimmune rheumatoid arthritis. A rat anti-mouse CD83 IgG2a monoclonal antibody, DCR-5, was developed and functionally tested in mixed leukocyte reactions, demonstrating depletion of CD83+ conventional (c)DC, induction of regulatory DC (DCreg), and suppression of allogeneic T cell proliferation. DCR-5 injection into mice caused partial splenic cDC depletion for 2-4 days (mostly CD8+ and CD83+ cDC affected) with a concomitant increase in DCreg and regulatory T cells (Treg). Mice with collagen induced arthritis (CIA) treated with 2 or 6 mg/kg DCR-5 at baseline and every three days thereafter until euthanasia at day 36 exhibited significantly reduced arthritic paw scores and joint pathology compared to isotype control or untreated mice. While both doses reduced anti-collagen antibodies, only 6 mg/kg achieved significance. Treatment with 10 mg/kg DCR-5 was ineffective. Immunohistological staining of spleens at the end of CIA model with CD11c, CD83, and FoxP3 showed greater DC depletion and Treg induction in 6 mg/kg compared to 10 mg/kg DCR-5 treated mice. In conclusion, DCR-5 conferred protection from arthritis by targeting CD83, resulting in selective depletion of mature cDC and subsequent increases in DCreg and Treg. This highlights the potential for anti-CD83 antibodies as a targeted therapy for autoimmune diseases.

Moving on From Sipuleucel-T: New Dendritic Cell Vaccine Strategies for Prostate Cancer.

Tumors evade the immune system though a myriad of mechanisms. Using checkpoint inhibitors to help reprime T cells to recognize tumor has had great success in malignancies including melanoma, lung, and renal cell carcinoma. Many tumors including prostate cancer are resistant to such treatment. However, Sipuleucel-T, a dendritic cell (DC) based immunotherapy, improved overall survival (OS) in prostate cancer. Despite this initial success, further DC vaccines have failed to progress and there has been limited uptake of Sipuleucel-T in the clinic. We know in prostate cancer (PCa) that both the adaptive and the innate arms of the immune system contribute to the immunosuppressive environment. This is at least in part due to dysfunction of DC that play a crucial role in the initiation of an immune response. We also know that there is a paucity of DC in PCa, and that those there are immature, creating a tolerogenic environment. These attributes make PCa a good candidate for a DC based immunotherapy. Ultimately, the knowledge gained by much research into antigen processing and presentation needs to translate from bench to bedside. In this review we will analyze why newer vaccine strategies using monocyte derived DC (MoDC) have failed to deliver clinical benefit, particularly in PCa, and highlight the emerging antigen loading and presentation technologies such as nanoparticles, antibody-antigen conjugates and virus co-delivery systems that can be used to improve efficacy. Lastly, we will assess combination strategies that can help overcome the immunosuppressive microenvironment of PCa.

Targeting CD83 in mantle cell lymphoma with anti-human CD83 antibody.

OBJECTIVES: Effective antibody-drug conjugates (ADCs) provide potent targeted cancer therapies. CD83 is expressed on activated immune cells including B cells and is a therapeutic target for Hodgkin lymphoma. Our objective was to determine CD83 expression on non-Hodgkin lymphoma (NHL) and its therapeutic potential to treat mantle cell lymphoma (MCL) which is currently an incurable NHL. METHODS: We analysed CD83 expression on MCL cell lines and the lymph node/bone marrow biopsies of MCL patients. We tested the killing effect of CD83 ADC in vitro and in an in vivo xenograft MCL mouse model. RESULTS: CD83 is expressed on MCL, and its upregulation is correlated with the nuclear factor κB (NF-κB) activation. CD83 ADC kills MCL in vitro and in vivo. Doxorubicin and cyclophosphamide (CP), which are included in the current treatment regimen for MCL, enhance the NF-κB activity and increase CD83 expression on MCL cell lines. The combination of CD83 ADC with doxorubicin and CP has synergistic killing effect of MCL. CONCLUSION: This study provides evidence that a novel immunotherapeutic agent CD83 ADC, in combination with chemotherapy, has the potential to enhance the efficacy of current treatments for MCL.

Distinguishing human peripheral blood CD16+ myeloid cells based on phenotypic characteristics.

Myeloid lineage cells present in human peripheral blood include dendritic cells (DC) and monocytes. The DC are identified phenotypically as HLA-DR+ cells that lack major cell surface lineage markers for T cells (CD3), B cells (CD19, CD20), NK cells (CD56), red blood cells (CD235a), hematopoietic stem cells (CD34), and Mo that express CD14. Both DC and Mo can be phenotypically divided into subsets. DC are divided into plasmacytoid DC, which are CD11c- , CD304+ , CD85g+ , and myeloid DC that are CD11c+ . The CD11c+ DC are readily classified as CD1c+ DC and CD141+ DC. Monocytes are broadly divided into the CD14+ CD16- (classical) and CD14dim CD16+ subsets (nonclassical). A population of myeloid-derived cells that have DC characteristics, that is, HLA-DR+ and lacking lineage markers including CD14, but express CD16 are generally clustered with CD14dim CD16+ monocytes. We used high-dimensional clustering analyses of fluorescence and mass cytometry data, to delineate CD14+ monocytes, CD14dim CD16+ monocytes (CD16+ Mo), and CD14- CD16+ DC (CD16+ DC). We sought to identify the functional and kinetic relationship of CD16+ DC to CD16+ Mo. We demonstrate that differentiation of CD16+ DC and CD16+ Mo during activation with IFNγ in vitro and as a result of an allo-hematopoietic cell transplant (HCT) in vivo resulted in distinct populations. Recovery of blood CD16+ DC in both auto- and allo-(HCT) patients after myeloablative conditioning showed similar reconstitution and activation kinetics to CD16+ Mo. Finally, we show that expression of the cell surface markers CD300c, CCR5, and CLEC5a can distinguish the cell populations phenotypically paving the way for functional differentiation as new reagents become available.

CD83: Activation Marker for Antigen Presenting Cells and Its Therapeutic Potential.

CD83 is a member of the immunoglobulin (Ig) superfamily and is expressed in membrane bound or soluble forms. Membrane CD83 (mCD83) can be detected on a variety of activated immune cells, although it is most highly and stably expressed by mature dendritic cells (DC). mCD83 regulates maturation, activation and homeostasis. Soluble CD83 (sCD83), which is elevated in the serum of patients with autoimmune disease and some hematological malignancies is reported to have an immune suppressive function. While CD83 is emerging as a promising immune modulator with therapeutic potential, some important aspects such as its ligand/s, intracellular signaling pathways and modulators of its expression are unclear. In this review we discuss the recent biological findings and the potential clinical value of CD83 based therapeutics in various conditions including autoimmune disease, graft-vs.-host disease, transplantation and hematological malignancies.

CD83 is a new potential biomarker and therapeutic target for Hodgkin lymphoma.

Chemotherapy and hematopoietic stem cell transplantation are effective treatments for most Hodgkin lymphoma patients, however there remains a need for better tumor-specific target therapy in Hodgkin lymphoma patients with refractory or relapsed disease. Herein, we demonstrate that membrane CD83 is a diagnostic and therapeutic target, highly expressed in Hodgkin lymphoma cell lines and Hodgkin and Reed-Sternberg cells in 29/35 (82.9%) Hodgkin lymphoma patient lymph node biopsies. CD83 from Hodgkin lymphoma tumor cells was able to trogocytose to surrounding T cells and, interestingly, the trogocytosing CD83+T cells expressed significantly more programmed death-1 compared to CD83-T cells. Hodgkin lymphoma tumor cells secreted soluble CD83 that inhibited T-cell proliferation, and anti-CD83 antibody partially reversed the inhibitory effect. High levels of soluble CD83 were detected in Hodgkin lymphoma patient sera, which returned to normal in patients who had good clinical responses to chemotherapy confirmed by positron emission tomography scans. We generated a human anti-human CD83 antibody, 3C12C, and its toxin monomethyl auristatin E conjugate, that killed CD83 positive Hodgkin lymphoma cells but not CD83 negative cells. The 3C12C antibody was tested in dose escalation studies in non-human primates. No toxicity was observed, but there was evidence of CD83 positive target cell depletion. These data establish CD83 as a potential biomarker and therapeutic target in Hodgkin lymphoma.

The Analysis of CD83 Expression on Human Immune Cells Identifies a Unique CD83+-Activated T Cell Population.

CD83 is a member of the Ig gene superfamily, first identified in activated lymphocytes. Since then, CD83 has become an important marker for defining activated human dendritic cells (DC). Several potential CD83 mRNA isoforms have been described, including a soluble form detected in human serum, which may have an immunosuppressive function. To further understand the biology of CD83, we examined its expression in different human immune cell types before and after activation using a panel of mouse and human anti-human CD83 mAb. The mouse anti-human CD83 mAbs, HB15a and HB15e, and the human anti-human CD83 mAb, 3C12C, were selected to examine cytoplasmic and surface CD83 expression, based on their different binding characteristics. Glycosylation of CD83, the CD83 mRNA isoforms, and soluble CD83 released differed among blood DC, monocytes, and monocyte-derived DC, and other immune cell types. A small T cell population expressing surface CD83 was identified upon T cell stimulation and during allogeneic MLR. This subpopulation appeared specifically during viral Ag challenge. We did not observe human CD83 on unstimulated human natural regulatory T cells (Treg), in contrast to reports describing expression of CD83 on mouse Treg. CD83 expression was increased on CD4+, CD8+ T, and Treg cells in association with clinical acute graft-versus-host disease in allogeneic hematopoietic cell transplant recipients. The differential expression and function of CD83 on human immune cells reveal potential new roles for this molecule as a target of therapeutic manipulation in transplantation, inflammation, and autoimmune diseases.

New insights into the phenotype of human dendritic cell populations.

HLDA10 is the Tenth Human Leukocyte Differentiation Antigen (HLDA) Workshop. The HLDA Workshops provide a mechanism to allocate cluster of differentiation (CD) nomenclature by engaging in interlaboratory studies. As the host laboratory, we invited researchers from national and international academic and commercial institutions to submit monoclonal antibodies (mAbs) to human leukocyte surface membrane molecules, particularly those that recognised molecules on human myeloid cell populations and dendritic cells (DCs). These mAbs were tested for activity and then distributed as a blinded panel to 15 international laboratories to test on different leukocyte populations. These populations included blood DCs, skin-derived DCs, tonsil leukocytes, monocyte-derived DCs, CD34-derived DCs, macrophage populations and diagnostic acute myeloid leukaemia and lymphoma samples. Each laboratory was provided with enough mAb to perform five repeat experiments. Here, we summarise the reactivity of different mAb to 68 different cell-surface molecules expressed by human myeloid and DC populations. Submitted mAbs to some of the molecules were further validated to collate data required to designate a formal CD number. This collaborative process provides the broader scientific community with an invaluable data set validating mAbs to leukocyte-surface molecules.

FLT3-ligand treatment of humanized mice results in the generation of large numbers of CD141+ and CD1c+ dendritic cells in vivo.

We established a humanized mouse model incorporating FLT3-ligand (FLT3-L) administration after hematopoietic cell reconstitution to investigate expansion, phenotype, and function of human dendritic cells (DC). FLT3-L increased numbers of human CD141(+) DC, CD1c(+) DC, and, to a lesser extent, plasmacytoid DC (pDC) in the blood, spleen, and bone marrow of humanized mice. CD1c(+) DC and CD141(+) DC subsets were expanded to a similar degree in blood and spleen, with a bias toward expansion of the CD1c(+) DC subset in the bone marrow. Importantly, the human DC subsets generated after FLT3-L treatment of humanized mice are phenotypically and functionally similar to their human blood counterparts. CD141(+) DC in humanized mice express C-type lectin-like receptor 9A, XCR1, CADM1, and TLR3 but lack TLR4 and TLR9. They are major producers of IFN-λ in response to polyinosinic-polycytidylic acid but are similar to CD1c(+) DC in their capacity to produce IL-12p70. Although all DC subsets in humanized mice are efficient at presenting peptide to CD8(+) T cells, CD141(+) DC are superior in their capacity to cross-present protein Ag to CD8(+) T cells following activation with polyinosinic-polycytidylic acid. CD141(+) DC can be targeted in vivo following injection of Abs against human DEC-205 or C-type lectin-like receptor 9A. This model provides a feasible and practical approach to dissect the function of human CD141(+) and CD1c(+) DC and evaluate adjuvants and DC-targeting strategies in vivo.

CD300 molecule regulation of human dendritic cell functions.

Dendritic cells (DC) are a heterogeneous population of leucocytes which play a key role in initiating and modulating immune responses. The human CD300 family consists of six immunoregulatory leucocyte membrane molecules that regulate cellular activity including differentiation, viability, cytokine and chemokine secretion, phagocytosis and chemotaxis. Recent work has identified polar lipids as probable ligands for these molecules in keeping with the known evolutionary conservation of this family. CD300 molecules are all expressed by DC; CD300b, d, e and f are restricted to different subpopulations of the myeloid DC lineage. They have been shown to regulate DC function both in vitro and in vivo. In addition DC are able to regulate their CD300 expression in an autocrine manner. The potential to form different CD300 heterodimers adds further complexity to their role in fine tuning DC function. Expression of CD300 molecules is altered in a number of diseases including many where DC are implicated in the pathogenesis. CD300 antibodies have been demonstrated to have significant therapeutic effect in animal models. The mechanisms underlying the immunoregulatory effects of the CD300 family are complex. Deciphering their physiology will allow effective targeting of these molecules as novel therapies in a wide variety of inflammatory diseases.

Human CD1c (BDCA-1)+ myeloid dendritic cells secrete IL-10 and display an immuno-regulatory phenotype and function in response to Escherichia coli.

Human blood myeloid DCs can be subdivided into CD1c (BDCA-1)(+) and CD141 (BDCA-3)(+) subsets that display unique gene expression profiles, suggesting specialized functions. CD1c(+) DCs express TLR4 while CD141(+) DCs do not, thus predicting that these two subsets have differential capacities to respond to Escherichia coli. We isolated highly purified CD1c(+) and CD141(+) DCs and compared them to in vitro generated monocyte-derived DCs (MoDCs) following stimulation with whole E. coli. As expected, MoDCs produced high levels of the proinflammatory cytokines TNF, IL-6, and IL-12, were potent inducers of Th1 responses, and processed E. coli-derived Ag. In contrast, CD1c(+) DCs produced only low levels of TNF, IL-6, and IL-12 and instead produced high levels of the anti-inflammatory cytokine IL-10 and regulatory molecules IDO and soluble CD25. Moreover, E. coli-activated CD1c(+) DCs suppressed T-cell proliferation in an IL-10-dependent manner. Contrary to their mouse CD8(+) DC counterparts, human CD141(+) DCs did not phagocytose or process E. coli-derived Ag and failed to secrete cytokines in response to E. coli. These data demonstrate substantial differences in the nature of the response of human blood DC subsets to E. coli.

Screening of the HLDA9 panel on peripheral blood dendritic cell populations.

Dendritic cells (DC) are a heterogeneous population of bone marrow derived leucocytes that are essential in the initiation of primary T lymphocyte responses. DC are identified as Lineage negative, HLA-DR(+) blood cells that can be further subdivided by CD11c to distinguish CD11c(+) DC and the CD11c(-) plasmacytoid DC. Plasmacytoid DC are the primary IFNα producing cells and express CD303, CD304 and CD123. The CD11c(+) myeloid DC can be divided into populations by CD1c, CD16 and CD141 expression. Despite DC being a functionally unique population, they share many cell surface antigens with myeloid lineage cells and B lymphocytes. We used flow cytometry to screen fresh human blood DC populations with the HLDA9 panel of 63 directly labelled mAb which included mAb specific for a number of B lymphocyte antigens. Of this panel, 23 mAb did not bind Lin(-)HLA-DR(+) DC and 10 bound all four populations. Eight mAb bound to the three CD11c(+) DC populations whilst no mAb tested bound to only pDC. Some of the mAb expected to bind to DC populations failed in this analysis. Overall, this screening highlighted similarities between the CD11c(+) DC subsets and the relatively immature state of peripheral blood DC.

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens.

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141+ DC subset. CD141+ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-beta, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c+ DC subset. Polyinosine-polycytidylic acid (poly I:C)-activated CD141+ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8+ cytotoxic T lymphocytes than poly I:C-activated CD1c+ DCs. Importantly, CD141+ DCs, but not CD1c+ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141+ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8alpha+ DC subset. The data demonstrate a role for CD141+ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

Review of human DC subtypes.

Dendritic cells (DC) are critical to the induction and regulation of the innate and adaptive immune responses. They have been implicated in the pathogenesis of many autoimmune and chronic inflammatory diseases as well as contributing to the development of tumours by their lack of appropriate function. As such, understanding human DC biology provides the insight needed to develop applications for their use in the treatment of diseases. Currently, studies on mouse DC outnumber those on human cells; however, the comparison between mouse and human models has been somewhat misleading due to the basic biological and practical differences between the two models. In this review, we summarise the current understanding of human DC subtypes by describing the phenotype of the populations and how this relates to function. We also hope to clarify the differences in nomenclature between the human and mouse models that have arisen by way of the different experimental models.

The CD300 molecules regulate monocyte and dendritic cell functions.

The CD300 glycoproteins are a family of related leucocyte surface molecules that modulate a diverse array of cell processes via their paired triggering and inhibitory receptor functions. All family members have a single Ig-V like domain and they share a common evolutionary pathway. At least one member of the family has undergone significant positive selection (ranked second in the top 50) indicating a need to maintain some crucial function. Here we have reviewed the CD300 family members, and their expression on cells of the monocyte and dendritic cell lineages. The consequences of CD300 molecule expression by these leucocyte lineages are only now beginning to be understood. The ability to fine tune monocyte and dendritic cell function and immune responses highlights several potential options to exploit these molecules as therapeutic targets in chronic inflammatory diseases, allergy and other disease states.

The CD300 family of molecules are evolutionarily significant regulators of leukocyte functions.

The CD300 glycoproteins are a family of cell surface molecules that modulate a diverse array of cell processes via their paired triggering and inhibitory receptor functions. Family members share a common evolutionary pathway and at least one member of the family has undergone significant positive selection, indicating their crucial value to the host. This review clarifies the occasionally confusing usage of nomenclature for the CD300 family and summarizes our current understanding of their genomics, expression and function. Their ability to fine tune leukocyte function and immune responses highlights several potential options to exploit the CD300 molecules as therapeutic targets in chronic inflammatory diseases, allergy and other disease states.

CD300a/c regulate type I interferon and TNF-alpha secretion by human plasmacytoid dendritic cells stimulated with TLR7 and TLR9 ligands.

Activation of human plasmacytoid dendritic cells (pDCs) with ligands for Toll-like receptors (TLRs) 7 and 9 induces the secretion of type I interferons and other inflammatory cytokines as well as pDC differentiation. Transcripts for 2 members of the CD300 gene family, CD300a and CD300c, were identified on pDCs during gene expression studies to identify new immunoregulatory molecules on pDCs. We therefore investigated the expression of CD300a and CD300c and their potential regulation of pDC function. CD300a/c RNA and surface expression were downregulated after stimulation of pDCs with TLR7 and TLR9 ligands. Exogenous interferon (IFN)-alpha down-regulated CD300a/c expression, whereas neutralizing IFN-alpha abolished TLR ligand-induced CD300a/c down-regulation. This implicates IFN-alpha in regulating CD300a/c expression in pDCs. In addition, IFN-alpha favored tumor necrosis factor (TNF)-alpha secretion by CpG-induced pDCs. CD300a/c triggering by cross-linking antibody reduced TNF-alpha and increased IFN-alpha secretion by pDCs. Furthermore, CD300a/c triggering, in the presence of neutralizing IFN-alpha, further reduced TNF-alpha secretion. These data indicate that CD300a and CD300c play an important role in the cross-regulation of TNF-alpha and IFN-alpha secretion from pDCs.

Novel human CD4+ T lymphocyte subpopulations defined by CD300a/c molecule expression.

The CD300c (CMRF-35A) and CD300a (CMRF-35H) molecules are leukocyte surface proteins that are part of a larger family of immunoregulatory molecules encoded by a gene complex on human chromosome 17. The CMRF-35 monoclonal antibody binds to an epitope common to both molecules, expressed on most human leukocyte populations, apart from B lymphocytes and a subpopulation of CD4(+) and CD8(+) T lymphocytes. We describe the CMRF-35(pos) and CMRF-35(-) fractions of CD4(+) T lymphocytes. The CMRF-35(pos) fraction can further be divided into CMRF-35(++) and CMRF-35(+)CD4(+) T lymphocyte subpopulations. Resting peripheral CD4(+) T lymphocytes express CD300a mRNA and very low amounts of CD300c. Activation results in an initial decrease in CD300a gene expression before an increase in both CD300a and CD300c gene expression. The up-regulated expression of these genes was associated with increased CMRF-35 binding to activated T lymphocytes. The CMRF-35(-) fraction of CD4(+) T lymphocytes proliferated to a greater extent than the CMRF-35(pos) fraction, in response to mitogens or allogeneic antigen. The poor proliferation of the CMRF-35(pos) CD4(+) in response to mitogens was explained by increased apoptosis within this subpopulation. The recall antigen, tetanus toxoid, stimulated the CMRF-35(++)CD4(+)CD45RO(+) but not the CMRF-35(-)CD4(+)CD45RO(+) subpopulation. Resting CMRF-35(++) CD4(+) lymphocytes express low levels of IFN-gamma mRNA. Within 18 h following in vitro activation, CMRF-35(++) CD4(+) lymphocytes express more IFN-gamma mRNA and protein compared with the CMRF-35(-)CD4(+) lymphocytes, however, after 24 h, both the CMRF-35(+) and CMRF-35(-)CD4(+) T lymphocytes were able to produce IFN-gamma. The CMRF-35(++)CD4(+) T lymphocyte population contains the Th(1) memory effector cells.

Transforming growth factor beta1 up-regulates interferon regulatory factor 8 during dendritic cell development.

Langerhans cells (LC) represent the cutaneous contingent of dendritic cells (DC). Their development critically depends on transforming growth factor beta1 (TGF-beta1) as demonstrated by analysis of TGF-beta1(-/-) mice, which lack LC. Here we used a two-step culture system and transcriptional profiling by DNA microarrays to search for TGF-beta1 target genes in DC. The study identified interferon regulatory factor 8 (IRF-8) as a novel target gene of TGF-beta1 signaling in DC. TGF-beta1 effectively induced Smad2/3 phosphorylation and IRF-8 RNA and protein expression. Blocking the TGF-beta1/Smad pathway by ectopic expression of inhibitory Smad7 and by SB431542 inhibitor abolished TGF-beta1 induced up-regulation of IRF-8. Furthermore, TGF-beta1-dependent induction of IRF-8 occurred in the absence of protein biosynthesis, suggesting a direct action of TGF-beta1/Smad signaling on IRF-8 gene activity. TGF-beta1 also induced expression of the chemokine receptor CCR7 and enhanced DC migration towards CCR7 ligand ELC. DC of IRF-8(-/-) mice show reduced CCR7 expression and migratory activity, thereby implicating the TGF-beta1/Smad/IRF-8 signaling pathway in CCR7 regulation. Thus, this study identified a novel TGF-beta1/Smad/IRF-8 signaling pathway with an impact on DC phenotype and function.