Jun Diao

Characterization of Distinct Conventional and Plasmacytoid Dendritic Cell-Committed Precursors in Murine Bone Marrow

The developmental pathways and differentiation relationship of dendritic cell (DC) subsets remain unclear. We report that murine CD11c+MHC II− bone marrow cells, which are immediate DC precursors of CD8α+, CD8α−, and B220+ DC in vivo, can be separated into B220+ and B220− DC precursor subpopulations. Purified B220− DC precursors expand, and generate exclusively mature CD11c+CD11b+B220− DC in vitro and after adoptive transfer. B220+ DC precursors, which resemble plasmacytoid pre-DC, have a lower proliferative potential than B220− DC precursors and generate both CD11b− B220+ and CD11b+B220− DC populations. Both DC precursor populations can give rise to CD8α+ and CD8α− DC subtypes. Our findings indicate that CD11c+MHC II−B220+ and CD11c+MHC II−B220− bone marrow cells are distinct DC lineage-restricted precursors.

Targeted Deletion of fgl2 Leads to Impaired Regulatory T Cell Activity and Development of Autoimmune Glomerulonephritis

Mice with targeted deletion of fibrinogen-like protein 2 (fgl2) spontaneously developed autoimmune glomerulonephritis with increasing age, as did wild-type recipients reconstituted with fgl2−/− bone marrow. These data implicate FGL2 as an important immunoregulatory molecule and led us to identify the underlying mechanisms. Deficiency of FGL2, produced by CD4+CD25+ regulatory T cells (Treg), resulted in increased T cell proliferation to lectins and alloantigens, Th 1 polarization, and increased numbers of Ab-producing B cells following immunization with T-independent Ags. Dendritic cells were more abundant in fgl2−/− mice and had increased expression of CD80 and MHCII following LPS stimulation. Treg cells were also more abundant in fgl2−/− mice, but their suppressive activity was significantly impaired. Ab to FGL2 completely inhibited Treg cell activity in vitro. FGL2 inhibited dendritic cell maturation and induced apoptosis of B cells through binding to the low-affinity FcγRIIB receptor. Collectively, these data suggest that FGL2 contributes to Treg cell activity and inhibits the development of autoimmune disease.

In Situ Replication of Immediate Dendritic Cell (DC) Precursors Contributes to Conventional DC Homeostasis in Lymphoid Tissue

The developmental biology of dendritic cells (DC) under physiological conditions remains unclear. In this study, we show that mouse CD11c+ MHC class II−lineage− cells are immediate precursors of conventional DC and are widely distributed in both bone marrow and lymphoid tissues. These precursors have a high clonal efficiency, and when cocultured on a supportive stromal monolayer or adoptively transferred in vivo, generate a population CD11c+MHC class II+ DC that retain limited proliferation capacity. During steady state conditions, a small proportion of immediate DC precursors (DCp) and DCs are dividing actively in bone marrow and spleen. Cytokines that initiate and support proliferation of immediate DCp were defined. Collectively, our findings provide evidence of a distinct development pathway for conventional DC in both bone marrow and lymphoid tissues and highlight the importance of in situ replication of immediate DCp and DC in maintaining conventional DC populations.

The FGL2-FcγRIIB pathway: A novel mechanism leading to immunosuppression

Fibrinogen‐like protein 2 (FGL2) is a multifunctional protein, which has been implicated in the pathogenesis of allograft and xenograft rejection. Previously, FGL2 was shown to inhibit maturation of BM‐derived DC and T‐cell proliferation. The mechanism of the immunosuppressive activity of FGL2 remains poorly elucidated. Here, we focus on identification of FGL2‐specific receptor(s) and their ability to modulate APC activity and allograft survival. Using flow cytometry and surface plasmon resonance analysis, we show that FGL2 binds specifically to Fc gamma receptor (FcγR)IIB and FcγRIII receptors, which are expressed on the surface of APC, including B lymphocytes, macrophages and DC. Antibody to FcγRIIB and FcγRIII, or deficiency of these receptors, abrogated FGL2 binding. FGL2 inhibited the maturation of BMDC from FcγRIIB+/+ mice but not from FcγRIIB−/− mice and induced apoptosis in the FcγRIIB+ mouse B‐cell line (A20) but not the A20IIA1.6 cell line that does not express FcγRIIB. Recombinant FGL2 infused into FcγRIIB+/+ (C57BL/6J, H‐2b) mice but not FcγRIIB−/− mice inhibited rejection of fully mismatched BALB/cJ (H‐2d) skin allografts. The identification of specific receptor binding has important implications for the pathogenesis of immune‐mediated disease and suggests a potential for targeted FGL2 therapy.

Breast cancer cell CD200 expression regulates immune response to EMT6 tumor cells in mice

CD200 has been characterized as an important immunoregulatory molecule, increased expression of which can lead to decreased transplant rejection, autoimmunity, and allergic disease. Elevated CD200 expression has been reported to be associated with poor prognosis in a number of human malignancies. We have found that cells of the transplantable EMT6 mouse breast cancer line growing in vitro express low levels of CD200, but levels increase markedly during growth in immunocompetent mice. Similar increased in vivo expression does not occur in NOD-SCID.IL-2γr−/− mice or mice with generalized over-expression of a CD200 transgene. In both mice, tumor growth occurs faster. Altered CD200 expression in control versus transgenic mice is accompanied by reproducible changes in tumor-infiltrating host cells, and altered cell composition in lymph nodes draining the tumor (DLN). Neutralization of expressed CD200 by anti-CD200mAbs leads to decreased tumor growth in immunocompetent mice, with improved detection of cytotoxic anti-tumor immune cells in DLN. Finally, we report that tumor growth in vivo can be monitored by levels of soluble CD200 (sCD200) in serum of tumor-bearing animals.

Toll-like Receptor 2 Activation Promotes Tumor Dendritic Cell Dysfunction by Regulating IL-6 and IL-10 Receptor Signaling

Although dendritic cell (DC) dysfunction in cancer is a well-recognized consequence of cancer-associated inflammation that contributes to immune evasion, the mechanisms that drive this process remain elusive. Here, we show the critical importance of tumor-derived TLR2 ligands in the generation of immunosuppressive IL-10-producing human and mouse DCs. TLR2 ligation induced two parallel synergistic processes that converged to activate STAT3: stimulation of autocrine IL-6 and IL-10 and upregulation of their respective cell surface receptors, which lowered the STAT3 activation threshold. We identified versican as a soluble tumor-derived factor that activates TLR2 in DCs. TLR2 blockade in vivo improved intra-tumor DC immunogenicity and enhanced the efficacy of immunotherapy. Our findings provide a basis for understanding the molecular mechanisms of DC dysfunction in cancer and identify TLR2 as a relevant therapeutic target to improve cancer immunotherapy.

Immunostimulatory conventional dendritic cells evolve into regulatory macrophage-like cells

Dendritic cell (DC) homeostasis in peripheral tissues reflect a balance between DC generation, migration, and death. The current model of DC ontogeny indicates that pre-cDCs are committed to become terminal conventional DCs (cDCs). Here, we report the unexpected finding that proliferating immunostimulatory CD11c(+) MHC class II(+) cDCs derived from pre-cDCs can lose their DC identity and generate progeny that exhibit morphologic, phenotypic, and functional characteristics of regulatory macrophages. DC-derived-macrophages (DC-d-Ms) potently suppress T-cell responses through the production of immunosuppressive molecules including nitric oxide, arginase, and IL-10. Relative deficiency of granulocyte-macrophage colony stimulating factor (GM-CSF) provided a permissive signal for DC-d-M generation. Using a transgenic mouse model that allows tracking of CD11c(+) cells in vivo, we found that DC-d-M development occurs commonly in cancer, but not in lymphoid or nonlymphoid tissues under steady-state conditions. We propose that this developmental pathway serves as an alternative mechanism of regulating DC homeostasis during inflammatory processes.

Recruitment and Differentiation of Conventional Dendritic Cell Precursors in Tumors

The origin of dendritic cells (DCs) in tumors remains obscure. Recent studies indicate that conventional DCs (cDCs) in lymphoid tissues arise from a distinct population of committed cDC precursors (pre-cDCs) that originate in bone marrow and migrate via blood. In this study, we show that pre-cDCs are precursors for cDCs in tumors. Pre-cDCs from tumors, bone marrow, and spleen exhibit similar morphologic, immunophenotypic, and functional properties. Adoptive transfer studies show that bone marrow pre-cDCs migrate from blood into the tumor where they generate cDCs. The chemokine CCL3, which is markedly upregulated in tumors, promotes pre-cDC recruitment. Both pre-cDCs and their cDC progeny actively proliferate within the tumor. cDCs that arise from pre-cDCs in tumors express lower levels of CD11c and MHC class II as compared with those in spleen; however, there was no difference in their abilities to respond to maturation stimuli or activate Ag-specific lymphocytes in vitro. Our study provides the first evidence supporting a role for pre-cDCs in DC development in tumors and suggests a potential target for cancer immunotherapy.

Antigen transmission by replicating antigen-bearing dendritic cells

During steady-state conditions, conventional spleen dendritic cells (DC) turn over every 2–3 days. Recent evidence indicates that in situ proliferation of DC arising from immediate conventional DC precursors is an important contributor to their homeostasis. In this study, we report that replication-competent conventional DC precursors and DC can internalize and transfer model particulate and soluble Ags directly to their DC progeny during cell division. Real-time confocal microscopy and flow cytometry indicated that Ag transmission to progeny was symmetrical, and suggested that other mechanisms of inter-DC Ag transfer were not involved. Soluble protein Ags inherited by DC progeny were presented effectively to Ag-specific T lymphocytes. Furthermore, we show that the number of DC, and the proportion that are actively proliferating, expands several-fold during an immune response against a viral infection. Our results point to an unanticipated mechanism in which DC are continuously replaced from Ag-bearing replication-competent precursor cells that pass Ag molecules onto their progeny through successive cell divisions. Our findings help explain how Ag may persist in a population of DC despite the brief lifespan of individual mature DC.

Tumors Suppress In Situ Proliferation of Cytotoxic T Cells by Promoting Differentiation of Gr-1+ Conventional Dendritic Cells through IL-6

Cancers are often accompanied by inflammation, which can promote tumor growth, invasion, and metastases. We show that the tumor microenvironment induces the development of a Gr-1+ conventional dendritic cell (cDC) subpopulation that is functionally defective. Gr-1+cDCs differentiated from recruited immediate precursors of cDCs, a process supported by the inflammatory cytokine milieu in tumors. Inhibition of Gr-1+cDC differentiation enhanced intratumor expansion of cytotoxic CD8+ T cells (CTLs), resulting in suppression of tumor growth. Diphtheria toxin treatment of CD11c–diphtheria toxin receptor chimeras revealed the importance of intratumor cDCs in stimulating CTL proliferation in situ. Our study demonstrates a key role of intratumor cDCs in determining antitumor CTL responses and suggests that they may be an appropriate target for tumor immunotherapy.