The antigenic targets recognized by naturally occurring CD4(+) CD25(+) regulatory T cells (T reg cells) have been elusive. We have serologically defined a series of broadly expressed self-antigens derived from chemically induced mouse sarcomas by serological identification of antigens by recombinant expression cloning (SEREX). CD4(+) CD25(+) T cells from mice immunized with SEREX-defined self-antigens had strong suppressive activity on peptide-specific proliferation of CD4(+) CD25(-) T cells and CD8(+) T cells. The suppressive effect was observed without in vitro T cell stimulation. Foxp3 expression in these CD4(+) CD25(+) T cells from immunized mice was 5-10 times greater than CD4(+) CD25(+) T cells derived from naive mice. The suppressive effect required cellular contact and was blocked by anti-glucocorticoid-induced tumor necrosis factor receptor family-related gene antibody. In vitro suppressive activity essentially disappeared 8 wk after the last immunization. However, it was regained by in vitro restimulation with cognate self-antigen protein but not with control protein. We propose that SEREX-defined self-antigens such as those used in this study represent self-antigens that elicit naturally occurring CD4(+) CD25(+) T reg cells.

Cancer immunoediting is the process whereby immune cells protect against cancer formation by sculpting the immunogenicity of developing tumors. Although the full process depends on innate and adaptive immunity, it remains unclear whether innate immunity alone is capable of immunoediting. To determine whether the innate immune system can edit tumor cells in the absence of adaptive immunity, we compared the incidence and immunogenicity of 3'methylcholanthrene-induced sarcomas in syngeneic wild-type, RAG2(-/-), and RAG2(-/-)x γc(-/-) mice. We found that innate immune cells could manifest cancer immunoediting activity in the absence of adaptive immunity. This activity required natural killer (NK) cells and interferon γ (IFN-γ), which mediated the induction of M1 macrophages. M1 macrophages could be elicited by administration of CD40 agonists, thereby restoring editing activity in RAG2(-/-)x γc(-/-) mice. Our results suggest that in the absence of adaptive immunity, NK cell production of IFN-γ induces M1 macrophages, which act as important effectors during cancer immunoediting.

Retention of lymphocytes in the intestinal mucosa requires specialized chemokine receptors and adhesion molecules. We find that both CD4(+)CD8(+) and CD4(+) T cells in the intestinal epithelium, as well as CD8(+) T cells in the intestinal mucosa and mesenteric lymph nodes, express the cell adhesion molecule class I-restricted T cell-associated molecule (Crtam) upon activation, whereas the ligand of Crtam, cell adhesion molecule 1 (Cadm1), is expressed on gut CD103(+)DCs. Lack of Crtam-Cadm1 interactions in Crtam(-/-) and Cadm1(-/-) mice results in loss of CD4(+)CD8(+) T cells, which arise from mucosal CD4(+) T cells that acquire a CD8 lineage expression profile. After acute oral infection with Toxoplasma gondii, both WT and Crtam(-/-) mice mounted a robust TH1 response, but markedly fewer TH17 cells were present in the intestinal mucosa of Crtam(-/-) mice. The almost exclusive TH1 response in Crtam(-/-) mice resulted in more efficient control of intestinal T. gondii infection. Thus, Crtam-Cadm1 interactions have a major impact on the residency and maintenance of CD4(+)CD8(+) T cells in the gut mucosa in the steady state. During pathogenic infection, Crtam-Cadm1 interactions regulate the dynamic equilibrium between newly formed CD4(+) T cells and their retention in the gut, thereby shaping representation of disparate CD4(+) T cell subsets and the overall quality of the CD4(+) T cell response.

Cancer immunoediting is the process whereby the immune system suppresses neoplastic growth and shapes tumor immunogenicity. We previously reported that type I interferon (IFN-α/β) plays a central role in this process and that hematopoietic cells represent critical targets of type I IFN's actions. However, the specific cells affected by IFN-α/β and the functional processes that type I IFN induces remain undefined. Herein, we show that type I IFN is required to initiate the antitumor response and that its actions are temporally distinct from IFN-γ during cancer immunoediting. Using mixed bone marrow chimeric mice, we demonstrate that type I IFN sensitivity selectively within the innate immune compartment is essential for tumor-specific T cell priming and tumor elimination. We further show that mice lacking IFNAR1 (IFN-α/β receptor 1) in dendritic cells (DCs; Itgax-Cre(+)Ifnar1(f/f) mice) cannot reject highly immunogenic tumor cells and that CD8α(+) DCs from these mice display defects in antigen cross-presentation to CD8(+) T cells. In contrast, mice depleted of NK cells or mice that lack IFNAR1 in granulocytes and macrophage populations reject these tumors normally. Thus, DCs and specifically CD8α(+) DCs are functionally relevant targets of endogenous type I IFN during lymphocyte-mediated tumor rejection.

The double-stranded RNA (dsRNA) analogue poly(I:C) is a promising adjuvant for cancer vaccines because it activates both dendritic cells (DCs) and natural killer (NK) cells, concurrently promoting adaptive and innate anticancer responses. Poly(I:C) acts through two dsRNA sensors, Toll-like receptor 3 (TLR3) and melanoma differentiation-associated protein-5 (MDA5). Here, we investigated the relative contributions of MDA5 and TLR3 to poly(I:C)-mediated NK cell activation using MDA5(-/-), TLR3(-/-), and MDA5(-/-)TLR3(-/-) mice. MDA5 was crucial for NK cell activation, whereas TLR3 had a minor impact most evident in the absence of MDA5. MDA5 and TLR3 activated NK cells indirectly through accessory cells and induced the distinct stimulatory cytokines interferon-alpha and interleukin-12, respectively. To identify the relevant accessory cells in vivo, we generated bone marrow chimeras between either wild-type (WT) and MDA5(-/-) or WT and TLR3(-/-) mice. Interestingly, multiple accessory cells were implicated, with MDA5 acting primarily in stromal cells and TLR3 predominantly in hematopoietic cells. Furthermore, poly(I:C)-mediated NK cell activation was not notably impaired in mice lacking CD8alpha DCs, providing further evidence that poly(I:C) acts through diverse accessory cells rather than solely through DCs. These results demonstrate distinct yet complementary roles for MDA5 and TLR3 in poly(I:C)-mediated NK cell activation.

Circulating murine monocytes comprise two largely exclusive subpopulations that are responsible for seeding normal tissues (Gr-1-/CCR2-/CX3CR1high) or responding to sites of inflammation (Gr-1+/CCR2+/CX3CR1lo). Gr-1+ monocytes are recruited to the site of infection during the early stages of immune response to the intracellular pathogen Toxoplasma gondii. A murine model of toxoplasmosis was thus used to examine the importance of Gr-1+ monocytes in the control of disseminated parasitic infection in vivo. The recruitment of Gr-1+ monocytes was intimately associated with the ability to suppress early parasite replication at the site of inoculation. Infection of CCR2-/- and MCP-1-/- mice with typically nonlethal, low doses of T. gondii resulted in the abrogated recruitment of Gr-1+ monocytes. The failure to recruit Gr-1+ monocytes resulted in greatly enhanced mortality despite the induction of normal Th1 cell responses leading to high levels of IL-12, TNF-alpha, and IFN-gamma. The profound susceptibility of CCR2-/- mice establishes Gr-1+ monocytes as necessary effector cells in the resistance to acute toxoplasmosis and suggests that the CCR2-dependent recruitment of Gr-1+ monocytes may be an important general mechanism for resistance to intracellular pathogens.

After oral ingestion, Toxoplasma gondii crosses the intestinal epithelium, disseminates into the deep tissues, and traverses biological barriers such as the placenta and the blood-brain barrier to reach sites where it causes severe pathology. To examine the cellular basis of these processes, migration of T. gondii was studied in vitro using polarized host cell monolayers and extracellular matrix. Transmigration required active parasite motility and the highly virulent type I strains consistently exhibited a superior migratory capacity than the nonvirulent type II and type III strains. Type I strain parasites also demonstrated a greater capacity for transmigration across mouse intestine ex vivo, and directly penetrated into the lamina propria and vascular endothelium. A subpopulation of virulent type I parasites exhibited a long distance migration (LDM) phenotype in vitro, that was not expressed by nonvirulent type II and type III strains. Cloning of parasites expressing the LDM phenotype resulted in substantial increase of migratory capacity in vitro and in vivo. The potential to up-regulate migratory capacity in T. gondii likely plays an important role in establishing new infections and in dissemination upon reactivation of chronic infections.

Plasmacytoid dendritic cells (pDCs) specialize in the secretion of type I interferons (IFN-I) and thus are considered critical mediators of antiviral responses. We recently reported that pDCs have a very early but limited and transient capacity to curtail viral infections. Additionally, pDC numbers are not sustained in human infections caused by Hepatitis B or C viruses (HBV and HCV) and HIV. Thus, the numbers and/or function of pDCs appear to be regulated during the course of viral infection. In this study, we show that splenic pDCs are reduced in vivo during several systemic viral infections and after administration of synthetic toll-like receptor ligands. We demonstrate that IFN-I, regardless of the source, contributes to this decline and mediates pDC death via the intrinsic apoptosis pathway. These findings demonstrate a feedback control mechanism by which IFN-I modulates pDC numbers, thus fine-tuning systemic IFN-I response to viruses. IFN-I-mediated control of pDCs may explain the loss of pDCs during human infections caused by HBV, HCV, or HIV and has important therapeutic implications for settings in which IFN-I is used to treat infections and autoimmune diseases.

The requirement of type I interferon (IFN) for natural killer (NK) cell activation in response to viral infection is known, but the underlying mechanism remains unclear. Here, we demonstrate that type I IFN signaling in inflammatory monocytes, but not in dendritic cells (DCs) or NK cells, is essential for NK cell function in response to a mucosal herpes simplex virus type 2 (HSV-2) infection. Mice deficient in type I IFN signaling, Ifnar-/- and Irf9-/- mice, had significantly lower levels of inflammatory monocytes, were deficient in IL-18 production, and lacked NK cell-derived IFN-γ. Depletion of inflammatory monocytes, but not DCs or other myeloid cells, resulted in lower levels of IL-18 and a complete abrogation of NK cell function in HSV-2 infection. Moreover, this resulted in higher susceptibility to HSV-2 infection. Although Il18-/- mice had normal levels of inflammatory monocytes, their NK cells were unresponsive to HSV-2 challenge. This study highlights the importance of type I IFN signaling in inflammatory monocytes and the induction of the early innate antiviral response.