Carsten Wiethe

Myeloid dendritic cell precursors generated from bone marrow suppress T cell responses via cell contact and nitric oxide production in vitro

Tolerogenic activity of myeloid dendritic cells (DC) has so far been attributed mostly to immature or semi‐mature differentiation stages but never to their precursor cells. Although myeloid suppressor cells (MSC) have been isolated ex vivo, their developmental relationship to DC and their precise phenotype remained elusive. Here, we describe the generation of MSC as myeloid DC precursors with potent suppressive activity on allogeneic and OVA‐specific CD4+ and CD8+ T cell responses in vitro. These MSC appear transiently in DC cultures of bone marrow (BM) cells after 8–10 days under low GM‐CSF conditions or after 3–4 days under high GM‐CSF conditions. They represent CD11c– myeloid precursor cells with ring‐shaped nuclei and are Gr‐1low (i.e. Ly‐6C+, Ly‐6Glow), CD11b+, CD31+, ER‐MP58+, asialoGM1+ and F4/80+. Sorted MSC develop into CD11c+ DC within 6 days. Their suppressor activity partially depends on IFN‐γ stimulation. Suppression is mediated through mechanisms requiring cell contact and nitric oxide but is independent of TNF, CD1d and TGF‐β. Together, our data describe the generation of MSC with distinct suppressor mechanisms in vitro preceding their development into immature DC.

Dendritic cells matured with TNF can be further activated in vitro and after subcutaneous injection in vivo which converts their tolerogenicity into immunogenicity.

Dendritic cell (DC) maturation can occur by different types of stimuli. Previously, we described that murine DC matured with tumor necrosis factor (TNF) up-regulate surface MHC and costimulatory molecules but lack cytokine release, and therefore termed them semi-mature DC. These TNF/DC-induced tolerance after intravenous (IV) injection in a model of experimental autoimmune encephalomyelitis (EAE). Here, we show that TNF/DC are not terminally differentiated but can still respond to the microbial stimulus lipopolysaccharide. Subcutaneously injected TNF/DC induce an unpolarized TH1/TH2 response and are not protective in the experimental autoimmune encephalomyelitis model. Although TNF/DC home to the draining lymph node, they remain negative for intracellular cytokine stainings. However, the nonmigrating, endogenous DC started to produce interleukin (IL)-12p40, TNF and little IL-6, IL-10, and MCP-1 in a bystander fashion. Together, DC matured with the inflammatory stimulus TNF remains responsive to further signals in vitro and in vivo. These signals can be provided by pathogens or the subcutaneous injection route, which can convert them from tolerogenic to immunogenic DC. These findings are important for selecting the appropriate injection route of human DC for tumor immunotherapy.

Provision of 4-1BB Ligand Enhances Effector and Memory CTL Responses Generated by Immunization with Dendritic Cells Expressing a Human Tumor-Associated Antigen

Up-regulation of receptor-ligand pairs during interaction of an MHC-presented epitope on dendritic cells (DCs) with cognate TCR may amplify, sustain, and drive diversity in the ensuing T cell immune response. Members of the TNF ligand superfamily and the TNFR superfamily contribute to this costimulatory molecule signaling. In this study, we used replication deficient adenoviruses to introduce a model tumor-associated Ag (the E7 oncoprotein of human papillomavirus 16) and the T cell costimulatory molecule 4-1BBL into murine DCs, and monitored the ability of these recombinant DCs to elicit E7-directed T cell responses following immunization. Splenocytes from mice immunized with DCs expressing E7 alone elicited E7-directed effector and memory CTL responses. Coexpression of 4-1BBL in these E7-expressing DCs increased effector and memory CTL responses when they were used for immunization. 4-1BBL expression up-regulated CD80 and CD86 second signaling molecules in DCs. We also report an additive effect of 4-1BBL and receptor activator of NF-κB/receptor activator of NF-κB ligand coexpression in E7-transduced DC immunogens on E7-directed effector and memory CTL responses and on MHC class II and CD80/86 expression in DCs. Additionally, expression of 4-1BBL in E7-transduced DCs reduced nonspecific T cell activation characteristic of adenovirus vector-associated immunization. The results have generic implications for improved or tumor Ag-expressing DC vaccines by incorporation of exogenous 4-1BBL. There are also specific implications for an improved DC-based vaccine for human papillomavirus 16-associated cervical carcinoma.

Simultaneous Induction of CD4 T Cell Tolerance and CD8 T Cell Immunity by Semimature Dendritic Cells

Previous studies suggested that depending on their maturation state, dendritic cells (DC) could either induce T cell tolerance (immature and semimature DC) or T cell activation (mature DC). Pretreatment of C57BL/6 mice with encephalitogenic myelin oligodendrocyte glycoprotein (MOG)35–55 peptide-loaded semimature DC protected from MOG-induced autoimmune encephalomyelitis. This protection was mediated by IL-10-producing CD4 T cells specific for the self Ag. Here we show that semimature DC loaded with the MHC class II-restricted nonself peptide Ag (OVA) induce an identical regulatory T cell cytokine pattern. However, semimature DC loaded simultaneously with MHC class II- and MHC class I-restricted peptides, could efficiently initiate CD8 T cell responses leading to autoimmune diabetes in a TCR-transgenic adoptive transfer model. Double-peptide-loaded semimature DC also induced simultaneously in the same animal partially activated CD8 T cells with cytolytic function as well as protection from MOG-induced autoimmune encephalomyelitis. Our study suggests that the decision between tolerance and immunity not only depends on the DC, but also on the type and activation requirements of the responding T cell.

CD83 Expression in CD4+ T Cells Modulates Inflammation and Autoimmunity

The transmembrane protein CD83 has been initially described as a maturation marker for dendritic cells. Moreover, there is increasing evidence that CD83 also regulates B cell function, thymic T cell maturation, and peripheral T cell activation. Herein, we show that CD83 expression confers immunosuppressive function to CD4+ T cells. CD83 mRNA is differentially expressed in naturally occurring CD4+CD25+ regulatory T cells, and upon activation these cells rapidly express large amounts of surface CD83. Transduction of naive CD4+CD25− T cells with CD83 encoding retroviruses induces a regulatory phenotype in vitro, which is accompanied by the induction of Foxp3. Functional analysis of CD83-transduced T cells in vivo demonstrates that these CD83+Foxp3+ T cells are able to interfere with the effector phase of severe contact hypersensitivity reaction of the skin. Moreover, adoptive transfer of these cells prevents the paralysis associated with experimental autoimmune encephalomyelitis, suppresses proinflammatory cytokines IFN-γ and IL-17, and increases antiinflammatory IL-10 in recipient mice. Taken together, our data provide the first evidence that CD83 expression can contribute to the immunosuppressive function of CD4+ T cells in vivo.

Dendritic Cell Differentiation State and Their Interaction with NKT Cells Determine Th1/Th2 Differentiation in the Murine Model of Leishmania major Infection

Recent reports demonstrated that dendritic cells (DC) sense inflammatory and microbial signals differently, redefining their classical subdivision into an immature endocytic and a mature Ag-presenting differentiation stage. Although both signals induce DC maturation by up-regulating MHC class II and costimulatory molecules, only TLR signals such as LPS are able to trigger proinflammatory cytokine secretion by DCs, including Th1-polarizing IL-12. Here, we explored the murine Leishmania major infection model to examine the CD4+ T cell response induced by differentially matured DCs. When partially matured TNF-DCs were injected into BALB/c mice before infection, the mice failed to control L. major infection and developed a Th2 response which was dependent on IL-4Rα signaling. In contrast, injections of fully matured LPS+CD40-DCs induced a Th1 response controlling the infection. Pulsing DCs with a lysate of L. major did not affect DC maturation with TNF-α or LPS+anti-CD40. When the expression of different Notch ligands on DCs was analyzed, we found increased expression of Th2-promoting Jagged2 in TNF-DCs, whereas LPS+CD40-DCs up-regulated the Th1-inducing Delta4 and Jagged1 molecules. The Th2 polarization induced by TNF-DCs required interaction with CD1d-restricted NKT cells. However, NKT cell activation by L. major lysate-pulsed DCs was not affected by blockade of the endogenous glycolipid, suggesting exchange with exogenous parasite-derived CD1 glycolipid Ag. In sum, the differentiation stage of DCs as well as their interaction with NKT cells determines Th1/Th2 differentiation. These results have generic implications for the understanding of DC-driven Th cell responses and the development of improved DC vaccines against leishmaniasis.

Enhanced Effector and Memory CTL Responses Generated by Incorporation of Receptor Activator of NF-κB (RANK)/RANK Ligand Costimulatory Molecules into Dendritic Cell Immunogens Expressing a Human Tumor-Specific Antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-κB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8+ T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-γ-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.

Interdependency of MHC Class II/Self-Peptide and CD1d/Self-Glycolipid Presentation by TNF-Matured Dendritic Cells for Protection from Autoimmunity

Dendritic cells (DC) are key regulators of T cell immunity and tolerance. NKT cells are well-known enhancers of Th differentiation and regulatory T cell function. However, the nature of the DC directing T and NKT cell activation and polarization as well as the role of the respective CD1d Ags presented is still unclear. In this study, we show that peptide-specific CD4+IL-10+ T cell-mediated full experimental autoimmune encephalomyelitis (EAE) protection by TNF-treated semimatured DCs was dependent on NKT cells recognizing an endogenous CD1d ligand. NKT cell activation by TNF-matured DCs induced high serum levels of IL-4 and IL-13 which are absent in NKT cell-deficient mice, whereas LPS plus anti-CD40-treated fully mature DCs induce serum IFN-γ. In the absence of IL-4Rα chain signaling or NKT cells, no complete EAE protection was achieved by TNF-DCs, whereas transfer of NKT cells into Jα281−/− mice restored it. However, activation of NKT cells alone was not sufficient for EAE protection and early serum Th2 deviation. Simultaneous activation of NKT cells and CD4+ T cells by the same DC was required for EAE protection. Blocking experiments demonstrated that NKT cells recognize an endogenous glycolipid presented on CD1d on the injected DC. Together, this indicates that concomitant and interdependent presentation of MHC II/self-peptide and CD1d/self-isoglobotrihexosylceramide to T and NKT cells by the same partially or fully matured DC determines protective and nonprotective immune responses in EAE.

Minor role of bystander tolerance to fetal calf serum in a peptide-specific dendritic cell vaccine model against autoimmunity: comparison with serum-free cultures.

Dendritic cells (DCs) are currently considered as promising tools for vaccination against tumors and also autoimmune responses. A major point of concern has been the use of fetal calf serum (FCS) as a source of heterologous antigen in DC cultures. FCS peptides can be presented by the DCs and cause T-cell responses in the recipient. We investigated the role of FCS in an autoimmune model where DC injections can prevent peptide-specifically from experimental autoimmune encephalomyelitis (EAE). We show that murine bone marrow-derived DCs generated in FCS-containing or serum-free media resulting in a similar phenotype, maturation potential, and functions. Peptide-specific protection could be achieved similarly with FCS-DC or serum-free DCs. Although FCS-DC induced strong CD4+ T cell proliferation and cytokine production against FCS, these T cells lack antigenic recall during EAE. Even if FCS was reinjected, the effect on EAE resulted only in a 3-day delay of disease onset. Together, our data show that presentation of bystander antigens by peptide-specific DC vaccinations may have little influence on T-cell responses in vivo if the bystander antigen cannot be recalled by specific T cells.