Tim Sparwasser

Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells

Bacterial DNA and immunostimulatory (i.s.) synthetic CpG‐oligodeoxynucleotides (ODN) act as adjuvants for Th1 responses and cytotoxic T cell responses to proteinaceous antigens. Dendritic cells (DC) can be referred to as “natures adjuvant” since they display the unique capacity to sensitize naive T cells. Here, we demonstrate that bacterial DNA or i.s. CpG‐ODN cause simultaneous maturation of immature DC and activation of mature DC to produce cytokines. These events are associated with the acquisition of professional antigen‐presenting cell (APC) function. Unfractionated murine bone marrow‐derived DC and FACS®‐fractionated MHC class IIlow (termed immature DC) or MHC class IIhigh populations (termed mature DC) were stimulated with bacterial DNA or i.s. CpG‐ODN. Similar to lipopolysaccharide, i.s. CpG‐ODN caused up‐regulation of MHC class II, CD40 and CD86, but not CD80 on immature and mature DC. In parallel both DC subsets were activated to produce large amounts of IL‐12, IL‐6 and TNF‐α. CpG‐ODN‐activated DC displayed professional APC function in allogeneic mixed lymphocyte reaction and in staphylococcal enterotoxin B‐driven naive T cell responses. We interpret these findings to mean that bacterial DNA and i.s. CpG‐ODN cause maturation (first step) and activation (second step) of DC to bring about conversion of immature DC into professional APC.

CpG‐DNA‐specific activation of antigen‐presenting cells requires stress kinase activity and is preceded by non‐specific endocytosis and endosomal maturation

Unmethylated CpG motifs in bacterial DNA, plasmid DNA and synthetic oligodeoxynucleotides (CpG ODN) activate dendritic cells (DC) and macrophages in a CD40‐CD40 ligand‐independent fashion. To understand the molecular mechanisms involved we focused on the cellular uptake of CpG ODN, the need for endosomal maturation and the role of the stress kinase pathway. Here we demonstrate that CpG‐DNA induces phosphorylation of Jun N‐terminal kinase kinase 1 (JNKK1/SEK/MKK4) and subsequent activation of the stress kinases JNK1/2 and p38 in murine macrophages and dendritic cells. This leads to activation of the transcription factor activating protein‐1 (AP‐1) via phosphorylation of its constituents c‐Jun and ATF2. Moreover, stress kinase activation is essential for CpG‐DNA‐induced cytokine release of tumor necrosis factor α (TNFα) and interleukin‐12 (IL‐12), as inhibition of p38 results in severe impairment of this biological response. We further demonstrate that cellular uptake via endocytosis and subsequent endosomal maturation is essential for signalling, since competition by non‐CpG‐DNA or compounds blocking endosomal maturation such as chloroquine or bafilomycin A prevent all aspects of cellular activation. The data suggest that endosomal maturation is required for translation of intraendosomal CpG ODN sequences into signalling via the stress kinase pathway, where p38 kinase activation represents an essential step in CpG‐ODN‐triggered activation of antigen‐presenting cells.

Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity

Fungal infections are increasing worldwide due to the marked rise in immunodeficiencies including AIDS; however, immune responses to fungi are poorly understood. Dectin-1 is the major mammalian pattern recognition receptor for the fungal component zymosan. Dectin-1 represents the prototype of innate non-Toll-like receptors (TLRs) containing immunoreceptor tyrosine-based activation motifs (ITAMs) related to those of adaptive antigen receptors. Here we identify Card9 as a key transducer of Dectin-1 signalling. Although being dispensable for TLR/MyD88-induced responses, Card9 controls Dectin-1-mediated myeloid cell activation, cytokine production and innate anti-fungal immunity. Card9 couples to Bcl10 and regulates Bcl10–Malt1-mediated NF-κB activation induced by zymosan. Yet, Card9 is dispensable for antigen receptor signalling that uses Carma1 as a link to Bcl10–Malt1. Thus, our results define a novel innate immune pathway and indicate that evolutionarily distinct ITAM receptors in innate and adaptive immune cells use diverse adaptor proteins to engage selectively the conserved Bcl10–Malt1 module.

DC activated via dectin-1 convert Treg into IL-17 producers

Th cells producing IL‐17 play a pro‐inflammatory role at mucosal surfaces. Treg at the same sites dampen inflammation and prevent immunopathology. Th cells producing IL‐17 (Th17) and Treg are thought to be distinct populations defined by expression of the transcription factors ROR‐γt and Foxp3, respectively. Here, we show that mouse CD25+Foxp3+ Treg can be converted into a hybrid T‐cell population characterized by the expression of Foxp3 and ROR‐γt and the production of IL‐17. Conversion was observed upon coculture with DC selectively activated via dectin‐1, a C‐type lectin receptor involved in fungal recognition, and depended on IL‐23 produced by DC. Within the Foxp3+ population, only Foxp3+ROR‐γt+ T cells but not Foxp3+ROR‐γt‐–T cells become Foxp3+IL‐17+ T cells. These results indicate that some Foxp3+ T cells can produce IL‐17 while retaining Foxp3 expression and suggest that Treg could play an unexpected pro‐inflammatory role in some settings.

Adjuvant IL-7 antagonizes multiple cellular and molecular inhibitory networks to enhance immunotherapies

Identifying key factors that enhance immune responses is crucial for manipulating immunity to tumors. We show that after a vaccine-induced immune response, adjuvant interleukin-7 (IL-7) improves antitumor responses and survival in an animal model. The improved immune response is associated with increased IL-6 production and augmented T helper type 17 cell differentiation. Furthermore, IL-7 modulates the expression of two ubiquitin ligases: Casitas B-lineage lymphoma b (Cbl-b), a negative regulator of T cell activation, is repressed, and SMAD-specific E3 ubiquitin protein ligase-2 (Smurf2) is enhanced, which antagonizes transforming growth factor-β signaling. Notably, we show that although short term IL-7 therapy potently enhances vaccine-mediated immunity, in the absence of vaccination it is inefficient in promoting antitumor immune responses, despite inducing homeostatic proliferation of T cells. The ability of adjuvant IL-7 to antagonize inhibitory networks at the cellular and molecular level has major implications for immunotherapy in the treatment of tumors.

Bacterial CpG-DNA activates dendritic cells in vivo: T helper cell-independent cytotoxic T cell responses to soluble proteins.

Receptors for conserved molecular patterns associated with microbial pathogens induce synthesis of co‐stimulatory molecules and cytokines in immature dendritic cells (DC), as do antigen‐reactive CD4 T helper cells via CD40 signaling. Once activated, antigen‐presenting DC may activate CD8 T cell responses in a T helper cell‐independent fashion. Using immunostimulatory CpG‐oligonucleotides (ODN) mimicking bacterial CpG‐DNA, we tested whether CpG‐DNA bypasses the need for T helper cells in CTL responses towards proteins by directly activating antigen‐presenting DC to transit into professional APC. We describe that immature DC in situ constitutively process soluble proteins and generate CD8 T cell determinants yet CD8 T cell responses remain abortive. Induction of primary antigen‐specific CD8 cytotoxic T lymphocyte (CTL)‐mediated responses becomes initiated in wild‐type as well as T helper cell‐deficient mice, provided soluble protein and CpG‐ODN are draining into the same lymph node. Specifically we show that CpG‐ODN trigger antigen‐presenting immature DC within the draining lymph node to acutely up‐regulate co‐stimulatory molecules and produce IL‐12. These results provide new insights for generating in vivo efficient CTL responses to soluble proteins which may influence vaccination strategies.

CpG-DNA-Mediated Transient Lymphadenopathy Is Associated with a State of Th1 Predisposition to Antigen-Driven Responses

Infections can influence concurrent and subsequent Th1 vs Th2 immune responses to Ags. Through pattern recognition of foreign unmethylated CpG dinucleotides, the vertebrate innate immune system can sense infectious danger and typically replies with a Th1-polarized adaptive immune response. We examined whether CpG-DNA exposure would influence subsequent responses to infection and soluble Ags. CpG-DNA injection led to local lymphadenopathy characterized by maintenance of cellular composition with some biasing toward elevated dendritic cell composition. Sustained local production of IL-12 and IFN-γ from dendritic cells and T cells was shown. Prior injection by up to 2 wk with CpG-DNA protected BALB/c mice from Th2 driven lethal leishmaniasis. CpG-DNA injection by up to 5 wk before soluble Ag challenge resulted in the generation of Ag-specific CTL, Th1 recall responses to Ag, and Th1-polarized Ag-specific Abs. Thus, CpG-DNA instigated a local predisposition for intense CTL responses and Th1-polarized immune responses to subsequent infections or Ag challenge. The induction by the innate immune system of a locally contained hypersensitivity could represent a capacitating immune reaction yielding rapid conditioned responses to secondary infections.

Identification and expansion of highly suppressive CD8 +FoxP3 + regulatory T cells after experimental allogeneic bone marrow transplantation

FoxP3(+) confers suppressive properties and is confined to regulatory T cells (T(reg)) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4(+) T(reg) are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8(+) population of FoxP3(+) T(reg) that convert from CD8(+) conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8(+) T(reg) undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4(+)FoxP3(+) population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8(+)FoxP3(+) T(reg) are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8(+)FoxP3(+) T(reg) thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation.

Selective depletion of Foxp3+ Treg during sensitization phase aggravates experimental allergic airway inflammation

Recent studies highlight the role of Treg in preventing unnecessary responses to allergens and maintaining functional immune tolerance in the lung. We investigated the role of Treg during the sensitization phase in a murine model of experimental allergic airway inflammation by selectively depleting the Treg population in vivo. DEpletion of REGulatory T cells (DEREG) mice were depleted of Treg by diphtheria toxin injection. Allergic airway inflammation was induced using OVA as a model allergen. Pathology was assessed by scoring for differential cellular infiltration in bronchoalveolar lavage, IgE and IgG1 levels in serum, cytokine secretion analysis of lymphocytes from lung draining lymph nodes and lung histology. Use of DEREG mice allowed us for the first time to track and specifically deplete both CD25+ and CD25− Foxp3+ Treg, and to analyze their significance in limiting pathology in allergic airway inflammation. We observed that depletion of Treg during the priming phase of an active immune response led to a dramatic exacerbation of allergic airway inflammation in mice, suggesting an essential role played by Treg in regulating immune responses against allergens as early as the sensitization phase via maintenance of functional tolerance.

BAC to immunology – bacterial artificial chromosome-mediated transgenesis for targeting of immune cells

Thirty years after the first transgenic mouse was produced, a plethora of genetic tools has been developed to study immune cells in vivo. A powerful development is the bacterial artificial chromosome (BAC) transgenic approach, combining advantages of both conventional transgenic and knock‐in gene‐targeting strategies. In immunology the potential of BAC transgenic technology has yet to be fully harvested and, combined with a variety of elegant genetic tools, it will allow the analysis of complex immunological processes in vivo. In this short review, we discuss the applications of BACs in immunology, such as identification of regulatory regions, expression and cell‐fate mapping, cell ablation, conditional mutations and the generation of humanized mice.