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The major challenge in the treatment of autoimmune diseases is the restoration of the impaired peripheral immune tolerance that always accompanies the development of such diseases. Here, we show that small splenic peptides (SSPs) of whole spleen extract efficiently suppress the development of psoriatic arthritis in vivo, even in the presence of sustained levels of pro-inflammatory cytokines. SSPs target dendritic cells (DCs) and convert them into tolerogenic cells, which in turn differentiate naive CD4+cells into Foxp3-expressing T regulatory cells (Tregs). The latter requires direct contact between SSP-activated DCs and naive CD4+T cells via PD-1 and CTLA4 immune checkpoint receptors of T cells. Finally, depletion of Foxp3+Tregs in vivoabrogated the protective effect of SSPs on psoriatic arthritis development. We hypothesize that SSPs represent an intrinsic component of the adaptive immune system responsible for the physiological maintenance of peripheral tolerance and that therapeutically administered SSPs are able to restore imbalanced peripheral tolerance in autoimmune diseases.

Inflammation is initiated by innate immune cell activation after contact with pathogens or tissue injury. An increasing number of observations have suggested that cellular stress, in the absence of infection or evident damage, can also induce inflammation. Thus, inflammation can be triggered by exogenous pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)—so-called classic inflammation—or by endogenous stress resulting from tissue or cellular dysfunction. External triggers and cellular stress activate the same molecular pathways, possibly explaining why classic and stress-induced inflammation have similar clinical manifestations. In some systemic autoinflammatory diseases (SAIDs), inflammatory cells exhibit reduction–oxidation (redox) distress, having high levels of reactive oxygen species (ROS), which promote proinflammatory cytokine production and contribute to the subversion of mechanisms that self-limit inflammation. Thus, SAIDs can be viewed as a paradigm of stress-related inflammation, being characterized by recurrent flares or chronic inflammation (with no recognizable external triggers) and by a failure to downmodulate this inflammation. Here, we review SAID pathophysiology, focusing on the major cytokines and DAMPs, and on the key roles of redox distress. New therapeutic opportunities to tackle SAIDs by blocking stress-induced pathways and control the response to stress in patients are also discussed.

ABSTRACTLymph nodes (LNs) are important sentinel organs where antigen-presenting cells interact with T cells to induce adaptive immune responses. In cutaneous infection of mice with Leishmania major, resistance depends on the induction of a T-helper-cell-1 (Th1)-mediated cellular immune response in draining, peripheral LNs. We investigated whether draining, peripheral LNs are absolutely required for resistance against L. majorinfection. We investigated the course of experimental leishmaniasis in wild-type (wt) mice lacking peripheral LNs (pLNs), which we generated by in utero blockade of membrane-bound lymphotoxin, and in mice lacking pLNs or all LNs due to genetic deletion of lymphotoxin ligands or receptors. wt mice of the resistant C57BL/6 strain without local skin-draining LNs were still able to generate specific T-cell responses, but this yielded Th2 cells. This switch to a Th2 response resulted in severe systemic infection. We also confirmed these results with mice lacking pLNs due to genetic depletion of lymphotoxin-β. The complete absence of LNs due to a genetic depletion of the lymphotoxin-β receptor also resulted in a marked deterioration of disease and a Th2 response. Thus, in the absence of pLNs, an L. major-specific Th2 response is induced in the remaining secondary lymphoid organs, such as the spleen and non-skin-draining LNs. This indicates a critical requirement for pLNs to induce protective Th1 immunity and suggests that whether Th1 or Th2 priming to the same antigen occurs depends on the site of the primary antigen recognition.

Lymph nodes (LNs) are important sentinel organs where antigen-presenting cells interact with T cells to induce adaptive immune responses. In cutaneous infection of mice with Leishmania major, resistance depends on the induction of a T-helper-cell-1 (Th1)-mediated cellular immune response in draining, peripheral LNs. We investigated whether draining, peripheral LNs are absolutely required for resistance against L. major infection. We investigated the course of experimental leishmaniasis in wild-type (wt) mice lacking peripheral LNs (pLNs), which we generated by in utero blockade of membrane-bound lymphotoxin, and in mice lacking pLNs or all LNs due to genetic deletion of lymphotoxin ligands or receptors. wt mice of the resistant C57BL/6 strain without local skin-draining LNs were still able to generate specific T-cell responses, but this yielded Th2 cells. This switch to a Th2 response resulted in severe systemic infection. We also confirmed these results with mice lacking pLNs due to genetic depletion of lymphotoxin-β. The complete absence of LNs due to a genetic depletion of the lymphotoxin-β receptor also resulted in a marked deterioration of disease and a Th2 response. Thus, in the absence of pLNs, an L. major-specific Th2 response is induced in the remaining secondary lymphoid organs, such as the spleen and non-skin-draining LNs. This indicates a critical requirement for pLNs to induce protective Th1 immunity and suggests that whether Th1 or Th2 priming to the same antigen occurs depends on the site of the primary antigen recognition.

Glucocorticoids (GC) are still the most widely used immunosuppressive agents in clinical medicine. Surprisingly, little is known about the mechanisms of GC action on monocytes, although these cells exert pro‐ and anti‐inflammatory effects. We have shown recently that GC induce a specific monocyte phenotype with anti‐inflammatory properties in humans. We now investigated whether this also applies for the murine system and how this subset would relate to recently defined murine subtypes. After treatment with dexamethasone for 48 h, monocytes up‐regulated scavenger receptor CD163 and Gr‐1, down‐regulated CX3CR1, and shared with human GC‐treated monocytes functional features such as low adhesiveness but high migratory capacity. They specifically up‐regulated anti‐inflammatory IL‐10, but not TGF‐β, and in contrast to their human counterparts, they down‐regulated IL‐6. Although GC‐induced monocytes down‐regulated CX3CR1, a distinctive marker for classical/proinflammatory human and murine monocytes (CX3CR1loCCR2+Ly6Chi), they differed from this physiologically occurring subset, as they remained Ly6Cmedand unactivated (CD62 ligand++). In addition to their immunosuppressive effects, they were CD11b+Gr‐1+and expressed the IL‐4Rα chain (CD124), a recently described, signature molecule of tumor‐induced myeloid‐derived suppressor cells (MDSC). We therefore generated murine MDSC in B16 melanoma‐bearing mice and indeed found parallel up‐regulation of CD11b+Gr‐1+and CD124 on GC‐induced monocytes and MDSC. These data allow us to speculate that the GC‐induced subtype shares with inflammatory monocytes the ability to migrate quickly into inflamed tissue, where they, however, exert anti‐inflammatory effects and that similarities between GC‐induced monocytes and MDSC may be involved in progression of some tumors observed in patients chronically treated with GC.

The β2 integrins are important for transendothelial migration of leukocytes as well as for T-cell activation during antigen presentation. Despite abundant expression of β2 integrins on antigen-presenting cells (APCs), their functional relevance for antigen presentation is completely unclear. We show here that dendritic cells (DCs) from CD18-deficient mice, which lack all functional β2 integrins, have no defect in antigen presentation. Moreover, DCs from normal mice express inactive β2 integrins that do not become activated on contact with T cells, at least in vitro. Pharmacologic activation of β2 integrins on DCs results in a significant reduction of their T cell–activating capacity. This effect is mediated by Mac-1 (CD11b/CD18) on DCs because it could be reversed via blocking antibodies against CD18 and CD11b. Furthermore, the antigen-presenting capacity of macrophages, which express constitutively active β2 integrins, is significantly enhanced on Mac-1 blockade. We therefore conclude that active CD11b/CD18 (Mac-1) on APCs directly inhibits T-cell activation.

For activation T cells engage antigen-presenting cells (APCs) in lymphatic tissues. The contact duration and kinetics (static versus dynamic) vary considerably in different model systems; however, it is unclear whether T cells, APCs, or the environment are responsible for the observed discrepancies. Using 3-D collagen matrices as structural scaffold, we directly compared the kinetics of T-cell engagement and activation by functionally major APC types, ie, dendritic cells (DCs) and resting or activated B cells. Resting B cells engaged T cells in long-lived (several hours), adhesive, and leukocyte function-associated antigen-1 (LFA-1)-dependent conjugates in 3-D collagen as well as in intact lymph nodes in vivo. DCs and preactivated B cells, however, supported predominantly dynamic, short-lived (minutes), and sequential contacts to T cells that were dependent on high cytoskeletal activity of the APCs but could not be inhibited by anti-LFA-1 treatment. Naive T cells were most strongly activated by DCs and activated B cells, whereas resting B cells were 100-fold less efficient to induce T-cell proliferation. Thus, in the same 3-D environment, naive T cells respond with a spectrum of different interaction modes dependent on the type and activation state of the APCs. Thereby, more dynamic interaction kinetics is positively correlated with higher T-cell priming efficiency. (Blood. 2004;104: 2801-2809)

Intravascular injection of adenoviral vectors may result in a toxic and potentially lethal reaction, the mechanism of which is poorly understood. We noted that mice demonstrated a transient change in behavior that was characterized by inactivity and lethargy within minutes after intravenous injection of relatively low doses of adenoviral vectors (including high-capacity gutless vectors). Moreover, immediately after vector injection a significant drop in blood pressure was measured that most probably was caused by the systemic activation of endothelial cells as monitored by detection of phosphorylated Akt/PKB kinase, activated endothelial nitric oxide synthase (eNOS), and nitrotyrosine. The activation of the endothelium was the result of the interaction of viral particles with Kupffer cells, which are resident macrophages of the liver representing the first line of defense of the innate immune system. Surprisingly, the uptake of vector particles by Kupffer cells not only resulted in their strong activation, but also in their nearly complete disappearance from the liver. Our results suggest that the toxicity of intravenously injected adenoviral vectors may be directly linked to the activation and destruction of Kupffer cells.

Researchers and clinicians have tried for decades to use the mechanisms of immunity for the fight against cancer. Early attempts aimed at the instrumentation of soluble immune mediators such as antibodies or cytotoxic proteins for the therapy of malignancies. Major improvements in understanding the induction and regulation of cellular immunity have now made it possible to generate effector cells in cancer patients which are specific for the neoplastic disease. At the beginning of every cellular immune reaction against cancers tumor antigens have to be presented to T cells in order to activate them and drive them into clonal expansion. This is done by antigen presenting cells, the most powerful of which is the dendritic cell (DC). While DC were hard to isolate initially, they can be generated in large numbers in vitro today and manipulated in multiple ways before given back to a patient to induce tumor immunity. Thus, a great amount of hope lies in the use of DC as inducers of tumor immunity. However, the first clinical studies, which have now been completed with only limited success make clear, that still a lot of open questions remain to be answered. This review tries to give an overview of this rapidly developing field, mentioning the major conceptual approaches and techniques, but also discussing important caveats. The next years will show whether we can improve our understanding of DC biology and the mechanisms of immune induction strongly enough to effectively employ DC for immunotherapy of cancer.

CD40–CD40 ligand (L) interactions play a pivotal role in immune-mediated inflammatory responses via the activation of antigen-presenting cells (APCs). To investigate the effects of continuous activation of resident tissue APCs, in this case the Langerhans cells (LCs) of the skin, CD40L expression was targeted to the basal keratinocytes of the epidermis of mice using the keratin-14 promoter. Approximately 80% of the transgenic (Tg) mice spontaneously developed dermatitis on the ears, face, tail, and/or paws. Compared with littermates, Tgs had a >90% decrease in epidermal LCs yet increased numbers within the dermis suggestive of enhanced emigration of CD40-activated LCs. Tgs also displayed massive regional lymphadenopathy with increased numbers of dendritic cells and B cells. Moreover, a decrease in IgM and an increase in IgG1/IgG2a/IgG2b/IgE serum concentrations was detectable. Screening for autoantibodies revealed the presence of antinuclear antibodies and anti-dsDNA antibodies implicative of systemic autoimmunity. Accordingly, renal Ig deposits, proteinuria, and lung fibrosis were observed. Adoptive transfer of T cells from Tgs to nonTg recipients evoked the development of skin lesions similar to those found in the Tgs. Dermatitis also developed in B cell–deficient CD40L Tg mice. These findings suggest that in situ activation of LCs by CD40L in the skin not only leads to chronic inflammatory dermatitis but also to systemic mixed-connective-tissue-like autoimmune disorders, possibly by breaking immune tolerance against the skin.

ABSTRACTA novel technology combining replication- and integration-defective human immunodeficiency virus type 1 (HIV-1) vectors with genetically modified dendritic cells was developed in order to induce T-cell immunity. We introduced the vector into dendritic cells as a plasmid DNA using polyethylenimine as the gene delivery system, thereby circumventing the problem of obtaining viral vector expression in the absence of integration. Genetically modified dendritic cells (GMDC) presented viral epitopes efficiently, secreted interleukin 12, and primed both CD4+and CD8+HIV-specific T cells capable of producing gamma interferon and exerting potent HIV-1-specific cytotoxicity in vitro. In nonhuman primates, subcutaneously injected GMDC migrated into the draining lymph node at an unprecedentedly high rate and expressed the plasmid DNA. The animals presented a vigorous HIV-specific effector cytotoxic-T-lymphocyte (CTL) response as early as 3 weeks after a single immunization, which later developed into a memory CTL response. Interestingly, antibodies did not accompany these CTL responses, indicating that GMDC can induce a pure Th1 type of immune response. Successful induction of a broad and long-lasting HIV-specific cellular immunity is expected to control virus replication in infected individuals.

In this study the participation of Rho family GTPases in the regulation of IL‐1‐activated protein kinase cascades controlling IL‐2 synthesis was investigated in murine EL‐4 thymoma cells. The recombinant C3‐like chimeric toxin, which consists of the C3 toxin of Clostridium limosumand the N‐terminal part of Clostridiumbotulinum C2 toxin (C2IN‐C3) interacting with the C2II binding subunit to facilitate uptake into cells, and selectively inactivates Rho A by ADP‐ribosylation, prevented IL‐1‐stimulated activation of Jun‐NH2‐terminal‐kinases (JNK) and p38 mitogen‐activated‐protein kinases (MAPK). UDP‐monoglucosylation and concomitant inactivation of Rho A and of Rac‐2 by Clostridium difficile toxin B also inhibited IL‐1‐induced activation of JNK and p38 MAPK, but additionally inhibited activation of the extracellular‐regulated‐kinase pathway and DNA binding of the transcription factor NFκB. Accordingly, pre‐treatment of cells with C2IN‐C3 fusion toxin only decreased IL‐1‐stimulated IL‐2 synthesis by 50%, while in C. difficile toxin B‐treated cells IL‐1‐induced IL‐2 secretion was reduced by 90%. These results imply that together with Rho A an additional member of the Rho family G proteins, i.e.Rac‐2, is critically involved as an upstream regulator in IL‐1‐induced activation of different MAPK, stress‐activated protein kinases, and in NFκB activation controlling IL‐2 gene expression in response to IL‐1, acting in close proximity to the IL‐1‐receptor complex.

ABSTRACTNon-small-cell lung cancer (NSCLC) is the most frequent type of lung cancer and demonstrates high resistance to radiation and chemotherapy. These tumors evade immune system detection by promoting an immunosuppressive tumor microenvironment. Genetic analysis has revealed oncogenic activation of the Ras/Raf/MEK/ERK signaling pathway to be a hallmark of NSCLCs, which promotes influenza A virus (IAV) infection and replication in these cells. Thus, we aimed to unravel the oncolytic properties of IAV infection against NSCLCs in an immunocompetent model in vivo. Using Raf-BxBtransgenic mice that spontaneously develop NSCLCs, we demonstrated that infection with low-pathogenic IAV leads to rapid and efficient oncolysis, eliminating 70% of the initial tumor mass. Interestingly, IAV infection of Raf-BxBmice caused a functional reversion of immunosuppressed tumor-associated lung macrophages into a M1-like pro-inflammatory active phenotype that additionally supported virus-induced oncolysis of cancer cells. Altogether, our data demonstrate for the first time in an immunocompetent in vivomodel that oncolytic IAV infection is capable of restoring and redirecting immune cell functions within the tumor microenvironment of NSCLCs.

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