Bernard Pajak

Role of CD8alpha+ and CD8alpha- dendritic cells in the induction of primary immune responses in vivo.

Data from adoptive transfer of mature dendritic cells (DC) indicate that they are responsible for the induction of primary immunity. Two subclasses of DC have been recently identified in spleen that differ in their phenotype and in certain regulatory features. In vitro, both subsets have the capacity to activate naive T cells, although CD8α+ DC have been shown to induce T cell apoptosis and to stimulate lower levels of cytokines compared with CD8α− DC. The objective of this study was to analyze the function of these distinct DC types in vivo. Our results show that both subsets, pulsed extracorporeally with antigen and injected in the footpads of syngeneic mice, sensitize an antigen‐specific T cell primary response. However, CD8α+ cells trigger the development of Th1‐type cells, whereas CD8α− DC induce a Th2‐type response. These observations suggest that the Th1/Th2 balance in vivo is regulated by the antigen‐presenting‐cells of the primary immune responses. J. Leukoc. Biol. 66: 242–246; 1999.

Selective Blockade of NF-κB Activity in Airway Immune Cells Inhibits the Effector Phase of Experimental Asthma

Knockout mice studies have revealed that NF-κB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-κB also plays a role in the effector phase of airway allergy and whether inhibiting NF-κB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-κB activity in the lungs during OVA challenge. Intratracheal administration of NF-κB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-κB activity in the airways upon OVA provocation. NF-κB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-κB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-κB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.

Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is phosphorylated by Ins(1,4,5)P3 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). The physiological function of Ins(1,3,4,5)P4 is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P3 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P3 amounts or calcium mobilization in Itpkb−/− thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P4 define an essential signaling pathway for T cell precursor responsiveness and development.

TLR4 and Toll-IL-1 Receptor Domain-Containing Adapter-Inducing IFN-β, but Not MyD88, Regulate Escherichia coli-Induced Dendritic Cell Maturation and Apoptosis In Vivo

Dendritic cells (DC) are short-lived, professional APCs that play a central role in the generation of adaptive immune responses. Induction of efficient immune responses is dependent on how long DCs survive in the host. Therefore, the regulation of DC apoptosis in vivo during infection remains an important question that requires further investigation. The impact of Escherichia coli bacteremia on DCs has never been analyzed. We show here that i.v. or i.p. administration of live or heat-killed E. coli in mice induces splenic DC migration, maturation, and apoptosis. We further characterize which TLR and Toll-IL-1R (TIR)-containing adaptor molecules regulate these processes in vivo. In this model, DC maturation is impaired in TLR2−/−, TLR4−/− and TIR domain-containing adapter-inducing IFN-β (TRIF)−/− mice. In contrast, DC apoptosis is reduced only in TLR4−/− and TRIF−/− mice. As expected, DC apoptosis induced by the TLR4 ligand LPS is also abolished in these mice. Injection of the TLR9 ligand CpG-oligodeoxynucleotide (synthetic bacterial DNA) induces DC migration and maturation, but only modest DC apoptosis when compared with LPS and E. coli. Together, these results suggest that E. coli bacteremia directly impacts on DC maturation and survival in vivo through a TLR4-TRIF-dependent signaling pathway.

CD8α− and CD8α+ subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen‐presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of naïve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse‐DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8α expression. We have studied the phenotype and maturation state of mouse splenic CD8α− and CD8α+ DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM‐CSF‐containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T‐cell priming capacity, transforming CD8α− and CD8α+ DCs into potent and equally efficient activators of naïve CD4+ and CD8+ T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.

T Cell-Dependent Maturation of Dendritic Cells in Response to Bacterial Superantigens

Dendritic cells (DC) express a set of germline-encoded transmembrane Toll-like receptors that recognize shared microbial products, such as Escherichia coli LPS, termed pathogen-associated molecular patterns. Analysis of the in vivo response to pathogen-associated molecular patterns has uncovered their ability to induce the migration and the maturation of DC, favoring thus the delivery of Ag and costimulatory signals to naive T cells in vivo. Bacterial superantigens constitute a particular class of pathogen-derived molecules known to induce a potent inflammatory response in vivo, secondary to the activation of a large repertoire of T cells. We demonstrate in this work that Staphylococcal superantigens induce migration and maturation of DC populations in vivo. However, in contrast to LPS, superantigens failed to induce DC maturation in RAG or MHC class II-deficient mice, suggesting that T cell activation was a prerequisite for DC maturation. This conclusion was further supported by the finding that T cell activation induced by 1) mitogenic anti-CD3 mAbs, 2) allo-MHC determinants, or 3) nominal Ag in a TCR-transgenic model induces DC maturation in vivo. These studies also revealed that DC that matured in response to T cell mitogens display, comparatively to LPS, a distinctive phenotype characterized by high expression of the MHC class II, CD40, and CD205 markers, but only moderate (CD86) to minimal (CD80) expression of CD28/CTLA4 ligands. This work demonstrates that activation of a sufficient number of naive T cells in vivo constitutes a novel form of immune danger, functionally linked to DC maturation.

The adjuvant OM-174 induces both the migration and maturation of murine dendritic cells in vivo.

The aim of this study was to test the capacity of the novel adjuvant OM-174, a lipid A analog, to induce the migration and the maturation of murine dendritic cells (DC) in vivo, a step which is considered as the initiation of the adaptive immune response. BALB/c mice were injected intravenously or subcutaneously with OM-174. The spleen and popliteal lymph nodes were harvested, and analyzed for DC localization and phenotype. The data presented here clearly show that, OM-174 induces the migration of DC from the periphery to the T cell areas of lymphoid organs, and their maturation into cells expressing high levels of MHC class II and co-stimulatory molecules, with a potency close to that of Escherichia coli lipopolysaccharide (LPS).

Alteration of Migration and Maturation of Dendritic Cells and T-Cell Depletion in the Course of Experimental Trypanosoma cruzi Infection

Trypanosoma cruzi, the etiologic agent of Chagas disease, induces infection that affects most immunocompetent cells. However, its effect on dendritic cells (DC) is still unknown in vivo. In this report, we show, by immunohistochemical staining, that T. cruzi infection triggers a huge increase in the number of CD11c+ DC in the spleen of infected mice at Days 14 and 21 post-inoculation (pi). In mice reaching the chronic phase (starting on Day 35 pi), the number of splenic DC (sDC) returned progressively to normal (ending on Day 98 pi). In the spleens of noninfected mice, most of the CD8α+CD11c+ and CD8α−CD11c+ DC were found in the red pulp and the marginal and T-cell zones. However, starting on Day 14 pi, a progressive decline of CD8α+CD11c+ was observed. In addition, sDC expressed low levels of the costimulatory molecule B7.2 at Days 14 and 21 pi, suggesting that they remained immature in the course of the infection. As expected, in lipopolysaccharide-treated and noninfected mice, the expression of B7.2 molecules was sharply up-regulated on sDC that migrated toward the T-cell zone. In contrast, upon lipopolysaccharide stimulation, sDC from T. cruzi-infected mice did not migrate toward the T-cell zone nor did they undergo maturation. Finally, white pulp was severely depleted in both CD4+ and CD8+ T cells at the peak of infection. Taken together, these results indicate that profound alterations of migration and maturation of sDC and depletion/redistribution of T cells occur during the acute phase of T. cruzi infection and could be part of another strategy to escape immune surveillance and to persist in the host.

Injection of lipopolysaccharide induces the migration of splenic neutrophils to the T cell area of the white pulp: role of CD14 and CXC chemokines.

There is increasing evidence that neutrophils are involved in the regulation of adaptive immunity. We therefore tested whether these cells may colocalize with T lymphocytes in lymphoid organs. Our results demonstrate that administration of the microbial product LPS induces the migration of neutrophils in the spleen from the red pulp and the marginal zone to the area of the white pulp where T cells reside. This movement is CD14‐dependent, whereas the recruitment of neutrophils in the peritoneal cavity is increased in the absence of CD14. Our data further suggest the involvement of the chemokine MIP‐2 and keratinocyte‐derived chemokine and their receptor CXCR2. We conclude that neutrophils may interact with naïve T cells upon infection/inflammation and that the migration of neutrophils in the lymphoid organs and in the periphery is regulated differently by a signal transduced by CD14

Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs

Aims—To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. Methods—This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. Results—Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessels. Conclusions—These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur.