Stefania Citterio

Coordinated events during bacteria-induced DC maturation.

Abstract The dendritic cell (DC) system represents a class of professional antigen-presenting cells whose primary function is to regulate the initiation of immune responses. Here, Maria Rescigno and colleagues describe the kinetics and molecular events of bacteria-induced DC maturation.

Differential effects of corticosteroids during different stages of dendritic cell maturation

Dendritic cell (DC) maturation is a complex process involving many cell functions. We have studied how the exposure of DC to corticosteroids at different stages of DC maturation affects priming and the expansion of different subsets of CD4+ T cells. Growth factor‐ dependent DC lines and fresh bone marrow‐derived DC were used. When exposed to inflammatory stimuli, immature DC previously treated with dexamethasone were unable to undergo full maturation and were unable to prime Th1 cells efficiently. There was specific and significant reduction in the number of IFN‐γ‐producing effector cell (shown by intracellular cytokine staining) and also in the amount of IFN‐γ produced. Interestingly, the number of IL‐4‐producing T cells and the amount of IL‐4 synthesis was not significantly altered. Furthermore, multiple restimulation of T cells with these DC gave rise to a subpopulation of T regulatory cells (Tr1) which were negative for IFN‐γ and IL‐4 but were IL‐10 positive. In contrast, when DC were activated with lipopolysaccharide prior to dexamethasone treatment, the suppressive effect of glucocorticoids was not significant. Thus, the stage of DC maturation influences the inhibitory effect of corticosteroids. By arresting DC maturation, corticosteroids strongly reduce cell‐mediated Th1 responses and allow the selective expansion of Tr1 cells.

Microglia induce myelin basic protein-specific T cell anergy or T cell activation, according to their state of activation

Microglial cells are non‐professional antigen‐presenting cells (APC) the function of which is still controversial. Here, we studied the function of microglia derived from H‐2u mice. We show that these microglia express a low level of B7.2 and CD40 and, interestingly, lack surface expression of B7.1. Resting and IFN‐γ‐activated microglia were unable to activate naive and primed myelin basic protein (MBP)‐specific CD4+ T cells in the presence of MBP and encephalomyelitic MBP Ac1‐11 peptide. Furthermore, in the presence of Ac1‐11 peptide, CD4+ TCR‐transgenic T cells became anergized. Microglia became professional APC only after a multistep activation process involving both stimulation through cytokines [granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and IFN‐γ] and cognate signaling (B7‐CD28 and CD40‐CD40 ligand interactions). As such they were able to present MBP to both unprimed and primed T cells. Co‐culture of microglia with GM‐CSF up‐regulated co‐stimulatory molecules, in particular B7.1. Additional activation with IFN‐γ induced MHC class II and CD40 up‐regulation. CD40‐CD40 ligand interaction significantly enhanced microglial ability to prime TCR‐transgenic T cells and was essential for presentation of MBP to in vivo primed non‐transgenic T cells. We propose that microglia may serve different functions under different inflammatory conditions, depending on the cytokine milieu and the type of cognate interaction they are involved in.

Differential activation of NF-κB subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide

Dendritic cell (DC) maturation is essential for the initiation of T-dependent immune responses. Nuclear factor kappa B/Rel (NF kappa B/Rel) transcription factors are ubiquitously expressed signalling molecules, known to regulate the transcription of a large number of genes involved in immune responses, including cytokines such as IL-1, IL-6, TNF-alpha and cell surface molecules (MHC class I and II, B7.2). In this study, we have compared the activation of five members of the NF-kappa B family, p65, c-Rel, p50, RelB and p52, during DC maturation in response to lipopolysaccharide (LPS) and to Salmonella typhimurium. We have shown that although the translocation of NF-kappa B occurred very early, 30 min after treatment with both S. typhimurium and LPS, bacteria-induced NF-kappa B activation was more pronounced. Four out of five members, i.e. p65, c-Rel, p50 and RelB, were similarly activated upon the two stimuli but with different kinetics. Indeed, we have observed that p65, c-Rel and p50 were translocated early, whereas RelB was translocated later in DC activation. This differential regulation suggests that the various members of NF-kappa B family can mediate distinct functions of DC physiology.

Induction of Peripheral T Cell Tolerance by Antigen-Presenting B Cells. I. Relevance of Antigen Presentation Persistence

Various mechanisms of peripheral T cell tolerization have evolved to avoid responses mediated by autoreactive T cells that have not been eliminated in the thymus. In this study, we investigated the peripheral conditions of Ag presentation required to induce T cell tolerance when the predominant APCs are B cells. We show that transient Ag presentation, in absence of inflammation and in a self-context, induces CD4+ T cell activation and memory formation. In contrast, chronic Ag presentation leads to CD4+ T cell tolerance. The importance of long-lasting Ag presentation in inducing tolerance was also confirmed in the herpes stromal keratitis autoimmune disease model. Keratogenic T cells could be activated or tolerized depending on the APC short or long persistence. Thus, when APCs are B cells, the persistence of the Ag presentation itself is one of the main conditions to have peripheral T cell tolerance.

Induction of Peripheral T Cell Tolerance by Antigen-Presenting B Cells. II. Chronic Antigen Presentation Overrules Antigen-Presenting B Cell Activation

Ag presentation in the absence of danger signals and Ag persistence are the inductive processes of peripheral T cell tolerization proposed so far. Nevertheless, it has never been definitively shown that chronic Ag presentation per se can induce T cell tolerance independent of the state of activation of APCs. In the present work, we investigated whether chronic Ag presentation by either resting or activated B cells can induce tolerance of peripheral Ag-specific T cells. We show that CD4+ T cells that re-encounter the Ag for a prolonged period, presented either by resting or activated Ag-presenting B cells, become nonfunctional and lose any autoimmune reactivity. Thus, when the main APCs are B cells, the major mechanism responsible for peripheral T cell tolerization is persistent Ag exposure, independent of the B cell activation state.

CHAPTER 34 – Interaction of dendritic cells with bacteria

Dendritic cells (DCs) synthesize a number of cytokines and chemokines after few hours of bacterial infection. The production of both TNFα and IL-6 has been detected in DCs infected with either gram-positive or gram-negative bacteria. Activation of DCs is achieved particularly when bacteria are alive, indeed, heat-inactivated bacteria although phenotypically induce the maturation of DCs, fail to induce a high production of inflammatory cytokines. TNFα production is rapidly induced following infection. It is likely that the phenotypical and functional maturation, which occurs in DCs within 24 h of bacteria uptake, is the result of cytokine amplification during this response. DC activation by TNF-α alone mimics the phenotypical maturation observed after bacterial infection, although the addition of anti-TNF-α antibodies only partly inhibits DC phenotypical and functional maturation. Maturation obtained by whole bacteria is quantitatively and qualitatively more pronounced indicating the induction of several transducing pathways, likely via receptors, which recognize distinct bacterial components.

BCR/ABL1 and BCR are under the transcriptional control of the MYC oncogene

BackgroundChronic Myeloid Leukaemia (CML) is caused by the BCR/ABL1 fusion gene. Both the presence and the levels of BCR/ABL1 expression seem to be critical for CML progression from chronic phase (CP) to blast crisis (BC). After the oncogenic translocation, the BCR/ABL1 gene is under the transcriptional control of BCR promoter but the molecular mechanisms involved in the regulation of oncogene expression are mostly unknown.MethodsA region of 1443bp of the functional BCR promoter was studied for transcription factor binding sites through in-silico analysis and Chromatin Immunoprecipitation experiments. BCR and BCR/ABL1 expression levels were analysed in CML cell lines after over-expression or silencing of MYC transcription factor. A luciferase reporter assay was used to confirm its activity on BCR promoter.ResultsIn the present study we demonstrate that MYC and its partner MAX bind to the BCR promoter, leading to up-regulation of BCR and BCR/ABL1 at both transcriptional and protein levels. Accordingly, silencing of MYC expression in various BCR/ABL1 positive cell lines causes significant downregulation of BCR and BCR/ABL1, which consequently leads to decreased proliferation and induction of cell death.ConclusionsHere we describe a regulatory pathway modulating BCR and BCR/ABL1 expression, showing that the BCR promoter is under the transcriptional control of the MYC/MAX heterodimer. Since MYC is frequently over-expressed in BC, this phenomenon could play a critical role in BCR/ABL1 up-regulation and blast aggressiveness acquired during CML evolution.

Generation of murine growth factor-dependent long-term dendritic cell lines to investigate host-parasite interactions.

Substantial progress has been made over the last several years in the development of protocols for the isolation of large numbers of dendritic cells (DCs) from different tissues and their short-term culture. Indeed, several stable DC lines and clones have been established from various tissues of mice and humans, providing useful experimental tools for studying the biology of DCs at both molecular and biochemical levels and for the establishment of new DC-based immunotherapies. In this chapter, we will describe the development of long-term DC lines that maintain the growth factor dependence and their immature functional state, thus providing a unique opportunity to study the mechanisms of the initiation of the immune response to infectious agents.

Green fluorescent protein transgene driven by Kit regulatory sequences is expressed in hematopoietic stem cells

The expression of Kit in multiple types of stem cells suggests that common transcriptional programs might regulate this gene in different stem cells. In this work, the authors used mouse lines expressing transgenic green fluorescent protein under the control of Kit promoter/first intron regulatory elements. This study provides the basis for the elucidation of DNA sequences regulating a stem cell gene in multiple types of stem cells. Background The transcriptional regulation of stem cell genes is still poorly understood. Kit, encoding the stem cell factor receptor, is a pivotal molecule for multiple types of stem/progenitor cells. We previously generated mouse lines expressing transgenic green fluorescent protein under the control of Kit promoter/first intron regulatory elements, and we demonstrated expression in hematopoietic progenitors. Design and Methods In the present work we investigated whether the transgene is also expressed in hematopoietic stem cells of adult bone marrow and fetal liver. To this purpose, we tested, in long-term repopulating assays, cell fractions expressing different levels of green fluorescent protein within Kit-positive or SLAM-selected populations. Results The experiments demonstrated transgene expression in both fetal and adult hematopoietic stem cells and indicated that the transgene is transcribed at distinctly lower levels in hematopoietic stem cells than in pluripotent and committed progenitors. Conclusions These results, together with previous data, show that a limited subset of DNA sequences drives gene expression in number of stem cell types (hematopoietic stem cells, primordial germ cells, cardiac stem cells). Additionally, our results might help to further improve high level purification of hematopoietic stem cells for experimental purposes. Finally, as the Kit/green fluorescent protein transgene is expressed in multiple stem cell types, our transgenic model provides powerful in vivo system to track these cells during development and tissue regeneration.