Huguette Bausinger

Measles Virus Induces Abnormal Differentiation of CD40 Ligand-Activated Human Dendritic Cells

Measles virus (MV) infection induces a profound immunosuppression responsible for a high rate of mortality in malnourished children. MV can encounter human dendritic cells (DCs) in the respiratory mucosa or in the secondary lymphoid organs. The purpose of this study was to investigate the consequences of DC infection by MV, particularly concerning their maturation and their ability to generate CD8+ T cell proliferation. We first show that MV-infected Langerhans cells or monocyte-derived DCs undergo a maturation process similarly to the one induced by TNF-α or LPS, respectively. CD40 ligand (CD40L) expressed on activated T cells is shown to induce terminal differentiation of DCs into mature effector DCs. In contrast, the CD40L-dependent maturation of DCs is inhibited by MV infection, as demonstrated by CD25, CD69, CD71, CD40, CD80, CD86, and CD83 expression down-regulation. Moreover, the CD40L-induced cytokine pattern in DCs is modified by MV infection with inhibition of IL-12 and IL-1α/β and induction of IL-10 mRNAs synthesis. Using peripheral blood lymphocytes from CD40L-deficient patients, we demonstrate that MV infection of DCs prevents the CD40L-dependent CD8+ T cell proliferation. In such DC-PBL cocultures, inhibition of CD80 and CD86 expression on DCs was shown to require both MV replication and CD40 triggering. Finally, for the first time, MV was shown to inhibit tyrosine-phosphorylation level induced by CD40 activation in DCs. Our data demonstrate that MV replication modifies CD40 signaling in DCs, thus leading to impaired maturation. This phenomenon could play a pivotal role in MV-induced immunosuppression.

Evidence of a trimolecular complex involving LPS, LPS binding protein and soluble CD14 as an effector of LPS response

The kinetics of the interaction of lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP) and CD14 was studied using surface plasmon resonance. The association and dissociation rate constants for the binding of LPS and rsCD14 were 2.9×104 M−1 s−1 and 0.07 s−1 respectively, yielding a binding constant of 4.2×105 M−1. Significantly, the presence of LBP increased not only the association rate but also the association constant for the interaction between LPS and CD14 by three orders of magnitude. Our experimental results suggest that LBP interacts with LPS and CD14 to form a stable trimolecular complex that has significant functional implications as it allows monocytes to detect the presence of LPS at a concentration as low as 10 pg/ml or 2 pM, and to respond by secreting interleukin‐6. Thus, LBP is not merely transferring LPS to CD14 but it forms an integral part of the LPS–rLBP–rsCD14 complex.

Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells

Priming of CTL by means of heat shock proteins (hsp) is dependent on antigen‐presenting cells (APC), which present the hsp‐associated peptides, via their cell surface MHC class I molecules, toCD8+ T cells. It has not yet been established how human (hu) hsp70 interacts with the major (hu)APC, the dendritic cells (DC). Here we show that (hu)hsp70 is specifically internalized intoCD14–, Toll‐like receptor 4– monocyte‐derived (hu)DC by receptor‐mediated endocytosis. We further demonstrate that (hu)hsp70 and (hu)hsp60 share the same receptors on (hu)monocyte‐derived DC. Both molecules as well as MHC class I molecules are spontaneously internalized and reach the MHC class II‐enriched compartments. Finally, freshly isolated (hu) epidermal Langerhans cells (LC), the DC of the skin, as well as CD34+‐derived LC do not bind hsp60 or hsp70. Given the likely importance of the internalization of hsp70 by APC in the induction of the immune responses, the finding that hsp60 and hsp70 are internalized through the same receptor(s) may explain why microbial hsp60 represents a major T cell antigen. This may rationalize the use of microbial hsp60 to primeimmune responses against microbes. The lack of hsp60/70 receptors on epidermal LC raises the crucial question as to whether absence of priming of the skin and mucosal immune systems by hsp‐polypeptide complexes could account for some tissue‐specific diseases. This work also points to a potential advantage of using monocyte‐derived DC in human immunotherapeutic applications of hsp60/70.

Gene induction during differentiation of human monocytes into dendritic cells: an integrated study at the RNA and protein levels.

Abstract. Changes in gene expression occurring during differentiation of human monocytes into dendritic cells were studied at the RNA and protein levels. These studies showed the induction of several gene classes corresponding to various biological functions. These functions encompass antigen processing and presentation, cytoskeleton, cell signalling and signal transduction, but also an increase in mitochondrial function and in the protein synthesis machinery, including some, but not all, chaperones. These changes put in perspective the events occurring during this differentiation process. On a more technical point, it appears that the studies carried out at the RNA and protein levels are highly complementary.

Soluble CD16/FcγRIII Induces Maturation of Dendritic Cells and Production of Several Cytokines Including IL-12

Fc gamma RIII (CD16), a low affinity FcR which binds IgG-containing immune-complexes, exists under membrane-associated forms and under a soluble form (sFc gamma RIII). The latter, present in biological fluids (serum, saliva), is generated by proteolytic cleavage of the two membrane-associated Fc gamma RIII isoforms, Fc gamma RIII-A (expressed by macrophages and NK cells) and Fc gamma RIII-B (expressed exclusively by neutrophils). Herein we demonstrate that dendritic cells (DCs), generated by culturing monocytes with GM-CSF and IL-4, bind biotinylated recombinant sFc gamma RIII. This binding is specific and involves the complement receptor CR3 (CD11b/CD18) and CR4 (CD11c/CD18). Indeed, preincubation of DCs with anti-CD11b and anti-CD11c mAbs decreased by 52% and 62% respectively the binding with sFc gamma RIII. Moreover, electron microscopy showed that binding of gold-labeled sFc gamma RIII to DCs maintained at 4 degrees C occurred within clathrin-coated pits. Once internalized, at 37 degrees C, sFc gamma RIII entered the endocytic pathway and reached the MHC class II compartments. Furthermore, DCs incubated for 48 h with multivalent sFc gamma RIII expressed increased levels of CD40, CD80, CD86, CD54, CD58, HLA class I and class II molecules and decreased levels of CD23 and CD32. These effects result in an increased capacity of DCs to trigger proliferative responses by CD4+ CD45RA+ allogeneic T cells. RT-PCR amplification demonstrated that incubation of DCs for 20 h in the presence of multivalent sFc gamma RIII induced the appearance of GM-CSF and IL-12 p40 mRNA. Among the cytokines constitutively expressed, IL-1 beta and IL-8 were strongly up-regulated whereas IL-6 and IL-12 p35 mRNA were increased to a lesser extent and the expression of MIP-1 alpha mRNA remained constant. Finally, ELISA tests demonstrated that DCs incubated with multivalent sFc gamma RIII secreted the cytokines IL-1 beta, IL-6, IL-8, GM-CSF and IL-12 p75. Thus, while becoming internalized sFc gamma RIII could affect the capacity of DCs to present antigens and, via the induction of accessory molecules and the release of the IL-12 p75 protein, could initiate Th1 type immune response.

Functions of Fc Receptors on Human Dendritic Langerhans Cells

Immature dendritic cells are antigen presenting cells highly specialized for capturing and processing foreign protein antigens. These cells express Fc gamma RII and Fc epsilon RI which, by their ability to internalize and use the endocytic pathway, increase their capacity to process antigens. Immature dendritic cells, such as epidermal Langerhans cells, also release soluble forms of Fc gamma RII. These latter molecules are likely to compete with the membrane-associated Fc gamma R to diminish or abrogate the capacity of dendritic cells to present immune complexes, as suggested by our in vitro experiments using both human and mouse epidermal Langerhans cells. However, when dendritic cells mature in vitro and become efficient stimulators of resting T cells, they rapidly down-regulate and sometimes completely abolish the expression of their membrane-associated Fc gamma R and Fc epsilon RI. Consequently, they lose or at least strongly diminish their capacity to capture immune complexes. At this stage, the release of soluble Fc gamma R by dendritic cells is also markedly diminished. One can hypothesize that the membrane-associated Fc gamma RII and the soluble Fc gamma RII are molecules expressed when dendritic cells are potent capturing and processing cells, the soluble Fc gamma RII molecule acting by competition as a negative regulatory element on the Fc gamma RII-mediated internalization of IgG-containing immune complexes. Thus, the expression of membrane-associated Fc gamma R and Fc epsilon RI, as well as the release of soluble Fc gamma R, would seem to characterize the immature stage of dendritic cells.

Phenotypic Studies of Natural Killer Cell Subsets in Human Transporter Associated with Antigen Processing Deficiency

Peripheral blood natural killer (NK) cells from patients with transporter associated with antigen processing (TAP) deficiency are hyporesponsive. The mechanism of this defect is unknown, but the phenotype of TAP-deficient NK cells is almost normal. However, we noticed a high percentage of CD56bright cells among total NK cells from two patients. We further investigated TAP-deficient NK cells in these patients and compared them to NK cells from two other TAP-deficient patients with no clinical symptoms and to individuals with chronic inflammatory diseases other than TAP deficiency (chronic lung diseases or vasculitis). Peripheral blood mononuclear cells isolated from venous blood were stained with fluorochrome-conjugated antibodies and the phenotype of NK cells was analyzed by flow cytometry. In addition, 51Chromium release assays were performed to assess the cytotoxic activity of NK cells. In the symptomatic patients, CD56bright NK cells represented 28% and 45%, respectively, of all NK cells (higher than in healthy donors). The patients also displayed a higher percentage of CD56dimCD16− NK cells than controls. Interestingly, this unusual NK cell subtype distribution was not found in the two asymptomatic TAP-deficient cases, but was instead present in several of the other patients. Over-expression of the inhibitory receptor CD94/NKG2A by TAP-deficient NK cells was confirmed and extended to the inhibitory receptor ILT2 (CD85j). These inhibitory receptors were not involved in regulating the cytotoxicity of TAP-deficient NK cells. We conclude that expansion of the CD56bright NK cell subtype in peripheral blood is not a hallmark of TAP deficiency, but can be found in other diseases as well. This might reflect a reaction of the immune system to pathologic conditions. It could be interesting to investigate the relative distribution of NK cell subsets in various respiratory and autoimmune diseases.