CD1a molecules traffic through the early recycling endosomal pathway in human Langerhans cells.

In this work, we studied the localization and traffic of CD1a molecules in human epidermal Langerhans cells and the ability of these cells to stimulate CD1a-restricted T cell clones. We found that CD1a was spontaneously internalized into freshly isolated Langerhans cells, where it was rapidly distributed to the early/sorting endosomes and then to the early/recycling endosomes. In the latter compartments, CD1a colocalized with Rab11, a small GTPase known to be involved in the recycling of transmembrane proteins from early endosomes to the cell surface. In the steady state, intracellular CD1a was mainly located in Rab11+ recycling endosomal compartments. When endocytosis was blocked, intracellular CD1a moved rapidly from the early/recycling endosomes to the cell surface where it accumulated. The resultant increase in the cell surface expression of CD1a enhanced the capacity of Langerhans cells to stimulate a CD1a-restricted T cell clone. These findings are consistent with a dynamic exchange of CD1a between recycling compartments and the plasma membrane and suggest that the antigen-presenting function of CD1a depends on its traffic through the early/recycling endosomal pathway.

Modulation of MHC class II transport and lysosome distribution by macrophage-colony stimulating factor in human dendritic cells derived from monocytes.

The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (M(PHI)) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-(alpha). These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-(alpha) or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.

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

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.

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-alpha 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-1alpha/beta 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.

Human peptide transporter deficiency: importance of HLA-B in the presentation of TAP-independent EBV antigens.

Two siblings with a peptide TAP deficiency were recently described. Despite poor cell surface expression of HLA class I molecules, these patients were not unusually susceptible to viral infections. The majority of the cell surface-expressed class I molecules were HLA-B products as assessed by cytofluorometry and biochemical analysis. Analysis of two peptides eluted from the class I molecules expressed by TAP-deficient EBV B lymphoblastoid cell lines indicated that both were derived from cytosolic proteins and presented by HLA-B molecules. Peripheral alphabeta CD8+ T cells were present and their TCR repertoire was polyclonal. Most of the alphabeta CD8+ T cell clones studied (21 of 22) were nonreactive against cells expressing normal levels of the same HLA alleles as those of the TAP-deficient patients. However, it was possible to isolate one cytotoxic CD8+ alphabeta T cell clone recognizing the EBV protein LMP2 presented by HLA-B molecules on TAP-deficient cells. These observations suggest that in the TAP-deficient patients, CD8+ alphabeta T cells could mature and be recruited in immune responses to mediate HLA class I-restricted cytotoxic defense against viral infections. They also strengthen the physiologic importance of a TAP-independent processing pathway of the LMP2 protein, which was previously shown to contain several other TAP-independent epitopes.

Soluble CD16/Fc gamma RIII 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.

An intronic mutation responsible for a low level of expression of an HLA-A*24 allele.

HLA class I typing performed in parallel by molecular biology and serology has revealed cases where an HLA class I allele was identified but the corresponding antigen on the cell surface was not detected. In the present report, we describe three members of a family in whom an HLA-A24 allele identified at the molecular level was typed as A "blank" by lymphocytotoxicity. This serologically blank antigen was nevertheless faintly detectable by isoelectric focusing (IEF) and FACS analyses. Sequencing of the HLA-A*24 allele from the promoter region to the eighth exonic region revealed a point mutation in the acceptor site of the second intron as compared to the normal HLA-A*24 allele. This mutation could lead to incorrect processing of mRNA through a cryptic acceptor site located at the beginning of the third exon and hence to alternative splicing with a frame shift introducing an early stop codon into the fourth exon.

CD1 expression is not affected by human peptide transporter deficiency.

Conventional major histocompatibility complex class I molecules are highly polymorphic and present peptides to cytotoxic T cells. These peptides derive from the proteolytic degradation of endogenous proteins in the cytosol and are translocated into the endoplasmic reticulum by a peptide transporter consisting of two transporter associated with antigen processing (TAP) molecules. Absence of this transporter leads to the synthesis of unstable peptide free class I molecules that are weakly expressed on the cell surface. Mouse nonconventional class I molecules (class Ib) may also present TAP-dependent peptides. In humans, CD1 antigens are nonconventional class I molecules. Recently, we characterized a human HLA class I deficiency resulting from a homozygous TAP deficiency. We show here that CD1a and -c are normally expressed on epidermal Langerhans cells of the TAP-deficient patients, as are CD1a, -b, and -c on dendritic cells differentiated in vitro from monocytes. Moreover, the CD1a antigens present on the surface of the dendritic cells are functional, since they internalize by receptor-mediated endocytosis gold-labeled F(ab')2 fragments of an anti-CD1a mAb. This suggests either that CD1 molecules are empty molecules, that they are more stable than empty conventional class I proteins, or that CD1 molecules present TAP-independent peptides.

Homozygous human TAP peptide transporter mutation in HLA class I deficiency.

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8+ alpha beta T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4+CD8+ T cells and gamma delta T cells.