Jean-Luc Teillaud

Structural Bases of Fcγ Receptor Functions

Three classes of receptors for the Fc region of IgG (Fey receptors (FcyR)) have been identified in mice and humans (Anderson & Looney 1986). FcyRI bind monomeric IgG with high affinity (Ka= 10^-10 M), whereas FcyRII and FcyRIII only bind IgG-containing immune complexes. The analysis of cDNAs and genes coding for FcyR has revealed that they all derive from a common ancestral gene and are members of the Ig superfamily. They are composed of homologous Ig-Iike extracellular (EC) domains and divergent transmembrane (TM) and intracytoplasmic (IC) regions (reviewed in Ravetch & Kinet 1991). In mice and humans, FcyR genes are clustered on chromosome 1 (Ravetch & Kinet 1991). In humans, at least two genes code for FcyRI, three genes code for FcyRII and two genes code for the transmembrane and GPI-linked forms of FcyRIII respectively. Additional diversity in the FcyR family is provided by alternative splicing of FcyRII mRNA giving rise to proteins with identical EC domains and different TM and IC regions. In mice, only three FcyR genes have been isolated, each coding for one class of receptor, and only the transmembrane form of FcyRIII has been detected (Ravetch & Kinet 1991). Although distributed on all cell types of the hematopoietic lineage, the various forms of FcyR have a certain specificity of expression. Thus, FcyRI are found on monocytes, neutrophils and macrophages, while FcyRII are present on virtually all hematopoietic, cells, but different FcyRII isoforms, generated by RNA splicing, are preferentially expressed in certain cells. In contrast, FcyRIII have a restricted expression since they have been found so far only on macrophages, NK and mast cells (Weinshank et al. 1988, Daeron et al. 1990). This type of cell-specific distribution of receptors with a similar binding capacity for the ligand (IgG-containing immune com-

The Fc Receptor for IgG Expressed in the Villus Endothelium of Human Placenta Is FcγRIIb2

To evaluate the potential role of human placental endothelial cells in the transport of IgG from maternal to fetal circulation, we studied Fcγ receptor (FcγR) expression by immunohistology and immunoblotting. Several pan-FcγRII Abs that label the placental endothelium displayed a distribution pattern that correlated well with transport functions, being intense in the terminal villus and nil in the cord. In contrast, the MHC class 1-like IgG transporter, FcRn, and the classical FcγRIIa were not expressed in transport-related endothelium of the placenta. Our inference, that FcγRIIb was the likely receptor, we confirmed by analyzing purified placental villi, enriched in endothelium, by immunoblotting with a new Ab specific for the cytoplasmic tail of FcγRIIb. These experiments showed that the FcγRII expressed in villus endothelium was the b2 isoform whose cytoplasmic tail is known to include a phosphotyrosyl-based motif that inhibits a variety of immune responses. We suggest that this receptor is perfectly positioned to transport IgG although as well it may scavenge immune complexes.

Generation of phagocytic MAK and MAC-DC for therapeutic use: characterization and in vitro functional properties.

Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures. Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC). A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low-intermediate phagocytosis capacity by MAK. Both CD14+ FCgammaR+ (FcgammaRI/CD64, FcgammaRII/CD32, FcgammaRIII/CD16) MAK and CD1a+/CD86+, CD14- MAC-DC were able to phagocytose whole tumor cells. However, only MAK phagocytosis was enhanced by FcgammaR engagement. MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcgammaRI. Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.

In vivo induction of functional FcγRI (CD64) on neutrophils and modulation of blood cytokine mRNA levels in cancer patients treated with G-CSF (rMetHuG-CSF)

Neutrophils from 13 children who received G‐CSF for the collection of peripheral blood progenitors while they were in haematological steady state were studied at various times after G‐CSF injection for FcγR expression (FcγRI or CD 64, FcγRII or CD32, and FcγRIII or CD16) and for their ability to exert antibody‐dependent cell cytotoxicity (ADCC) through FcγRI. Changes in IFNγ, IL8, IL10, MCP1 and TNFα mRNA levels in peripheral blood cells were also studied 4u2003h and 24u2003h after the first G‐CSF injection. FcγRI expression increased strongly after 24u2003h and then remained at the same level throughout treatment. In contrast, FcγRIII expression sharply decreased at day 1 and diminished even further thereafter. No change in FcγRII was observed. ADCC exerted by neutrophils through FcγRI started to increase after 24u2003h with the peak level at day 5. Cytokine mRNA analyses indicated a reproducible and strong increase of IL8 mRNA (11/13 children) after 24u2003h, whereas the changes in the mRNA levels of the other cytokines tested were more heterogenous (IFNγ: three; IL10: six; MCP1: five; TNFα: four, of the 13 children). Therefore this study opens the way to an optimized therapeutic schedule for the combined use of G‐CSF and monoclonal antibodies in adjuvant immuno‐intervention.

Detection and quantification of secreted soluble FcγRIIA in human sera by an enzyme-linked immunosorbent assay

Abstract FcγRIIA can be produced in a soluble form that contains both the extracellular and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-coding exon. We have developed an enzyme-linked immunosorbent assay (ELISA) that permits the specific detection and quantification in human sera of this secreted soluble FcγRIIA. It uses the monoclonal antibody (MAb) IV.3 as capture antibody and rabbit polyclonal IgG directed against the intracellular region of FcγRIIA as detector antibodies. The enzymatic reaction was amplified using an NADH/NAD + amplification system. As little as 0.8–1.5 ng/ml (20–38 pM) of purified recombinant secreted FcγRIIA could be detected. The serum levels of secreted sFcγRIIA ranged from 0 to 30 ng/ml in sera from 51 healthy donors. The mean value was 11.9 ng/ml ± 6.55 (297 pM ± 163) and the median value was 10.6 ng/ml (265 pM) (range: 0–764 pM).

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.

Possibilities of interference with the immune system of tumor bearers by non-lymphoid FcγRII expressing tumor cells

The ectopic expression of Fc gamma RII by PyV transformed 3T3 cells derived from tumors of long latency has been established. It was suggested that this expression is one of several changes conferring upon the cells an increased capacity for survival. We found that in one case cells expressing a very high level of Fc gamma RII had also a very high metastatic phenotype as compared to FcR negative cells. Direct evidence that Fc gamma RIIbl functions as a progression factor was provided by transfection experiments. The transfected gene conferred an increased malignancy and invasive phenotype upon PyV or c-Ha-ras transformed cells. In the present study we tested the possibility that Fc gamma RII expressing tumor cells could interfere with the immune system. The following subjects were investigated: 1) The ability of Fc gamma R on the tumor cells to bind the ligand and/or release IBF. 2) The effect of a local accumulation of ligand and/or IBF (assumed to take place in situ in the tumor) on Fc gamma RII expressing T cells. It was found that both tumor-derived receptor positive and beta l transfected PyV transformed cells were capable of binding aggregated mouse IgG. The binding of bivalent ligand was followed by an increase in membrane Fc gamma RII expression. Also both types of cells were capable of releasing IBF. We then tested the possibility that a local accumulation of IgG within the tumor could effect Fc gamma R expressing T cells. It was found that aggregated mouse IgG (as well as IgGl) could stimulate the proliferation of the T cell hybridoma (T2D4) and other Fc gamma RII expressing T cells. We also found that the expression of beta Fc gamma RII specific mRNA peaked at the logarithmic phase of T2D4 cultures, in parallel with their maximal potential to release IBF. Several pathways for interference with the immune system are suggested.

Soluble Fcgamma receptor, Fc gammaRIIa2, is present in two forms in human serum and is increased in patients: with stage C chronic lymphocytic leukemia

Human Fcgamma receptor type II (FcgammaRII/CD32) can be produced in a soluble form, termed FcgammaRIIa2, which contains the extra- and intracellular regions of the receptor, due to an alternative splicing of the transmembrane domain-encoding exon. We show that human sera contain two forms of FcgammaRIIa2. A full-length secreted protein, which has a 32 kDa backbone, can be detected only in some sera while all sera contain a C-terminal truncated form, lacking part of the intracytoplasmic tail, and exhibiting a 24 kDa protein backbone. The 24 kDa form is significantly increased in sera from stage C patients with B chronic lymphocytic leukemia, compared to healthy donors, stage A and B CLL patients, or CLL patients in complete remission.

In vitro inhibition of tumor B cell growth by IgG-BF-producing FcγRII+T cell hybridoma and by immunoglobulin G-binding factors

The growth-modulating effect on mouse hybridoma B cells of IgG-BF-producing FcγRII+mouse T cell hybridomas and of the IgG-BF isolated from the culture supernatants of these cells has been examined. Cocultures of IgG-secreting hybridoma B cells with IgG-BF-producing T hybridomas or with partially purified IgG-BF demonstrated a reproducible inhibition of the tumor B cell growth. The inhibition was due to a cytostatic and not to a cytotoxic effect. Hybridoma B cells cultured in liquid medium in the presence of soluble IgG-BF, or cocultured in semisolid agarose assays with IgG-BF-producing hybridoma T cells did not undergo immediate cytolysis but were prevented from proliferating. Thus, our data indicate that IgG-BF-producing FcγRII+T cells interfere with the proliferation of transformed B cells, possibly through soluble IgG-BF.

Cytokine production and T-cell activation by macrophage-dendritic cells generated for therapeutic use.

Clinical grade ex vivo‐generated antigen‐presenting cells, macrophage–dendritic cells (MAC–DCs) or macrophage‐activated killers (MAKs) were derived from peripheral blood mononuclear cells (PBMCs). Cultures (7u2003d) were performed in non‐adherent conditions in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and either interleukin 13 (IL‐13) or dihydroxy‐vitamin D3 respectively. MAKs were activated during the last 24u2003h with interferon γ (IFNγ). Reverse transcription polymerase chain reaction (RT‐PCR) and enzyme‐linked immunosorbent assay (ELISA) analyses indicated that IL‐1β and tumour necrosis factor α (TNFα) were produced by both cells. Higher pro‐inflammatory cytokine (IL‐1β and TNFα) amounts were detected on average in MAK supernatants. In contrast, IL‐12 p40 was found only in MAC–DC supernatants, but the biologically active IL‐12 form (p70) was never detected. T‐cell cytokines (IL‐2, IL‐4, IL‐10) were not produced in culture conditions in which T cells were nevertheless present. At du20037, TNFα or lipopolysaccharide (LPS) upregulated IL‐12 p40 production by MAC–DCs, while IL‐12 p70 remained undetectable. LPS stimulation also increased TNFα production by these cells. Allogeneic mixed lymphocyte reactions (MLR) showed that MAKs are poor stimulatory cells compared with MAC–DCs. The MAC–DC stimulatory capacity was enhanced by LPS, although the expression of HLA class II, CD83, CD80 and CD86 was unmodified. Thus, MAC–DCs represent a tool for triggering adaptative immunity, while MAK should be primarily used as effector killer cells.