Cornelis de Groot

Follicular dendritic cells carry MHC class II-expressing microvesicles at their surface

Follicular dendritic cells (FDCs) present in lymphoid follicles play a critical role in germinal center reactions. They trap native Ags in the form of immune complexes providing a source for continuous stimulation of specific B lymphocytes. FDCs have been reported to express MHC class II molecules, suggesting an additional role in the presentation of not only native, but also processed Ag in the form of peptide-loaded MHC class II. Adoptive bone marrow transfer experiments have shown that MHC class II molecules are only passively acquired. Up to now the origin of these MHC class II molecules was not clear. Here we show by cryoimmunogold electron microscopy that MHC class II molecules are not present at the plasma membrane of FDCs. In contrast, microvesicles attached to the FDC surface contain MHC class II and other surface proteins not expressed by FDCs themselves. The size and marker profiles of these microvesicles resemble exosomes. Exosomes, which are secreted internal vesicles from multivesicular endosomes, have been shown earlier to stimulate proliferation of specific T lymphocytes in vitro, but their target in vivo remained a matter of speculation. We demonstrate here that isolated exosomes in vitro bind specifically to FDCs and not to other cell types, suggesting that FDCs might be a physiological target for exosomes.

Human follicular dendritic cells: function, origin and development.

Follicular dendritic cells (FDCs) have important functions in the selection of memory B lymphocytes during germinal center reactions (GCR). They present native antigens to potential memory cells, of which only B cells with high affinity B cell receptors (BCR) can bind. These B lymphocytes survive, whereas nonbinding B cells undergo apoptotic cell death. FDCs are present in follicles of any secondary lymphoid organ and belong to the stromal cells of these organs. Ectopic FDC-formation can be found in a number of autoimmune diseases and/or chronic inflammatory situations. This indicates that the development of FDCs is not restricted to secondary lymphoid organs, but that it is rather a matter of local conditions that drives a precursor cell type into FDC-maturation. A precursor of FDCs has presently not been identified, but phenotypic marker studies, in vitro experiments with fibroblast-like cell lines, and recent data on mesenchymal precursor cells from the peripheral blood suggest a close relation to fibroblast-like cells.

Triple check for antigen specificity of B cells during germinal centre reactions

Somatic hypermutation of the immunoglobulin variable genes during germinal reactions might permit the expansion of B-cell clones with unwanted (e.g. autoreactive) specificity. Here, Ernst Lindhout and colleagues propose three antigen-specific checkpoints that ensure the appropriate antigen specificity of activated B cells is maintained by regulating the activation, selection and further differentiation of B cells.

Death-receptor contribution to the germinal-center reaction

Both helper T cells and follicular dendritic cells play crucial roles in the germinal-center (GC) reaction. One of their key functions is to provide GC B cells with anti-apoptotic signals during their growth, diversification of antibody repertoire and positive selection. Dysregulation of the mechanisms that control B-cell apoptosis in the GC could cause hyperplasia, endanger self-tolerance or impair dramatically the efficiency of the humoral response. This article discusses how the death receptor Fas and components of its signaling machinery contribute to the GC reaction.

Cutting Edge: Cellular Fas-Associated Death Domain-Like IL-1-Converting Enzyme-Inhibitory Protein Protects Germinal Center B Cells from Apoptosis During Germinal Center Reactions

During germinal center (GC) reactions, follicular dendritic cells are believed to select memory B lymphocytes by switching off apoptosis in the successfully binding B cells. The cellular signals involved in this process are largely unknown. Here, we show that GC B lymphocytes have a long isoform of the cellular homologue of the viral Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (cFLIPL), which is capable of inhibiting death receptor-induced caspase activation. In isolated GC B cells, cFLIPL decays rapidly even without Fas ligation, and this results in activation of caspase activity and apoptosis. Contact with follicular dendritic cells prevents cFLIPL degradation and blocks all signs of apoptosis, even in the presence of anti-Fas Abs. cFLIPL expression is sustained by CD40 ligation as well, suggesting that at least at some stage of the GC reaction activated T cells may help selected B cells to leave the follicular dendritic cell network without becoming apoptotic.

Follicular dendritic cells and apoptosis: Life and death in the germinal centre

SummaryThe germinal centre forms a specialized microenvironment thought to play a key role in the induction of antibody synthesis, affinity maturation of B cells and memory B cell formation. Clonal-expanded follicular B lymphocytes with mutated antigen receptors (centrocytes) have to be selected on the basis of their capacity to compete for binding to antigen held in limited amounts on the follicular dendritic cells. In this way, only high-affinity B cells are selected. Binding to a follicular dendritic cell is an unconditional prerequisite for centrocytes to survive. Cells that do not succeed in binding to a follicular dendritic cell die rapidly by apoptosis. Apoptosis is a common form of cell death characterized by the activation of an endonuclease culminating in nuclear destruction. The pathway by which apoptosis is triggered varies from cell type to cell type. However, for germinal centre B cells this process is still poorly understood.

Lysosomal Destabilization Contributes to Apoptosis of Germinal Center B-lymphocytes

During germinal center (GC) reactions, B-lymphocytes with high-affinity B-cell receptors are selected. Regulation of apoptosis is a key process in selecting such wanted B-cells and in eliminating B-cells with unwanted specificities. In this paper, we show that apoptosis in human GC B-cells involves lysosomal destabilization, which is strictly controlled by caspase-8 activity, but not by caspase-9 activity. Ligation of CD40 provides resistance to lysosomal destabilization. Experimental lysosomal rupture by the lysosomotropic drug O-methyl-L-serine dodecylamide hydrochloride (MSDH) induces apoptosis in GC B-cells, including phosphatidyl serine exposure, mitochondrial inactivation, and DNA fragmentation. These apoptotic features occur in the absence of caspase-3 activity. Follicular dendritic cells (FDCs) protect binding B-lymphocytes from lysosomal destabilization, in both the absence and the presence of MSDH. Our study demonstrates that lysosomal leakage induces apoptosis of GC B-cells in a caspase-3-independent manner and that high-affinity binding to FDCs prevents lysosomal leakage and apoptosis in GC B-cells.

Proteinases and their inhibitors in the immune system.

The most important roles of proteinases in the immune system are found in apoptosis and major histocompatibility complex (MHC) class II-mediated antigen presentation. A variety of cysteine proteinases, serine proteinases, and aspartic proteinases as well as their inhibitors are involved in the regulation of apoptosis in neutrophils, monocytes, and dendritic cells, in selection of specific B and T lymphocytes, and in killing of target cells by cytotoxic T cells and natural killer cells. In antigen presentation, endocytosed antigens are digested into antigenic peptides by both aspartic and cysteine proteinases. In parallel, MHC class II molecules are processed by aspartic and cysteine proteinases to degrade the invariant chain that occupies the peptide-binding site. Proteinase activity in these processes is highly regulated, particularly by posttranslational activation and the balance between active proteinases and specific endogenous inhibitors such as cystatins, thyropins, and serpins. This article discusses the regulation of proteolytic processes in apoptosis and antigen presentation in immune cells and the consequences of therapeutic interference in the balance of proteinases and their inhibitors.

An efficient procedure for the generation of human monoclonal antibodies based on activation of human B lymphocytes by a murine thymoma cell line

A new, efficient procedure for the generation of human monoclonal antibodies has been developed. The procedure is based on the activation of human B cells in microwells by murine thymoma EL4B5 cells. This mode of B cell stimulation leads to proliferation of at least one per eight of human B cells and to a high rate of antibody production. Subsequently, supernatants of the microwells are screened by ELISA for the presence of antibody of the desired specificity and B cells from selected wells are hybridized by electroporation. To optimize the procedure, the kinetics of the B cell expansion induced by EL4B5 cells were analysed. Counting and phenotyping of cultured cells at different time points indicated that the peak of B cell expansion occurred at day 5 for tonsil B cells (16-fold increase) and at day 7 for peripheral blood B cells (20-fold increase). The B cells did not merely proliferate but also differentiated, as indicated by loss of CD20 expression and increase of CD38 expression. At the peak of B cell expansion, B cells could be hybridized efficiently with myeloma cells. The majority of the resultant hybridomas secreted human immunoglobulin. The efficiency of the procedure is exemplified by the generation of hybridomas secreting human IgG against Haemophilus influenzae from limited numbers of either human tonsil B lymphocytes or peripheral blood B lymphocytes.

Improvement of EBV transformation and cloning efficiency of human B cells using culture supernatants from lymphoblastoid cell lines.

Exposure of human B cells to Epstein-Barr virus (EBV) usually results in low frequencies of transformed cells. The transformed cells can be cloned poorly by limiting dilution, even when feeder cells are used. In recent years it has become clear that growth and antibody production of EBV-transformed cells are influenced by auto- and paracrine growth factors. Therefore, supernatants from the lymphoblastoid B cell lines JY and Raji were used as a source of growth factors to investigate their effect during EBV transformation of human B cells and consequently on cloning by limiting dilution of these transformed cells. Initial experiments to clone three established EBV-transformed B cell lines showed a strong increase in outgrowth of the number of cells in the presence of the supernatant (range: 1 per 2-8 of the originally plated cells) as compared to cells cultured without the supernatant (range: 1 per 17-100 of the originally plated cells). Transformation efficiencies of freshly isolated tonsil B cells were not influenced by the supernatant and were generally less than 1%. In contrast, transformation efficiency was increased up to 9.4% if B cells were both transformed and cultured in the presence of the supernatant. Cloning efficiencies increased if the cells used were transformed in the presence of the supernatant. Best results were seen when the supernatant was present during transformation and cloning of the cells. The presence of the supernatant during transformation and/or cloning of the B cells dramatically enhanced the number of B cells secreting IgG. Cloning of two established tonsil B cell lines resulted in a large number of B cell clones.(ABSTRACT TRUNCATED AT 250 WORDS)