We have analyzed the murine CD40 ligand promoter with regard to stimulation of transcriptional activity in Jurkat T cells after signaling via the TCR and the costimulatory molecules CD28 and CD2. TCR engagement was necessary for the induction of transcriptional activity from the CD40 ligand promoter, and costimulation through either CD28 or CD2 further increased the activity. Analysis of promoter deletants showed that the DNA elements needed for transcriptional activity induced by costimulatory molecules were located within two regions containing previously identified transcription factor NFAT sites. Further studies of the proximal NFAT site showed that it was not dependent on AP-1 binding for transcriptional activity induced by costimulation through CD28. Instead, a region between the TATA box and the proximal NFAT site was shown to bind proteins of the early growth response family and to contribute to NFAT-mediated transcriptional activation.

A group of bacterial Ig-binding surface proteins were studied: protein H and M1 are from Streptococcus pyogenes and interact with IgG, protein L is expressed by Peptostreptococcus magnus and shows affinity for Ig light chains, whereas protein LG is a chimeric construction combining the binding properties of protein L with the IgG-binding activity of protein G from group C and G streptococci. Proteins L and H coupled to Sepharose were mitogenic for human peripheral blood lymphocytes (PBL) and mouse splenic B cells, but not when added in soluble form. Differentiation to Ig secretion was induced by protein H-Sepharose in mouse splenic B cells but not in human PBLs. In FACS analysis FITC-labelled protein H stained virtually all CD19+ cells in human peripheral blood as well as a majority of the CD3+ population. Protein L bound the majority of the CD19+ population, but also a fraction of the CD19-/CD3 population. Protein M1 was not mitogenic but stained the entire CD19+ population and 70% of the CD3+ population. Identical staining patterns were observed with mouse splenocytes using B220 and T-cells receptor as lineage markers. The chimeric protein LG was a potent mitogen for mouse splenic B cells when added either coupled to Sepharose or in soluble form. In addition, protein LG induced differentiation to Ig secretion of the responding mouse splenic B cells. In FACS analysis, protein LG stained the entire CD19+ and the majority of the CD19-/CD3 lymphocyte population as well as all B220+ mouse splenocytes and a fraction of the splenic T cells. These data indicate that the bacterial proteins studied interact with surface structures of several leucocyte populations and can hence interfere with the immune system at multiple levels.

Stimulation in vitro of murine splenic B cells by lipopolysaccharide, anti-kappa Sepharose, anti-CD40 or allo-reactive T helper cells all up-regulated CD21 and CD23 surface expression. Neither anti-CD21 nor anti-CD23 antibodies induced B cell growth or differentiation when added in soluble form or coupled to Sepharose. However, anti-CD40-stimulated B cells showed increased proliferation in the presence of anti-CD21 antibodies coupled to Sepharose; co-stimulation via CD21 also induced differentiation to immunoglobulin secretion in a fraction of anti-CD40-stimulated B cells. Furthermore, anti-CD40 antibodies inhibited differentiation to immunoglobulin secretion induced by lipopolysaccharide and, hence, appears to be a dominant negative signal for B cell differentiation.

Both anti-CD40 antibodies and anti-immunoglobulin (Ig) coupled to Sepharose induced proliferation of resting B cells and suppressed lipopolysaccharide (LPS)-induced B-cell differentiation to immunoglobulin secretion at comparable levels determined with the plaque-forming assay and Ig RNA steady state levels. Anti-CD40 antibodies also increased the proliferation of B cells stimulated by T helper cells in vitro while suppressing their differentiation to Ig secretion. Further, B cells preactivated by anti-Ig, anti-CD40 or a combination of the two mitogens could be restimulated by anti-CD40 but not by anti-Ig antibodies. Phenotypic divergence of Ig and CD40 signals regarding surface expression of activation markers was observed. Restimulation of anti-Ig- or anti-CD40-prestimulated cells with anti-Ig induced apoptosis whereas apoptosis could be inhibited when cells were recultivated with anti-CD40.

. taken up by lymphocytes and transported to the perinuclear/nuclear compartment. A consequence of this process is interference with lymphocyte functions, which should add selective advantages to protein H-expressing bacteria. Results Protein H interacts with human lymphocytes and granulocytesProtein H is released from the streptococcal surface through the action of a cysteine proteinase produced by the bacteria (Berge and Björck, 1995). The Escherichia coli-produced fragment of protein H used in this study is similar in size to the fragment released by the streptococcal enzyme ( Fig. 1) and, in the following text, protein H refers to this C-terminally truncated fragment expressed by and purified from E. coli. The interaction of protein H with the surface of T cells and granulocytes was analysed by flow cytometry. Human peripheral blood lymphocytes were incubated with protein H, and the majority (>90%) of the CD3 + , CD4 + and CD8 + cells interacted with protein H ( Fig. 2A). When the binding of protein H to cells within the granulocyte gate was analysed, these cells were also © 2002 Blackwell Science Ltd, Molecular Microbiology, 44, 917-934 918 I.-M. Frick et al. Fig. 1. Schematic representation of streptococcal protein H. The binding sites for IgGFc and fibronectin type III domains are located in the A-B domains, which are followed by three homologous albuminbinding C domains (Frick et al., 1994;. The C-terminal part of the D domain anchors protein H to the bacterial cell wall. Fragment I was produced in E. coli and used throughout this study, whereas fragment II is released from the bacterial cell surface by the action of a cysteine proteinase secreted by the Streptococcus. Numbers refer to amino acid residue positions. Uptake and transportation of streptococcal protein H 919 fluorescent (data not shown). Protein H binds to IgGFc, and previous work has also indicated affinity for human major histocompatibility complex (MHC) class II antigens (Åkesson et al., 1994). The human Jurkat T cell line was chosen for subsequent experiments, as these cells do not express MHC class II antigens, which was confirmed by flow cytometry (data not shown). More than 97% of the Jurkat T cells were found to interact with protein H. Furthermore, as shown in Fig. 2B, Jurkat cells were stained by anti-CD45 but not with unspecific g1 fluorescein isothiocyanate (FITC)/g2 PE mouse monoclonal antibodies (mAbs). Protein H is taken up by lymphocytes and transported to the perinuclear regionThe cellular interaction of protein H demonstrated by flow cytometry raised the question whether the protein is also internalized. To investigate this, Jurkat cells were incubated with fluorescently labelled protein H at a concentration of 0.17 mg ml -1 for different periods of time, and its cellular localization was determined by laser scanning confocal microscopy. As shown in Fig. 3, protein H is taken up by the cells, and a gradual intracellular accumulation takes place. The internalization process is rapid and, after 30 min, protein H is found a...