Eva Källberg

Expansion, Selection and Mutation of Antigen-Specific B Cells in Germinal Centers

There are many points of decision in the life of a B cell the outcome of which is a matter of life or death. In other words, B cells, like the rest of the immune system, are constantly under selective pressure from the outside world. In most situations a B cell passes a selective barrier due to the properties exhibited by its receptor for antigen, cell surface immunoglobulin (sig). It has the capacity to influence the result of this selection test by intemal manipulation of its sIg as well as other cell surface molecules. For instance it can change the affinity of its sIg receptor via the mechanism of somatic mutation; it can enhance signalling via sIg by alteration in the level and/or coupling of accessory molecules (Hombach et al. 1990, Venkituraman et al. 1991); and it might bypass the selection process altogether by the use of other surface molecules such as CD40 (Liu et al. 1989, Banchereau et al. 1991) and regulation of oncogene products (e.g. bc!-2; McDonnel et al. 1989, Nunez et al. 1991).

S100A9 Interaction with TLR4 Promotes Tumor Growth

By breeding TRAMP mice with S100A9 knock-out (S100A9−/−) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b+ S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68+ macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9−/− and TLR4−/−, but not in RAGE−/− animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b+ cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.

Somatic Mutation of Immunoglobulin V Genes in Vitro

The molecular mechanism behind affinity maturation is the introduction of point mutations in immunoglobulin (Ig) V genes, followed by the selective proliferation of B cells expressing mutants with increased affinity for antigen. An in vitro culture system was developed in which somatic hypermutation of Ig V genes was sustained in primed B cells. Cognate T cell help and cross-linking of the surface Ig were required, whereas the addition of lipopolysaccharide or a CD40 ligand to drive proliferation was insufficient. This system should facilitate understanding of the molecular and cellular mechanisms that regulate somatic mutation and B cell selection.

Induction of NFκB responses by the S100A9 protein is TLR4-dependent.

Interactions between danger‐associated molecular patterns (DAMP) and pathogen‐associated molecular patterns (PAMP) and pattern recognition receptors such as Toll‐like receptors (TLRs) are critical for the regulation of the inflammatory process via activation of nuclear factor‐κB (NF‐κB) and cytokine secretion. In this report, we investigated the capacity of lipopolysaccharide (LPS) ‐free S100A9 (DAMP) protein to activate human and mouse cells compared with lipoprotein‐free LPS (PAMP). First, we showed that LPS and S100A9 were able to increase NF‐κB activity followed by increased cytokine and nitric oxide (NO) secretion both in human THP‐1 cells and in mouse bone marrow‐derived dendritic cells. Surprisingly, although S100A9 triggered a weaker cytokine response than LPS, we found that S100A9 more potently induced IκBα degradation and hence NF‐κB activation. Both the S100A9‐induced response and the LPS‐induced response were completely absent in TLR4 knockout mice, whereas it was only slightly affected in RAGE knockout mice. Also, we showed that LPS and S100A9 NF‐κB induction were strongly reduced in the presence of specific inhibitors of TLR‐signalling. Chloroquine reduced S100A9 but not LPS signalling, indicating that S100A9 may need to be internalized to be fully active as a TLR4 inducer. This was confirmed using A488‐labelled S100A9 that was internalized in THP‐1 cells, showing a raise in fluorescence after 30 min at 37°. Chloroquine treatment significantly reduced the fluorescence. In summary, our data indicate that both human and mouse S100A9 are TLR4 agonists. Importantly, S100A9 induced stronger NF‐κB activation albeit weaker cytokine secretion than LPS, suggesting that S100A9 and LPS activated NF‐κB in a qualitatively distinct manner.

Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9.MethodsFlow cytometry, confocal microscopy and real-time PCR of flow cytometry-sorted cells were used to measure cell surface S100A8/A9, intracellular S100A8/A9 and mRNA levels of S100A8 and S100A9, respectively.ResultsCell surface S100A8/A9 was detected on all leukocyte subpopulations investigated except for T cells. By confocal microscopy, real-time PCR and stimulation assays, we could demonstrate that pDCs, monocytes and PMNs could synthesize S100A8/A9. Furthermore, pDC cell surface S100A8/A9 was higher in patients with active disease as compared to patients with inactive disease. Upon immune complex stimulation, pDCs up-regulated the cell surface S100A8/A9. SLE patients had also increased serum levels of S100A8/A9.ConclusionsPatients with SLE had increased cell surface S100A8/A9, which could be important in amplification and persistence of inflammation. Importantly, pDCs were able to synthesize S100A8/A9 proteins and up-regulate the cell surface expression upon immune complex-stimulation. Thus, S100A8/A9 may be a potent target for treatment of inflammatory diseases such as SLE.

Ly6C expression differentiates plasma cells from other B cell subsets in mice.

Plasma cell differentiation is induced in vitro by lipopolysaccharide (LPS) stimulation but can be blocked by including anti‐CD40 antibodies. Using subtractive cDNA hybridization we have identified the cell surface protein Ly6C as differentially expressed on B cells stimulated with LPS only. Ly6C has been shown to be expressed on certain T cell subsets and on subsets of macrophages and NK cells, but not on resting B cells. We show that Ly6C is up‐regulated upon LPS stimulation of B cells in vitro and that this up‐regulation is blocked by anti‐CD40 or anti‐Ig antibodies. Furthermore, ELISPOT analysis of cells sorted by magnetic‐activated cell sorting show that Ly6C is expressed on ex vivo plasma cells from the spleen and bone marrow. Flow cytometric analysis showed that Ly6C is expressed on splenic plasma cells as well as on lamina propria plasma cells. Finally, Ly6C cross‐linking positively up‐regulated the amount of immunoglobulin produced by LPS‐stimulated splenic B cells in vitro.

Mice lacking NCF1 exhibit reduced growth of implanted melanoma and carcinoma tumors.

The NADPH oxidase 2 (NOX2) complex is a professional producer of reactive oxygen species (ROS) and is mainly expressed in phagocytes. While the activity of the NOX2 complex is essential for immunity against pathogens and protection against autoimmunity, its role in the development of malignant tumors remains unclear. We compared wild type and Ncf1 m1J mutated mice, which lack functional NOX2 complex, in four different tumor models. Ncf1 m1J mutated mice developed significantly smaller tumors in two melanoma models in which B16 melanoma cells expressing a hematopoietic growth factor FLT3L or luciferase reporter were used. Ncf1 m1J mutated mice developed significantly fewer Lewis Lung Carcinoma (LLC) tumors, but the tumors that did develop, grew at a pace that was similar to the wild type mice. In the spontaneously arising prostate carcinoma model (TRAMP), tumor growth was not affected. The lack of ROS-mediated protection against tumor growth was associated with increased production of immunity-associated cytokines. A significant increase in Th2 associated cytokines was observed in the LLC model. Our present data show that ROS regulate rejection of the antigenic B16-luc and LLC tumors, whereas the data do not support a role for ROS in growth of intrinsically generated tumors.

Indoleamine 2,3‐dioxygenase (IDO) activity influence tumor growth in the TRAMP prostate cancer model

Indoleamine 2,3‐dioxygenase (IDO) activity has been shown to be expressed in local lymph nodes and induce immune suppression of tumor immunity. Here we analyze the effect of IDO expression on prostate tumor growth using the transgenic adenocarcinoma of mouse prostate (TRAMP) animal model.

Identification of a novel thioredoxin-related protein, PC-TRP, which is preferentially expressed in plasma cells.

Using cDNA subtraction we have identified a previously unknown cDNA, termed plasma cell thioredoxin‐related protein (PC‐TRP), which was overexpressed in plasma cells in vitro. The full‐length 2636‐bp PC‐TRP cDNA contained an open reading frame coding for 417 amino acids. The cDNA sequence was mapped to a region on mouse chromosome 13 and the PC‐TRP gene consists of ten exons distributed over a 28‐kb region. The PC‐TRP was found to contain three atypical thioredoxin domains and showed functional thioredoxin activity in vitro. A more detailed analysis revealed that PC‐TRP was expressed at a high level in plasma cells, both at the mRNA and protein level, while present at very low levels in all other cells and tissues examined. Immunohistological analysis revealed that PC‐TRP was expressed at high levels in all plasma cells isolated from bone marrow or lamina propria, irrespective of their Ig isotype. In the spleen, however, 60% of the IgG‐expressing plasma cells showed only a dull staining at day 14 after immunization, while >90% of the IgG‐secreting cells in the spleen expressed PC‐TRP at day 21 after immunization. In addition, an IgG‐secreting, PC‐TRPdull plasma cell population could also be observed after B cell activation in vitro.

Human S100A9 Protein Is Stabilized by Inflammatory Stimuli via the Formation of Proteolytically-Resistant Homodimers

S100A8 and S100A9 are Ca2+-binding proteins that are associated with acute and chronic inflammation and cancer. They form predominantly heterodimers even if there are data supporting homodimer formation. We investigated the stability of the heterodimer in myeloid and S100A8/S100A9 over-expressing COS cells. In both cases, S100A8 and S100A9 proteins were not completely degraded even 48 hrs after blocking protein synthesis. In contrast, in single transfected cells, S100A8 protein was completely degraded after 24 h, while S100A9 was completely unstable. However, S100A9 protein expression was rescued upon S100A8 co-expression or inhibition of proteasomal activity. Furthermore, S100A9, but not S100A8, could be stabilized by LPS, IL-1β and TNFα treatment. Interestingly, stimulation of S100A9-transfected COS cells with proteasomal inhibitor or IL-1β lead to the formation of protease resistant S100A9 homodimers. In summary, our data indicated that S100A9 protein is extremely unstable but can be rescued upon co-expression with S100A8 protein or inflammatory stimuli, via proteolytically resistant homodimer formation. The formation of S100A9 homodimers by this mechanism may constitute an amplification step during an inflammatory reaction.