Deciphering systemic lupus erythematosus-associated serum biomarkers reflecting apoptosis and disease activity.

Systemic lupus erythematosus (SLE) is a severe chronic inflammatory autoimmune connective tissue disease. Despite major efforts, SLE remains a poorly understood disease with unpredictable course, unknown etiology and complex pathogenesis. Apoptosis combined with deficiency in clearing apoptotic cells is an important etiopathogenic event in SLE, which could contribute to the increased load of potential autoantigen(s); however, the lack of disease-specific protein signatures deciphering SLE and the underlying biological processes is striking and represents a key limitation. In this retrospective pilot study, we explored the immune system as a specific sensor for disease, in order to advance our understanding of SLE. To this end, we determined multiplexed serum protein expression profiles of crude SLE serum samples, using antibody microarrays. The aim was to identify differential immunoprofiles, or snapshots of the immune response modulated by the disease, reflecting apoptosis, a key process in the etiology of SLE and disease activity. The results showed that multiplexed panels of SLE-associated serum biomarkers could be decoded, in particular reflecting disease activity, but potentially the apoptosis process as well. While the former biomarkers could display a potential future use for prognosis, the latter biomarkers might help shed further light on the apoptosis process taking place in SLE.

Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack.

The multibillion-dollar global antibody industry produces an indispensable resource but that is generated using millions of animals. Despite the irrefutable maturation and availability of animal-friendly affinity reagents (AFAs) employing naïve B lymphocyte or synthetic recombinant technologies expressed by phage display, animal immunisation is still authorised for antibody production. Remarkably, replacement opportunities have been overlooked, despite the enormous potential reduction in animal use. Directive 2010/63/EU requires that animals are not used where alternatives exist. To ensure its implementation, we have engaged in discussions with the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) and the Directorate General for Environment to carve out an EU-led replacement strategy. Measures must be imposed to avoid outsourcing, regulate commercial production, and ensure that antibody producers are fully supported.

Phenotypic protein profiling of different B cell sub-populations using antibody CD-microarrays.

Antibody microarrays enable extensive protein expression profiling, and provide a valuable complement to DNA microarray-based gene expression profiling. In this study, we used DotScan antibody microarrays that contain antibodies against 82 different cell surface antigens, to determine phenotypic protein expression profiles for human B cell sub-populations. We then demonstrated that the B cell protein profile can be used to delineate the relationship between normal B cells and malignant counterparts. Principle component analysis showed that the lymphomas did not cluster with the normal memory B cells or germinal centre B cells, but they did cluster with germinal centre founder cells and naïve B cells.

Individuals with occupational allergy to detergent enzymes display a differential transcriptional regulation and cellular immune response.

BACKGROUND: In spite of significant safety measures, allergy to industrial enzymes remains a major concern. The increasing prevalence of occupational allergy emphasizes the need to investigate the functional properties of enzyme-exposed dendritic cells (DCs), as DCs possess a potent ability to activate allergen-specific T cells. OBJECTIVE: This study aims at elucidating the molecular mechanisms underlying allergic immune responses to lipase, an industrial enzyme. For this purpose, we studied the effect of both hypoallergenic and wild-type lipase on the transcriptional regulation in DCs and their stimulatory effect on memory CD4+ T cells. METHODS: Five individuals with documented lipase allergy were tested for specific serum IgE. DCs from these individuals, stimulated with lipases, were assayed for their ability to affect proliferation and polarization of memory T cells. The effect of lipases on transcriptional activity in DCs was evaluated using global expression analysis. RESULTS: Lipase-specific IgE levels varied considerably between donors, with donor 4 exhibiting highest levels, and a potent specific CD4+ T cell recall response was demonstrated only for donor 4. No difference was detected in cytokine profile when T cells from donor 4 were co-cultured with DCs pulsed with either hypoallergenic or wild-type lipase, as demonstrated by high IL-4 and IL-13, and low IFN-gamma production. However, the lipases induced different genetic signatures in DCs from donor 4, as compared with the non-responders. CONCLUSIONS: DCs from individuals with clinically diagnosed allergy to lipase displayed a differential response to stimulation with hypoallergenic and wild-type lipase in vitro. Only allergen-pulsed DCs from donor 4 were able to induce CD4+ T cell proliferation. The lipase-specific T cells displayed a T-helper type 2 phenotype, which was not altered by hypoallergenic lipase-pulsed DCs. Furthermore, DCs derived from donor 4 and stimulated with either of the lipases displayed different transcriptional profiles, as compared with the other donors. These signatures represent genes of potential importance for an immunoregulatory role of DC in an ongoing allergic response.

Affinity and epitope profiling of mouse anti-CD40 monoclonal antibodies.

The CD40-CD40L interaction plays a critical role in both humoral and cellular immune responses and interfering antibodies have been suggested as an effective approach for the treatment of lymphomas and autoimmune diseases. In this study we have profiled a panel of mouse antihuman CD40 monoclonal antibodies (MoAbs), regarding their CD40 binding affinity and epitope-specificity relative to the CD40L binding in relation to their cellular activating potential. Despite a rather similar domain-recognition profile, the MoAbs blocked the CD40L binding to a varying degree, with MoAb 5C3 being the poorest inhibitor. There was no correlation between affinity and cellular activation potential. In contrast, a correlation between the ability to block CD40L-binding and activation potential could be seen. We believe that this analysis of several mouse anti-CD40 antibodies can be used to develop strategies for producing new human anti-CD40 antibodies that can more effectively induce or block B-cell proliferation.

Expression of CD137 (4-1BB) on human follicular dendritic cells.

Follicular dendritic cells (FDCs) are the antigen (Ag)-trapping accessory cells of the germinal centres (GCs), essential for the development of humoral immune responses and memory. FDCs reside in the microenvironment of secondary lymphoid tissue where Ag-activated B cells expand, and undergo isotype switching and affinity maturation prior to becoming memory B cells. In addition to delivering Ag, FDCs also provide potent nonspecific accessory signals to the B cells, which are important for the GC reaction. In this report, we show that human tonsilar FDCs express the costimulatory molecule CD137. Surface expression of CD137 on FDCs was confirmed by immunofluorescent labelling and fluorescence-activated cell sorter analysis. CD137 was also highly expressed by the human cell line HK, which displays many characteristics of in vivo FDCs. The interaction between B cells and FDCs is essential for the GC reactions, and our finding suggests that CD137 plays a role in FDC-regulated B-cell responses.

Characterization of specific IgE response in vitro against protein and drug allergens using atopic and normal donors.

BACKGROUND: As the incidence of allergy to different compounds increases in society, the need to understand and characterize specific IgE responses becomes obvious. Different cell culture systems have been evaluated for their ability to support such IgE secretion. METHODS: One system employed human peripheral lymphocytes (PBL) from normal donors stimulated with anti-CD3 activated T cells with or without the presence of allergens like benzylpenicillin (BP) and Phlenum pratense (PP). Secretion of IgE was analyzed in ELISA and compared to the IgG response to the nonallergenic antigen tetanus toxoid (TT). Another system employed stimulation of T and B cells with a heterotope, consisting of a T helper cell epitope derived from TT, and a B cell allergen epitope derived from BP. The specific IgE secretion was compared, using lymphocytes from normal as well as BP-allergic donors. RESULTS: Anti-CD3 stimulated T cells supported BP-specific IgE secretion in six of 11 normal donors. This response was inhibited in four donors and enhanced in two donors by the addition of the BP-allergen to the culture. In contrast, addition of the protein allergen (PP) or antigen (TT) to the same culture system inhibited both IgE and IgG synthesis in all experiments. Cells from the majority (10/16) of the BP-allergic donors failed to produce BP-specific IgE in vitro, when cultured in the presence of allergen. CONCLUSIONS: An allergen specific immune response is readily generated in vitro. The differential response against benzylpenicillin between different donor categories most probably reflects the level of pre-exposure to this allergen in vivo.

CD40 employs p38 MAP kinase in IgE isotype switching.

IgE switching requires the prior induction of C epsilon germline transcripts which is mediated by the concerted binding of STAT-6 and NF kappa B to the C epsilon promoter. These transcription factors are regulated by IL-4 and CD40, respectively. However the latter can effect other signaling pathways and the present study explores the role of p38 MAPK in induction of C epsilon germline transcripts. CD40 and IL-4, both alone and in synergy, were initially shown to activate the C epsilon promoter in a B cell lymphoma cell line. Under the same conditions CD40 caused activation of p38 MAPK, whereas IL-4 was ineffective. The p38 MAPK inhibitor, SB203580, and a dominant negative form of p38 MAPK decreased the CD40 activation of the C epsilon promoter by reducing the ability of CD40 to increase the transactivation potential of NF kappa B. This study suggests that p38 MAPK is crucially important in mediating CD40 activation of NF kappa B which acts to induce C epsilon germline transcripts, ultimately facilitating IgE switching.

Human therapeutic antibodies.

The development of genetic engineering technologies has today advanced to the point where the generation of high-affinity human antibodies against therapeutic targets is not a major hurdle. Rather, it is the selection of target molecules in, for example, cancer therapy that poses a challenge. Targets that are not merely passive acceptors but those that signal into the cell are preferred. Recent advances in the clinical use of antibody-based therapy--such as anti-CD20 (rituximab) for the treatment of non-Hodgkin's lymphoma and anti-tumour-necrosis-factor-alpha for Crohn's disease--as well as novel antibody designs and improved understanding of the mode of action of current antibodies lend great hope to the future of this therapeutic approach.

The immune diversity in a test tube--non-immunised antibody libraries and functional variability in defined protein scaffolds.

Technologies to develop and evolve the function of proteins and, in particular, antibodies have developed rapidly since the introduction of phage display. Importantly, it has become possible to identify molecules with binding properties that cannot be found by other means. A range of different approaches to create general libraries that are useful for the selection of such molecules specific for essentially any kind of target have emerged. We herein review some of the most prominent approaches in the field and in particular discuss specific features related to the development of antibody libraries based on single antibody framework scaffolds. This approach not only permits identification of a range of specific binders, but also facilitates further evolution of initially derived molecules into molecules with optimised functions.

Protein chips based on recombinant antibody fragments: a highly sensitive approach as detected by mass spectrometry.

With the human genome in a first sequence draft and several other genomes being finished this year, the existing information gap between genomics and proteomics is becoming increasingly evident. The analysis of the proteome is, however, much more complicated because the synthesis and structural requirements of functional proteins are different from the easily handled oligonucleotides, for which a first analytical breakthrough already has come in the use of DNA chips. In comparison with the DNA microarrays, the protein arrays, or protein chips, offer the distinct possibility of developing a rapid global analysis of the entire proteome. Thus, the concept of comparing proteomic maps of healthy and diseased cells may allow us to understand cell signaling and metabolic pathways and will form a novel base for pharmaceutical companies to develop future therapeutics much more rapidly. This report demonstrates the possibilities of designing protein chips based on specially constructed, small recombinant antibody fragments using nano-structure surfaces with biocompatible characteristics, resulting in sensitive detection in the 600-amol range. The assay readout allows the determination of single or multiple antigen-antibody interactions. Mass identity of the antigens, currently with a resolution of 8000, enables the detection of structural modifications of single proteins.

A central core structure in an antibody variable domain determines antigen specificity.

Antibody binding sites provide an adaptable surface capable of interacting with essentially any molecular target. Using CDR shuffling, residues important for the assembly of mucin-1 specific paratopes were defined by random recombination of the complementarity determining regions derived from a set of mucin-1 specific clones, previously selected from an antibody fragment library. It was found that positions 33 and 50 in the heavy chain and 32, 34, 90, 91 and 96 in the light chain were conserved in many of the clones. These particular residues seem to be located centrally in the binding site as indicated by a structure model analysis. The importance of several of these conserved residues was supported by their presence in a mouse monoclonal antibody with a known structure and the same epitope specificity. Several of these corresponding residues in the mouse monoclonal antibody are known to interact with the antigen. In conclusion, critical residues important for maintaining a human antigen-specific binding site during the process of in vitro antibody evolution were defined. Furthermore, an explanation for the observed restricted germline gene usage in certain antibody responses against protein epitopes is provided.

Rapid polarization of Th2 cells during induction of antigen-specific IgE antibodies in vitro.

BACKGROUND: Type 2 T-helper cells (Th2) are involved in the regulation of the humoral immune response against antigens and allergens and directly affect which isotype will be produced. The mechanism that regulates antigen-specific IgE secretion and immune deviation is still not known. OBJECTIVES: To delineate mechanisms behind antigen-specific IgE secretion we have used in vitro immunization and focused on T-cell phenotype and the activation status of the transcription factor NFkappaB. METHODS: Peripheral blood lymphocytes (PBMC) from seronegative donors were immunized in vitro with a peptide consisting of both a T-cell and a B-cell epitope. RESULTS: Antigen-specific IgE antibodies could be detected after a primary immunization, during which T-helper cells secreted type 2 cytokines. Specific IgE was also detected in the secondary immunization, but due to a rapid polarization from Th2 to Th1 phenotype, exogenous IL-4 was required for the specific IgE secretion. Analysis of NFkappaB activation in B and T cells during primary and secondary immunization showed that NFkappaB could be detected in both B and T cells during primary immunization, but was dependent on exogenous IL-4 in the secondary immunization. CONCLUSION: This is the first evidence of antigen-specific IgE induction in vitro using naive B cells, demonstrating the involvement of T-helper cell phenotype and NFkappaB and demonstrates the usefulness of in vitro cultures to study the effect of antigens on human immunocytes.

Signals sustaining human immunoglobulin V gene hypermutation in isolated germinal centre B cells.

Affinity maturation of antibody responses depends on somatic hypermutation of the immunoglobulin V genes. Hypermutation is initiated specifically in proliferating B cells in lymphoid germinal centres but the signals driving this process remain unknown. This study identifies signals that promote V gene mutation in human germinal centre (GC) B cells in vitro. Single GC B cells were cultured by limiting dilution to allow detection of mutations arising during proliferation in vitro. Cells were first cultured in the presence of CD32L cell transfectants and CD40 antibody (the 'CD40 system') supplemented with combinations of cytokines capable of supporting similar levels of CD40-dependent GC B-cell growth [interleukin (IL)-10 + IL-1beta + IL-2 and IL-10 + IL-7 + IL-4]. Components of the 'EL4 system' were then added to drive differentiation, providing sufficient immunoglobulin mRNA for analysis. Analysis of VH3 genes from cultured cells by reverse transcription-polymerase chain reaction (RT-PCR)-based single-strand conformation polymorphism indicated that the combination IL-10 + IL-1beta + IL-2 promoted active V gene mutation whereas IL-10 + IL-7 + IL-4 was ineffective. This was confirmed by sequencing which also revealed that the de novo generated mutations were located in framework and complementarity-determining regions and shared characteristics with those arising in vivo. Somatic mutation in the target GC B-cell population may therefore be actively cytokine driven and not simply a consequence of continued proliferation. The experimental approach we describe should facilitate further studies of the mechanisms underlying V gene hypermutation.

In vitro induction of somatic mutations in human peripheral B lymphocytes.

The major cellular and molecular factors that lead to the induction of somatic mutations in human B cells remain unclear. This study describes an approach which allowed us to single-cell culture antigen-specific B cells after in vitro immunization. Using single strand conformational polymorphism analysis on the descendant cultures we were able to demonstrate that the Ig gene of B cells was induced to mutate. Three clones were isolated from a single cell culture of naive B cells immunized against the semi-conserved region of V3 of HIV-1, which contained two point mutations at the CDR2 region leading to amino acid codon change. Two clones were also obtained from a single cell culture of memory B cells immunized against the recall antigen tetanus toxoid. These clones showed one point mutation at the CDR1-FR2 border region leading to amino acid codon change. Fab constructs from the mutated culture of naive B cells immunized against V3 were cloned to a procaryotic expression vector. The expression products of two clones showed anti-V3 specificity in ELISA using three different conjugates. Our results suggest that somatic mutations can be induced in human peripheral B cells through specific interaction with antigen and antigen-activated T cells.

Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries.

We constructed a single-chain Fv antibody library that permits human complementarity-determining region (CDR) gene fragments of any germline to be incorporated combinatorially into the appropriate positions of the variable-region frameworks VH-DP47 and VL-DPL3. A library of 2 x 109 independent transformants was screened against haptens, peptides, carbohydrates, and proteins, and the selected antibody fragments exhibited dissociation constants in the subnanomolar range. The antibody genes in this library were built on a single master framework into which diverse CDRs were allowed to recombine. These CDRs were sampled from in vivo-processed gene sequences, thus potentially optimizing the levels of correctly folded and functional molecules, and resulting in a molecule exhibiting a lower computed immunogenicity compared to naive immunoglobulins. Using the modularized assembly process to incorporate foreign sequences into an immunoglobulin scaffold, it is possible to vary as many as six CDRs at the same time, creating genetic and functional variation in antibody molecules.

Antibodies in diagnostics - from immunoassays to protein chips.

Antibodies are used extensively as diagnostic tools in a wide array of different analyses. Monoclonal and recombinant antibodies provide a never ending source of molecules and can produce endless possibilities for novel genetic constructs. Antibodies are still very much in vogue and are now also being used in microarray analysis of the proteome using protein chips. Here, recent opportunities presented by antibodies as diagnostic tools are reviewed.

Immature B cells in bone marrow express Fas/FasL.

Negative selection is a process by which autoreactive lymphocytes are eliminated from the developing antigen receptor repertoire. The mechanisms regulating negative selection of immature B lymphocytes in the bone marrow are poorly elucidated. Human bone marrow cells were examined in order to investigate the presence of the members of the Fas (APO-1/CD95) system. Here we demonstrate the expression of Fas in immature B lymphocytes (CD10/CD19+/CD40+/sIg+), and the presence of Fas natural ligand (FasL) in CD19+ bone marrow cells. The observed expression of apoptosis-related molecules might indicate how negative selection of autoreactive B cells can occur in human bone marrow.