Gammadelta T cell non-responsiveness in Campylobacter jejuni-associated Guillain-Barré syndrome patients.

To seek evidence for a role of molecular mimicry in the induction of Guillain-Barré syndrome (GBS), the authors studied Campylobacter jejuni-reactive T lymphocytes in patients with GBS. In contrast to controls, gammadelta T cells of patients with GBS with antecedent C jejuni infections failed to respond to C jejuni. Supplementing cell cultures with the cytokines interleukin-2 or interleukin-15 resulted in restoration of the gammadelta T cell proliferative response. Gammadelta T cell non-responsiveness may lead to defective regulation of antibody production, and in this way an (auto)immune response against ganglioside-like epitopes on peripheral nerve may cause GBS.

Expansion of human gammadelta T cells after in vitro stimulation with Campylobacter jejuni.

Campylobacter jejuni is currently the prime cause of food-borne bacterial gastro-enteritis. An important complication of C. jejuni enteritis is Guillain-Barré syndrome (GBS), an immune-mediated disorder of peripheral nerve tissue. Because little is known about T cell reactivity to C. jejuni, we have analyzed the in vitro immune response of normal individuals against five isolates of C. jejuni representing five different serotypes. We found a preferential expansion of peripheral blood gammadelta T cells after exposure to crude sonicates of all five C. jejuni serotypes. Expansion of gammadelta T cells was dependent on the presence of CD4+/alphabeta+ T cells in the cultures or addition of exogenous IL-2 or IL-15. C. jejuni stimulation was mediated via the TCR and appeared to be induced by a non-proteinaceous bacterial antigen, most likely of phosphoantigenic origin.

Human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis show the imprints of an antigen-dependent process of somatic hypermutation and clonal selection.

The persistent presence of rheumatoid factors (RFs) in the circulation is a characteristic phenomenon in patients with rheumatoid arthritis (RA). Recent data indicate that RFs associated with seropositive RA are derived from terminally differentiated CD20-, CD38+ plasma cells (PCs) present in synovial fluids of the inflamed joints. These cells were shown to secrete RFs actively and are thought to originate from germinal centre (GC)-like structures present in the inflamed synovium. To obtain a representative image of the structural properties of IgM and IgG RFs associated with RA, phage antibody display libraries were constructed from CD38+ PCs isolated from the inflamed joints of RF-seropositive patients with RA. Subsequently, human IgG Fc-binding monoclonal phage antibodies were selected and analysed. The data suggest that RA-associated RFs are encoded by a diverse set of VL and a more restricted set of VH regions. VH gene family usage of PC-derived IgM- and IgG-RFs was found to be restricted to the VH1 and 3 gene families, with a preference for VH3, and many different VL genes were shown to contribute to RF specificity. Clonally related VH as well as VL sequences were identified, based on the presence of identical CDR3 regions and shared somatic mutations. In this B cell selection process base-pair substitutions as well as deletions of triplets in CDR regions, leaving the transcripts in frame, were involved. Together, these data provide further evidence for an Ag-driven immune response in the terminal differentiation into RF-producing PCs in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a GC reaction.

Polyreactivity of human IgG Fc-binding phage antibodies constructed from synovial fluid CD38+ B cells of patients with rheumatoid arthritis.

Recent data indicate that rheumatoid factors (RFs) that occur in patients with rheumatoid arthritis (RA) are derived from Ig-producing terminally differentiated CD20-, CD38+ plasma cells present in synovial fluids (SFs). Phage antibody display libraries were constructed using CD38+ plasma cells isolated from SFs of two RF-seropositive RA patients. The libraries were enriched for phage antibodies (Phabs) binding to human IgG (HuIgG) Fc fragments and the sequences of their V genes were analysed. These data provided further evidence for an Ag-driven immune response in patients with RA, including expansion of clonally related B cells, selection and isotype switching, all hallmarks of a germinal center reaction. In the present study, the functional characteristics of these HuIgG Fc-binding monoclonal (mo) Phabs were further analysed in order to provide more insight into the specificity of HuIgG Fc-binding Phabs. Remarkably, all HuIgG Fc-binding moPhabs tested (n=48; derived from four different libraries) displayed polyreactivity. Structural analysis of the CDR3 regions revealed characteristic features of polyreactive Igs. Most H chain CDR3 regions harboured tryptophan/tyrosine-rich parts and approximately 60% of the L chain CDR3 regions of both RA patients displayed an identical stretch of amino acids (W/Y-D-S-S). Supportive for a dominant role of VH in specificity, exchange of VL regions with a single VH region yielded moPhabs with similar specificities. All together, the data suggest the presence of an Ag-driven process in the joints of patients with RA, including somatic mutation and clonal selection entailing isotype switching, resulting in the differentiation of B cells into polyreactive RF-secreting plasma cells.

Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease.

Since targeting of recombinant adenovirus vectors to defined cell types in vivo is a major challenge in gene therapy and vaccinology, we explored the natural diversity in human adenovirus tissue tropism. Hereto, we constructed a library of Ad5 vectors carrying fibers from other human serotypes. From this library, we identified vectors that efficiently infect human cells that are important for diverse gene therapy approaches and for induction of immunity. For several medical applications (prenatal diagnosis, artificial bone, vaccination, and cardiovascular disease), we demonstrate the applicability of these novel vectors. In addition, screening cell types derived from different species revealed that cellular receptors for human subgroup B adenoviruses are not conserved between rodents and primates. These results provide a rationale for utilizing elements of human adenovirus serotypes to generate chimeric vectors that improve our knowledge concerning adenovirus biology and widen the therapeutic window for vaccination and many different gene transfer applications.

Tumor cell killing by in vitro affinity-matured recombinant human monoclonal antibodies.

We have developed a method that allows the rapid improvement of the affinity of phage-displayed antibody fragments by selection on intact eukaryotic cells. A single chain Fv fragment, specific for the tumor-associated Ep-Cam molecule, was mutagenized by shuffling of the immunoglobulin light chain variable region and DNA shuffling of both heavy and light chain variable regions. Higher-affinity mutants were selected from small phage display libraries by cell panning under stringent conditions. When converted to an intact fully human antibody, the mutagenized anti-tumor monoclonal antibody displayed an affinity of 0.4 nM, a 15-fold improvement over the affinity of the original antibody. Compared to previously reported affinity maturation schemes, panning on intact cells does not require purified targets for selection and may be particularly useful when the target molecule can not be expressed as a recombinant molecule or easily purified without disrupting its native configuration. In vitro tumor cell killing assays demonstrated an improved performance of the higher-affinity antibody in complement-mediated tumor cell killing. In contrast, the lower-affinity antibody performed somewhat better in antibody-dependent cellular cytotoxicity assays and penetrated better in multicell spheroids of tumor cells, an in vitro model for the tumor penetration capacity of antibodies.

Sural nerve T-cell receptor Vbeta gene utilization in chronic inflammatory demyelinating polyneuropathy and vasculitic neuropathy.

OBJECTIVE: To investigate the utilization of T-cell receptor (TCR) variable (V) regions in infiltrates of sural nerve biopsies of patients with chronic inflammatory demyelinating polyneuropathy (CIDP) and vasculitic neuropathy. BACKGROUND: The presence of infiltrating T lymphocytes in sural nerve biopsies may suggest a T cell-mediated immune mechanism in the pathogenesis of CIDP and vasculitic neuropathy. PATIENTS AND METHODS: The utilization of TCR Vbeta regions in sural nerves of 13 patients with CIDP and five patients with vasculitic neuropathy was determined by immunohistochemistry, reverse-transcription PCR, and nucleotide sequence analysis. These techniques were also applied in four patients with chronic idiopathic axonal polyneuropathy (CIAP) who acted as noninflammatory controls, and in five autopsy controls. RESULTS: The TCR Vbeta utilization of infiltrating T cells in sural nerves of patients with CIDP, vasculitic neuropathy, and noninflammatory controls is heterogeneous. A dominant TCR Vbeta utilization was not found in any of the patients or controls. CONCLUSION: There is no evidence for the presence of clonally expanded T cells in sural nerves of patients with CIDP and vasculitic neuropathy.

Expression of accessory molecules for T-cell activation in peripheral nerve of patients with CIDP and vasculitic neuropathy.

Vasculitic neuropathy and chronic inflammatory demyelinating polyneuropathy (CIDP) are neuropathies characterized by a T-lymphocyte infiltrate in the peripheral nerves. The microenvironment in which these T cells become activated, and the molecules and cells that play a role in this process are incompletely understood. Using immunohistochemical analysis, we studied the effect of the presence of adhesion, costimulatory and antigen-presenting molecules on different cell types as a precondition for local T-cell activation in human sural nerve biopsies of seven patients with CIDP, three patients with vasculitic neuropathy and three healthy controls. In biopsies from CIDP and vasculitic neuropathy patients, but not in those from healthy controls, Schwann cells expressed the adhesion/T-cell stimulatory molecule CD58 (LFA-3). The CD58 molecule was also present on endothelial cells of all vasculitic neuropathy patients and one CIDP patient. In biopsies from normal controls and patients, CD54 (ICAM-1) expression was detectable on microvascular endothelial cells. In addition, expression of the costimulatory molecule CD86 was detected on vascular tissue in patients with vasculitic neuropathy. Although macrophages were always present in all subjects, expression of the major histocompatibility complex (MHC)-like molecule CD1a by macrophages was restricted to biopsies from two CIDP patients and one vasculitic neuropathy patient. Unexpectedly, Schwann cells of a single vasculitis patient strongly expressed CD1b, a molecule involved in the presentation of self-glycolipids to T cells. Schwann cells in biopsies from patients and normal controls expressed high levels of the invariant chain, CD74, a molecule involved in the intracellular sorting of MHC class II molecules. There was no evidence for the presence of dendritic cells in sural nerve biopsies. These findings support a model in which T-cell activation can be initiated and/or perpetuated locally in sural nerve biopsies of patients with CIDP and vasculitic neuropathy, and predict an important role for Schwann cells and endothelial cells.

Monitoring of neutrophil priming in whole blood by antibodies isolated from a synthetic phage antibody library.

Neutrophil activation is a multistep process. In vitro activation of neutrophils with semiphysiological activators is optimal only after preactivation or priming with cytokines, chemotaxins, and/or bacterial products. Until now, no antibodies have been developed that can distinguish between resting and (cytokine) primed neutrophils with a sufficient dynamic range necessary for screening clinical samples. We have isolated two human phage antibodies, designated MoPhab A17 and A27, from a synthetic bacteriophage antibody library. These phage antibodies recognize epitopes that are upregulated on neutrophils present in whole blood treated with low priming concentrations of cytokines, such as GM-CSF and TNF-alpha. This induction was time- and concentration-dependent and optimal at concentrations that are sufficient for priming functional responses in neutrophils: GM-CSF (10 pM) and TNF-alpha (100 IU/ml). PMNs, isolated from the peripheral blood of chronic obstructive pulmonary disease (COPD) patients with a clinical exacerbation, exhibited a partial in vivo primed phenotype. These antibodies promise to be an ideal tool to monitor disease activity in whole blood of patients with inflammatory diseases.

Functional human monoclonal antibodies of all isotypes constructed from phage display library-derived single-chain Fv antibody fragments.

We have constructed a series of eukaryotic expression vectors that permit the rapid conversion of single chain (sc) Fv antibody fragments, derived from semi-synthetic phage display libraries, into intact fully human monoclonal antibodies (mAb) of each isotype. As a model, a scFv fragment specific for sheep red blood cells (SRBC) was isolated from a semi-synthetic phage antibody (Ab) display library, and used to produce human mAbs of IgM, IgG1-IgG4, IgA1, IgA2m(1) and IgE isotype in vitro in stably transfected cells. N-terminal protein sequence analysis of purified immunoglobulin heavy (H) and light (L) chains revealed precise proteolytic removal of the leader peptide. Biochemical analysis of purified recombinant human mAbs demonstrated that properly glycosylated molecules of the correct molecular size were produced. The IgG and IgA mAbs retained SRBC-binding activity, interacted with different Fc receptor-transfectants, and induced complement-mediated hemolysis and Ab-dependent phagocytosis of SRBC by neutrophils in a pattern consistent with the immunoglobulin (Ig) H chain isotype. We conclude that in vitro produced recombinant human mAbs constructed from phage display library-derived scFv fragments mirror their natural counterparts and may represent a source of mAbs for use in human therapy.

A splice variant of human ephrin-A4 encodes a soluble molecule that is secreted by activated human B lymphocytes.

Ephrin-A4 is a ligand for the erythropoietin-producing hepatocellular (Eph) receptor family of tyrosine kinases. We have identified a secreted form of ephrin-A4, denoted ephrin-A4 (s), which is encoded by an alternatively spliced mRNA and is produced by in vivo activated B cells in tonsils. Blood B cells secrete ephrin-A4 (s) upon stimulation via the B-cell antigen receptor. A subpopulation of tonsil cells in the crypts with a dendritic cell phenotype was shown to express EphA2, an Eph receptor tyrosine kinase that was found to be capable of binding an ephrin-A4 immunoglobulin chimeric protein. We conclude that ephrin-A4 (s) may play a role in the interaction between activated B lymphocytes and dendritic cells in human tonsils. (Blood. 2000;95:221-230)

Antitumor immune effector mechanisms recruited by phage display-derived fully human IgG1 and IgA1 monoclonal antibodies.

We have constructed a recombinant, fully human IgA1 monoclonal antibody, UBS-54/IgA1, against the tumor-associated Ep-CAM molecule and compared its tumor-killing capacity with its IgG1 counterpart in in vitro assays. The data show that phage display-derived fully human IgA1 antibodies efficiently recruit immune effector cells that express the Fc receptor for IgA, FcalphaRI (CD89). UBS-54/IgA1-mediated killing of tumor cells by isolated polymorphonuclear cells (PMNs) and in whole blood was found to proceed without the necessity to preactivate effector cells with cytokines. In addition, the IgA1 anti-Ep-CAM human monoclonal antibody (huMab) triggered phagocytosis of tumor cells by monocyte-derived macrophages. Strikingly, simultaneous addition of IgA1 and IgG1 anti-Ep-CAM antibodies did not result in enhancement of tumor cell killing unless the effector cells were stimulated with granulocyte colony-stimulating factor. The lack of an additive effect could be attributed to an inhibitory effect of IgG on IgA-mediated tumor cell killing through binding of IgG1 to the inhibitory FcgammaRIIb receptor expressed by PMNs. These results show that IgA1 antitumor huMabs are capable of recruiting the large population of peripheral blood PMNs for tumor cell killing. This population is not effectively recruited by IgG type antibodies, currently the antibodies most frequently used for clinical application. In addition, the data suggest that a combination of IgG1 and IgA1 antitumor huMabs may collaborate in tumor cell killing in patients treated with granulocyte colony-stimulating factor.

A phage antibody identifying an 80-kDa membrane glycoprotein exclusively expressed on a subpopulation of activated B cells and hairy cell leukemia B cells.

We have isolated a phage display library-derived monoclonal antibody, phab V-3, that identifies a membrane glycoprotein of approximately 80 kDa which is expressed on a subpopulation of activated B lymphocytes in secondary lymphoid organs. In agreement with their activated phenotype, phab V-3(+) B cells display a blast-like morphology, and are prone to spontaneous apoptosis in vitro, unless rescued by stimulation with CD40 ligand (CD40L). The expression of the phab V-3 molecule coincides with B cells that produce high levels of IgM, IgG and IgA in vitro upon stimulation with CD40L in combination with IL-2 and IL-10. Immunofluorescent analysis of B cell malignancies unveiled that the phab V-3 molecule was uniquely expressed on hairy cell leukemia (HCL) B cells. Similar to phab V-3(+) tonsils B cells, HCL B cells have been reported to express CD11c, CD95 and CD27, which might indicate that the phab V-3(+) B cells in HCL are the malignant counterpart of the phab V-3(+) B cell subpopulation.

Limitations of the semisynthetic library approach for obtaining human monoclonal autoantibodies to the thyrotropin receptor of Graves' disease.

Graves' disease (GD) is characterized by the presence of autoantibodies against the TSH-receptor (TSH-R) which are pathogenic and, upon binding to the receptor, trigger intracellular signal transduction. The autoantibodies are oligoclonal and as they are responsible for disease activity, their characterization would lead to a better understanding of the development of GD. Attempts to isolate anti-TSH-R antibodies from patients have proved to be difficult due to the exceedingly low serum levels due to rarity of these B cells, together with difficulties in obtaining purified TSH-R capable of interacting with patients autoantibodies. We employed phage antibody display technology and performed selection with a previously characterized semisynthetic antibody library on the purified extracellular ectodomain of the TSH-R. We report the isolation of six different anti-TSH-R monoclonal phage antibodies (moPhabs) from this library. All the moPhabs recognized TSH-R and its recombinant fragments by Western blotting, but failed to recognize the native TSH-R by flow cytometry. Consequently, the moPhabs did not lead to TSH-R activation. As these were the first moPhabs to TSH-R, they were analysed in terms of nucleotide and amino acid sequence and epitope specificity on the receptor. The moPhabs used immunoglobulin VH1 and VH3 germ line genes, all associated with Vlambda3 genes. Interestingly, the CDR3 regions of all moPhabs were remarkably similar, though not identical. In light of the common CDR3 usage, the epitopes recognized on TSH-R appeared to be restricted to amino acids residues 405-411 and 357-364. In summary, our results show that semisynthetic libraries may be limited in isolating human monoclonal antibodies that resemble pathogenic antithyrotropin receptor autoantibodies present in patients with GD. It is likely that until preparations of purified TSH-R that can be recognized by patients autoantibodies become available, similar to the recently described glycosylphosphatidylinositol (GPI) anchored TSH-R ectodomain, monoclonal antibodies from phage antibody display to TSH-R will be limited for isolating the rare, pathogenic antibodies of GD.

Leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is differentially expressed during human B cell differentiation and inhibits B cell receptor-mediated signaling.

Leukocyte-associated Ig-like receptor-1 (LAIR-1) belongs to the growing family of immunoreceptor tyrosine-based inhibitory motif-bearing receptors and is expressed on the majority of peripheral mononuclear cells, including NK cells, T cells, B cells, monocytes, and dendritic cells. In this study, we investigated the distribution and the capacity of LAIR-1 to function as an inhibitory receptor on human B cells. LAIR-1 is expressed from early on during B cell differentiation, but is absent on approximately half of the memory B cells, and all germinal center B cells, plasmablasts, and terminally differentiated plasma cells. In vitro stimulation of naive B cells via the B cell receptor (BCR) or CD40, triggering proliferation and differentiation into Ig-producing plasma cells, is accompanied by loss of LAIR-1 expression. We previously reported that LAIR-1 can function as an inhibitory receptor on NK cells and T cells. Here, we demonstrate that it can also function as a negative regulator of BCR-mediated signaling, since simultaneous cross-linking of LAIR-1 and the BCR reduces the increase of intracellular Ca(2+) evoked by BCR ligation. Taken together, this suggests that the inhibitory mechanism of LAIR-1 is functional in multiple components of the hematopoietic system.

New strategies in anti-vascular cancer therapy.

Angiogenesis is a critical step in the progression of tumours from dormancy to a clinical relevant cancer. Inhibition of this process is one of the most promising new anti-cancer strategies. To develop new drugs that interfere with the cascade of events required for the formation of new blood vessels, insight into this process is essential. Here, we discuss the molecular basis of angiogenesis and the concepts of vascular targeting. Furthermore new strategies will be discussed to discover surface markers on endothelial cells that confer sufficient specificity for targeted intervention in the tumour vasculature.

Dissecting the human peripheral B-cell compartment with phage display-derived antibodies.

Previously we have employed a large semisynthetic phage antibody display library, in combination with subtractive selection by flow cytometry to isolate phage antibodies specific for subpopulations of leucocytes. In this study, human tonsillar B cells were incubated with the phage library and IgD- CD38- memory B lymphocytes and attached phage antibodies were selected by cell sorting. In a panel of 17 monoclonal phage antibodies obtained, five displayed binding to cells of multiple haematopoietic lineages or broadly reacted with B-lineage cells. Immunofluorescent, immunohistochemical and biochemical studies permitted the characterization of the target molecules recognized by these phage antibodies. The remaining 12 antibodies displayed restricted binding to small subpopulations of peripheral human B cells. These results show that subtractive selections with phage antibody display libraries in combination with flow cytometry yield antibodies that bind to differentially expressed molecules on closely related cell populations, and can be used as a tool in a variety of assays.

The diagnostic value of sural nerve T cells in chronic inflammatory demyelinating polyneuropathy.

BACKGROUND: T-cell infiltrates in sural nerve biopsy specimens of patients with inflammatory neuropathies have been reported, suggesting a role for T cells in the pathogenesis, but the specificity of the presence and localization of sural nerve T cells in chronic inflammatory demyelinating polyneuropathy (CIDP) is unknown. OBJECTIVE: To study the diagnostic value of the number and distribution of sural nerve T cells in CIDP. METHODS: We performed a quantitative immunohistochemical examination of T cells in sural nerve biopsy specimens taken from 23 patients with a CIDP and compared them with sural nerves of 15 patients with a chronic idiopathic axonal polyneuropathy (CIAP), 5 patients with a vasculitic neuropathy, and 10 normal controls. RESULTS: T cells were found in sural nerves of all CIDP patients as well as in all disease and normal controls. Only six CIDP patients had increased numbers and densities of T cells compared with CIAP patients and controls. Based on the distribution of endoneurial or epineurial T cells, it was not possible to differentiate CIDP patients from CIAP patients or normal controls. In patients and controls perivascular epineurial T cells predominated. Increased numbers and densities of sural nerve T cells in patients with CIDP were associated with female sex, a more severe disease course, worse outcome, highly elevated CSF protein level, and a larger sural nerve area, but not with loss of myelinated nerve fibers in the sural nerve biopsy sample or demyelinating features on electrophysiologic examination. CONCLUSIONS: In the majority of CIDP patients, the number and distribution of T cells in sural nerve biopsy samples were similar to patients with noninflammatory neuropathies and normal controls. Only large numbers of sural nerve T cells are specific for inflammatory neuropathies and therefore of diagnostic value for CIDP.

Synergy between tumor suppressor APC and the beta-catenin-Tcf4 target Tcf1.

Mutations in APC or beta-catenin inappropriately activate the transcription factor Tcf4, thereby transforming intestinal epithelial cells. Here it is shown that one of the target genes of Tcf4 in epithelial cells is Tcf1. The most abundant Tcf1 isoforms lack a beta-catenin interaction domain. Tcf1(-/-) mice develop adenomas in the gut and mammary glands. Introduction of a mutant APC allele into these mice substantially increases the number of these adenomas. Tcf1 may act as a feedback repressor of beta-catenin-Tcf4 target genes and thus may cooperate with APC to suppress malignant transformation of epithelial cells.