Frontiers in clinical immunology 2008.

In recent years, investigations in immunology have led to progress in clinical medicine, including understanding transplant rejection, autoimmune diseases, immune deficiencies, inflammation, transplantation, cancer and the development of new vaccines. At a meeting recently held on the Mediterranean shore, advances in several facets of clinical immunology were the focus of discussion. Here, we highlight some of the debates that reflected advances in a variety of human immune disorders.

A novel tissue engineering-based assay for immunological infertility.

Antisperm antibodies (ASA) are present in 9-36% of infertile couples, a condition called immunological infertility. The variability of ASA in terms of antigenic specificity and biological effects has made it difficult to design a test able to distinguish reliably between ASA that contribute to infertility and those that do not. To develop a reliable and reproducible method able to detect sperm antibodies, we took advantage of recent progress made in tissue engineering techniques. We used collagen gel as a bio-scaffold for the production of engineered sperm analogues. The advantages of using collagen gels include biocompatibility, ease of fabrication and low cost. We found that this tissue engineering-based assay is more specific and more sensitive than a conventional test routinely used for ASA detection. In addition, it exhibited low intra- and inter-variations. We envision the use of this novel approach for the detection of a variety of autoantibodies in autoimmune diseases. In addition to diagnostic purposes, tissue-engineering based tests could be useful in monitoring treatments with bio-drugs.

Paraneoplastic autoimmunity.

Loss of homeostasis is a hallmark of malignant tumorigenesis and autoimmune diseases. Recent studies have implicated apoptotic cell death pathways in initiating and propagating autoimmune diseases in susceptible individuals. During malignancy, however, there is an accumulation of cells resistant to apoptosis. Intriguingly, some patients with malignant tumors develop symptoms that cannot be explained solely on the basis of the effects produced by either the primary tumor or its metastases. The mechanisms responsible for these complex symptoms, known as paraneoplastic autoimmunity, remain the focus of investigation.

Silent antibodies.

Over the past years, progress has been made in understanding B cells and antibody recognition functions, particularly in the context of autoimmune diseases. In addition to the existence of "natural antibodies", recent studies suggest the existence of immunoglobulins with no apparent specificity that may acquire polyreactivity following a mild denaturation in inflammatory sites. They are called "silent antibodies". Together with related observations on B cell development, selection and signaling, the recent insights are providing clues into our understanding of autoimmunity.

[Werner's syndrome and endocrine disorders]. / Syndrome de Werner et atteintes endocriniennes.

Werner's syndrome is a rare autosomal recessive disease caused by the mutation of DNA helicase gene (WRN), characterized by the premature onset of multiple age-related disorders and skin changes similar to those observed in scleroderma. Some endocrinologic and metabolic disorders have been described in patients with Werner's syndrome. We report one case in a 41-year-old man issuing from consanguineous parents, who presented for exploration of hypoglycemic episodes and sexual impotence. Werner's syndrome was diagnosed on the basis of his characteristic clinical appearance. Metabolic disorders were insulin-requiring diabetes and hypertriglyceridemia. Endocrinologic investigation revealed nodular goiter, sub clinical primary hypothyroidism, hypergonadotrophic hypogonadism,adrenal cortical hypofunction and GH deficiency. Pathology examination of the skin biopsy showed a scleroderma-like aspect. Finally, osteoporosis, atherosclerosis and sub-capsular cataract were associated. Thus, in Werner's syndrome metabolic and endocrinologic investigation is necessary in order to treat these disorders and improve the patient's prognosis and life.

Probing the specificity of human myeloma proteins with a random peptide phage library.

Human myeloma proteins (HMPs) from 10 patients with multiple myeloma (MM) were used to affinity-select peptides from a random phage-display peptide library. Binding peptides were identified for the 10 analysed antibodies (eight, immunoglobulin G (IgG), and two, immunoglobulin A (IgA)). The specificity of the binding was confirmed by competitive experiments using phages and chemically synthesized peptides. Interestingly, some phage-displayed peptides were immuno-selected with HMPs isolated from different patients. Sequence alignments and homology searches revealed a significant homology with human proteins (e.g. neural cell adhesion proteins) and pathogen-derived proteins (e.g. herpes simplex virus capsid proteins). The selected peptides could be useful as targeting agents for myeloma cells expressing surface immunoglobulins.

Subversion of B lymphocyte signaling by infectious agents.

Infectious agents and their hosts interact in a complex manner, involving not only superficially apparent mechanisms, but also the signaling machinery that governs host cells responses. Thus, signaling events, surface molecule expression, and transcriptional control may be affected in various cell types, with profound consequences for the function of individual cells and organ systems. Studies of the biochemistry of cell signaling and cell invasion by infectious agents have begun to detail the interplay between elements of infectious organisms and the host at the molecular level. Consequently, the resulting interferences with lymphocyte signaling may disturb the function of the immune system. In B cells, alterations of immune receptor signaling has implications for human diseases. By affecting the mechanisms of the host's immune defense, this may not only lead to inadequate elimination of an infectious agent, but also to autoimmunity or neoplasia.

Receptor revision and systemic lupus.

Studies over the past 10 years have shown that B cells can undergo secondary heavy- or light-chain immunoglobulin (Ig) rearrangements at various stages of their normal development, a process termed receptor editing. In the bone marrow, this mechanism is important to maintain tolerance because it can extinguish a self-reactive specificity without having to physically eliminate a potentially autoreactive B cell. In the periphery, secondary rearrangements may also play a role in the diversification and maturation of an immune response, although conclusive evidence for this process is still required. Individuals with systemic autoimmune diseases, such as lupus, show evidence of intricate abnormalities in receptor editing. On the one hand, decreased editing may not eliminate the self-reactive specificities that emerge during B-cell development in the bone marrow. Conversely, excessive secondary rearrangements, especially in the periphery where tolerance mechanisms are less effective, can result in the production of autoantibodies by edited B cells. It will be important to assess whether the complex editing defects observed during lupus are a primary susceptibility factor to this disease or if they are secondary to other abnormalities of lymphocyte development in these autoimmune patients.

Expression of B cell receptor-associated signaling molecules in human lupus.

B cell receptor (BcR) signaling requires a tight regulation of several protein tyrosine kinases and phosphatases, and associated co-receptors. Mounting evidence indicates that abnormal BcR signaling, such as occurs in SHP-1 and Lyn-deficient mice, results in production of pathogenic autoantibodies and lupus-like glomerulonephritis, suggesting that altered signaling thresholds could underlie the development of systemic autoimmunity. To test this hypothesis, we investigated expression of BcR-associated signaling molecules in lymphocytes from patients with systemic lupus erythematosus (SLE) during inactive phases of the disease. We found that the transmembrane regulatory protein tyrosine phosphatase CD45 is expressed at abnormal levels. Strikingly, this reduction persisted during four months of follow-up. By contrast, despite its potent role as a regulator of thymus-independent immune responses and of B cell life span, the CD22 co-receptor is expressed at normal levels in B lymphocytes isolated ex vivo from SLE patients. We also noted unusual levels of the cytosolic protein tyrosine kinase Lyn and the protein tyrosine phosphatase SHP-1 in the lymphocytes of the patients. Since in normal B cells Lyn and SHP-1 act in concert within a common negative pathway in which CD45 counteracts SHP-I regulatory role, we propose that this feedback regulatory pathway is crippled to different degrees in human SLE B cells. Break of the balance between positive and negative signaling molecules likely modifies the BcR signaling thresholds. Such alterations, together with other factors, may contribute to the disruption of self-tolerance in this disease.

B cell receptor signaling and autoimmunity.

The immune receptors of lymphocytes are able to sense the nature of bound ligands. Through coupled signaling pathways the generated signals are appropriately delivered to the intracellular machinery, allowing specific functional responses. A central issue in contemporary immunology is how the fate of B lymphocytes is determined at the successive developmental stages and how the B cell receptor distinguishes between signals that induce immune response or tolerance. Experiments with mice expressing transgenes or lacking signal transduction molecules that lead to abnormal lymphocyte development and/or response are providing important clues to the mechanisms that regulate signaling thresholds at different developmental stages. The studies are also revealing novel potential mechanisms of induction of autoimmunity, which may have a bearing on the understanding of human diseases.

Molecular determinants of the human antibody response to HIV-1: implications for disease control.

Various aspects of the immune response to HIV-1 infection remain unclear. While seropositive subjects generally mount a strong humoral response, the antibodies produced are not effective in halting disease progression. Molecular characterization of the antibody repertoire specific for HIV-1 antigens represents an approach to further our understanding of the mechanisms involved in mounting a humoral immunity in this infection. Recently, the content, structure, and organization of the human immunoglobulin-variable gene loci have been elucidated and a number of laboratories have characterized the variable gene elements of human anti-HIV-1 antibodies derived from infected persons by cell fusion or by Epstein-Barr virus transformation. The results show evidence for extensive somatic mutations that lead to preferential amino acid substitutions in the hypervariable regions, an indication of an antigen-driven process. Multiple other molecular events also are engaged in generating antibody diversity, including various types of fusions of variable genes, usage of inverted diversity genes, and addition of extragenomic nucleotides. Most importantly, there is a paucity of antibodies expressing the major V(H)3 gene family, which could result from the capacity of gp120 to act as superantigen for human B cells. This V(H)3+ antibody deficit also has been observed in B cells isolated ex vivo from the patients. Since V(H)3+ antibodies play an essential role in immune defense against infections, the abnormalities observed in HIV-1 infection may predispose to opportunistic infections and further compromise the immune defense mechanisms of the subjects.

B cell tolerance to self in systemic autoimmunity.

After a century of research and despite intensive scrutiny, the origin of autoantibody production remains an enigma. Recently, the essential role of B cells in promoting systemic autoimmunity in mice seems more important than previously thought: self-reactive B cells can be subject to positive selection and a deficiency in serum IgM predisposes to the development of IgG antibodies to autoantigens. Studies of the B cell repertoire expressed in systemic autoimmune diseases have provided important clues. In human lupus, quantitation of this repertoire reveals the presence of an expansion of IgG clonotypes that impart reactivity with disease-related autoantigens. The nucleotide sequences of autoantibodies derived from these patients and expressing nephritogenic idiotopes (present in immune complexes and renal eluates of subjects with active disease) show features of diversification with a high rate of replacement/silent mutations and clustering of the mutations in the hypervariable regions, suggesting than an antigen-driven process plays a role in the generation of pathogenic autoantibodies. Currently, the contributions of apoptosis and of cell receptor signaling to this triggering are being appreciated. Pursuing these and related issues will have an important impact on autoimmune research.

Tracking immunoglobulin variable-gene expression in HIV infection.

B-cell superantigens (SAgs) interact with normal human nonimmune immunoglobulins (Igs) independently of the light-chain isotype, and activate a large proportion of the B-cell repertoire. Recently, the major envelope protein of human immunodeficiency virus 1 (HIV-1), gp120, was found to exhibit SAg-like properties for B cells with potential pathological consequences for the infected host, including accelerated apoptosis and progressive loss of B cells. This unconventional mode of interaction contrasts with its binding to immunization-induced antibodies, which requires the tertiary structure of the heavy- and light-chain variable regions. Examining the temporal development of V(H)3+ antibodies in HIV-1-infected subjects over a 7-yr period showed that V(H)3+ antibodies specific for the gp120 SAg-binding site are deficient. Quantification of V(H)3+ antibodies, which impart protective responses to infectious agents, in serum samples from HIV-seropositive slow progressors and from patients who progressed to AIDS-related manifestations reveals that paucity in V(H)3+ antibodies is a marker of rapid clinical decline. Remarkably, anti-gp160 V(H)3+ antibodies show a gradual decrease in progressors and vary with time, depending on the viral load. Thus, V(H)3+ antibodies could play an important role in protection, and their underexpression may accelerate disease progression. Investigation of the structural basis of the interaction between human Igs and gp120 shows that the viral gp120 SAg can interact only with a subset of human V(H)3+ Igs. A number of amino acid-positions present primarily in the first and third framework regions of the Ig heavy-chain variable regions correlate with gp120 binding. These residues partially overlap with the Staphylococcus aureus protein A-binding site for V(H)3+ Igs. Overall, these interactions could represent a novel mechanism of humoral deficiency resulting from the capacity of a viral SAg to impact an important subset of the B-cell repertoire and to induce B-cell death by apoptosis.

Analysis of immunoglobulin VH and TCR cbeta polymorphisms in a large family with thyroid autoimmune disorder.

In order to investigate the association of TCR Cbeta and immunoglobulin (Ig) VH polymorphisms with thyroid autoimmune diseases (TAD), we analyzed restriction-endonuclease-generated polymorphisms using T-cell receptor (TCR) Cbeta and VH gene-family-specific probes. We tested genomic DNAs of patients isolated from a large family affected with Graves' disease and Hashimoto's thyroiditis as well as the genomic DNA of unrelated Tunisian controls. Hybridization of BglII-digested DNA with a TCR Cbeta probe revealed two alleles of 9.2 and 10 kb. These Cbeta polymorphisms have already been found in the Caucasian population. However, there was no abnormal distribution of this polymorphism in patients with TAD, compared to related healthy individuals and to unrelated Tunisian controls. Besides, there was a low VH polymorphism in members of the family affected with TAD. Analysis of the Ig gene families revealed no restriction site polymorphism pattern specific for TAD.

Abnormal DNA methylation and deoxycytosine-deoxyguanine content in nucleosomes from lymphocytes undergoing apoptosis.

Systemic lupus erythematosus (SLE) is characterized by an accelerated apoptosis of peripheral lymphocytes and an impairment of the clearance of apoptotic cells. Since changes in DNA methylation and in deoxycytosine and deoxyguanine (GC) content have been shown to enhance the potential of DNA to activate murine and human B lymphocytes, we tested the capacity of lymphocytes undergoing apoptosis (under conditions that mimic the deletion of self-reactive cells after antigen receptor engagement) to generate nucleosomes with a particular base composition. Using two cell culture systems and four apoptosis triggers, we found an increase of deoxymethylcytosine in fragmented chromosomal DNA of apoptotic B and T lymphocytes. However, this increase was not associated with modulation of DNA (cytosine-5) methyltransferase, the enzyme that methylates eukaryotic DNA, which suggests that the changes in DNA methylation patterns are not linked to the process of de novo DNA methylation during cell death. In addition, we could not detect a unique methylation pattern in highly repetitive Alu sequences present in the human genome of SLE subjects, as compared with controls. However, the abnormal DNA methylation of apoptotic nucleosomes was associated with an unusual pattern of nuclease-resistant, GC-rich regions in these DNA fragments. We propose that the combination of an accelerated apoptosis with a defect in the clearance of apoptotic cells results in release of increased amounts of nucleosomes with abnormally methylated, GC-rich DNA and provides an autologous stimulation that could bypass tolerance to self in systemic autoimmune diseases. These findings support the concept that the structure and dynamics of nucleosomes are critical in determining their immunogenicity in SLE.

Structural basis of the gp120 superantigen-binding site on human immunoglobulins.

B cell superantigens (SAg) interact with normal human nonimmune Igs (Igs), independently of the light chain isotype, and activate a large proportion of the B cell repertoire. Recently, the major envelope protein of HIV-1, gp120, was found to exhibit SAg-like properties for B cells with potential pathologic consequences for the infected host. This unconventional mode of interaction contrasts with its binding to immunization-induced Abs, which requires the tertiary structure of the heavy and light chain variable regions. In this report, we have examined the structural basis of the interaction between human Igs and gp120. We found that gp120 binding is restricted to Igs from the V(H)3 gene family and that the two V(H) genes 3-23 and 3-30, known to be overutilized during all stages of B cell development, frequently impart gp120 binding. We also provide evidence that the viral gp120 SAg can interact with only a subset of the human V(H)3+ Igs that can convey binding to the prototypic bacterial B cell SAg protein A from Staphylococcus aureus. Finally, we have identified amino acid positions present primarily in the first and third framework regions of the Ig heavy chain variable region, outside the conventional hypervariable loops, which correlate with gp120 binding. In a three-dimensional sequence-homology model, these residues partially overlap with the predicted SAg protein A binding site for V(H)3+ Igs.

High-density expression of CD95 on B cells and underrepresentation of the B-1 cell subset in human lupus.

Recent evidence indicates that B cell receptor signaling plays a role in the generation of the B-1 cell lineage that expresses the CD5 marker, and the CD95-mediated death plays an essential role in maintaining B cell tolerance. We therefore probed CD5 and CD95 expression on B cells from systemic lupus erythematosos (SLE) patients and control subjects. Firstly, in agreement with previous studies, we found that CD5 expression (11%) was relatively constant among control individuals. We also noted that the activation of B cells up-regulates this marker. Unexpectedly, we found that the B-1 cell subset is under-represented (3.9+/-0.3%) in SLE patients in an inactive stage of the disease. Together with related studies, these findings suggest that there is a correlation between CD5 expression and disease activity. Secondly, we found that CD95(+) B cells can be divided into two subsets expressing a high- (CD95(high)) and a low-density (CD95(low)) of CD95. There was no difference in the proportion of total CD95(+) B cells (23.5+/-2.8) in the two groups, but SLE patients in an inactive phase of the disease characteristically expressed a relatively high proportion (50%) of CD95(high) B cells. This finding would mean that a large fraction of B lymphocytes are sensitive to apoptosis, implying that autoantibody-producing B cells are derived from CD95(low) B cells and are relatively resistant to apoptosis.