The B cell repertoire

The hallmark of the immune system is its ability to produce a seemingly infinite variety of antigen-binding receptors. This is made possible by molecular and cellular mechanisms uniquely suited to continuously generate a large number of individual receptor molecules and to select some for further expansion. The well-studied genetic rearrangement that results in the juxtaposition of germ line-encoded variable, diversity, and joining elements remains the foundation for diversification on which the repertoire is built. Many of the rules that regulate this phenomenon have been described, although the underlying enzymatic machinery responsible for these events remains to be elucidated. Recent progress in categorizing the immunoglobulin heavy-chain variable region genes into families as well as studies establishing their utilization in both fetal and adult life is helping to further refine these rules. Subsequent cellular interactions 1) permit the discriminant expansion of clones expressing relevant antibody molecules, 2) allow the active affinity alterations needed for effective ongoing immune responses, and 3) limit the potential deleterious effect of autoreactive cells.     

Shaping of the autoreactive regulatory T cell repertoire by thymic cortical positive selection

The main function of regulatory T lymphocytes is to keep autoimmune responses at bay. Accordingly, it has been firmly established that the repertoire of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) is enriched in autospecific cells. Differences in thymic-positive and/or -negative selection may account for selection of the qualitatively distinct regulatory and conventional T cell (Tconv) repertoires. It has previously been shown that precursors for Tregs are less sensitive to negative selection than Tconv precursors. Studies with TCR/ligand doubly transgenic mice suggested that an agonist ligand might induce positive selection of Treg (but not Tconv) cells. However, massive deletion of Tconv (but not Treg) cell precursors observed in these mice renders interpretation of such data problematic and a potential role for positive selection in generation of the autospecific Treg repertoire has remained therefore incompletely understood. To study this important unresolved issue and circumvent use of TCR/ligand-transgenic mice, we have developed transgenic mice expressing a single MHC class II/peptide ligand on positively selecting thymic cortical epithelial cells. We found that functional Treg (but not Tconv) cells specific for the single ligand were preferentially selected from the naturally diverse repertoire of immature precursors. Our data therefore demonstrate that thymic cortical positive selection of regulatory and Tconv precursors is governed by distinct rules and that it plays an important role in shaping the autoreactive Treg repertoire.     

Expression of human endogenous retroviruses in pemphigus vulgaris patients

Molecular Biology Reports - Pemphigus is a severe, potentially life-threatening autoimmune blistering mucocutaneous disease which establishes with autoreactive IgG antibodies that target cellular...     

T cell recognition of desmoglein 3 peptides in patients with pemphigus vulgaris and healthy individuals

Pemphigus vulgaris is a severe autoimmune disease caused by autoantibodies against the cutaneous adhesion molecule, desmoglein 3 (Dsg3). The aim of this study was to characterize the specificity of autoreactive Th cells, which presumably regulate Dsg3-specific autoantibody production. Ninety-seven Th1 and Th2 clones isolated from 16 pemphigus patients and 12 HLA-matched healthy donors recognized the Dsg3 peptides, DG3(78-94), DG3(96-112), DG3(189-205), DG3(205-221), and DG3(250-266). Peptide DG3(96-112), and to a lesser extent DG3(250-266), was recognized by the majority of T cells from patients and healthy donors in association with HLA-DRB1*0402 and DQB1*0503 which were prevalent in the pemphigus patients and Dsg3-responsive healthy donors. Analyzing the Vbeta-chain of the TCR of the DG3(96-112)-specific T cells showed no restricted TCR usage. Peptides DG3(342-358) and DG3(376-392) were exclusively recognized by T cell clones (n=13) from patients while DG3(483-499) was only recognized by T cell clones (n=3) from a healthy donor. All Dsg3 peptides contained conserved amino acids at relative positions 1, 4, and 6; amino acids with a positive charge at position 4 presumably represent anchor motifs for DRB1*0402. These findings demonstrate that T cell recognition of distinct Dsg3 peptides is restricted by distinct HLA class II molecules and is independent from the development of pemphigus vulgaris.     

Tolerance and B cell repertoire establishment

We have probed the mechanism by which immature B cells are uniquely susceptible to antigen-induced inactivation. Our studies have demonstrated that this tolerance trigger is an active process that requires both energy metabolism and the biosynthesis of various macromolecules, including protein, RNA, and DNA. However, the tolerance trigger is resistant to inhibitors of patching and capping, as well as an inhibitor of mitosis. The tolerance trigger requires a high-affinity interaction between a multivalent antigen and the cells' Ig receptor, but apparently does not require interactions with other cell surface molecules, or interactions with T cells or macrophages. Our efforts to demonstrate the physiological applicability of this tolerance trigger have concentrated on an attempt to demonstrate potentially self-reactive cells within the immature bone marrow population that do not appear in the mature splenic B cell population. To date we have identified prereceptor B cells of several specificities whose frequency is much lower in the spleen and whose elimination appears to involve tolerance rather than antiidiotypic regulation. However, the demonstration that such cells are eliminated by contact with self-antigens has not as yet been accomplished.     

Dichotomy of autoreactive Th1 and Th2 cell responses to desmoglein 3 in patients with pemphigus vulgaris (PV) and healthy carriers of PV-associated HLA class II alleles

Pemphigus vulgaris (PV) is the most severe autoimmune bullous skin disorder and is primarily associated with circulating autoantibodies (autoAb) against desmoglein 3 (Dsg3). In light of recent evidence that autoreactive T cells are critical for the induction and regulation of Ab production, the goal of this study was to characterize and quantitate autoreactive T cells in patients with PV and healthy controls. Peripheral Dsg3-reactive Th cells from 28 patients with acute-onset, chronic active, and remittent PV were quantitated by MACS secretion assay. Dsg3-reactive Th2 cells were detected at similar frequencies in all studied PV patients, while the number of autoreactive Th1 cells exceeded those of Th2 cells in chronic active PV. In contrast, healthy carriers of the PV-associated HLA class II alleles, DRB1*0402 and DQB1*0503, exhibited exclusively Dsg3-reactive Th1 cell responses, while healthy carriers of other HLA class II alleles did not. Moreover, the presence of IgG1 and IgG4 against Dsg3 was directly related to the ratio of Dsg3-reactive Th1/Th2 cells. T cell recognition of Dsg3 was restricted by HLA-DRB1*0402 and DQB1*0503 in PV patients and Dsg3-responsive healthy donors. These observations strongly suggest 1) that the appearance of Dsg3-reactive Th2 cells is restricted to patients with PV; 2) that specific HLA class II alleles that are prevalent in PV are critical for T cell recognition of Dsg3 in PV patients and Dsg3-responsive healthy donors; and 3) that autoAb production is associated with both Th1 and Th2 cells.     

Heterogeneity of the human phosphocholine-specific B cell repertoire

We have utilized a highly efficient method of culturing small numbers of Epstein Barr virus (EBV)-infected cells to analyze the heterogeneity of human antibodies specific for phosphocholine (PC). Lymphocytes from peripheral blood or tonsils of individuals who had no evidence of recent pneumococcal infection were infected with EBV and cultured at limiting dilution. After correction for the cloning efficiency, between 1/1500 and 1/10,000 B cells produced specific anti-PC antibodies by our criteria. Examination of the heterogeneity of these antibodies revealed that most individuals had an overwhelming predominance of anti-PC antibodies with kappa-light chain. Fine specificity analysis of 39 monoclonal anti-PC antibodies demonstrated that the IgM antibodies examined displayed significant binding site diversity, whereas the IgA PC-specific clones were much less heterogeneous. In general, the human anti-PC antibodies had a much higher relative affinity (Krel) for choline and glycerophosphocholine than the murine antibody families. Through examination of the human PC-specific B cell repertoire we have drawn some interesting parallels with the well-defined murine clonotype families and have begun to dissect the human response to this naturally occurring antigenic determinant.     

Possible apoptotic mechanism in epidermal cell acantholysis induced by pemphigus vulgaris autoimmunoglobulins

Through a still unclear mechanism, pemphigus vulgaris autoantibodies (PV-IgG) induce intra-epidermal acantholytic lesions responsible for severe to fatal skin wounding. We present evidence that PV lesions contain apoptotic keratinocytes, and that cell death is induced in the lesional tissue apparently before cell separation. These data suggest that apoptosis could be the cause of the acantholytic phenomenon. We show that PV-IgG and an antibody against Fas receptor (anti-FasR) induce lesions in vitro in a similar way, causing: (1) secretion of soluble FasL; (2) elevated cellular amounts of FasR, FasL (soluble and membranal), Bax and p53 proteins; (3) reduction in levels of cellular Bcl-2; (4) enrichment in caspase 8, and activation of caspases 1 and 3; (5) co-aggregation of FasL and FasR with caspase 8 in membranal death-inducing signaling complex (DISC). Hence, the Fas-mediated death signaling pathway seems to be involved in lesion formation. Moreover, we have shown that in skin organ cultures and in keratinocyte cultures, PV-IgG can induce caspase activation and DNA fragmentation, and caspase inhibitors can prevent the formation of PV-IgG-induced epidermal lesions. Altogether, these results suggest that PV-IgG-induced acantholysis may proceed through the death-signaling pathway. They highlight new perspectives on mechanisms of tissue damage in autoimmune diseases.     

mTOR pathway regulates the differentiation of peripheral blood Th2/Treg cell subsets in patients with pemphigus vulgaris

Pemphigus vulgaris (PV) is a chronic and potentially life-threatening autoimmune blistering disease.Aberrant mTOR pathway activity is involved in many autoimmun...     

Quantitative clonal analysis of the B cell repertoire in human lupus

To gain further insight into the origin of autoantibody hyperproduction in human lupus, we quantitated the B cell repertoire toward exogenous and self-antigens. Using the Spot-ELISA method and two panels of nine exogenous and 10 self-antigens, we found that the normal human immune repertoire comprises a high frequency of B cell precursors secreting IgM antibodies to self- and exogenous determinants. This repertoire was markedly deficient in precursors producing IgG able to bind self-antigens. In lupus patients, the absolute numbers of clone precursors of the immune repertoire expressing IgM receptors whose paratopes impart affinity to self- and exogenous determinants were higher than in control individuals. Additionally, IgG antibody-forming cell precursors with binding specificity for lupus-associated antigens were detectable in the repertoire of these patients. Based on these results, we propose that hyperproduction of human lupus-associated autoantibodies arises in a two-stage mechanism whereby a general activation of the multireactive immune B cell repertoire precedes an oligospecific expansion of selected B cell clonotypes.     

Changes in desmoglein 1 expression and subcellular localization in cultured keratinocytes subjected to anti-desmoglein 1 pemphigus autoimmunity

The complexity of pemphigus acantholysis together with the weak expression of desmoglein 1 (Dsg1) in cultured keratinocytes have made the study on the pathogenic action of anti-Dsg1 antibodies quite difficult. The pathophysiology of the acantholytic phenomenon could depend on the reduction of Dsg1 adhesion function occurring after its massive internalization or decrease of its synthesis. Here, we have investigated this hypothesis by using sera of patients having antibodies against Dsg1 or monoclonal anti-Dsg1 antibodies to simulate pemphigus autoimmunity in Dsg1-rich keratinocytes. Similar to pemphigus foliaceus (PF) and vulgaris (PV) sera, monoclonal anti-Dsg1 antibodies induced transient internalization of Dsg1 and reduced the adhesion strength among keratinocytes. However, binding of IgG to Dsg1 did not determine its early depletion from the adhesion complexes but reduced the amount of Dsg1 found in the Triton X-100 soluble pool of proteins. Taken together, our results represent the first demonstration that anti-Dsg1 antibodies induce similar alterations on the subcellular distribution of Dsg1 irrespective of the disease where they come from. Furthermore, the present study provides insight into the mechanisms underlying epithelial blistering observed in the skin type of pemphigus.     

The diversity of the secondary Salmonella typhimurium-specific B cell repertoire

This report describes the first analysis of the expressed B cell repertoire specific for a bacterium. In this study, responses to an acetone-killed and dried preparation of Salmonella typhimurium strain TML (AKD-TML) are described. The results show that AKD-TML can stimulate splenic B cells from primed CBA/Ca mice over a wide dose range. The average frequency of secondary TML-specific B cells is 16.4 per 10(5) splenic B cells. This frequency is similar to that observed for another complex, natural antigen, the hemagglutinin of influenza virus. The majority of all secondary TML-specific B cells (greater than 70%) secrete immunoglobulin M, but most of these clones also secrete other isotypes of which immunoglobulins G2 and A are the most prevalent. Analysis of the specificity of secondary TML-specific B cells showed that the vast majority of these B cells were specific for the lipopolysaccharide (LPS) molecule. Moreover, fine specificity analysis demonstrated that approximately two-thirds of these anti-LPS-specific B cell clones are directed against the core polysaccharides or lipid A regions of the LPS molecule, while only about one-third are directed toward the O antigen region. Since anti-S. typhimurium serum antibodies are directed primarily against the O antigens, these studies suggest that the serum levels of antibodies to a given epitope on a bacterial antigen may not be a true reflection of the expressed B cell repertoire when analyzed at the single B cell level. These studies also suggest that the role of antibodies to lipid A molecules in the development of protective immunity to S. typhimurium be reevaluated.     

Intermittent cyclophosphamide in pemphigus vulgaris and bullous pemphigoid

Cyclophosphamide, given in widely spaced doses, was used in the treatment of a patient with pemphigus vulgaris and a patient with bullous pemphigoid. To our knowledge, this form of therapy has not previously been reported in these two diseases. The distinct advantages of the larger intermittent dose method of cyclophosphamide therapy over the more conventional daily dose regimen are discussed.     

Modeling of pemphigus vulgaris in guinea pigs]

In 146 guinea pigs with body weight of 150-200 g pemphigus vulgaris (PV) was induced using screening techniques. The animals were injected with PV patients' IgG (IgGPV) and blister fluid (PVBF) with the PV patient mononuclears. Intraperitoneal administration of IgGPV caused dystrophy in epidermis of experimental animals while intracutaneous injection of PVBF resulted in balloon dystrophy. Clinical manifestations of -PV were obtained after two-day intraperitoneal IgGPV administration in a total dose of 1.5 g (6 mg/g) following a cycle of intracutaneous injections of 2.0 ml PVBF in 10-15 sites of guinea pig back. The presence of acantholysis, intraepidermal localization of blisters, pemphigus antibodies fixation on spinous cell surface, and almost 100% mortality of experimental animals were the major criteria for the developed model of PV in guinea pigs. Experimental PV in guinea pigs was abolished due to treatment of PVBF with dexamethasone, contrykal, heating (56 degrees C, 30 min), preincubation with human skin samples, and PVBF cell-free supernatant application. The conclusion was made that for occurrence of pemphigus in laboratory animals a combined effect of pemphigus antibodies, sensitized mononuclears, complement and endogenous proteinases is required.     

Preferential autoantibody reactivity of the preimmune B cell repertoire in normal mice

Naturally occurring autoantibodies are frequently found in the sera of healthy individuals. They usually exhibit low binding affinities for autoantigens and often react with multiple antigenic determinants. To determine whether their frequencies have been overestimated by sensitive testing procedures, the germ-line B cell repertoire of strain A mice was examined for reactivity with a panel of auto- and foreign Ag. If the high frequencies of autoantibodies result from testing procedures, equally high frequencies would be expected for foreign Ag specificities detected in the same manner. The presence of specific autoantibodies was confirmed in this study by the disparate frequencies observed for antibodies reactive with individual Ag. The frequencies were highest for autoantigens associated with SLE, indicating a bias toward autoreactivity in the preimmune repertoire. Analysis of VH gene usage did not indicate any selection in V gene expression with autoreactivity.     

Development of the autoimmune B cell repertoire in MRL-lpr/lpr mice

The processes responsible for the production of autoantibodies have been shown to include both Ag-specific and generalized (polyclonal) forms of B cell activation. The relative contribution and temporal association of these processes to the genesis of systemic autoimmunity are incompletely understood. To study this relationship, the B cell repertoires of MRL-lpr/lpr mice were analyzed by ELISA spot assay over an 8-mo period. Between 6 and 12 wk of age, the number of splenic lymphocytes producing antibodies reactive with both autoantigens and conventional Ag increased proportionately. The repertoires of MRL-lpr/lpr mice under 12 wk were dominated by IgM-secreting B cells that showed no bias toward the production of specific autoantibodies. From 12 to 38 wk of age, an increasing proportion of animals developed repertoires dominated by IgG-secreting B cells that were skewed toward reactivity against one or very few (auto)antigens. Although there was no single Ag against which all mice developed skewed reactivity, 55% of MRL-lpr/lpr adults had increased numbers of B cells producing antibodies to the Sm Ag and 13 to 16% developed increased reactivity toward DNA, myosin, histone, thyroglobulin, or T cells. These data indicate that generalized (polyclonal) B cell activation dominates early repertoire development whereas (auto)-antigen-specific responses become increasingly important during the latter stages of disease in these autoimmune-prone mice.