Oral administration of lipopolysaccharides activates B-1 cells in the peritoneal cavity and lamina propria of the gut and induces autoimmune symptoms in an autoantibody transgenic mouse

About a half of the antierythrocyte autoantibody transgenic (autoAb Tg) mice, in which almost all B cells are detected in the spleen, lymph nodes, and Peyer's patches, but not in the peritoneal cavity, suffer from autoimmune hemolytic anemia. The occurrence of this disease is strongly linked to production of autoAb by activated peritoneal B-1 cells in the Tg mice. In this study, we have shown that oral administration of lipopolysaccharides (LPS) activated B-1 cells in the lamina propria of the gut as well as the peritoneal cavity in the healthy Tg mice and induced the autoimmune symptoms in all the Tg mice. The activation of peritoneal and lamina propria B-1 cells by enteric LPS is found not only in the anti-RBC autoAb Tg mice and normal mice but also in the aly mice which congenitally lack lymph nodes and Peyer's patches. These results suggest that B-1 cells in the two locations may form a common pool independent of Peyer's patches and lymph nodes, and can be activated by enteric thymus-independent antigens or polyclonal activators such as LPS. The induction of autoimmune hemolytic anemia in the Tg mice by enteric LPS through the activation of B-1 cells in the lamina propria of gut and in the peritoneal cavity suggests that B-1 cells and bacterial infection may play a pathogenic role in the onset of autoimmune diseases.     

Activation and differentiation of autoreactive B-1 cells by interleukin 10 induce autoimmune hemolytic anemia in Fas-deficient antierythrocyte immunoglobulin transgenic mice

The Fas (CD95) gene is among critical genetic factors in some autoimmune diseases, which are characterized by autoantibody (autoAb) productions. In mice, mutations in the Fas gene cause lymphoproliferation (lpr) which predominantly develops glomerulonephritis, whereas the mutations in human cause autoimmune lymphoproliferative syndrome (ALPS) characterized by autoimmune hemolytic anemia (AIHA) and thrombocytopenia. Although the mechanism of antinuclear Ab in Fas-deficient background has been well characterized, that of antierythrocyte Ab production in ALPS has been still unclear. To investigate this mechanism, we developed a mouse line by crossing the antierythrocyte antibody transgenic mice (H+L6 mice) and Fas-deficient mice. Although Fas deficiency did not break tolerance of autoreactive B-2 cells in H+L6 mice, autoreactive B-1 cells in Fas-deficient H+L6 homozygous mice became activated and differentiated into autoAb-producing cells in mesenteric lymph nodes and lamina propria of intestine, resulting in severe anemia. In addition, serum levels of interleukin (IL)-10 significantly increased in Fas-/- x H+L6 homozygous mice and administration of anti-IL-10 Ab prevented exacerbation of autoAb production and AIHA. These results suggest that activation of B-1 cells is responsible for induction of AIHA in Fas-deficient condition and that IL-10 plays a critical role in terminal differentiation of B-1 cells in these mice.     

Migration and differentiation of autoreactive B-1 cells induced by activated gamma/delta T cells in antierythrocyte immunoglobulin transgenic mice

Using normal and transgenic (Tg) mice, we have shown that peritoneal B-1 cells are activated by administration of cytokines or lipopolysaccharide and migrate to other lymphoid organs where they differentiate into antibody-secreting cells. However, little is known about the process of B-1 cell migration and differentiation in vivo. We developed a mouse line by crossing the antierythrocyte antibody Tg mice (HL mice) with TCR-gamma/delta Tg mice specific for a self-thymus leukemia (TL) antigen in the recombination activating gene (RAG)2(-/-) background. In the presence of the self-antigen, Tg gamma/delta T cells increased in number and manifested activated phenotypes. Peritoneal B-1 cells in these mice migrated into mesenteric lymph nodes and differentiated into autoantibody-secreting cells, resulting in strong autoimmune hemolytic anemia. Furthermore, transfer of RAG2(-/-) x HL bone marrow or peritoneal cells into the peritoneal cavity of RAG2(-/-) x TCR-gamma/delta Tg mice gave rise to donor-derived B-1 cells in mesenteric lymph nodes, and these cells produced the autoantibody. Thus, this study demonstrates that the migration of B-1 cells and differentiation into the antibody-secreting cells can be induced by noncognate T cell help and implies the possibility that gamma/delta T cells may induce B-1 cell differentiation in vivo.     

Development of B-1 cells: segregation of phosphatidyl choline-specific B cells to the B-1 population occurs after immunoglobulin gene expression

Adult mice have two easily recognizable subsets of B cells: the predominant resting population of the spleen, called B-2, and those called B-1, which predominate in coelomic cavities and can express CD5. Some antibody specificities appear to be unique to the B-1 population. Cells expressing antibody specific for phosphatidyl choline (PtC) are the most frequent, comprising 2-10% of peritoneal B cells in normal mice. To understand the basis for the segregation of the anti-PtC specificity to this population, we have produced transgenic (Tg) mice expressing the rearranged VH12 and V kappa 4 genes of a PtC-specific B- 1 cell lymphoma. We find that VH12-Tg and VH12/V kappa 4 double-Tg mice develop very high numbers of PtC-specific peritoneal and splenic B cells. These cells have the characteristics of B-1 cells; most are CD5+, and are all IgMhi, B220lo, and CD23-. In the peritoneum these cells are also CD11b+. In addition, adult mice have many splenic B cells (up to one third of Tg+ cells) that express the VH12 Tg but do not bind PtC, presumably because they express a V kappa gene other than V kappa 4. These cells appear to be B-2 cells; they are CD23+, CD11b-, IgMlo, B220hi, and CD5-. Thus, mice given either the VH12 Tg alone or together with the V kappa 4 Tg develop a large population of PtC- specific B cells which belong exclusively to the B-1 population. Since B-2 cells can express the VH12 and V kappa 4 gene separately, we interpret these data to indicate that the events leading to the segregation of PtC-specific B cells to the B-1 population in normal mice are initiated after Ig gene rearrangement and expression. These data are discussed with regard to hypotheses of the origin of B-1 cells. We also find that VH12-Tg mice have a marked decrease in the generation of Tg-expressing B cells in adult bone marrow, but not newborn liver. We speculate that this may be related to positive selection of VH12-expressing B cells during differentiation.     

自身免疫性溶血性贫血患者外周血B-1a细胞的研究

目的：研究自身免疫性溶血性贫血患者外周血B-1a细胞的变化。方法：用CD5CD19双标抗体和流式细胞仪检测20例正常对照、13例自身免疫性溶血性贫血（AIHA）发病初期患者、4例脾切除术后AIHA患者以及10例外伤后脾切除患者的外周血B细胞和13-1a细胞占外周血单个核细胞的百分比。结果：正常对照外周血昏1a细胞占外周血单个核细胞的比例为（0．78±0．16）％，AIHA患者发病初期外周血B-1a细胞百分比为（2．58±0．63）％，AIHA患者脾切除术后外周血B-1a细胞百分比降至（0．58±0．37）％，外伤后脾切除患者外周血B-1a细胞百分比为（0．65±0．43）％。结论：AIHA患者外周血B-1a细胞比例明显增高，且AIHA患者接受脾切除手术溶血得到控制后，B-1a细胞比例明显下降。     

Expression levels of B cell surface immunoglobulin regulate efficiency of allelic exclusion and size of autoreactive B-1 cell compartment.

Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens.     

Immune tolerance to autoantibody-derived peptides delays development of autoimmunity in murine lupus.

Mechanisms that initiate and maintain autoantibody (autoAb) production in individuals with autoimmune diseases like SLE are poorly understood. Inadequate suppression of autoreactive T cells and/or unusual activation of T and B cells may underlie the persistence of pathogenic autoAbs in lupus. Here, we examine the possibility that in mice with lupus, autoAb molecules may be upregulating their own production by activating self-reactive T cells via their own processed peptides; downregulation of this circuit may decrease autoAb production and delay the development of lupus. We found that before the onset of clinical disease, lupus-prone (NZB/NZW) F1 [BWF1] (but not MHC-matched nonautoimmune mice) developed spontaneous T cell autoimmunity to peptides from variable regions of heavy chains (VH) of syngeneic anti-DNA mAbs but not to peptides from the VH region of an mAb to an exogenous antigen. Tolerizing young BWF1 mice with intravenous injections of autoAb-derived determinants substantially delayed development of anti-DNA antibodies and nephritis and prolonged survival. Thus, in such an autoAb-mediated disease, the presence of autoreactive T cells against VH region determinants of autoAbs may represent an important mechanism involved in the regulation of autoimmunity. Our findings show that tolerizing such autoreactive T cells can postpone the development of an autoimmune disease like SLE.     

Conventional B cells, not B-1 cells, are responsible for producing autoantibodies in lpr mice

Mice homozygous for the lpr gene develop autoantibodies and polyclonal B cell activation similar to what is seen in human systemic lupus erythematosus patients. We have previously shown that an lpr-specific intrinsic B cell defect was necessary for autoantibody production in this model. In the current study, we have further defined these autoantibody-producing B cells. Two major subsets of B cells have been described. B-1 cells (CD5+ B cells) can be distinguished from conventional B cells on the basis of phenotype, cytokine secretion, gene expression, anatomical location, and function. In addition, B-1 cells have been implicated in autoimmunity in several murine and human studies. To address the question of which B cell subset produces autoantibodies in lpr mice, we used immunoglobulin heavy chain (Igh) allotype-marked peritoneal (B-1 cell source) and bone marrow (conventional B cell source) cells from lpr mice to establish B cell chimeras. We used two general approaches. In one, we reconstituted sublethally irradiated mice with B-1 cells of one allotype and bone marrow cells of another allotype. In the second method, we suppressed endogenous B cells in neonatal mice with allotype-specific anti-IgM antibody, and injected peritoneal cells of another allotype. After antibody treatment was stopped, the mouse's conventional B cells recovered, but the B-1 subset was only reconstituted by the donor. In both types of chimeras, antichromatin, rheumatoid factor, and anti- single stranded DNA (ssDNA) autoantibodies were produced by the conventional B cell bone marrow source. In addition, an age-related decrease in peritoneal B-1 cells was seen, even in unmanipulated lpr mice. These data show that lpr B-1 cells are not important producers of autoantibodies. Conventional B cells are the source of autoantibodies directed at chromatin, ssDNA, and IgG.     

An acetylcholine receptor alpha subunit promoter confers intrathymic expression in transgenic mice. Implications for tolerance of a transgenic self-antigen and for autoreactivity in myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor (AChR). Although the autoantigen is present in the thymus, it is not tolerated in MG patients. In addition, the nature of the cell bearing the autoantigen is controversial. To approach these questions, we used two lineages of transgenic mice in which the beta-galactosidase (beta-gal) gene is under the control of a 842-bp (Tg1) or a 3300-bp promoter fragment (Tg2) of the chick muscle alpha subunit AChR gene. In addition to expression in muscle cells, thymic expression was observed in both mouse lines (mainly in myoid cells in Tg1 and myoid cells and epithelial cells in Tg2). After challenge with beta-gal, Tg1 mice produced Th2-dependent anti-beta-gal antibodies, while Tg2 mice were almost unresponsive. By contrast, in a proliferation assay both Tg lines were unresponsive to beta-gal. Cells from Tg1 mice produce Th2-dependent cytokine whereas cells from Tg2 mice were nonproducing in response to beta-gal. These data indicate that the level of expression in Tg1 mice could be sufficient to induce tolerance of Th1 cells but not of Th2 cells, while both populations are tolerated in Tg2 mice. These findings are compatible with the hypothesis that AChR expression is not sufficiently abundant in MG thymus to induce a full tolerance.     

Association of BAFF/BLyS overexpression and altered B cell differentiation with Sjögren’s syndrome

BAFF (BLyS, TALL-1, THANK, zTNF4) is a member of the TNF superfamily that specifically regulates B lymphocyte proliferation and survival. Mice transgenic (Tg) for BAFF develop an autoimmune condition similar to systemic lupus erythematosus. We now demonstrate that BAFF Tg mice, as they age, develop a secondary pathology reminiscent of Sj?gren's syndrome (SS), which is manifested by severe sialadenitis, decreased saliva production, and destruction of submaxillary glands. In humans, SS also correlates with elevated levels of circulating BAFF, as well as a dramatic upregulation of BAFF expression in inflamed salivary glands. A likely explanation for disease in BAFF Tg mice is excessive survival signals to autoreactive B cells, possibly as they pass through a critical tolerance checkpoint while maturing in the spleen. The marginal zone (MZ) B cell compartment, one of the enlarged B cell subsets in the spleen of BAFF Tg mice, is a potential reservoir of autoreactive B cells. Interestingly, B cells with an MZ-like phenotype infiltrate the salivary glands of BAFF Tg mice, suggesting that cells of this compartment potentially participate in tissue damage in SS and possibly other autoimmune diseases. We conclude that altered B cell differentiation and tolerance induced by excess BAFF may be central to SS pathogenesis.     

Interleukin 9-induced in vivo expansion of the B-1 lymphocyte population

The activity of interleukin (IL)-9 on B cells was analyzed in vivo using transgenic mice that constitutively express this cytokine. These mice show an increase in both baseline and antigen-specific immunoglobulin concentrations for all isotypes tested. Analysis of B cell populations showed a specific expansion of Mac-1(+) B-1 cells in the peritoneal and pleuropericardial cavities, and in the blood of IL-9 transgenic mice. In normal mice, the IL-9 receptor was found to be expressed by CD5(+) as well as CD5(-) B-1 cells, and repeated injections of IL-9 resulted in accumulation of B-1 cells in the peritoneal cavity, as observed in transgenic animals. Unlike other mouse models, such as IL-5 transgenic mice, in which expansion of the B-1 population is associated with high levels of autoantibodies, IL-9 did not stimulate the production of autoantibodies in vivo, and most of the expanded cells were found to belong to the B-1b subset (IgM+Mac-1(+)CD5(-)). In addition, we found that these IL-9-expanded B-1b cells do not share the well-documented antibromelain-treated red blood cell specificity of CD5(+) B-1a cells. The increase of antigen-specific antibody concentration in immunized mice suggests that these B-1 cells are directly or indirectly involved in antibody responses in IL-9 transgenic mice.     

Augmented TLR9-induced Btk activation in PIR-B–deficient B-1 cells provokes excessive autoantibody production and autoimmunity

Pathogens are sensed by Toll-like receptors (TLRs) expressed in leukocytes in the innate immune system. However, excess stimulation of TLR pathways is supposed to be connected with provocation of autoimmunity. We show that paired immunoglobulin (Ig)-like receptor B (PIR-B), an immunoreceptor tyrosine-based inhibitory motif–harboring receptor for major histocompatibility class I molecules, on relatively primitive B cells, B-1 cells, suppresses TLR9 signaling via Bruton''s tyrosine kinase (Btk) dephosphorylation, which leads to attenuated activation of nuclear factor κB p65RelA but not p38 or Erk, and blocks the production of natural IgM antibodies, including anti-IgG Fc autoantibodies, particularly rheumatoid factor. The autoantibody production in PIR-B–deficient (Pirb^−/−) mice was further augmented in combination with the Fas^lpr mutation, which might be linked to the development of autoimmune glomerulonephritis. These results show the critical link between TLR9-mediated sensing and a simultaneously evoked, PIR-B–mediated inhibitory circuit with a Btk intersection in B-1 cells, and suggest a novel way toward preventing pathogenic natural autoantibody production.The emergence of autoimmunity is often coupled with aging, and is suggested to be linked to activation of the innate immune system in individuals suffering from bacterial and viral infections (Baccala et al., 2007; Groom et al., 2007; Krieg and Vollmer 2007; Rothlin et al., 2007). Toll-like receptors (TLRs) expressed in leukocytes of the innate immune system play indispensable roles in the sensing of viral and bacterial invasion through binding pathogen-associated molecular patterns, which leads to efficient T cell–mediated inflammatory responses (Akira et al., 2001; Iwasaki and Medzhitov, 2004). The TLR-mediated priming of inflammation and production of neutralizing antibodies against pathogens should be strictly regulated, otherwise there is the possibility of the development of autoimmune diseases (Marsland and Kopf, 2007). The mechanisms underlying the efficient TLR-mediated activation of the innate and adaptive immune systems with prevention of reactivity to autologous tissues remain elusive.Examples of critical cells that express TLRs and could potentially link the innate and adaptive immune systems are relatively primitive B cells, B-1 cells, found mainly in the peritoneal and pleural cavities. In contrast to recirculating follicular B cells (or conventional B or B-2 cells), B-1 cells are characterized by B220^lowIgM^highCD23⁻CD43⁺IgD^low cells (Berland and Wortis, 2002; Tung and Herzenberg, 2007). Although it has been pointed out by many researchers that innate B-1 cells but not conventional B cells are producers of natural antibodies against pathogens (Ochsenbein et al., 1999), accumulating lines of evidence suggest that a major source of autoantibodies is also those B-1 cells (Baumgarth et al., 2005; Carroll and Holers, 2005), but it has been a matter of debate. By stimulation via different TLRs, the B-1 cell population in the peritoneal cavity has been enlarged and B-1 cell–mediated autoantibody production augmented (Murakami et al., 1995). This could be partly because B-1 cells express a set of TLRs, including TLR4, TLR7, and TLR9 (Gururajan et al., 2007), and are more prone to differentiate into plasma cells than B-2 cells upon TLR-mediated stimulation, although B-2 cells similarly possess a range of TLRs (Genestier et al., 2007). For example, Murakami et al. (1995) have shown, in anti–red blood cell autoantibody transgenic mice, that the susceptibility to autoimmune hemolytic anemia was significantly increased when the mice were transferred from germ-free or specific pathogen-free conditions to conventional conditions or injected with a TLR4 ligand, LPS, with a concomitant increase in the peritoneal B-1 cell population, whereas almost all B-2 cells are constitutively deleted in the transgenic mice. These findings again suggest the importance of the regulation of TLR signaling in B-1 cells, which prevents overstimulation of TLRs so as not to evoke overproduction of natural antibodies, including potentially harmful autoantibodies. Therefore, what mechanisms may regulate the overstimulation of the TLR signal, particularly in B-1 cells?We speculated that paired Ig-like receptor B (PIR-B; Hayami et al., 1997; Kubagawa et al., 1997) could participate in the regulation of B-1 cells. Recruitment of SH2 domain–containing tyrosine phosphatase 1 (SHP-1) to phosphotyrosylated immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic portion of PIR-B was shown to be critical for PIR-B–mediated inhibitory signaling in general (Ho et al., 1999; Maeda et al., 1999), and this inhibition is achieved, at least in part, via constitutive binding of PIR-B to its ligand, i.e., MHC class I molecules, expressed on the same cell surface (Masuda et al., 2007). Interestingly, in PIR-B–deficient (Pirb^−/−) mice, the peritoneal B-1 cell population significantly increased, particularly with aging, compared with wild-type mice (Ujike et al., 2002). However, it has not been determined how the B-1 cell compartment is physiologically regulated by PIR-B or what the physiological or pathological consequence of the expanded B-1 cells is in the contexts of infection and autoimmunity.In this paper, we show that PIR-B can inhibit TLR9-mediated signaling via regulation of Bruton''s tyrosine kinase (Btk) phosphorylation in peritoneal B-1 cells. We found that TLR9 activation immediately activates an Src family kinase, Lyn, which then phosphorylates PIR-B cytoplasmic ITIMs. In the absence of PIR-B, B-1 cells become hyperproducers of natural IgM antibodies, including anti-IgG Fc autoantibodies, i.e., rheumatoid factor (RF), via unmethylated CpG-B oligodeoxynucleotide (CpG-B) stimulation in vitro and in vivo or upon aging. These phenotypes caused by PIR-B deficiency were further exaggerated in combination with the Fas^lpr mutation, which caused the Pirb^−/−Fas^lpr mutant mice to be short-lived mainly because of autoimmune glomerulonephritis with immune complex depositions. Our findings may provide a novel strategy for preventing autoimmunity by reducing the production of pathogenic autoantibodies by B-1 cells, such as through down-regulation of Btk activation or enhancement of PIR-B–mediated B-1 cell regulation.     

Diagnosis and treatment of autoimmune haemolytic anaemias in adults: a clinical review

Autoimmune haemolytic anaemia (AIHA) is an immune disorder caused by antibodies directed against unmodified autologous red cells. The disorder may be a primary (idiopathic) or a secondary disease. The diagnosis is based on the presence of anaemia, signs of haemolysis with reticulocytosis, low haptoglobin, increased lactate dehydrogenase, elevated indirect bilirubin, and a positive direct antiglobulin test (Coombs test). Sometimes, not all of these typical features are present. Most AIHA are caused by warm antibodies, whereas cold antibodies are less commonly detected. While half of the warm antibody-based AIHA are idiopathic anaemias, almost all cold antibody AIHA are secondary anaemias. Underlying diseases are Non Hodgkin's lymphomas and systemic autoimmune disorders, and less frequently organ transplantation, infections, or solid tumors. Moreover, AIHA is an important complication of treatment with nucleoside analogs. Most patients with AIHA require therapy. In warm antibody AIHA, standard first line therapy are glucocorticosteroids with or without high dose immunoglobulins, whereas splenectomy is considered second-line therapy. Response rates of primary AIHA to corticosteroid therapy are high. After initial remission, the dose should be tapered down slowly and with caution, and in some cases, low-dose maintenance therapy is required. The efficacy of standard therapy is low in secondary AIHA that develops in lymphoma patients, posttransplant patients, or tumor patients. Among other immunosuppressive treatments, rituximab (anti-CD20) appears to be highly effective in patients with warm antibody AIHA refractory to standard therapy. Mycophenolate mofetil is quite effective in AIHA patients with an underlying autoimmune or lymphoproliferative disease. Patients with cold agglutinins are refractory to steroids and splenectomy. Half of these patients may respond to rituximab, although responses usually are short-lived. Sometimes, AIHA that is associated with malignant lymphomas or tumors, disappears after successful anti-lymphoma or anti-tumor therapy.     

B Lymphocytes Producing Demyelinating Autoantibodies: Development and Function in Gene-targeted Transgenic Mice

We studied the cellular basis of self tolerance of B cells specific for brain autoantigens using transgenic mice engineered to produce high titers of autoantibodies against the myelin oligodendrocyte glycoprotein (MOG), a surface component of central nervous system myelin. We generated “knock-in” mice by replacing the germline J_H locus with the rearranged immunoglobulin (Ig) H chain variable (V) gene of a pathogenic MOG-specific monoclonal antibody. In the transgenic mice, conventional B cells reach normal numbers in bone marrow and periphery and express exclusively transgenic H chains, resulting in high titers of MOG-specific serum Igs. Additionally, about one third of transgenic B cells bind MOG, thus demonstrating the absence of active tolerization. Furthermore, peritoneal B-1 lymphocytes are strongly depleted. Upon immunization with MOG, the mature transgenic B cell population undergoes normal differentiation to plasma cells secreting MOG-specific IgG antibodies, during which both Ig isotype switching and somatic mutation occur. In naive transgenic mice, the presence of this substantial autoreactive B cell population is benign, and the mice fail to develop either spontaneous neurological disease or pathological evidence of demyelination. However, the presence of the transgene both accelerates and exacerbates experimental autoimmune encephalitis, irrespective of the identity of the initial autoimmune insult.     

B-1 Cell Development: Evidence for an Uncommitted Immunoglobulin (Ig)M+ B Cell Precursor in B-1 Cell Differentiation

Murine phosphatidyl choline (PtC)–specific B cells in normal mice belong exclusively to the B-1 subset. Analysis of anti-PtC (V_H12 and V_H12/Vκ4) transgenic (Tg) mice indicates that exclusion from B-0 (also known as B-2) occurs after immunoglobulin gene rearrangement. This predicts that PtC-specific B-0 cells are generated, but subsequently eliminated by either apoptosis or differentiation to B-1. To investigate the mechanism of exclusion, PtC-specific B cell differentiation was examined in mice expressing the X-linked immunodeficiency (xid) mutation. xid mice lack functional Bruton's tyrosine kinase (Btk), a component of the B cell receptor signal transduction pathway, and are deficient in B-1 cell development. We find in C57BL/ 6.xid mice that V_H12 pre-BII cell selection is normal and that PtC-specific B cells undergo modest clonal expansion. However, the majority of splenic PtC-specific B cells in anti-PtC Tg/xid mice are B-0, rather than B-1 as in their non-xid counterparts. These data indicate that PtC-specific B-0 cell generation precedes segregation as predicted, and that Btk function is required for efficient segregation to B-1. Since xid mice exhibit defective B cell differentiation, not programmed cell death, these data are most consistent with an inability of PtC-specific B-0 cells to convert to B-1 and a single B cell lineage.     

BAFF and MyD88 signals promote a lupuslike disease independent of T cells

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the production of autoantibodies. However, the underlying cause of disease appears to relate to defects in T cell tolerance or T cell help to B cells. Transgenic (Tg) mice overexpressing the cytokine B cell-activating factor of the tumor necrosis factor family (BAFF) develop an autoimmune disorder similar to SLE and show impaired B cell tolerance and altered T cell differentiation. We generated BAFF Tg mice that were completely deficient in T cells, and, surprisingly, these mice developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did, however, require B cell-intrinsic signals through the Toll-like receptor (TLR)-associated signaling adaptor MyD88, which controlled the production of proinflammatory autoantibody isotypes. TLR7/9 activation strongly up-regulated expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), which is a receptor for BAFF involved in B cell responses to T cell-independent antigens. Moreover, BAFF enhanced TLR7/9 expression on B cells and TLR-mediated production of autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, but requires TLR signaling and is independent of T cell help. It is possible that SLE patients with elevated levels of BAFF show a similar basis for disease.     

T-cell activation and receptor downmodulation precede deletion induced by mucosally administered antigen

The fate of antigen-specific T cells was characterized in myelin basic protein (MBP) T-cell receptor (TCR) transgenic (Tg) mice after oral administration of MBP. Peripheral Th cells are immediately activated in vivo, as indicated by upregulation of CD69 and increased cytokine responses (Th1 and Th2). Concurrently, surface TCR expression diminishes and internal TCR levels increase. When challenged for experimental autoimmune encephalomyelitis during TCR downmodulation, Tg mice are protected from disease. To characterize Th cells at later times after antigen feeding, it was necessary to prevent thymic release of naive Tg cells. Therefore, adult Tg mice were thymectomized before treatment. TCR expression returns in thymectomized Tg mice 3 days after MBP feeding and then ultimately declines in conjunction with MBP-specific proliferation and cytokine responses (Th1-type and Th2-type). The decline correlates with an increase in apoptosis. Collectively, these results demonstrate that a high dose of fed antigen induces early T-cell activation and TCR downmodulation, followed by an intermediate stage of anergy and subsequent deletion.     

Autoimmune hemolytic anemia

Diagnosis of autoimmune hemolytic anemia (AIHA) requires both serologic evidence of an autoantibody and hemolysis. Based on the characteristic temperature reactivity of the autoantibody to red cell membranes, AIHA is classified into warm AIHA or cold AIHA (cold agglutinin disease and paroxysmal cold hemoglobinuria). Sensitized RBCs are destructed by intravascular and/or extravascular hemolysis. On the basis of etiology, AIHA are classified as idiopathic or secondary. The common cause of secondary AIHA is lymphoproliferative disorders, autoimmune diseases, and infections. The first line therapy of patients with warm AIHA is glucocorticoids and primary treatment for cold AIHA is avoiding cold exposure. The other standard treatments include splenectomy and immunosuppressive drugs. Recently, rituximab, a monoclonal anti-CD20 antibody, has been used in refractory AIHA with excellent responses.     

Autoantigen, innate immunity, and T cells cooperate to break B cell tolerance during bacterial infection

Autoantibody production during infections is considered to result from nonspecific activation of low-affinity autoreactive B cells. Whether this can lead to autoimmune disease remains uncertain. We show that chronic infection by Borrelia burgdorferi of Tg animals expressing human rheumatoid factor (RF) B cells (of low or intermediate affinities) in the absence or in the constitutive presence of the autoantigen (represented here by chimeric IgG with human constant region) breaks their state of immunological ignorance, leading to the production of RFs. Surprisingly, this production was more pronounced in intermediate-affinity RF Tg mice co-expressing the autoantigen. This overproduction was mediated by immune complexes and involved synergistic signaling between the B cell receptor and Toll-like receptors and T cell help. These findings indicate that chronic infection can activate autoreactive B cells with significant affinity and creates conditions that can drive them to differentiate into memory cells. Such cells may have some physiological yet undetermined role, but in autoimmune-prone individuals, this scenario may initiate autoimmunity.     

The regulation of T cell homeostasis and autoimmunity by T cell–derived LIGHT

Costimulatory molecules on antigen-presenting cells (APCs) play an important role in T cell activation and expansion. However, little is known about the surface molecules involved in direct T-T cell interaction required for their activation and expansion. LIGHT, a newly discovered TNF superfamily member (TNFSF14), is expressed on activated T cells and immature dendritic cells. Here we demonstrate that blockade of LIGHT activity can reduce anti-CD3-mediated proliferation of purified T cells, suggesting that T cell-T cell interaction is essential for this proliferation. To test the in vivo activity of T cell-derived LIGHT in immune homeostasis and function, transgenic (Tg) mice expressing LIGHT in the T cell lineage were generated. LIGHT Tg mice have a significantly enlarged T cell compartment and a hyperactivated peripheral T cell population. LIGHT Tg mice spontaneously develop severe autoimmune disease manifested by splenomegaly, lymphadenopathy, glomerulonephritis, elevated autoantibodies, and severe infiltration of various peripheral tissues. Furthermore, the blockade of LIGHT activity ameliorates the severity of T cell-mediated diseases. Collectively, these findings establish a crucial role for this T cell-derived costimulatory ligand in T cell activation and expansion; moreover, the dysregulation of T cell-derived LIGHT leads to altered T cell homeostasis and autoimmune disease.