Generation and selection of an IgG-driven autoimmune repertoire during B-lymphopoiesis in Igmicro-deficient/lpr mice

Class switch recombination (CSR) is a well-regulated process that occurs in peripheral lymphoid tissue, and is thought of as an important factor constructing the memory repertoire. We have recently shown that CSR normally occurs during bone marrow (BM) development, and these isotype-switched B cells are negatively selected by Fas signaling. This novel pathway of B cell development may generate a primary repertoire driven by gamma-heavy receptors, the nature of which is yet unknown. To study this gammaH-driven repertoire we used mice lacking IgM-transmembrane tail exon ( micro MT), where B cell development is limited by their ability to undergo CSR. We already showed that lack of Fas signaling rescues development of a significant population of isotype-switched B cells and production of high titers of non-IgM serum antibodies in micro MT mice deficient in Fas ( micro MT/lpr), thereby providing a mouse model allowing the assessment of gammaH-driven repertoire. Using a tissue array and phage display epitope library we report here that IgG repertoire in micro MT/lpr mice is oligo-monoclonal, bearing self-tissue reactivity. This is supported by analysis of the Vkappa utilization in peripheral B cells from micro MT/lpr mice, which revealed a strikingly restricted repertoire. In contrast, micro MT/lpr B cells that are grown in non-selective BM cultures utilize a wide repertoire. These results suggest that the Fas pathway is an important regulator in the generation and selection of an autoimmune gammaH-driven repertoire in vivo.     

Increased plasma cell frequency and accumulation of abnormal syndecan-1plus T-cells in Igmu-deficient/lpr mice

The expression of muH chain is an important checkpoint in B cell development. In mice deficient for IgM transmembrane tail exons (muMT mice) B cell development is blocked at the pro-B stage. However, we showed that Fas-deficient muMT mice (muMT/lpr) develop a very small population of isotype-switched B cells and produce high titers of self-reactive serum antibodies. In addition, muMT/lpr mice develop severe lymphoproliferation and both pathologic processes occur at young ages. This may suggest that lack of Fas-Fas ligand signaling exacerbates murine lupus in B cell lymphopenic mice. To test this we analyzed antibody and plasma cell formation, and accumulation of abnormal T cells in muMT/lpr mice. Our results show that the muMT/lpr mouse is particularly permissive for the development and accumulation of antibody-producing cells, thereby explaining the high titers of serum antibodies in these mice. In addition, we found that accumulating cells in spleen and lymph nodes of muMT/lpr mice are alphabeta T cells expressing the abnormal B220+/CD3+ surface markers, a phenotype also described for other Fas-deficient mouse models. Strikingly, we found that accumulating cells in muMT/lpr mice express the membrane proteoglycan syndecan-1, a known plasma cell marker. Development of these cells is blocked in mice deficient for TCRbeta and TCRdelta. We also found that both antibody production and lymphoproliferation in muMT/lpr mice are Th1 regulated. Our results, therefore, suggest that in the muMT/lpr mouse model a small population of isotype-switched B cells is sufficient for the initiation and propagation of Th1-regulated murine lupus.     

Severe impairment of B cell function in lpr/lpr mice expressing transgenic Fas selectively on B cells.

Transgenic lpr/lpr mice expressing functional Fas selectively on B cells were produced in an attempt to elucidate the role of Fas on B cells in the regulation of autoantibody production. The homozygous lpr/lpr mice carrying the transgene did not produce anti-double-stranded DNA antibodies throughout their lives, whereas the development of abnormal lpr T cells (double negative, B220(+)) was not suppressed. Further analyses, however, revealed that the expression of the transgenic Fas on B cells of lpr/lpr homozygous mice resulted in severe impairment of the B cell function. The defect was characterized by a decrease in the number of mature peripheral B cells, a reduction in the serum Ig level and the total failure of B cells to mount antibody responses to stimulations of T-dependent as well as T-independent antigens. Such a defect was prominent only when the transgene was expressed on the lpr/lpr homozygous background. On the contrary, B cells of the transgenic lpr/lpr mice were shown to be capable of producing Ig when stimulated with anti-CD40 in the presence of IL-4 and IL-5. Furthermore, lpr/lpr T cells showed enhanced non-specific cytolytic activity. These observations suggested that the observed B cell defect was probably attributable to the destruction of activated B cells expressing transgenic Fas by aggressive lpr/lpr T cells.     

Contribution of secondary Igkappa rearrangement to primary immunoglobulin repertoire diversification

Abs reactive to DNA and DNA/histone complexes are a distinguished characteristic of primary immunoglobulin repertoires in autoimmune B6.MRL-Fas^lpr and MRL/MpJ-Fas^lpr mice. These mice are defective in Fas receptor, which is critical for the apoptosis of autoreactive B cells by an extrinsic pathway. In the present study, we explored the possibility that bone marrow small pre-B and immature B cells from adult B6.MRL-Fas^lpr mice and MRL/MpJ-Fas^lpr mice respectively, which contain autoreactive B-cell antigen receptors (BCR) and manifest autoimmune syndromes, exhibit enhanced receptor editing patterns. Indeed, FAS^lpr pre B and immature B cells were shown to possess more ongoing replacements of non-productive (nP) than productive (P) primary VκJκ rearrangements. Significantly, the P vs nP ratios of these replaced primary rearrangements were 1:2, thus indicating that κ light-chain production appears not to inhibit secondary rearrangements. In addition, we identified multiple atypical rearrangements, such as Vκ cRS (cryptic recombination signals) cleavages. These results suggest that the onset of light chain secondary rearrangements persists similarly as a non-selected mode and independent of BCR autoreactivity during certain developmental windows of bone marrow B cells in lupus-prone mice and control, and leads us to propose the function of secondary, de novo Igκ rearrangements to increase BCR diversity.     

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.     

Lupus-prone MRL/faslpr/lpr mice display increased AID expression and extensive DNA lesions,comprising deletions and insertions,in the immunoglobulin locus: Concurrent upregulation of somatic hypermutation and class switch DNA recombination

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of an array of pathogenic autoantibodies, including high-affinity anti-dsDNA IgG antibodies. These autoantibodies are mutated and class-switched, mainly to IgG, indicating that immunoglobulin (Ig) gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) are important in their generation. Lupus-prone MRL/fas^lpr/lpr mice develop a systemic autoimmune syndrome that shares many features with human SLE. We found that Ig genes were heavily mutated in MRL/fas^lpr/lpr mice and contained long stretches of DNA deletions and insertions. The spectrum of mutations in MRL/fas^lpr/lpr B cells was significantly altered, including increased dG/dC transitions, increased targeting of the RGYW/WRCY mutational hotspot and the WGCW AID-targeting hotspot. We also showed that MRL/fas^lpr/lpr greatly upregulated CSR, particularly to IgG2a and IgA in B cells of the spleen, lymph nodes and Peyer's patches. In MRL/fas^lpr/lpr mice, the significant upregulation of SHM and CSR was associated with increased expression of activation-induced cytidine deaminase (AID), which mediates DNA lesion, the first step in SHM and CSR, and translesion DNA synthesis (TLS) polymerase (pol) θ, pol η and pol ζ, which are involved in DNA synthesis/repair process associated with SHM and, possibly, CSR. Thus, in lupus-prone MRL/fas^lpr/lpr mice, SHM and CSR are upregulated, as a result of enhanced AID expression and, therefore, DNA lesions, and dysregulated DNA repair factors, including TLS polymerases, which are involved in the repair process of AID-mediated DNA lesions.     

Stochastic pairing of Ig heavy and light chains frequently generates B cell antigen receptors that are subject to editing in vivo

We examined the generation and selection of the B cell antibody repertoire through crossing of mice bearing distinct Ig heavy (H) and light (L) chain rearranged variable region transgenes. Ig gene knock-in and transgenic mice whose H and L chains pair to form a non-autoreactive, functional B cell antigen receptor (BCR) have significantly reduced pre-B cells in the bone marrow as their B cell progenitors rapidly differentiate into surface IgM(+) B cells. The presence of a pre-B cell compartment in these Ig transgenic mice, however, indicates the induction of receptor editing. Here, 18 distinct combinations of H and L chains were generated that we showed could pair in vitro to form BCRs of unknown specificities. Of these, nine induced receptor editing in vivo as evidenced by the presence of pre-B cells and endogenous L chain rearrangements in mice bearing these H and L chain transgenes. These data thus suggest that about half of the emerging antibody repertoire is negatively selected during B lymphopoiesis due to the likely encoding of autoreactive or non-functional BCRs.     

B-lineage cell deficits in bone marrow of lpr/lpr mice

Analyses of bone marrow (BM) lymphocytes in C57BL/6 mice homozygousfor the lpr mutation (BS.Ipr) disclosed low numbers of pre-Band B cells, as compared with age-matched control B6 mice. BMdepletion in B6.lpr mice was selective for B-lineage cells,appeared in young adults, and developed markedly with age anddisease progression, contrasting with the peripheral lymphocytehypercellularity. Normalization of pre-B and B cellularity inBM of B6.lprmice was observed after administration of polyclonalIg, that also markedly improved the clinical condition. Isolatedpre-B (B220⁺ IgM^–) cells from B6 or B6.lpr mice, however,showed essentially the same rates of IL-7-dependent proliferationand differentiation to B (lgM⁺) cells in culture, indicatingthat the BM B-lineage deficit is not the result of an intrinsicdefect inB cell generation.     

An MRL/MpJ-lpr/lpr substrain with a limited expansion of lpr double-negative T cells and a reduced autoimmune syndrome

The autosomal recessive mutant gene, lpr, has been shown toaccelerate the progression of lupus-like autoimmune disease,which is associated with a massive expansion of a unique CD4^–CD8^–double-negative T cell subset, in MRL/MpJ mice. Here we reporta substrain of MRL/MpJ-lpr/lpr (MRL-lpr) mice which live almosttwice as long with delayed development of glomerulonephritis,compared with conventional MRL-lpr mice. This substrain, termedMRL-lpr.II (II for long-lived), develops generalized lymphadenopathycharacteristically seen in MRL-lpr mice. However, the expansionof a double negative lpr T cell subset is markedly limited witha mean value of 15% in their lymph nodes compared to about 70%in conventional MRL-lpr mice. Overall production of autoantibodies,such as anti-DNA and rheumatoid factors, does not significantlydiffer between the two MRL-lpr mice. However, serum levels ofcryoglobulins, whose major component is lgG3, are markedly diminishedin MRL-lpr.ll mice with a parallel decrease in lgG3. Since MRL-lpr.llmice still carry the lpr mutation, as documented by the presenceof defects in the Fas antigen, a possible new mutation in thissubstrain may play a significant role in the pathogenesls oflupus-like autoimmune syndrome.     

Surface expression of TRAIL/Apo-2 ligand in activated mouse T and B cells

Like other members of the TNF family, TRAIL/Apo-2 ligand induces apoptosis in sensitive target cells in a caspase-dependent fashion. We recently found that TRAIL may be constitutively expressed on the surface of mouse and human tumor cells of T and B origin. To define the pattern of TRAIL expression in normal immune cells, freshly isolated splenocytes, Concanavalin A/IL-2-activated T cells and lipopolysaccharide-activated B cells were analyzed by surface staining with or without secondary stimulation. Activated, but not resting, CD3⁺ cells expressed TRAIL in an activation-dependent fashion. Conversely, freshly isolated B220⁺ cells displayed surface TRAIL and CD95L that were retained following activation. Restimulation with the protein kinase C activator phorbol 12-myristate 13-acetate and the calcium ionophore ionomycin or an agonistic anti-CD3 monoclonal antibody induced significant up-regulation of surface TRAIL and CD95L in CD3⁺ , TCRα β cells with CD4⁺ or CD8⁺ phenotype. Similarly to CD95L, TRAIL up-regulation was protein synthesis dependent and cyclosporin A sensitive. These results indicate that both TRAIL and CD95L are displayed on the cell surface of activated immune cells and may thus represent complementary effector pathways in the regulatory functions of T and B cells.     

Decrease of CD154 intensity on peripheral CD4+ T cells in autoimmune thyroid disease

To clarify immunological differences among patients with Graves' disease (GD) and Hashimoto's disease (HD) at various levels of severity, we examined the expression of the CD154 molecules on peripheral T cells, which regulate B cell activation, B cell differentiation, and T-cell survival. We found decreases in the intensities of CD154 on peripheral CD4(+) cells from euthyroid patients with GD and HD, but we did not find any differences between patients with different disease severities. CD8(+) cells did not express CD154 molecules. Thus, CD154 expression on CD4(+) cells may be related to the pathogenesis of the autoimmune thyroid diseases, not to the disease severity.     

IL‐10 production in murine IgM+CD138hi cells is driven by Blimp‐1 and downregulated in class‐switched cells

In contrast to antibody‐induced inflammatory responses, some B‐cell subpopulations suppress inflammation through the production of interleukin (IL)‐10. However, the mechanisms underlying Il10 gene expression during B‐cell development is elusive. Here, we identify IgM⁺B220^loCD138^hi cells responsible for marked IL‐10 production in the bone marrow and spleen of mice. These murine IL‐10‐producing cells predominantly secrete IgM and have unique characteristics of long‐lived plasma cells in spite of high expression of surface IgM. We found that IL‐10 production is strongly correlated with the expression level of Prdm1 (encoding the Blimp‐1 protein), an essential regulator of plasma cell development. Furthermore, overexpression of Prdm1 induces Il10 expression in naïve B cells. Immunoglobulin class‐switching recombination events resulted in the downregulation of both Il10 and Prdm1 expression in differentiating B cells. Thus, the prolonged elevation of Blimp‐1 expression during the formation of IgM⁺CD138^hi cells without class‐switching elicits IL‐10 production. Adoptive transfer of Il10‐deficient B cells into B‐cell‐deficient mice demonstrated that IgM⁺CD138^hi cell‐derived IL‐10 supports the survival of class‐switched plasma cells and their antibody production in response to antigen challenge. These findings reveal an important role for IL‐10 secretion by IgM⁺CD138^hi cells in the complete and efficient humoral response.     

Functional T cells in primary immune response to histoplasmosis

Functional T cells are critical to host defense against infection. It has been reported that functional T cells as determined by their cytokine production represent antigen-specific T cells in infectious disease models. In this study, we enumerated Histoplasma-specific interferon gamma-producing cells in bulk splenocyte culture and showed that infection with Histoplasma capsulatum, an intracellular pathogen of the macrophage, activated both CD4 and CD8 T cells. The magnitude of CD8 T cell response was lower than CD4 T cell, but the expansion and contraction of both cell types followed the same kinetics. Over 90% of interferon gamma-producing CD4 T cells and >85% of CD8 T cells expressed CD44(hi) phenotype. The strong correlation between interferon gamma production and CD44(hi) expression was observed not only at the peak of response but also throughout the course of infection. Moreover, a broad spectrum of Vbeta populations responded to systemic as well as pulmonary infections, suggesting no obvious T cell receptor bias in primary immune response to histoplasmosis. While each Vbeta population contributed to interferon gamma production, several specific Vbeta populations made up higher percentages of interferon gamma-producing cells. Our study laid the groundwork for further investigations in immune response to histoplasmosis.     

Regulatory T cells in B-cell-deficient and wild-type mice differ functionally and in expression of cell surface markers

NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) with chronic inflammation of thyroids by T and B cells. B-cell deficient (B^–/–) mice are resistant to SAT but develop SAT if regulatory T (Treg) cells are transiently depleted. We established a transfer model using splenocytes from CD28^–/– B^–/– mice (effector cells and antigen-presenting cells) cultured with or without sorted Treg cells from Foxp3-GFP wild-type (WT) or B^–/– mice. After transfer to mice lacking T cells, mice given Treg cells from B^–/– mice had significantly lower SAT severity scores than mice given Treg cells from WT mice, indicating that Treg cells in B^–/– mice are more effective suppressors of SAT than Treg cells in WT mice. Treg cells from B^–/– mice differ from WT Treg cells in expression of CD27, tumour necrosis factor receptor (TNFR) II p75, and glucocorticoid-induced TNFR-related protein (GITR). After transient depletion using anti-CD25 or diphtheria toxin, the repopulating Treg cells in B^–/– mice lack suppressor function, and expression of CD27, GITR and p75 is like that of WT Treg cells. If B^–/– Treg cells develop with B cells in bone marrow chimeras, their phenotype is like that of WT Treg cells. Addition of B cells to cultures of B^–/– Treg and T effector cells abrogates their suppressive function and their phenotype is like that of WT Treg cells. These results establish for the first time that Treg cells in WT and B^–/– mice differ both functionally and in expression of particular cell surface markers. Both properties are altered after transient depletion and repopulation of B^–/– Treg cells, and by the presence of B cells during Treg cell development or during interaction with effector T cells.     

Evidence for regulation of the autoimmune anti-erythrocyte response by idiotype-specific suppressor T cells in NZB mice

The present study demonstrates that specific CD8+, CD4- suppressor T cells (Ts) actively regulate the autoimmune anti-mouse red blood cell (MRBC) antibody response in spleen cell populations of young, Coombs-negative NZB mice. These Ts appear to bind a monoclonal NZB autoantibody (G-8 mAb) to unmodified MRBC which expresses a dominant idiotype (Id) in the spontaneous anti-MRBC autoantibody response of NZB mice. Treatment of normally nonauto-responsive spleen cells from young NZB mice with the G-8 mAb + C prior to culture allows these cells to develop, in 4-5 days, an autoantibody response to MRBC. The level of response obtained after depletion of the G-8-binding cells is comparable with that obtained after generalized depletion of Ts by treatment with anti-CD8 + C, suggesting that the G-8-binding cells make up a major portion of the regulatory Ts in this response. Yet, G-8 + C treatment depletes a very small subset of cells and not the total CD8+ T cell population. The regulatory cells appear to be neither isotype nor allotype specific, nor do they appear to have MRBC antigens bound to or expressed on their membranes. Rather, these cells are more likely G-8 idiotype specific. The regulatory G-8-binding cells are CD8+ T cells, not B cells. Furthermore, Ts-enriched populations when depleted of G-8-binding cells lose their ability to suppress in vitro anti-MRBC responses of spleen cells from Coombs-negative NZB mice depleted of CD8+ cells, as well as those of unfractionated spleen cells from Coombs-positive NZB mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of alphabeta and gammadelta T cells in the host response to Salmonella infection as demonstrated in T-cell-receptor-deficient mice of defined Ity genotypes.

Salmonella spp. are facultative intracellular bacteria which enter the body through the intestinal tract. We studied the roles of T cells expressing either the alpha and beta chains or the gamma and delta chains of the T-cell receptor (alphabeta T cells or gammadelta T cells, respectively) in the host defense against Salmonella using mice genetically deficient in either alphabeta T cells, gammadelta T cells, or both T-cell subsets. These mutant strains of mice were infected orally or intraperitoneally with Salmonella dublin, and the progression of the disease was monitored by determining bacterial numbers in the feces, gut wall, Peyer's patches, mesenteric lymph nodes, spleen, and liver. Since susceptibility to Salmonella infection in mice is strongly affected by the alleles at the Ity locus, T-cell-mutant mice with either the Ity-sensitive or Ity-resistant phenotype were tested for resistance to S. dublin infection. We found that even though large numbers of intraepithelial and mucosal alphabeta and gammadelta T cells populate the normal intestine, they have no role in controlling the invasion of S. dublin into the intestine or the subsequent bacterial replication in the Peyer's patches or gut wall. Furthermore, systemic infections were equally severe for the first 6 days in normal, alphabeta T-cell-deficient, and gammadelta T-cell-deficient mice, and alphabeta but not gammadelta T cells were required for clearance of S. dublin, regardless of the Ity phenotype. However, mice that lacked both T-cell subsets had higher bacterial counts in their livers 15 to 18 days after infection than did alphabeta T-cell-deficient mice, suggesting that gammadelta T cells can contribute to acquired immunity to S. dublin.     

T cells are required for the peripheral phase of B-cell maturation

B-lymphocyte maturation is considered to be independent of the thymus. However, there is circumstantial evidence suggesting that it may be impaired in nude animals that lack the thymus. Our study shows that the proportion of immature B-lymphocyte subsets (CD90(high) IgM(high) and CD90(high) IgM(low)) was significantly increased, whereas that of mature B-lymphocyte subsets (CD90- IgM(low) and CD90- IgM(high)) was decreased in the blood and lymph nodes of nude rats. In addition, the expression of major histocompatibility complex class II, intercellular adhesion molecule-1, CD44 and l-selectin was significantly down-regulated both on immature and mature B-lymphocyte subsets. After implantation of thymic tissue under the kidney capsule of nude rats the block in B-lymphocyte maturation was alleviated and the expression of surface molecules was normalized. Comparable effects were seen after the adoptive transfer of T lymphocytes. Thus, we show that in nude rats B cells do not mature properly because of the lack of T-cell help and that T lymphocytes are required for the peripheral phase of B-lymphocyte maturation, as well as for the appropriate expression of surface molecules. This should be considered when treating patients with T-cell deficiencies.     

A novel model of acute liver injury in mice induced by T cell-mediated immune response to lactosylated bovine serum albumin

The present study aimed to establish a murine liver injury model with T cell-mediated immune response to mimic the cellular immunological pathogenesis of hepatitis. Bovine serum albumin was conjugated with lactose by redox to prepare lactosylated bovine serum albumin (LacBSA). The liver injury was induced in C57BL/6 mice by a T cell-mediated immune response to LacBSA. After a systemic injection of LacBSA, which elicited immune response in mice sensitized twice with LacBSA, liver injury was observed with obvious increase of aminotransferase levels in serum and hepatocelluar damage in liver histology. However, the splenectomy before the elicitation significantly alleviated the liver injury. Direct contact between spleen T cells/nonparenchymal cells and hepatocytes was proved to be essential to induce the release of alanine aminotransferase in the culture supernatant. In addition, both levels of Fas mRNA in liver tissues and Fas Ligand in spleen cells were up-regulated at 6 h after the elicitation. The Fas mRNA expression was maintained in a relatively high level at 18 h while Fas Ligand began to decrease after 12 h. These results demonstrated that the T cell immune response to LacBSA could be successfully localized in the liver to induce an immunological liver injury in mice. The pathogenesis of the liver injury might involve the interaction between the liver-infiltrating lymphocytes and hepatocytes through Fas/FasL pathway.