Antigen-specific responses and ANA production in B6.Sle1b mice: a role for SAP

B6.Sle1b mice, which contain the Sle1b gene interval derived from lupus prone NZM2410 mice on a C57BL/6 background, present with gender-biased, highly penetrant anti-nuclear antibody (ANA) production. To obtain some insight into the possible induction mechanism of autoantibodies in these mice we compared antigen-specific T dependent (TD) and T independent (TI-II) responses between B6.Sle1b and B6 mice before the development of high ANA titers. Our results show that B6.Sle1b mice mount enhanced responses to a TI-II antigen. Additionally, the memory T cell response generated by a TD antigen also increased. This enhancement correlates with the greater ability of B cells from B6.Sle1b mice to present antigen to T cells. The SLAM Associated Protein (SAP) is critical for signaling of many of the molecules encoded by the SLAM/CD2 gene cluster, candidates for mediating the Sle1b phenotype; therefore, we also investigated the effect of sap deletion in these strains on the TD and TI-II responses as well as on ANA production. The results of these studies of responses to non-self-antigens provide further insight into the mechanism by which responses to self-antigens might be initiated in the context of specific genetic alterations.     

Association of extensive polymorphisms in the SLAM/CD2 gene cluster with murine lupus

Susceptibility to autoimmunity in B6.Sle1b mice is associated with extensive polymorphisms between two divergent haplotypes of the SLAM/CD2 family of genes. The B6.Sle1b-derived SLAM/CD2 family haplotype is found in many other laboratory mouse strains but only causes autoimmunity in the context of the C57Bl/6 (B6) genome. Phenotypic analyses have revealed variations in the structure and expression of several members of the SLAM/CD2 family in T and B lymphocytes from B6.Sle1b mice. T lymphocytes from B6.Sle1b mice have modified signaling responses to stimulation at 4-6 weeks of age. While autoimmunity may be mediated by a combination of genes in the SLAM/CD2 family cluster, the strongest candidate is Ly108, a specific isoform of which is constitutively upregulated in B6.Sle1b lymphocytes.     

The role of NK cells in the development of autoantibodies

The systemic lupus erythematosus (Sle1) interval from the NZM2410 mouse strain has been shown to be responsible for high levels of autoantibody production against antinuclear antibodies (ANA) when transferred into C57BL/6 mice. B cells derived from the B6.Sle1 strain are required for the production but help from both T-dependent and independent sources have been documented. Using radiation chimeras constructed in a strain of mice that is chronically depleted of Natural killer (NK) cells, but not NKT cells, we have examined the role of NK cells in the development of ANA in this context. Our results show that in the presence of intact T cell help depletion of NK cells does not affect ANA production. However, when T cell help is compromised, the prevalence of animals producing ANA is significantly decreased suggesting that NK cells can provide help for the T-independent production of ANA. Further experiments provide a possible mechanism for the NK-cell dependence.     

The SLAM family member CD48 (Slamf2) protects lupus-prone mice from autoimmune nephritis

Polymorphisms in the SLAM family of leukocyte cell surface regulatory molecules have been associated with lupus-like phenotypes in both humans and mice. The murine Slamf gene cluster lies within the lupus-associated Sle1b region of mouse chromosome 1. Non-autoreactive C57BL/6 (B6) mice that have had this region replaced by syntenic segments from other mouse strains (i.e. 129, NZB and NZW) are B6 congenic strains that spontaneously produce non-nephritogenic lupus-like autoantibodies. We have recently reported that genetic ablation of the SLAM family member CD48 (Slamf2) drives full-blown autoimmune disease with severe proliferative glomerulonephritis (CD48GN) in B6 mice carrying 129 sequences of the Sle1b region (B6.129CD48^−/−). We also discovered that BALB/c mice with the same 129-derived CD48-null allele (BALB.129CD48^−/−) have neither nephritis nor anti-DNA autoantibodies, indicating that strain specific background genes modulate the effects of CD48 deficiency. Here we further examine this novel model of lupus nephritis in which CD48 deficiency transforms benign autoreactivity into fatal nephritis. CD48GN is characterized by glomerular hypertrophy with mesangial expansion, proliferation and leukocytic infiltration. Immune complexes deposit in mesangium and in sub-endothelial, sub-epithelial and intramembranous sites along the glomerular basement membrane. Afflicted mice have low-grade proteinuria, intermittent hematuria and their progressive renal injury manifests with elevated urine NGAL levels and with uremia. In contrast to the lupus-like B6.129CD48^−/− animals, neither BALB.129CD48^−/− mice nor B6 × BALB/c F1.129CD48^−/− progeny have autoimmune traits, indicating that B6-specific background genes modulate the effect of CD48 on lupus nephritis in a recessive manner.     

Defective B-cell response to T-dependent immunization in lupus-prone mice

Lupus anti-nuclear Ab show the characteristics of Ag-driven T-cell-dependent (TD) humoral responses. If autoAg elicit the same response as exogenous Ag, lupus should enhance humoral responses to immunization. Blunted responses to various immunizations have, however, been reported in a significant portion of lupus patients. In this study, we show that lupus-prone C57BL/6.Sle1.Sle2.Sle3 (B6.TC) mice produce significantly less Ab in response to TD immunization than congenic controls, while producing significantly more total Ig. This blunted Ab response to TD Ag could be reconstituted with B6.TC B and CD4+ T cells. Multiple defects were found in the B6.TC response to 4-hydroxy-3-nitrophenylacetyl-keyhole limpet hemocyanin (NP-KLH) compared with total Ig, including a smaller percentage of B cells participating in the NP-response, a reduced entry into germinal centers, and highly defective production of NP-specific long-lived plasma cells (PC) in the bone marrow. B6.TC PC expressed reduced levels of FcgammaRIIb, which suggests that reduced apoptosis in resident PC prevents the establishment of newly formed NP-specific PC in bone marrow niches. Overall, these results show that lupus-prone mice responded differently to auto- and exogenous Ag and suggest that low FcgammaRIIb, hypergammaglobulinemia, and high autoAb production would be predictive of a poor response to immunization in lupus patients.     

The role of NK cells in the development of autoantibodies

The systemic lupus erythematosus (Sle1) interval from the NZM2410 mouse strain has been shown to be responsible for high levels of autoantibody production against antinuclear antibodies (ANA) when transferred into C57BL/6 mice. B cells derived from the B6.Sle1 strain are required for the production but help from both T-dependent and independent sources have been documented. Using radiation chimeras constructed in a strain of mice that is chronically depleted of Natural killer (NK) cells, but not NKT cells, we have examined the role of NK cells in the development of ANA in this context. Our results show that in the presence of intact T cell help depletion of NK cells does not affect ANA production. However, when T cell help is compromised, the prevalence of animals producing ANA is significantly decreased suggesting that NK cells can provide help for the T-independent production of ANA. Further experiments provide a possible mechanism for the NK-cell dependence.     

Identification of a MHC I-restricted epitope of DsRed in C57BL/6 mice

Fluorescent proteins can be used to visualize cells and their constituents by various imaging techniques. Adoptive transfer of T cells from C57Bl/6 (B6) mice that expressed DsRed.T3 under the actin promoter lead to frequent rejection of transferred cells. In short term in vivo cytotoxicity assays these mice showed detectable, but weak lysis of DsRed spleen cells but their responses could be boosted by re-challenge with DsRed spleen cells. To determine whether DsRed protein may contain an H-2^b MHC I-restricted T cell epitope, B6 mice immune to DsRed spleen cells were examined for in vivo lysis of target cells coated with various DsRed-derived peptides selected by the SYFPEITHI epitope prediction program. This analysis identified one D^b-restricted peptide sequence within DsRed (SSLQDGCFI) that acted as an epitope for B6 target lysis. Knowledge of this epitope could allow DsRed to be used as a model antigen in B6 mice and cautions against using this fluorochrome, as well as several others containing the immunogenic sequence, in adoptive transfer studies where rejection is not desirable.     

Selenium supplementation suppresses immunological and serological features of lupus in B6.Sle1b mice

Systemic lupus erythematosus (SLE) is a debilitating multi-factorial immunological disorder characterized by increased inflammation and development of anti-nuclear autoantibodies. Selenium (Se) is an essential trace element with beneficial anti-cancer and anti-inflammatory immunological functions. In our previous proteomics study, analysis of Se-responsive markers in the circulation of Se-supplemented healthy men showed a significant increase in complement proteins. Additionally, Se supplementation prolonged the life span of lupus prone NZB/NZW-F1 mice. To better understand the protective immunological role of Se in SLE pathogenesis, we have investigated the impact of Se on B cells and macrophages using in vitro Se supplementation assays and the B6.Sle1b mouse model of lupus with an oral Se or placebo supplementation regimen. Analysis of Se-treated B6.Sle1b mice showed reduced splenomegaly and splenic cellularity compared to untreated B6. Sle1b mice. A significant reduction in total B cells and notably germinal center (GC) B cell numbers was observed. However, other cell types including T cells, Tregs, DCs and pDCs were unaffected. Consistent with reduced GC B cells there was a significant reduction in autoantibodies to dsDNA and SmRNP of the IgG2b and IgG2c subclass upon Se supplementation. We found that increased Se availability leads to impaired differentiation and maturation of macrophages from mouse bone marrow derived progenitors in vitro. Additionally, Se treatment during in vitro activation of B cells with anti-CD40L and LPS inhibited optimal B cell activation. Overall our data indicate that Se supplementation inhibits activation, differentiation and maturation of B cells and macrophages. Its specific inhibitory effect on B cell activation and GC B cell differentiation could be explored as a potential therapeutic supplement for SLE patients.     

Characterization of murine newborn inhibitory T lymphocytes: functional and phenotypic comparison with an adult T cell subset activated in vitro by alphafetoprotein

Inhibitory T lymphocytes isolated from the spleens of newborn CBA/J mice which have high intrinsic levels of alpha-fetoprotein (AFP) were compared, in terms of surface antigen phenotype and mode of immunosuppressive action, with a population of adult spleen T cells activated in vitro by a 4-day preculture in the presence of purified AFP. The suppressing ability of these two groups of T cells was assayed on in vitro antibody synthesis of adult spleen cells to the T-dependent (TD) and T independent (TI) antigens, sheep red blood cells and 2,4-dinitrophenylated Ficoll. Negative selection of T cell subpopulations with antisera and complement showed that the inhibitory activity of newborn and adult AFP-activated T cells on anti-sheep red blood cell responses was abrogated by anti-Thy-1 anti-Lyt 1, and by anti-Ia^k (A. TH anti-A. TL), whereas anti-Lyt 2 antiserum and complement had no effect. Cytotoxic pretreatment with a panel of alloantisera specific for distinct products of major histocompatibility complex I subregions indicated that the Ia antigens expressed on the inhibitory T cells included I-J, but not I-EC determinants. Newborn and adult AFP-activated inhibitory T cells could thus be shown to belong to a very similar, if not identical, subset of T lymphocytes characterized by the Thy-1⁺, Lyt-1⁺2^?, I-J⁺ phenotype. Both groups of T lymphocytes were shown to behave like efficient suppressor inducer cells in that they augmented TD antibody responses when added to purified B cell cultures which could not respond to TD antigen on their own, but caused strong inhibition of these responses when normal T cells were present. Dilution analyses of the two groups of inhibitory T cells showed that efficient suppressive activity was highly selective for TD responses, compared with a relatively weak inhibitory effect on TI responses. Passive administration of anti-I-J alloantibodies into newborn animals resulted in a significantly increased ability to respond to TD antigen, suggesting an anti-I-J-mediated disruptive effect on newborn suppressor mechanisms. Our findings, therefore, indicate that TD antibody responses in newborn mice are regulated by an I-J⁺ T cell-mediated feedback inhibition circuit. Based on functional and phenotypic analyses, our results further suggest the probable identity of the naturally occurring inhibitory T cells in newborn mice and a subset of adult spleen T cells activated in vitro by AFP. This supports the contention that AFP may function as an important immunoregulatory agent in vivo.     

Enhanced expression of stem cell antigen-1 (Ly-6A/E) in lymphocytes from lupus prone mice correlates with disease severity

B6.Sle1 mice, congenic for the NZM2410-derived lupus susceptibility locus, Sle1 on chromosome 1 exhibit many of the features seen in human lupus including activated lymphocytes and high titers of antinuclear autoantibodies. Among the different surface molecules that were aberrantly expressed on the B6.Sle1 lymphocytes was Ly-6A/E. Splenic B- and T-lymphocytes but not myeloid cells from B6.Sle1 mice exhibited enhanced levels of Ly-6A/E compared to B6 controls. In particular, MZ B cells, GC B cells and B-cell blasts expressed the highest levels of Ly-6A/E in both strains, with the levels being even higher on B6.Sle1 derived cells. Following stimulation with LPS or anti-IgM, there was a profound up-regulation in Ly-6A/E, particularly on MZ B cells and B-cell blasts. CD4 and CD8 T cells also up-regulated Ly-6A/E after stimulation with anti-CD3 and anti-CD28. These studies were extended to additional autoimmune strains including B6.Sle3, B6.Sle1.lpr and BXSB. Importantly, Ly-6A/E levels on lymphocytes were commensurate with the degree of disease exhibited by these lupus strains. Finally, it appears that increased interferon levels, in addition to antigen receptor stimulation, may also be a factor accounting for elevated Ly-6A/E in lupus. Given these observations it is important to elucidate the functional role of Ly-6A/E in lupus in future studies.     

Distinct and synergistic roles of FcγRIIB deficiency and 129 strain-derived SLAM family proteins in the development of spontaneous germinal centers and autoimmunity

The inhibitory IgG Fc receptor (FcγRIIB) deficiency and 129 strain-derived signaling lymphocyte activation molecules (129-SLAMs) are proposed to contribute to the lupus phenotype in FcγRIIB-deficient mice generated using 129 ES cells and backcrossed to C57BL/6 mice (B6.129.RIIBKO). In this study, we examine the individual contributions and the cellular mechanisms by which FcγRIIB deficiency and 129-derived SLAM family genes promote dysregulated spontaneous germinal center (Spt-GC) B cell and follicular helper T cell (Tfh) responses in B6.129.RIIBKO mice. We find that B6 mice congenic for the 129-derived SLAM locus (B6.129-SLAM) and B6 mice deficient in FcγRIIB (B6.RIIBKO) have increased Spt-GC B cell responses compared to B6 controls but significantly lower than B6.129.RIIBKO mice. These data indicate that both FcγRIIB deficiency and 129-SLAMs contribute to elevated Spt-GC B cell responses in B6.129.RIIBKO mice. However, only 129-SLAMs contribute significantly to augmented Tfh responses in B6.129.RIIBKO mice, and do so by a combination of T cell-dependent effects and enhanced B cell and DC-dependent antigen presentation to T cells. Elevated Spt-GC B cell responses in mice with FcγRIIB deficiency and polymorphic 129-SLAMs were associated with elevated metabolic activity, improved GC B cell survival and increased differentiation of naïve B cells into GC B cell phenotype. Our data suggest that the interplay between 129-SLAM expression on B cells, T cells and DCs is central to the alteration of the GC tolerance checkpoint, and that deficiency of FcγRIIB on B cells is necessary to augment Spt-GC responses, pathogenic autoantibodies, and lupus disease.     

Immunization against tumor and minor histocompatibility antigens by eluted cellular peptides loaded on antigen processing defective cells

Material eluted from RMA lymphoma or B6 spleen cells under acid conditions was fractionated by reverse phase high-performance liquid chromatography, and tested for ability to restore the sensitivity to cytotoxic T lymphocytes of the processing/presentation defective mutant line RMA-S. This allowed identification of three fractions (termed M1, M2 and M3) carrying B6 antigens recognized by cytotoxic T lymphocytes (CTL) elicited across the minor histocompatibility barrier A. BYanti-B6 (both H-2^b) and one fraction (termed TI) carrying a tumor antigen recognized by B6 anti-RMA CTL. By parallel runs of material from cell lysates over major histocompatibility complex class I affinity columns, the M2 and M3 antigens were defined as K^b restricted, and M1 and T1 as D^b restricted. Isolated fractions loaded onto RMA-S cells could be used to prime anti-minor histocompatibility antigen and tumor CTL in vivo. They could also be used for in vitro restimulation of spleen cells from mice that had been primed either by antigen-loaded RMA-S, or by wild-type RMA tumor cells and B6 splenocytes. The CTL generated by these methods were specific for the loading antigen, and they also recognized the antigen on the “physiological” target, i.e. RMA or B6 lymphoblasts. This system based on RMA-S as an immunization and target antigen reporter cell may be used for dissection of complex CTL responses, e.g. in studies of clonal composition and epitope dominance, or for studies of tumors that are poor stimulators of immunity.     

Immunogenicity of recombinant <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> bacille Calmette–Guèrin clones expressing T and B cell epitopes of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigens

Recombinant Mycobacterium bovis bacille Calmette–Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4⁺ and CD8⁺ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.     

T cell-independent and T cell-dependent immunoglobulin G responses to polyomavirus infection are impaired in complement receptor 2-deficient mice

Polyomavirus (PyV) infection induces protective T cell-independent (TI) IgM and IgG antibody responses in T cell-deficient mice, but these responses are not generated by immunization with viral proteins or virus like particles. We hypothesized that innate signals contribute to the generation of isotype-switched antiviral antibody responses. We studied the role of complement receptor (CR2) engagement in TI and T cell-dependent (TD) antibody responses to PyV using CR2-deficient mice. Antiviral IgG responses were reduced by 80-40% in CR2-/- mice compared to wild type. Adoptive transfer experiments demonstrated the need for CR2 not only in TD, but also in TI IgG responses to PyV. Transfer of CR2-/- B lymphocytes to SCID mice resulted in TI antiviral IgG responses that corresponded to 10% of that seen in wild-type B cell-reconstituted mice. Thus, our studies revealed a profound dependence of TI and TD antiviral antibody responses on CR2-mediated signals in PyV-infected mice, where the viral antigen is abundant and persistent.     

Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation

B and T lymphocytes develop normally in mice lacking the guanine nucleotide exchange factor Vav-2. However, the immune responses to type II thymus-independent antigen as well as the primary response to thymus-dependent (TD) antigen are defective. Vav-2-deficient mice are also defective in their ability to switch immunoglobulin class, form germinal centers and generate secondary immune responses to TD antigens. Mice lacking both Vav-1 and Vav-2 contain reduced numbers of B lymphocytes and display a maturational block in the development of mature B cells. B cells from Vav-1(-/-)Vav-2(-/-) mice respond poorly to antigen receptor triggering, both in terms of proliferation and calcium release. These studies show the importance of Vav-2 in humoral immune responses and B cell maturation.     

Vaccination with Trypomastigote Surface Antigen 1-Encoding Plasmid DNA Confers Protection against Lethal Trypanosoma cruzi Infection

DNA vaccination was evaluated with the experimental murine model of Trypanosoma cruzi infection as a means to induce antiparasite protective immunity, and the trypomastigote surface antigen 1 (TSA-1), a target of anti-T. cruzi antibody and major histocompatibility complex (MHC) class I-restricted CD8⁺ cytotoxic T-lymphocyte (CTL) responses, was used as the model antigen. Following the intramuscular immunization of H-2^b and H-2^d mice with a plasmid DNA encoding an N-terminally truncated TSA-1 lacking or containing the C-terminal nonapeptide tandem repeats, the antibody level, CTL response, and protection against challenge with T. cruzi were assessed. In H-2^b mice, antiparasite antibodies were induced only by immunization with the DNA construct encoding TSA-1 containing the C-terminal repeats. However, both DNA constructs were efficient in eliciting long-lasting CTL responses against the protective H-2K^b-restricted TSA-1_515–522 epitope. In H-2^d mice, inoculation with either of the two TSA-1-expressing vectors effectively generated antiparasite antibodies and primed CTLs that lysed T. cruzi-infected cells in an antigen-specific, MHC class I-restricted, and CD8⁺-T-cell-dependent manner. When TSA-1 DNA-vaccinated animals were challenged with T. cruzi, 14 of 22 (64%) H-2^b and 16 of 18 (89%) H-2^d mice survived the infection. The ability to induce significant murine anti-T. cruzi protective immunity by immunization with plasmid DNA expressing TSA-1 provides the basis for the application of this technology in the design of optimal DNA multicomponent anti-T. cruzi vaccines which may ultimately be used for the prevention or treatment of Chagas’ disease.

Chagas’ disease, caused by the intracellular protozoan parasite Trypanosoma cruzi, is a lifelong health problem in Central and South America, where an estimated 18 million people are infected with this parasite and 90 million are at risk of infection (35, 65). Following a short-lived acute-phase illness characterized by fever and a patent parasitemia, infected individuals enter a nearly aparasitemic asymptomatic chronic phase, where most remain for the remainder of their lifetime. However, at 10 to 20 years postinfection nearly 30% of infected individuals develop severe cardiomyopathy, which is responsible for most of the 50,000 deaths caused by Chagas’ disease each year (45). Although reduviid vector control and blood bank screening measures have had a major impact in reducing transmission of T. cruzi (65), the operational costs to maintain such control programs, behavioral differences among vector species, existence of animal reservoirs, persistence of parasites in chronically infected patients, and lack of adequate chemotherapies to treat the infection will likely prevent these control measures alone from completely eradicating T. cruzi. An additional approach that could contribute significantly to control the transmission of Chagas’ disease is the development of anti-T. cruzi vaccines. To date, however, vaccine production for T. cruzi has been a low priority despite the current knowledge about the protective roles that antibodies, type 1 cytokines, and CD8⁺ T cells play in resistance to experimental T. cruzi infections (53).During T. cruzi infection, both chagasic patients and experimental animals produce strong immune responses to molecules from the infective nonreplicative trypomastigote stage and the replicative amastigote forms (3, 4, 14, 29). Among these, trypomastigote surface antigen 1 (TSA-1) (15, 38), a major trypomastigote surface antigen and the first identified member of the trans-sialidase gene superfamily (48), is a target of protective immune responses in mice (61, 66). Immunization with an amino-proximal fragment of TSA-1 induces a strong antibody response and protects mice against an otherwise lethal challenge with T. cruzi (66). Our studies have recently identified TSA-1 as the first bona fide target of CD8⁺ cytotoxic T lymphocytes (CTL) in T. cruzi-infected mice and demonstrated that the adoptive transfer of TSA-1-specific gamma interferon (IFN-γ)- and tumor necrosis factor alpha-producing CTL lines protects naive animals against lethal T. cruzi infection (61). Moreover, we have recently determined that TSA-1 and amastigote surface protein-1 and -2 (33, 44), which are also recognized by murine CTL (32), represent three target molecules of T. cruzi-specific human CD8⁺ CTL (62). These studies demonstrated the validity of the mouse model to identify target antigens of protective anti-T. cruzi immune responses and provide a strong incentive for the development of vaccines as a potential control measure against Chagas’ disease. For this purpose, and given the success of plasmid DNA vaccination in specifically stimulating a broad spectrum of immune responses to the vector-encoded target antigen (12), we have chosen to investigate DNA-based immunization as a system to generate vaccine-induced resistance against T. cruzi and have used TSA-1 as a model antigen for its initial evaluation. In this report we document that intramuscular injection of BALB/c and C57BL/6J mice with TSA-1-encoding plasmid DNA induces antibodies, CTL, and significant protection against lethal challenge with T. cruzi.     

Influence of the nude and X-Iinked immune deficiency genes on expression of × and λ light chains

The relative amounts of Igx and Igλ₁ anti-2,4-dinitrophenyl antibodies were measured at various times after immunizing mice with prototype thymus-dependent (TD), thymus-independent type 1 (TI-1) and thymus-independent type 2 (TI-2) antigens. Similar amounts of Igλ₁ were produced after TD and TI-2 immunization and somewhat less was produced after a TI-1 stimulus. In contrast, Igx levels were much greater after TD than after TI-1 or TI-2 antigen. The amount of light chain isotype produced appeared to depend on the molecular form in which the hapten was presented, although possible adjuvant effects were not ruled out. Levels of Igx and Igλ present in nonimmune sera were measured in normal, xid and nude mice. The x/λ, ratio was higher in xid than in normal mice and the difference was demonstrated by F₁ analysis to be due to an X-linked gene. Conversely, the x/λ ratio was lower in nude than in normal mice. This was true for the CBA/Tufts (Igh^j), CBA. Igh^b and C57BL/10 strains. However, there were no detectable differences in the relative frequencies of surface Igx- and Igλ -bearing B cells in adult CBA/Tufts, CBA/N and nude mice. Hence, serum ratios may reflect differences at the level of B cell triggering. Two possible explanations for these differences are discussed. Igx and Igλ may be expressed on functionally distinct B cell subsets. (For instance Igλ -producing cells might be readily triggered by T1 antigens whereas Igx-producing cells are more dependent on T cell signals. Such functional subsets could be determined by light chain expression). Alternatively, cells producing Igx antibody are selected for because they have a higher affinity for antigen. If so, triggering of cells producing high affinity Igx or their subsequent selection is T cell-dependent.     

Lack of T cells in Act1-deficient mice results in elevated IgM-specific autoantibodies but reduced lupus-like disease

Act1 is a negative regulator of B‐cell activation factor of the TNF family (BAFF) and CD40L‐induced signaling. BALB/C mice lacking Act1 develop systemic autoimmunity resembling systemic lupus erythematosus (SLE) and Sjögren's syndrome (SjS). SLE and SjS are characterized by anti‐nuclear IgG autoantibody (ANA‐IgG) production and inflammation of peripheral tissues. As autoantibody production can occur in a T‐cell dependent or T‐cell independent manner, we investigated the role of T‐cell help during Act1‐mediated autoimmunity. Act1‐deficiency was bred onto C57Bl/6 (B6.Act1^?/?) mice and B6.TCRβ^?/?TCRδ^?/?Act1^?/? (TKO) mice were generated. While TCRβ/δ‐sufficient B6.Act1^?/? mice developed splenomegaly and lymphadenopathy, hypergammaglobulinemia, elevated levels of ANA‐IgG, and kidney pathology, TKO mice failed to develop any such signs of disease. Neither B6.Act1^?/? nor TKO mice developed SjS‐like disease, suggesting that epigenetic interactions on the BALB/C background are responsible for this phenotype in BALB/C.Act1^?/? mice. Interestingly, BAFF‐driven transitional B‐cell abnormalities, previously reported in BALB/C.Act1^?/? mice, were intact in B6.Act1^?/? mice and largely independent of T cells. In conclusion, T cells are necessary for the development of SLE‐like disease in B6.Act1^?/? mice, but not BAFF‐driven transitional B‐cell differentiation.     

Prevention of murine lupus by an I-E α chain transgene: Protective role of I-E α chain-derived peptides with a high affinity to I-Ab molecules

The expression of a transgene encoding the I-E α chain prevents a lupus-like autoimmune syndrome in BXSB mice. However, it had not been elucidated whether the Eα^d transgene-mediated protective effect results from I-E expression or from the generation of I-E α chain-derived peptides (Eα peptide) displaying high affinity for the I-A^b molecule. To address this question, two different BXSB lines expressing the transgene at low or high levels were crossed with lupus-prone MRL mice; this resulted in three types of (MRL × BXSB)F₁ mice, differing in the expression levels of I-E molecules and of Eα peptides presented by I-A^b molecules. Comparative analysis of these three (MRL × BXSB)F₁ mice as well as several BXSB transgenic lines showed that the Eα^d transgene-mediated protection paralleled the expression levels of Eα peptide presented by I-A^b molecules, but not of I-E molecules on B cells. In addition, use of transgenic and nontransgenic double bone marrow chimeras showed a selective activation of nontransgenic B cells during I-A^b-restricted T cell-dependent immune responses, while both transgenic and nontransgenic B cells were comparably activated during T cell-independent responses. These results favor a model of autoimmunity prevention based on competition for antigen presentation, in which excessive generation of Eα peptides prevents, because of their high affinity to the I-A molecules, activation of potential autoreactive T and B cells.     

Both positive and negative effects on immune responses by expression of a second class II MHC molecule

It is perplexing why vertebrates express a limited number of major histocompatibility complex (MHC) molecules when theoretically, having a greater repertoire of MHC molecules would increase the number of epitopes presented, thereby enhancing thymic selection and T cell response to pathogens. It is possible that any positive effects would either be neutralized or outweighed by negative selection restricting the T cell repertoire. We hypothesize that the limit on MHC number is due to negative consequences arising from expressing additional MHC. We compared T cell responses between B6 mice (I-A⁺) and B6.E⁺ mice (I-A⁺, I-E⁺), the latter expressing a second class II MHC molecule, I-E^b, due to a monomorphic Eα^k transgene that pairs with the endogenous I-Eβ^b chain. First, the naive T cell Vβ repertoire was altered in B6.E⁺ thymi and spleens, potentially mediating different outcomes in T cell reactivity. Although the B6 and B6.E⁺ responses to hen egg-white lysozyme (HEL) protein immunization remained similar, other immune models yielded differences. For viral infection, the quality of the T cell response was subtly altered, with diminished production of certain cytokines by B6.E⁺ CD4⁺ T cells. In alloreactivity, the B6.E⁺ T cell response was significantly dampened. Finally, we observed markedly enhanced susceptibility to experimental autoimmune encephalomyelitis (EAE) in B6.E⁺ mice. This correlated with decreased percentages of nTreg cells, supporting the concept of Tregs exhibiting differential susceptibility to negative selection. Altogether, our data suggest that expressing an additional class II MHC can produce diverse effects, with more severe autoimmunity providing a compelling explanation for limiting the expression of MHC molecules.