IgG Switch Research Articles

Orientation Regulation of Class-switch Recombination in Human B Cells.

We developed a linear amplification-mediated high-throughput genome-wide translocation sequencing method to profile Ig class-switch recombination (CSR) in human B cells in an unbiased and quantitative manner. This enables us to characterize CSR junctions resulting from either deletional recombination or inversion for each Ig class/subclass. Our data showed that more than 90% of CSR junctions detected in peripheral blood in healthy control subjects were due to deletional recombination. We further identified two major CSR junction signatures/patterns in human B cells. Signature 1 consists of recombination junctions resulting from both IgG and IgA switching, with a dominance of Sµ-Sγ junctions (72%) and deletional recombination (87%). Signature 2 is contributed mainly by Sµ-Sα junctions (96%), and these junctions were almost all due to deletional recombination (99%) and were characterized by longer microhomologies. CSR junctions identified in healthy individuals can be assigned to both signatures but with a dominance of signature 1, whereas almost all CSR junctions found in patients with defects in DNA-PKcs or Artemis, two classical nonhomologous end joining (c-NHEJ) factors, align with signature 2. Thus, signature 1 may represent c-NHEJ activity during CSR, whereas signature 2 is associated with microhomology-mediated alternative end joining in the absence of the studied c-NHEJ factors. Our findings suggest that in human B cells, the efficiency of the c-NHEJ machinery and the features of switch regions are crucial for the regulation of CSR orientation. Finally, our high-throughput method can also be applied to study the mechanism of rare types of recombination, such as switching to IgD and locus suicide switching.

IgG Subtype Response against Virulence-Associated Protein A in Foals Naturally Infected with Rhodococcus equi.

Rhodococcus equi is an intracellular bacterium that causes suppurative pneumonia in foals. T-helper (Th) 1 cells play an important role in the protective response against R. equi. In mice and humans, the directionality of IgG switching reflects the polarization of Th-cell responses, but this has not been fully elucidated in horses. In this 4-year study, we classified R. equi-infected foals into surviving and non-surviving group and investigated differences in IgG subclass response to virulence-associated protein A, the main virulence factor of R. equi, between the groups. IgGa, IgGb, and IgG(T) titers were significantly higher in the non-surviving group compared with the surviving group. The titers of IgGa and IgG(T), IgGb and IgG(T), and IgGa and IgGb, respectively, were positively correlated, and the IgG(T)/IgGb ratio in the non-surviving group was significantly higher than that in the surviving group. The IgG(T) titer tended to increase more than the IgGa and IgGb titers in the non-surviving group compared with the surviving group. Our findings suggest that the IgG(T) bias in IgG subclass responses reflects the immune status, which exacerbates R. equi infection.

HLA Awareness in tissue decellularization: A paradigm shift for enhanced biocompatibility, studied on the model of the human fascia lata graft

Last twenties, tissue engineering has rapidly advanced to address the shortage of organ donors. Decellularization techniques have been developed to mitigate immune rejection and alloresponse in transplantation. However, a clear definition of effective decellularization remains elusive. This study compares various decellularization protocols using the human fascia lata model. Morphological, structural and cytotoxicity/viability analyses indicated that all the five tested protocols were equivalent and met Crapo's criteria for successful decellularization. Interestingly, only the in vivo immunization test on rats revealed differences. Only one protocol exhibited Human Leucocyte Antigen (HLA) content below 1% residual threshold, the only criterion preventing rat immunization with an absence of rat anti-human IgG switch after one month (N=4 donors for each of the 7 groups, added by negative and positive controls, n=28). By respecting a refined set of criteria, i.e. lack of visible nuclear material, <50ng DNA/mg dry weight of extracellular matrix, and <1% residual HLA content, the potential for adverse host reactions can be drastically reduced. In conclusion, this study emphasizes the importance of considering not only nuclear components but also major histocompatibility complex in decellularization protocols and proposes new guidelines to promote safer clinical development and use of bioengineered scaffolds.

AFF3, a susceptibility factor for autoimmune diseases, is a molecular facilitator of immunoglobulin class switch recombination.

Immunoglobulin class switch recombination (CSR) plays critical roles in controlling infections and inflammatory tissue injuries. Here, we show that AFF3, a candidate gene for both rheumatoid arthritis and type 1 diabetes, is a molecular facilitator of CSR with an isotype preference. Aff3-deficient mice exhibit low serum levels of immunoglobulins, predominantly immunoglobulin G2c (IgG2c) followed by IgG1 and IgG3 but not IgM. Furthermore, Aff3-deficient mice show weak resistance to Plasmodium yoelii infection, confirming that Aff3 modulates immunity to this pathogen. Mechanistically, the AFF3 protein binds to the IgM and IgG1 switch regions via a C-terminal domain, and Aff3 deficiency reduces the binding of AID to the switch regions less efficiently. One AFF3 risk allele for rheumatoid arthritis is associated with high mRNA expression of AFF3, IGHG2, and IGHA2 in human B cells. These findings demonstrate that AFF3 directly regulates CSR by facilitating the recruitment of AID to the switch regions.

TLR7 Signaling Shapes and Maintains Antibody Diversity Upon Virus-Like Particle Immunization.

Virus-like particles (VLPs) are used in different marketed vaccines and are able to induce potent antibody responses. The innate pattern recognition receptors TLR7/8 recognize single stranded (ss) RNA naturally packaged into some VLPs and have been shown to enhance the production of IgG antibodies upon immunization. Here we demonstrate that, upon immunization with RNA-loaded bacteriophage-derived VLP Qβ, TLR7 signaling accelerates germinal center formation, promotes affinity/avidity maturation of VLP-specific IgG and isotype switching to IgG2b/2c. These findings extrapolated to antigens displayed on Qβ; as Fel d 1, the major cat allergen, chemically attached to Qβ also induced higher affinity/avidity IgG2b/2c antibodies in a TLR7-dependent fashion. Chimeric mice lacking TLR7-expression exclusively in B cells demonstrated that the enhanced IgG responses were driven by a B cell intrinsic mechanism. Importantly, deep sequencing of the BCR repertoire of antigen-specific B cells demonstrated higher diversity in mice with TLR7 signaling in B cells, suggesting that TLR7-signaling drives BCR repertoire development and diversity. Furthermore, the current data demonstrate that high levels of clonal diversity are reached early in the response and maintained by TLR7 signaling. In conclusion, TLR7 signaling enhances levels and quality of IgG antibodies, and this finding has major implications for vaccine design.

B Cell Subsets Differentially Contribute to the T Cell-Independent Memory Pool.

The roles distinct B cell subsets play in clonal expansion, isotype switching, and memory B cell differentiation in response to T cell-independent type 2 Ags (TI-2 Ags) has been understudied. Using sorted B cells from VHB1-8 knock-in mice, we evaluated B-1b, marginal zone, and follicular B cell responses to the TI-2 Ag, NP-Ficoll. All subsets extensively divided in response to NP-Ficoll. Nonetheless, B-1b cells exhibited significantly increased IgG switching and differentiation into Ab-secreting cells (ASC)-a finding that coincided with increased AgR signaling capacity and Blimp1 expression by B-1b cells. All subsets formed memory cells and expressed markers previously identified for T cell-dependent memory B cells, including CD80, PDL2, and CD73, although B-1b cells generated the greatest number of memory cells with higher frequencies of IgG- and CD80-expressing cells. Despite memory formation, secondary immunization 4 wk after primary immunization did not increase NP-specific IgG. However, boosting occurred in B-1b cell-recipient mice when IgG levels declined. CD80+ memory B-1b cells divided, class switched, and differentiated into ASC in response to Ag in vivo, but this was inhibited in the presence of NP-specific IgG. Furthermore, CD80 blockade significantly increased memory B-1b cell division and differentiation to ASC upon Ag restimulation. Collectively, these findings demonstrate B-1b, marginal zone B, and follicular B subsets significantly contribute to the TI-2 Ag-specific memory B cell pool. In particular, we show B-1b cells generate a functional CD80-regulated memory population that can be stimulated to divide and differentiate into ASC upon Ag re-encounter when Ag-specific IgG levels decline.

Food-Specific IgE and IgG Antibodies in Patients With Chronic Rhinosinusitis With Nasal Polyps: A Case-Control Study.

EPOS 2012 states that investigation is needed to study a possible role for food allergy in the initiation and perpetuation of chronic rhinosinusitis with nasal polyps (CRSwNP). Our main goal was to compare serum levels of food-specific immunoglobulin G (IgG) and IgE antibodies in patients with CRSwNP and controls. A prospective case-control study with 33 patients with CRSwNP and 31 controls without CRS was carried out. Clinical data were gathered through a systematic interview and blood sample was collected. Enzyme-linked immunosorbent assay tests using OmegaDiagnostics kit with 40 food allergens for detection of specific IgG antibodies were performed and food-specific IgE antibodies were determined by immunoassay using ImmunoCAP. Immunoglobulin classes and IgG subclasses levels were also evaluated. Statistical analysis was performed using SPSS v.23. The overall sum of food IgG antibodies was significantly lower in CRSwNP compared to control group, and this difference was also observed for different specific IgG antibodies (corn, soya, grain legumes, pear and apple, berries, citric fruit). In controls, a positive correlation between IgG1 and the sum of food IgG antibodies was seen, but in CRSwNP group a negative correlation was found. In addition, a significant higher level of IgG1 and lower IgG2 and IgG3 was found among patients with CRSwNP. Levels of serum-specific IgE antibodies against multiallergen food mix (fx5) and against shrimp, strawberry, orange, rye, or egg yolk, as well as the sum of food IgE antibodies, did not differ significantly between the groups. These findings suggest that food allergy does not have an important role in CRSwNP etiopathogenesis, whether it is IgG or IgE mediated. Moreover, the observed suppression of specific IgG antibodies against food allergens, its negative correlation with IgG1 and the IgG1 switching in CRSwNP, can be related to deviated IgG responses against other targets (eg, airborne particles) and warrants future investigation.

Bach2 Deficiency Leads to Spontaneous Expansion of IL-4-Producing T Follicular Helper Cells and Autoimmunity.

The transcription factor Bach2 is a susceptible gene for numerous autoimmune diseases including systemic lupus erythematosus (SLE). Bach2−/− mice can develop a lupus-like autoimmune disease. However, the exact cellular and molecular mechanisms via which Bach2 protects the hosts from developing autoimmunity remains incompletely understood. Here, we report that Bach2 ablation on T cells, but not B cells, resulted in humoral autoimmunity, and this was associated with expansion of T follicular helper (Tfh) cells and abnormal germinal centers. Bach2 was down-regulated in Tfh cells and directly suppressed by the Tfh-defining transcription factor BCL6. Mechanistically, Bach2 directly suppresses the transcription of Cxcr5 and c-Maf, two key regulators of Tfh cell differentiation. Bach2-deficient Tfh cells were skewed toward the IL-4-producing subset, which induced IgG1 and IgE isotype switching of B cells. Heterozygous Bcl6 deficiency reduced the formation of germinal center and autoantibodies, and ameliorated the pathology in Bach2-deficient mice. Our findings identify Bach2 as a crucial negative regulator of Tfh cells at steady state and prove that Bach2 controls autoimmunity in part by restraining accumulation of pathogenic Tfh cells.

IgG Responses to Porins and Lipopolysaccharide within an Outer Membrane-Based Vaccine against Nontyphoidal Salmonella Develop at Discordant Rates.

ABSTRACTAntibodies acquired after vaccination or natural infection with Gram-negative bacteria, such as invasive Salmonella enterica serovar Typhimurium, can protect against disease. Immunization with naturally shed outer membrane vesicles from Gram-negative bacteria is being studied for its potential to protect against many infections, since antigens within vesicles maintain their natural conformation and orientation. Shedding can be enhanced through genetic modification, and the resulting particles, generalized modules for membrane antigens (GMMA), not only offer potential as vaccines but also can facilitate the study of B-cell responses to bacterial antigens. Here we show that the response to immunization with GMMA from S. Typhimurium (STmGMMA) provides B-cell-dependent protection and induces antibodies to two immunodominant antigens, lipopolysaccharide (LPS) and porins. Antibodies to LPS O antigen (O-Ag) markedly enhance protection in the spleen, but this effect is less marked in the liver. Strikingly, IgG responses to LPS and porins develop with distinct kinetics. In the first week after immunization, there is a dramatic T-cell-independent B1b-cell-associated induction of all IgG isotypes, except IgG1, to porins but not to LPS. In contrast, production of IgG1 to either antigen was delayed and T cell dependent. Nevertheless, after 1 month, cells in the bone marrow secreting IgG against porins or LPS were present at a similar frequency. Unexpectedly, immunization with O-Ag-deficient STmGMMA did not substantially enhance the anti-porin response. Therefore, IgG switching to all antigens does not develop synchronously within the same complex and so the rate of IgG switching to a single component does not necessarily reflect its frequency within the antigenic complex.

Stat3 Is Important for Follicular Regulatory T Cell Differentiation.

The production of antibody is precisely controlled during the germinal center (GC) reaction. This process is dependent on the help from follicular T helper (Tfh) cells to germinal center (GC) B cells and is regulated by regulatory follicular T helper (Tfr) cells. How Tfr cells develop and how their suppressive activity functions are not well understood. Here, we found that Stat3 is indispensible for Tfr cell differentiation. After immunization with Sheep Red Blood Cells (SRBC), the loss of Tfr cells caused by deletion of Stat3 in Treg cells does not affect the size of Tfh or GC B cell population, but rather leads to strongly enhanced production of antigen-specific IgG1 and IgG2b. In Peyer’s patches (PPs) in the gut, we found that Stat3 expression in Treg cells is also required for Tfr cell formation to commensal organisms. However, loss of Tfr cells in the gut did not affect the numbers of Tfh cells and GC B cells, nor affect IgG1 or IgA switching by GC B cells. Overall, our study has uncovered unique roles of Stat3 in Tfr cell differentiation and the regulation of the antibody response.

Distinguishing benign from pathologic TH2 immunity in atopic children

Although most children with asthma and rhinitis are sensitized to aeroallergens, only a minority of sensitized children are symptomatic, implying the underlying operation of efficient anti-inflammatory control mechanisms. We sought to identify endogenous control mechanisms that attenuate expression of IgE-associated responsiveness to aeroallergens in sensitized children. In 3 independent population samples we analyzed relationships between aeroallergen-specific IgE and corresponding allergen-specific IgG (sIgG) and associated immunophenotypes in atopic children and susceptibility to asthma and rhinitis, focusing on responses to house dust mite and grass. Among mite-sensitized children across all populations and at different ages, house dust mite-specific IgG/IgE ratios (but not IgG4/IgE ratios) were significantly lower in children with asthma compared with ratios in those without asthma and lowest among the most severely symptomatic. This finding was mirrored by relationships between rhinitis and antibody responses to grass. Depending on age/allergen specificity, 20% to 40% of children with allergen-specific IgE (sIgE) of 0.35 kU/L or greater had negative skin test responses, and these children also expressed the high sIgG/sIgE immunophenotype. sIgG1 from these children inhibited allergen-induced IgE-dependent basophil activation in a dose-dependent fashion. Profiling of aeroallergen-specific CD4(+) TH memory responses revealed positive associations between sIgG/sIgE ratios and IL-10-dependent gene signatures and significantly higher IL-10/TH2 cytokine (protein) ratios among nonsymptomatic children. In addition to its role in blocking TH2 effector activation in the late-phase allergic response, IL-10 is a known IgG1 switch factor. We posit that its production during allergen-induced memory responses contributes significantly to attenuation of inflammation through promoting IgG1-mediated damping of the FcεRI-dependent acute-phase reaction. sIgG1/sIgE balance might represent a readily accessible therapeutic target for asthma/rhinitis control.

IGHV4-34 B-Cell Receptor Immunoglobulins from CLL Stereotyped Subset 4 React with Influenza A Virus: Requirement for IGHV-D-J/Iglv-J Rearrangement and Isotype Switching to IgG

B-cell chronic lymphocytic leukemia (CLL) clones from different patients can express stereotyped BCR immunoglobulins (IGs) that share very similar IGHV-D-J rearrangements and HCDR3s, often with similar IGLV gene usage, suggesting that they were selected by common antigenic epitope(s). Patients bearing IGHV4-34/D5-18/JH6/VK2-30/JK2 rearrangements belong to one of such stereotyped subsets, subset 4. In addition, subset 4 BCRs are always IgG switched and exhibit IGHVsomatic mutations.The germline IGHV4-34 gene is inherently autoreactive and is often found in antibodies that bind to the surfaces of erythrocytes and B lymphocytes through the linear i blood group antigen, poly-N-acetyllactosamine (NAL). To test the reactivity of subset 4 monoclonal antibodies (mAbs) to auto and foreign antigens, we expressed a panel of CLL BCR IGs carrying IGHV4-34, 1-69, 3-21, and 4-39 as recombinant human IgGs and tested the binding of these CLL mAbs to HEp-2 cells by intracellular immunostaining. Three subset 4 mAbs (183, 240 and 342) showed only weak binding to permeabilized HEp-2 cells compared to other CLL mAbs which often showed strong binding to cytoplasmic structures. Furthermore, because apoptotic cells are a source of autoantigens in autoimmune conditions, the reactivity of CLL mAbs to non-permeabilized, irradiated Ramos B and Jurkat T cell line cells was also tested by flow cytometry. Surprisingly, the average binding percentage to apoptotic Ramos B cells of subset 4 mAbs 183, 240, and 342 was only 6, 5, and 4%, respectively. Moreover, all three subset 4 mAbs exhibited minimum binding to live or apoptotic Jurkat T cells (1-10% and 4-6% to each population, respectively). We next tested the reactivity of subset 4 mAbs to i/I carbohydrate epitopes, the known targets of many non-stereotyped IGHV4-34 mAbs, using an i/I focused microarray containing 60 i/I related oligosaccharides. Notably, the subset 4 mAbs showed no binding at a concentration of 10 μg/mL. Together, the above results suggest that CLL subset 4 mAbs have weak or no interaction to the known autoantigens recognized by other non-stereotyped IGVH4-34 IGs. Hence we hypothesized that the restricted IGHV-D-Jrearrangement, somatic mutations, and IgG switch of subset 4 were acquired by selection for other auto or foreign antigens.To test this possibility, we screened CLL mAbs against a library of over 8,000 full length human proteins, including an influenza A virus and some human viral gene encoded proteins. Each subset 4 mAb showed selective and strong binding to influenza A virus H3N2 Texas 1/77 at concentration of 50 μg/mL; this binding was 6-15 times higher than that of other non-subset 4 mAbs, which included two IGHV4-34, non-stereotyped CLL mAbs. Because the binding to virus is usually mediated via its glycoprotein hemagglutinin (HA), we tested reactivity of subset 4 mAbs to the HA of influenza A H3N2 Texas 1/77. Subset 4 mAbs had on average 2 fold (1.8-2.2) higher binding signals than the two non-stereotyped IGHV4-34mAbs. To explore if subset 4 mAbs recognize other influenza A virus strains, we tested the binding of a representative subset 4 mAb 183 to HAs from 11 strains that belong to 3 subtypes: H1 (4 strains), H2 (1 strain) and H3 (6 strains). Clear preferential binding to all 6 H3s and to some of H1s was found. mAb 183 did not bind to H2 (A/Japan/305/1957 H2N2). The average binding signal ratio to H3s, H1s and H2 was 2.4:1.6:1. Testing to another hemagglutinin subtype H7 (A/Anhui/1/13 H7N9) showed that mAb 183 did not interact with H7.Intrigued by the observation that CLL subset 4 BCR IGs are always the IgG isotype, we tested if isotype switching contributed to antigen recognition by expressing CLL 183 IGHV-D-Jas divalent human IgM (mAb183M) and checked its reactivity to virus A H3N2 Texas 1/77. Switching isotype from IgG to IgM led to a 48% loss in reactivity to intact virus and 74% loss to hemagglutinin H3 (A/Philippines/2/1982 H3N2).Taken together, our results indicate that unlike other IGHV4-34 bearing IGs, CLL subset 4 mAbs have only limited reactivity to autoantigens, including linear NAL epitopes. Instead, this subset has acquired specific recognition of subtypes of an infectious agent, influenza A (H3 and H1), apparently via its envelope protein hemagglutinin. The shift in antigen recognition appears to be dependent upon both the stereotyped IGVH-D-Jrearrangement and isotype switching to IgG. DisclosuresNo relevant conflicts of interest to declare.

IRF5-mediated signaling and implications for SLE

Transcription of the type I IFN genes is regulated by members of the Interferon Regulatory Factor (IRF) family of transcription factors, composed in humans of 9 distinct proteins. In addition to IRF3 and IRF7, the transcription factor IRF5 has been shown to be involved in type I IFN production and interestingly, polymorphisms of the IRF5 gene in humans can result in risk or protective haplotypes with regard to SLE susceptibility. In addition to regulation of type I IFN expression, IRF5 is involved in other signaling pathways, including IgG switching in B cells, macrophage polarization and apoptosis, and its role in SLE pathogenesis may therefore not be limited to dysregulated control of IFN expression. In this review we will comprehensively discuss the role of IRF5 in immune-mediated responses and its potential multifaceted role in conferring SLE susceptibility.

Immunoglobulin isotype switching during early B cell development (IRM8P.702)

Abstract During B cell lymphopoiesis in the bone marrow (BM), V(D)J joining assembles immunoglobulin (Ig) VH and VL genes mediated by RAG recombinase. Mature splenic B cells undergo class switch recombination (CSR) and somatic hypermutation initiated by activation induced deaminase (AID). However, the strict ontological separation of these processes may breakdown under some circumstances. Several immunodeficiencies including Omenn syndrome display increased serum IgE despite undetectable numbers of B cells. We hypothesized that CSR occurs in BM pro- or pre-B cells. Earlier, we showed that Rag1 and Mb1 deficient pro-B cells can be induced to undergo robust CSR, albeit to a restricted subset of IgH isotypes, including IgG2b and IgE. Immunization of Rag1-/- mice with LPS, promoted IgG2b switching in BM pro-B cells. We demonstrated that V(D)J joining can be induced to follow CSR. We found a unique stage specific chromatin conformation of Igh locus in pro-B cells that may repress IgG3 and IgG1 switching (Genes Dev. 27:2439). Here we show that CSR is inducible in WT pro-B cells. Using an elegant mouse model to irreversibly mark AID expressing cells, a knockin carrying AIDcre/+RosaYFP (Nat Imm. 10:1292), we assess the frequency of AID expression in BM pro-B cells activated in vitro and in vivo with LPS. These findings have implications for the hyper-IgE phenotype in Omenn syndrome, autoimmune repertoire development and leukemagenesis via the co-expression of the RAG and AID recombinases.

Generation of IgA plasma cells during a secondary response in the absence of IgA memory B cells (LYM6P.800)

Abstract IgA antibodies are important for protection against infection at mucosal surfaces. Despite this importance in protective immunity, the differentiation of IgA B cells is not well understood. To address this issue, an antigen-based enrichment method was used to track polyclonal B cells specific for the model antigen R-Phycoerythrin (PE) during the course of a T-dependent immune response. While 10-40% of the PE-specific short lived plasma cells expressed IgA, less than 0.05% of the PE-specific germinal center and memory B cells expressed IgA. Despite the lack of IgA memory B cells, secondary antigen challenge resulted in the generation of large numbers of PE-specific IgA plasma cells that appeared at times prior to the appearance of IgA plasma cells during the primary response. Since IgA memory B cells were infrequently detected, we hypothesize that IgA plasma cells generated during the secondary challenge are the result of a secondary switch from memory B cells expressing other isotypes. We are currently examining the role that individual isotypes of memory B cells play in the generation of IgA plasma cells by looking for IgG switch remnants in the PE-specific IgA plasma cells generated after secondary challenge, and by testing the ability of purified isotypes of memory B cells to produce IgA plasma cells after transfer and challenge of recipient mice.

Is there B cell involvement in a rat model of spontaneous idiopathic nephrotic syndrome treated with LF15-0195?

The Buffalo/Mna (Buff/Mna) rat spontaneously develops idiopathic nephrotic syndrome (INS), and its nephropathy recurs after the renal transplantation of a healthy graft. Only LF15-0195 is able to cause regression of the Buff/Mna nephropathy and to induce regulatory T cells, which decrease proteinuria when transferred into proteinuric Buff/Mna rats. Based on previous research on B cells in human INS, we evaluated the involvement of B cells in our model and the impact of LF15-0195. We studied the effect of LF15-0195 on peripheral B cells by flow cytometry and quantitative reverse transcription-polymerase chain reaction. B cells were purified from LF15-0195-treated Buff/Mna rats in remission, and transferred into proteinuric Buff/Mna rats. We treated the Buff/Mna rats with mitoxantrone and measured the depletion of B/T cells in parallel with proteinuria. LF15-0195 changed the phenotype of B cells: the number of naïve mature B cells increased significantly, while the number of switched, transitional 1, and transitional 2 B cells decreased. There were no changes in the amount of memory, activated or regulatory B cells. We observed a significant increase of immunoglobulin (Ig)M mRNA transcripts in the LF15-0195-treated Buff/Mna B cells compared to controls, but no difference in the level of IgG. This profile is consistent with a block in B cell maturation at the IgM to IgG switch. The transfer of B cells from LF15-0195-treated rats into proteinuric Buff/Mna rats did not have an effect on proteinuria. Mitoxantrone, despite causing a significant depletion of B cells, did not reduce proteinuria. Despite LF15-0195 acting on B cells, the beneficial effects of this drug on nephrotic syndrome did not involve the induction of regulatory B cells. Moreover, the B cell depletion was not effective in reducing proteinuria, indicating that B cells are not a therapeutic target.

The CXCR4 mutations in WHIM syndrome impair the stability of the T-cell immunologic synapse

AbstractWHIM (warts, hypogammaglobulinemia, infections, myelokathexis) syndrome is a rare disease characterized by diverse symptoms indicative of aberrantly functioning immunity. It is caused by mutations in the chemokine receptor CXCR4, which impair its intracellular trafficking, leading to increased responsiveness to chemokine ligand and retention of neutrophils in bone marrow. Yet WHIM symptoms related to adaptive immunity, such as delayed IgG switching and impaired memory B-cell function, remain largely unexplained. We hypothesized that the WHIM-associated mutations in CXCR4 may affect the formation of immunologic synapses between T cells and antigen-presenting cells (APCs). We show that, in the presence of competing external chemokine signals, the stability of T-APC conjugates from patients with WHIM-mutant CXCR4 is disrupted as a result of impaired recruitment of the mutant receptor to the immunologic synapse. Using retrogenic mice that develop WHIM-mutant T cells, we show that WHIM-mutant CXCR4 inhibits the formation of long-lasting T-APC interactions in ex vivo lymph node slice time-lapse microscopy. These findings demonstrate that chemokine receptors can affect T-APC synapse stability and allow us to propose a novel mechanism that contributes to the adaptive immune response defects in WHIM patients.

ALKBH1 Is Dispensable for Abasic Site Cleavage during Base Excision Repair and Class Switch Recombination

Potential roles of the abasic site lyase activity associated with AlkB homolog 1 (ALKBH1) were assessed by studies focusing on the two cellular processes that create abasic sites as intermediates: base excision repair and class switch recombination. Alkbh1−/− pups (lacking exon 3) were born at a lower than expected frequency from heterozygous parents, suggesting a reduced survival rate and non-Mendelian inheritance, and they exhibited a gender bias in favor of males (70% males and 30% females). To study ALKBH1’s potential involvement in DNA repair, fibroblasts were isolated from Alkbh1−/− mice, spontaneously immortalized and tested for resistance to DNA damaging agents. Alkbh1−/− and isogenic cells expressing hALKBH1 showed no difference in survival to the DNA damaging agents methyl-methionine sulfate or H2O2. This result indicates that ALKBH1 does not play a major role in the base excision repair pathway. To assess ALKBH1’s role in class switch recombination, splenic B cells were isolated from Alkbh1−/− and Alkbh1+/+ mice and subjected to switching from IgM to IgG1. No differences were found in IgG1 switching, suggesting that Alkbh1 is not involved in class switch recombination of the immunoglobulin heavy chain during B lymphocyte activation.

Development of novel Sjogren's Syndrome susceptible mouse lines expressing floxed COX-2 within the major Aec2 disease susceptibility region in chromosome 1 (P4016)

Abstract SS patients exhibit impaired exocrine gland function and high risk for B cell lymphoma. IgG autoAbs to acetylcholine receptors, detectable in humans, impair salivation in SS-afflicted mice with NOD Aec2 and Aec1 susceptibility loci. COX-2 is a potential target for reducing SS pathogenic IgG Ab and B cell lymphoma, since (a) within in vitro cultures B cell-expressed COX-2 and PGE2 augment DNA-modifying AID and downstream IgG switching and (b) mice deficient in COX-2 have reduced IgG. If B cell specific COX-2 function is critical for SS disease, cell-targeted delivery of inhibitors might circumvent adverse side effects. With a downstream goal of examining whether B cell COX-2 contributes to autoimmunity, we introduced a floxed COX-2 gene and a CD19Cre gene independently onto the NOD.B10-H2b SS-susceptible background. To our surprise, early congenic lines HET for either CD19Cre or COX-2flox failed to show aging-related hyposalivation (xerostomia), in contrast to wild-type NOD.B10. Reduced disease in CD19cre HET mice might reflect haplodiploid CD19 or flanking non-NOD genes. Extended backcrossing of COX-2flox onto NOD.B10 yielded two congenic lines that are comparable to wild-type NOD.B10 in developing xerostomia upon aging. Fine genetic mapping of the chr 1 Aec2 region within the novel COX-2flox lines showed that (a) variations in COX-2 do not explain the observed diversity and (b) unidentified genes within a presently more narrowed Aec2 interval are important for xerostomia.

O007 IRF-5; Mediates cross talk between innate and adaptive immunity

Introduction IRF-5 is a transcription factors activated by TLR7 and TLR9 during innate immune responses. IRF-5 activates not only Type I IFN, but also inflammatory cytokines and chemokines. Most importantly a distinct genetic variations in the IRF-5 gene show a strong association with autoimmune diseases such as Lupus (SLE). Here we analyzed the role of IRF-5 in the induction of inflammatory cytokines and B cells activation, two hallmarks of SLE. Methods The two strains of IRF-5 knockout mice were evaluated for their rsponse to antigens in vivo. The IgG switching was anlayzed in isolated B cells in vitro. The molecular mechanism involved in the alteration of the antigenic response was determined on the molecular levels. Results Using two independently generated strains of IRF5-deficient mice Irf 5−/− mice we showed that these mice exhibit high susceptibility to VSV and HSV-1 infection, reduction in serum levels of type I IFN as well as inflammatory cytokines,including interferon in response to viral infection, high sussceptibilty to virus (VSV) and parasite (Laishmania donovai) infection and a defect in generation of the TH-1 response. IRF-5, but not IRF-7 is also critical for B cells differention to plasma cells, IgG2a/c responses to antigens and to polyoma virus infection. This defect is due to the intrinsic deletion of IRF-5 in B cells, as Rag1 knockout mice reconstituted with Irf5 −/− B cells show a decrease in IgG2a/c expression after viral infection. Irf 5−/−B cells in vitro have diminished TLR and cytokine-induced class switching to IgG2a/c. Addressing the molecular mechanism of these defects, we show that IRF-5 regulates Blimp-1 and Ikaros expression, factors essential for plasma cells differentiation and IgG2a/c expression respectively. Reconstitution of IRF-5 in Irf 5−/− B cells rescue Blimp-1 expression, down-regulates Ikaros levels and increases switching to IgG2a/c. The IRF site in Blimp-1 and ikzf1 promoters binds IRF-5. Conclusion Collectively these results identify the role of IRF-5 both in the innate and adaptive immune respose and indicate its a critical role in the cross talk between the innate and adaptive immune responses.