Ig Class Research Articles

B Cell Endosomal RAB7 Promotes TRAF6 K63 Polyubiquitination and NF-κB Activation for Antibody Class-Switching.

Upon activation by CD40 or TLR signaling, B lymphocytes activate NF-κB to induce activation-induced cytidine deaminase and, therefore, Ig class switch DNA recombination, as central to the maturation of the Ab and autoantibody responses. In this study, we show that NF-κB activation is boosted by colocalization of engaged immune receptors, such as CD40, with RAB7 small GTPase on mature endosomes, in addition to signals emanating from the receptors localized on the plasma membrane, in mouse B cells. In mature endosomes, RAB7 directly interacts with TRAF6 E3 ubiquitin ligase, which catalyzes K63 polyubiquitination for NF-κB activation. RAB7 overexpression in Cd19+/creRosa26fl-STOP-fl-Rab7 mouse B cells upregulates K63 polyubiquitination activity of TRAF6, enhances NF-κB activation and activation-induced cytidine deaminase induction, and boosts IgG Ab and autoantibody levels. This, together with the extensive intracellular localization of CD40 and the strong correlation of RAB7 expression with NF-κB activation in mouse lupus B cells, shows that RAB7 is an integral component of the B cell NF-κB activation machinery, likely through interaction with TRAF6 for the assembly of "intracellular membrane signalosomes."

Specific microbiota enhances intestinal IgA levels by inducing TGF-β in T follicular helper cells of Peyer's patches in mice.

In humans and mice, mucosal immune responses are dominated by IgA antibodies and the cytokine TGF-β, suppressing unwanted immune reactions but also targeting Ig class switching to IgA. It had been suggested that eosinophils promote the generation and maintenance of mucosal IgA-expressing plasma cells. Here, we demonstrate that not eosinophils, but specific bacteria determine mucosal IgA production. Co-housing of eosinophil-deficient mice with mice having high intestinal IgA levels, as well as the intentional microbiota transfer induces TGF-β expression in intestinal T follicular helper cells, thereby promoting IgA class switching in Peyer's patches, enhancing IgA+ plasma cell numbers in the small intestinal lamina propria and levels of mucosal IgA. We show that bacteria highly enriched for the genus Anaeroplasma are sufficient to induce these changes and enhance IgA levels when adoptively transferred. Thus, specific members of the intestinal microbiota and not the microbiota as such regulate gut homeostasis, by promoting the expression of immune-regulatory TGF-β and of mucosal IgA.

IL-21 in Conjunction with Anti-CD40 and IL-4 Constitutes a Potent Polyclonal B Cell Stimulator for Monitoring Antigen-Specific Memory B Cells.

Detection of antigen-specific memory B cells for immune monitoring requires their activation, and is commonly accomplished through stimulation with the TLR7/8 agonist R848 and IL-2. To this end, we evaluated whether addition of IL-21 would further enhance this TLR-driven stimulation approach; which it did not. More importantly, as most antigen-specific B cell responses are T cell-driven, we sought to devise a polyclonal B cell stimulation protocol that closely mimics T cell help. Herein, we report that the combination of agonistic anti-CD40, IL-4 and IL-21 affords polyclonal B cell stimulation that was comparable to R848 and IL-2 for detection of influenza-specific memory B cells. An additional advantage of anti-CD40, IL-4 and IL-21 stimulation is the selective activation of IgM+ memory B cells, as well as the elicitation of IgE+ ASC, which the former fails to do. Thereby, we introduce a protocol that mimics physiological B cell activation through helper T cells, including induction of all Ig classes, for immune monitoring of antigen-specific B cell memory.

Association of rheumatoid arthritis disease activity and antibodies to periodontal bacteria with serum lipoprotein profile in drug naive patients

Objective: We investigated lipid concentrations, particle sizes and antibodies binding to periodontal bacteria Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis and to malondialdehyde-acetaldehyde (MAA) modified low-density lipoprotein in immunoglobulin (Ig) class A, G and M among patients with newly diagnosed rheumatoid arthritis (RA) in a population-based cohort.Methods: Concentrations and sizes of lipoprotein particles analysed by proton nuclear magnetic resonance spectroscopy and antibody levels to MAA modified low-density lipoprotein were studied at baseline and after one-year of follow-up. Serum Ig A and G class antibodies to periodontal bacteria were determined at baseline.Results: Sixty-three patients were divided into tertiles according to disease activity by disease activity score with 28 joint count and erythrocyte sedimentation rate (ESR) (<3.9, 3.9–4.7, >4.7). Small low-density lipoprotein concentration was lowest in the tertile with the highest disease activity. In high-density lipoprotein, the concentrations of total, medium and small particles decreased with disease activity. The particle size in low-density lipoprotein associated with disease activity and the presence of antibodies to P. gingivalis. Ig G and M antibodies to MAA modified low-density lipoprotein correlated with disease activity. Inflammation associated changes faded by one year.Conclusions: Drug naive RA patients had proatherogenic changes in lipid profiles, but they were reversible, when inflammation diminished.Key messagesPatients with drug naive rheumatoid arthritis showed proatherogenic lipid profiles.Reversible changes in lipid profiles can be achieved as response to inflammation suppression.Active therapy aimed at remission is essential in all patients with rheumatoid arthritis.

The First Report of Immunoglobulin G, M, and A Concentrations in Serum of European Bison and Their Changes with Age.

The age-specific reference values for immunoglobulin (Ig) serum concentrations in European bison (Bison bonasus) are lacking. Identification of immune alterations that accompany normal physiological aging will help assist in development of better monitoring health programs. In the present study, the age-associated changes in concentration of IgG, IgM, and IgA in serum of apparently healthy European bison of various ages were studied. The quantities of IgA, IgM, and IgG were measured by the use of a commercial ELISA kit. The serum samples originating from apparently healthy European bison (n = 206) were divided into the following age categories: (1) <1 year of age; (2) animals between 1 and 3 years of age; and (3) animals which have reached sexual maturity: (3a) animals between 4 and 8 years of age, (3b) animals between 9 and 15 years of age, and (3c) animals > 15 years of age. IgG was found to be predominant Ig in the serum regardless of the age of the animals. The significant positive correlation between IgG absolute and relative concentration and the age of animals was found. The absolute concentration of IgM did not differ significantly during the lifespan; however, the negative correlation was observed between percentage of IgM and European bison's age. IgA represented the least class of serum Ig. Total serum concentration of analyzed Ig also significantly increased with age. No gender-related differences were detected. Our findings represent a meaningful contribution to the studies on the immunity of European bison and effect of age on the immunoglobulin level. Our results would be useful for veterinarians and researchers in the studies with this animal's species.

Sestrin2 Mediates IL-4-induced IgE Class Switching by Enhancing Germline ε Transcription in B Cells

Sestrin2 (Sesn2), a metabolic regulator, accumulates in response to a diverse array of cellular stresses. Sesn2 regulates cellular metabolism by inhibiting the mammalian target of rapamycin complex 1 through the AMP-activated protein kinase (AMPK) signaling pathway. Recently, researchers reported that Sesn2 regulates the differentiation and function of innate immune cells and T cells; however, the role of Sesn2 in B cells is largely unknown. In this study, we investigated the role of Sesn2 in Ig class switching and Ig production in mouse B cells. We observed that mouse B cells express Sesn2 mRNA. Interestingly, the expression of germline ε transcripts (GLTε) was selectively decreased in lipopolysaccharide-stimulated Sesn2−/− splenocytes. Overexpression of Sesn2 increased GLTε promoter activity in B cells. In addition, AICAR (an activator of AMPK) selectively increased IL-4-induced GLTε expression and surface IgE (sIgE) expression in splenocytes. Furthermore, AICAR selectively enhanced IL-4-induced GLTε expression, sIgE expression, and IgE production by anti-CD40-stimulated B cells. We observed that ovalbumin (OVA)-specific IgE concentration was reduced in OVA-challenged Sesn2−/− mice. Taken together, these results indicate that Sesn2-AMPK signaling selectively enhances IL-4-induced IgE class switching and IgE production by B cells, suggesting that this could be a therapeutic strategy targeting Sesn2 in IgE-mediated allergic diseases.

Mechanistic basis of co-stimulatory CD40-CD40L ligation mediated regulation of immune responses in cancer and autoimmune disorders.

Generation of an accurate humoral and a cell mediated adaptive immune responsesare dictated by binding of an antigen to a T- and a B-cell receptor, respectively (first signal) followed by ligation of costimulatory molecules (second signal). CD40, a costimulatory receptor molecule, expressed mainly on antigen presenting cells, some non-immune cells and tumors, binds to CD40 ligand molecule expressed transiently on T-cells and non-immune cells under inflammatory conditions. In the past decade, the CD40-CD40L interaction has emerged as an immune-potentiating system that governs and regulates host immune response against various diseases and pathogens, failing of which results in detrimental patho-physiologies including cancer and autoimmune disorders. CD40-CD40L transduces immune signals intracellularly via TRAF-dependent and independent mechanisms and further downstream by different MAPK pathways and transcription factors such as NF-κB, p38 etc. While CD40 signaling pathway through its cognate interaction between B and T cells promotes activation and proliferation of B-cells, Ig class switching, and generation of B cell memory; however, CD40-CD40L interaction involving other APCs and non-immune cells relay distinct cell signaling resulting in production of a variety of cytokines/chemokines and cell adhesion molecules ultimately conferring host defense against pathogen. In cancer and autoimmune disorders, CD40-CD40L interaction is also responsible for aberrant expression of many disease specific markers, class I/II MHC molecules and other co-stimulatory molecules such as B7 and CD28 in cell- and disease-specific manner. In the present review, the current state of understanding about the CD40-CD40L mediated regulation of immune and non-immune cells is presented. The current paradigm is to target CD40 using agonist anti-CD40 mAbs alone or in synergistic combination with chemotherapy in order to harness or confer anti-tumor and anti-inflammatory immunity.

Igh locus structure and evolution in Platyrrhines: new insights from a genomic perspective.

Non-human primates have been used as animal models because of their phylogeneticcloseness to humans. However, the genetic differences between humans and non-human primates must be considered to select the appropriate animal models. Recently, New World monkeys (Platyrrhines) have generated a higher interest in biomedical research, especially in assessing vaccine safety and immunogenicity. Given the continued and renewed interest in Platyrrhines as biomedical models, it is a necessary to have a better and more complete understanding of their immune system and its implications for research. Immunoglobulins (Ig) are the main proteins that mediate humoral immunity. These proteins have evolved as part of an adaptive immune response system derived from ancient vertebrates. There are at least four Ig classes in Prosimians, whereas five have been reported in Catarrhines. Information on the structure and evolution of the loci containing immunoglobulin heavy chain constant genes (Igh) in Platyrrhines, however, is limited. Here, Igh loci were characterized in 10 Platyrrhines using the available whole genome sequences. Human and Macaca Igh loci were also assessed to compare them with their Platyrrhines counterparts. Differences in Igh locus structure were observed between Platyrrhines and Catarrhines. Noteworthy changes occur in the γ gene, which encodes a key Ig involved in organism defense that would favor protection after vaccination. The remarkable differences between the immunoglobulin proteins of Platyrrhines and Catarrhines warrant a cautionary message to biomedical researchers.

More than one antibody of individual B cells revealed by single-cell immune profiling

Antibodies have a common structure consisting of two identical heavy (H) and two identical light (L) chains. It is widely accepted that a single mature B cell produces a single antibody through restricted synthesis of only one VHDJH (encoding the H-chain variable region) and one VLJL (encoding the L-chain variable region) via recombination. Naive B cells undergo class-switch recombination (CSR) from initially producing membrane-bound IgM and IgD to expressing more effective membrane-bound IgG, IgA, or IgE when encountering antigens. To ensure the “one cell — one antibody” paradigm, only the constant region of the H chain is replaced during CSR, while the rearranged VHDJH pattern and the L chain are kept unchanged. To define those long-standing classical concepts at the single-cell transcriptome level, we applied the Chromium Single-Cell Immune Profiling Solution and Sanger sequencing to evaluate the Ig transcriptome repertoires of single B cells. Consistent with the “one cell — one antibody” rule, most of the B cells showed one V(D)J recombination pattern. Intriguingly, however, two or more VHDJH or VLJL recombination patterns of IgH chain or IgL chain were also observed in hundreds to thousands of single B cells. Moreover, each Ig class showed unique VHDJH recombination pattern in a single B-cell expressing multiple Ig classes. Together, our findings reveal an unprecedented presence of multi-Ig specificity in some single B cells, implying regulation of Ig gene rearrangement and class switching that differs from the classical mechanisms of both the “one cell — one antibody” rule and CSR.

Bach2 deficiency leads autoreactive B cells to produce IgG autoantibodies and induce lupus through a T cell-dependent extrafollicular pathway

Class-switched IgG autoantibodies but not unswitched IgM autoantibodies play a crucial role in the development of systemic lupus erythematosus (SLE). Bach2 is known to be essential for class switch recombination of Ig genes, but recent genomic and clinical studies have suggested an association of Bach2 deficiency with SLE. This study was undertaken to examine the mechanism by which Bach2 regulates the development of SLE. Despite defects in Ig class switch recombination and germinal center formation when actively immunized, Bach2−/− mice spontaneously accumulated IgG autoantibody-secreting cells without germinal center reactions in a regulatory T cell-independent manner, and this phenomenon was accompanied by manifestations akin to SLE. Transcriptome analyses revealed that Bach2 regulated the expression of genes related to germinal center formation and SLE pathogenesis in B cells. B cell-specific deletion of Bach2 was sufficient to impair the development of germinal center B cells but insufficient to promote the production of IgG autoantibodies. Bach2 deficiency caused CD4+ T cells to overexpress Icos and differentiate into extrafollicular helper T cells in a cell-autonomous manner. These findings suggest that Bach2-deficient autoreactive B cells preferentially react at extrafollicular sites to give rise to IgG class-switched pathogenic plasma cells and that this effect requires the help of Bach2-Icoshi helper T cells. Thus, the cell-autonomous roles of Bach2 in B cells and in their cognate CD4+ T cells are required to maintain self-tolerance against SLE.

Immune receptor repertoires in pediatric and adult acute myeloid leukemia

BackgroundAcute myeloid leukemia (AML), caused by the abnormal proliferation of immature myeloid cells in the blood or bone marrow, is one of the most common hematologic malignancies. Currently, the interactions between malignant myeloid cells and the immune microenvironment, especially T cells and B cells, remain poorly characterized.MethodsIn this study, we systematically analyzed the T cell receptor and B cell receptor (TCR and BCR) repertoires from the RNA-seq data of 145 pediatric and 151 adult AML samples as well as 73 non-tumor peripheral blood samples.ResultsWe inferred over 225,000 complementarity-determining region 3 (CDR3) sequences in TCR α, β, γ, and δ chains and 1,210,000 CDR3 sequences in B cell immunoglobulin (Ig) heavy and light chains. We found higher clonal expansion of both T cells and B cells in the AML microenvironment and observed many differences between pediatric and adult AML. Most notably, adult AML samples have significantly higher level of B cell activation and more secondary Ig class switch events than pediatric AML or non-tumor samples. Furthermore, adult AML with highly expanded IgA2 B cells, which might represent an immunosuppressive microenvironment, are associated with regulatory T cells and worse overall survival.ConclusionsOur comprehensive characterization of the AML immune receptor repertoires improved our understanding of T cell and B cell immunity in AML, which may provide insights into immunotherapies in hematological malignancies.

Mucosal Antibodies: Defending Epithelial Barriers against HIV-1 Invasion.

The power of mucosal anti-HIV-1 envelope immunoglobulins (Igs) to block virus transmission is underappreciated. We used passive immunization, a classical tool to unequivocally prove whether antibodies are protective. We mucosally instilled recombinant neutralizing monoclonal antibodies (nmAbs) of different Ig classes in rhesus macaques (RMs) followed by mucosal simian–human immunodeficiency virus (SHIV) challenge. We gave anti-HIV-1 IgM, IgG, and dimeric IgA (dIgA) versions of the same human nmAb, HGN194 that targets the conserved V3 loop crown. Surprisingly, dIgA1 with its wide-open, flat hinge protected 83% of the RMs against intrarectal R5-tropic SHIV-1157ipEL-p challenge, whereas dIgA2, with its narrow hinge, only protected 17% of the animals—despite identical epitope specificities and in vitro neutralization curves of the two dIgA isotypes (Watkins et al., AIDS 2013 27(9):F13-20). These data imply that factors in addition to neutralization determine in vivo protection. We propose that this underlying protective mechanism is immune exclusion, which involves large nmAb/virion aggregates that prevent virus penetration of mucosal barriers. Future studies need to find biomarkers that predict effective immune exclusion in vivo. Vaccine development strategies against HIV-1 and/or other mucosally transmissible pathogens should include induction of strong mucosal Abs of different Ig classes to defend epithelial barriers against pathogen invasion.

Abstract P1108: Serum Immunoglobulins are Not Necessary for Experimental Hypertension

The role of B cells and serum immunoglobulins (Ig) in hypertension is unclear. Elevated serum IgG has been observed in some hypertensive patients and in animal models of hypertension. Mice with a severe B cell deficiency due to lack of B cell activating factor receptor (BAFF-R) and mice lacking the cytokine interleukin 21 (IL21) - which is a potent inducer of B cell Ig class switching and high affinity Ig production - are protected from Ang II-induced hypertension and exhibit reduced serum IgG levels. Thus, we sought to determine whether hypertensive IgGs play a causative role in hypertension and end-organ dysfunction. First, we purified serum IgG from normotensive and hypertensive wild type (WT) mice and adoptively transferred equal amounts of IgG by intraperitoneal injection into normotensive recipient mice followed by infusion of a sub-pressor dose of angiotensin II (Ang II) for 2 weeks. Flow cytometric analysis of aortas revealed a modest increase in F4/80+ macrophages (p=0.04) and CD8+ T cells (p=0.02) in mice that received hypertensive IgG. However, there was no difference in endothelial dependent relaxation, renal inflammation, albuminuria, and blood pressure (BP) between the two groups. As an alternative method to investigate the role of class-switched high affinity Igs, we studied mice with genetic deletion of activation-induced deaminase (AID) which is an enzyme necessary for somatic hypermutation (to generate high affinity Igs) and isotype class switching. AID -/- mice exhibit undetectable IgG and higher levels of IgM in serum compared to WT mice. Ang II infusion for 4 weeks revealed no difference in BP, aortic inflammation, renal inflammation, and albuminuria. We also studied mice with deficiency of the IgM heavy chain (uMT). These mice exhibit undetectable levels of all immunoglobulins and are severely deficient in mature B cells. uMT-/- mice are also not protected from hypertension induced by Ang II infusion or deoxycorticosterone acetate (DOCA-salt) treatment. Together, these results demonstrate that while elevated serum immunoglobulins seen in hypertensive animals and humans may represent a biomarker of aberrant immune activation, they are likely not playing a causal role in hypertension pathophysiology.

Convergent Evolution of Mucosal Immune Responses at the Buccal Cavity of Teleost Fish.

SummaryThe buccal mucosa (BM) is a critical first line of defense in terrestrial animals. To gain further insights into the evolutionary origins and primordial roles of BM in teleosts here we show that rainbow trout, a teleost fish, contains a diffuse mucosal associated lymphoid tissue (MALT) within its buccal cavity. Upon parasite infection, a fish immunoglobulin specialized in mucosal immunity (sIgT) was induced to a high degree, and parasite-specific sIgT responses were mainly detected in the buccal mucus. Moreover, we show that the trout buccal microbiota is prevalently coated with sIgT. Overall our findings revealed that the MALT is present in the BM of a non-tetrapod species. As fish IgT and mucus-producing cells are evolutionarily unrelated to mammalian IgA and salivary glands, respectively, our findings indicate that mucosal immune responses in the BM of teleost fish and tetrapods evolved through a process of convergent evolution.

Cigarette smoking differentially affects immunoglobulin class levels in serum and saliva: An investigation and review.

The aim of the present study was to compare concentrations of IgG, IgA, IgM and IgD in both serum and saliva samples from smoking and non-smoking individuals using a protein microarray assay. The findings were also compared to previous studies. Serum and saliva were collected from 48 smoking male individuals and 48 age-matched never-smoker male individuals. The protein microarray assays for detection of human IgG, IgM, IgA and IgD were established and optimized using Ig class-specific affinity-purified goat anti-human Ig-Fc capture antibodies and horseradish peroxidase (HRP)-conjugated goat anti-human Ig-Fc detection antibodies. The Ig class specificity of the microarray assays was verified, and the optimal dilutions of serum and saliva samples were determined for quantification of Ig levels against standard curves. We found that smoking is associated with reduced IgG concentrations and enhanced IgA concentrations in both serum and saliva. By contrast, smoking differentially affected IgM concentrations-causing increased concentrations in serum, but decreased concentrations in saliva. Smoking was associated with decreased IgD concentrations in serum and did not have a significant effect on the very low IgD concentrations in saliva. Thus, cigarette smoking differentially affects the levels of Ig classes systemically and in the oral mucosa. Although there is variation between the results of different published studies, there is a consensus that smokers have significantly reduced levels of IgG in both serum and saliva. A functional antibody deficiency associated with smoking may compromise the body's response to infection and result in a predisposition to the development of autoimmunity.

Recalling the Future: Immunological Memory Toward Unpredictable Influenza Viruses.

Persistent and durable immunological memory forms the basis of any successful vaccination protocol. Generation of pre-existing memory B cell and T cell pools is thus the key for maintaining protective immunity to seasonal, pandemic and avian influenza viruses. Long-lived antibody secreting cells (ASCs) are responsible for maintaining antibody levels in peripheral blood. Generated with CD4+ T help after naïve B cell precursors encounter their cognate antigen, the linked processes of differentiation (including Ig class switching) and proliferation also give rise to memory B cells, which then can change rapidly to ASC status after subsequent influenza encounters. Given that influenza viruses evolve rapidly as a consequence of antibody-driven mutational change (antigenic drift), the current influenza vaccines need to be reformulated frequently and annual vaccination is recommended. Without that process of regular renewal, they provide little protection against “drifted” (particularly H3N2) variants and are mainly ineffective when a novel pandemic (2009 A/H1N1 “swine” flu) strain suddenly emerges. Such limitation of antibody-mediated protection might be circumvented, at least in part, by adding a novel vaccine component that promotes cross-reactive CD8+ T cells specific for conserved viral peptides, presented by widely distributed HLA types. Such “memory” cytotoxic T lymphocytes (CTLs) can rapidly be recalled to CTL effector status. Here, we review how B cells and follicular T cells are elicited following influenza vaccination and how they survive into a long-term memory. We describe how CD8+ CTL memory is established following influenza virus infection, and how a robust CTL recall response can lead to more rapid virus elimination by destroying virus-infected cells, and recovery. Exploiting long-term, cross-reactive CTL against the continuously evolving and unpredictable influenza viruses provides a possible mechanism for preventing a disastrous pandemic comparable to the 1918-1919 H1N1 “Spanish flu,” which killed more than 50 million people worldwide.

CUL7 E3 Ubiquitin Ligase Mediates the Degradation of Activation-Induced Cytidine Deaminase and Regulates the Ig Class Switch Recombination in B Lymphocytes.

Activation-induced cytidine deaminase (AID) initiates class switch recombination and somatic hypermutation in Ig genes. The activity and protein levels of AID are tightly controlled by various mechanisms. In this study, we found that CUL7 E3 ubiquitin ligases specifically mediated AID ubiquitination. CUL7 overexpression or knockdown influenced the decay of AID, affecting AID protein levels and subsequently IgA class switching in CH12F3 cells, a mouse B lymphocyte cell line. Further analysis indicated that CUL7 mediated AID ubiquitination by forming a complex with FBXW11. In a CUL7 fl/fl CD19 cre+ mouse model, we demonstrated that CUL7 knockout significantly enhanced AID protein levels in B cells in the germinal center and increased both the IgG1 and IgA class switching. Collectively, our results reveal a subtle regulation mechanism for tightly controlling AID protein levels. The manipulation of this pathway may be useful for regulating AID abundance and efficiency of Ig class switching and is therefore a potential target for developing immunologic adjuvants for vaccines of various pathogens such as HIV-1 and influenza viruses.

Locus suicide recombination actively occurs on the functionally rearranged IgH allele in B-cells from inflamed human lymphoid tissues.

B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID). AID promotes affinity maturation of Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control of the 3’ regulatory region (3’RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sμ to “like-switch” DNA repeats that flank the 3’ super-enhancer can thus accomplish so-called “locus suicide recombination” (LSR) in mouse B-cells. Using an optimized LSR-seq high throughput method, we now show that AID-mediated LSR is evolutionarily conserved and also actively occurs in humans, providing an activation-induced cell death pathway in multiple conditions of B-cell activation. LSR either focuses on the functional IgH allele or is bi-allelic, and its signature is mainly detected when LSR is ongoing while it vanishes from fully differentiated plasma cells or from “resting” blood memory B-cells. Highly diversified breakpoints are distributed either within the upstream (3’RR1) or downstream (3’RR2) copies of the IgH 3’ super-enhancer and all conditions activating CSR in vitro also seem to trigger LSR although TLR ligation appeared the most efficient. Molecular analysis of breakpoints and junctions confirms that LSR is AID-dependent and reveals junctional sequences somehow similar to CSR junctions but with increased usage of microhomologies.

PS1593 AIHA IN HEMATOLOGICAL DISEASES - CHALLENGES FOR IMMUNOCHEMATOLOGY AND RISK-MANAGEMENT OF TRANSFUSION THERAPY

Background: Heavy AIHAs are a problem for transfusion medicine because they make it difficult to treat the underlying disease, and because of the high risk of hemolytic post-transfusion reactions. The safety and effectiveness of transfusion therapy in severe AIHAs depends entirely on: Ig class of auto-antierytrocite antibodies, t optimum of activity, activity, and specificity. Aims: Frequency, evolution, and severity of AIHA in hematological diseases: AML, ALL, CLL, NHL, MH, MM, MDS, MAA; An. Cooley, Myelofibrosis, ITP, B12-deficiency, choice of blood transfusion approach Methods: Materials: Number of AIHAs - 136 cases in adults for the period 2014–2018, evolution in the course of the underlying disease, Ig class of auto-antierytrocite antibodies, activity, specificity, transfusion therapy - with or without complications (presentation of 3 severe clinical cases of AIHA in MM, MDS, after allogeneic ABO-compatibility HSCT). Methods: Routine manual technique, column-agglutination technique for blood typing, screening and identification of anti-erythrocyte antibodies (agglutination, enzyme, indirect antiglobulin test), in vitro compatibility tests on all methods and techniques, absorption and elution by standard techniques, if necessary. Results: AIHA was found in 136 cases: male-39.7%, female-52.94%; AML-11.77%, ALL-3.69%, CLL-16.92%, NHL-25%, MH-3.67%, MM-6.62%, MDS-16.18%, An.Cooley-1.47%, MAA-4.41%, Myelofibrosis-2.21 %, B12 deficit-2.95%, ITP-3.67%; AIHA at the expense of IgG −41.91%, IgG + C3d-26.47%, C3d-12.5%, 19.2% are atypical; autoantibody specificity-22.8%; mixed autoimmune disease-5.15%, major approach to severe AIHA-retention of blood transfusion due to the effect of immunosuppressive therapy, transfusion therapy was performed only in severe AIHA (IgG + C3d, CAD) with Hb values <45 G / L and the blood transfusion without reactions and complications for the patients.Summary/Conclusion: The severe AIHA in the evolution of the underlying disease are the challenge of transfusion medicine and the choice of approach/risk management in transfusion therapy/retention of blood transfusion or blood transfusion only at low critical levels of Hb <45 G / L are favorable for the patient. The favorable evolution of hematological disease depends on good transfusion support.

T-bet expression is fine-tuned for balancing IFN-γ-producing Th1 and Tfh cell differentiation and IgG2a(c) production

Abstract T follicular helper (Tfh) cells and cytokine IFN-γ are involved in Ig class switching in B cells to produce IgG2a(c). Tfh cells mainly produce IL-21 as their signature cytokine, and several studies have also shown that some Tfh cells are capable of expressing Th1 signature cytokine IFN-γ without expressing the Th1 master transcription factor T-bet. In Toxoplasma gondii peptide AS15/Complete Freund’s Adjuvant (CFA)-induced immune response, we found antigen AS15-specific IgG2a(c) were abolished in CD4-Cre-mediated T-bet-deficient mice. By using T-bet-fate-mapping mouse strains, we reported that transient expression of T-bet epigenetically imprints the Ifng locus for cytokine production in a subset of Tfh cells; In germinal centers, multi-color tissue imaging revealed that the ex-T-bet Tfh cells express IFN-γ in situ; IFN-γ-expressing Tfh cells are absent in T-bet-deficient mice. More interestingly, we noticed that IFN-γ-expressing Tfh cells were fully present in mice with T-bet deletion at late stages of T cell differentiation, and antigen AS15-specific IgG2a(c) were even further up-regulated in such “T-bet late knockout” mice. Taken together, our findings demonstrate that IFN-γ expressed by T cells plays a dominant role in antibody IgG2a(c) production; T-bet expression is fine-tuned as spatiotemporal manner for balancing Th1 and Tfh cell differentiation in vivo and regulating the strength of antibody production.