The immunoglobulin heavy chain super enhancer controls class switch recombination in developing B cells.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only.

Isotype switching in human memory B cells sets intrinsic antigen-affinity thresholds that dictate antigen-driven fates.

Memory B cells (MBCs) play a critical role in protection against homologous and variant pathogen challenge by either differentiating to plasma cells (PCs) or to germinal center (GC) B cells. The human MBC compartment contains both switched IgG+ and unswitched IgM+ MBCs; however, whether these MBC subpopulations are equivalent in their response to B cell receptor cross-linking and their resulting fates is incompletely understood. Here, we show that IgG+ and IgM+ MBCs can be distinguished based on their response to κ-specific monoclonal antibodies of differing affinities. IgG+ MBCs responded only to high-affinity anti-κ and differentiated almost exclusively toward PC fates. In contrast, IgM+ MBCs were eliminated by apoptosis by high-affinity anti-κ but responded to low-affinity anti-κ by differentiating toward GC B cell fates. These results suggest that IgG+ and IgM+ MBCs may play distinct yet complementary roles in response to pathogen challenge ensuring the immediate production of high-affinity antibodies to homologous and closely related challenges and the generation of variant-specific MBCs through GC reactions.

A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome.

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

MCT1-governed pyruvate metabolism is essential for antibody class-switch recombination through H3K27 acetylation.

Monocarboxylate transporter 1 (MCT1) exhibits essential roles in cellular metabolism and energy supply. Although MCT1 is highly expressed in activated B cells, it is not clear how MCT1-governed monocarboxylates transportation is functionally coupled to antibody production during the glucose metabolism. Here, we report that B cell-lineage deficiency of MCT1 significantly influences the class-switch recombination (CSR), rendering impaired IgG antibody responses in Mct1f/fMb1Cre mice after immunization. Metabolic flux reveals that glucose metabolism is significantly reprogrammed from glycolysis to oxidative phosphorylation in Mct1-deficient B cells upon activation. Consistently, activation-induced cytidine deaminase (AID), is severely suppressed in Mct1-deficient B cells due to the decreased level of pyruvate metabolite. Mechanistically, MCT1 is required to maintain the optimal concentration of pyruvate to secure the sufficient acetylation of H3K27 for the elevated transcription of AID in activated B cells. Clinically, we found that MCT1 expression levels are significantly upregulated in systemic lupus erythematosus patients, and Mct1 deficiency can alleviate the symptoms of bm12-induced murine lupus model. Collectively, these results demonstrate that MCT1-mediated pyruvate metabolism is required for IgG antibody CSR through an epigenetic dependent AID transcription, revealing MCT1 as a potential target for vaccine development and SLE disease treatment.

RPA guides UNG to uracil in ssDNA to facilitate antibody class switching and repair of mutagenic uracil at the replication fork.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sµ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

Autoantibody subclass predominance is not driven by aberrant class switching or impaired B cell development.

A subset of autoimmune diseases is characterized by predominant pathogenic IgG4 autoantibodies (IgG4-AID). Why IgG4 predominates in these disorders is unknown. We hypothesized that dysregulated B cell maturation or aberrant class switching causes overrepresentation of IgG4+ B cells and plasma cells. Therefore, we compared the B cell compartment of patients from four different IgG4-AID with two IgG1-3-AID and healthy donors, using flow cytometry. Relative subset abundance at all maturation stages was normal, except for a, possibly treatment-related, reduction in immature and naïve CD5+ cells. IgG4+ B cell and plasma cell numbers were normal in IgG4-AID patients, however they had a (sub)class-independent 8-fold increase in circulating CD20-CD138+ cells. No autoreactivity was found in this subset. These results argue against aberrant B cell development and rather suggest the autoantibody subclass predominance to be antigen-driven. The similarities between IgG4-AID suggest that, despite displaying variable clinical phenotypes, they share a similar underlying immune profile.

Mechanism and regulation of secondary immunoglobulin diversification.

Secondary immunoglobulin diversification by somatic hypermutation and class switch recombination in B cells is instrumental for an adequate adaptive humoral immune response. These genetic events may, however, also introduce aberrations into other cellular genes and thereby cause B cell malignancies. While the basic mechanism of somatic hypermutation and class switch recombination is now well understood, their regulation and in particular the mechanism of their specific targeting to immunoglobulin genes is still rather mysterious. In this review, we summarize the current knowledge on the mechanism and regulation of secondary immunoglobulin diversification and discuss known mechanisms of physiological targeting to immunoglobulin genes and mistargeting to other cellular genes. We summarize open questions in the field and provide an outlook on future research.

Activation-induced cytidine deaminase an antibody diversification enzyme interacts with chromatin modifier UBN1 in B-cells.

Activation-induced cytidine deaminase (AID) is the key mediator of antibody diversification in activated B-cells by the process of somatic hypermutation (SHM) and class switch recombination (CSR). Targeting AID to the Ig genes requires transcription (initiation and elongation), enhancers, and its interaction with numerous factors. Furthermore, the HIRA chaperon complex, a regulator of chromatin architecture, is indispensable for SHM. The HIRA chaperon complex consists of UBN1, ASF1a, HIRA, and CABIN1 that deposit H3.3 onto the DNA, the SHM hallmark. We explored whether UBN1 interacts with AID using computational and in-vitro experiments. Interestingly, our in-silico studies, such as molecular docking and molecular dynamics simulation results, predict that AID interacts with UBN1. Subsequently, co-immunoprecipitation and pull-down experiments established interactions between UBN1 and AID inside B-cells. Additionally, a double immunofluorescence assay confirmed that AID and UBN1 were co-localized in the human and chicken B-cell lines. Moreover, proximity ligation assay studies validated that AID interacts with UBN1. Ours is the first report on the interaction of genome mutator enzyme AID with UBN1. Nevertheless, the fate of interaction between UBN1 and AID is yet to be explored in the context of SHM or CSR.

Three doses of COVID-19 mRNA vaccine induce class-switched antibody responses in inflammatory arthritis patients on immunomodulatory therapies.

Patients with inflammatory arthritis (IA) are at increased risk of severe COVID-19 due to medication-induced immunosuppression that impairs host defenses. The aim of this study was to assess antibody and B cell responses to COVID-19 mRNA vaccination in IA patients receiving immunomodulatory therapies. Adults with IA were enrolled through the Johns Hopkins Arthritis Center and compared with healthy controls (HC). Paired plasma and peripheral blood mononuclear cell (PBMC) samples were collected prior to and 30 days or 6 months following the first two doses of mRNA vaccines (D2; HC=77 and IA=31 patients), or 30 days following a third dose of mRNA vaccines (D3; HC=11 and IA=96 patients). Neutralizing antibody titers, total binding antibody titers, and B cell responses to vaccine and Omicron variants were analyzed. Anti-Spike (S) IgG and S-specific B cells developed appropriately in most IA patients following D3, with reduced responses to Omicron variants, and negligible effects of medication type or drug withholding. Neutralizing antibody responses were lower compared to healthy controls after both D2 and D3, with a small number of individuals demonstrating persistently undetectable neutralizing antibody levels. Most IA patients respond as well to mRNA COVID-19 vaccines as immunocompetent individuals by the third dose, with no evidence of improved responses following medication withholding. These data suggest that IA-associated immune impairment may not hinder immunity to COVID-19 mRNA vaccines in most individuals.

Unique repetitive nucleic acid structures mirror switch regions in the human IgH locus.

Immunoglobulin (Ig) genes carry the unique ability to be reshaped in peripheral B lymphocytes after these cells encounter a specific antigen. B cells can then further improve their affinity, acquire new functions as memory cells and eventually end up as antibody-secreting cells. Ig class switching is an important change that occurs in this context, thanks to local DNA lesions initiated by the enzyme activation-induced deaminase (AID). Several cis-acting elements of the Ig heavy (IgH) chain locus make it accessible to the AID-mediated lesions that promote class switch recombination (CSR). DNA repeats, with a non-template strand rich in G-quadruplexes (G4)-DNA, are prominent cis-targets of AID and define the so-called "switch" (S) regions specifically targeted for CSR. By analyzing the structure of the human IgH locus, we uncover that abundant DNA repeats, some with a putative G4-rich template strand, are additionally present in downstream portions of the IgH coding genes. These like-S (LS) regions stand as 3' mirror-images of S regions and also show analogies to some previously reported repeats associated with the IgH locus 3' super-enhancer. A regulatory role of LS repeats is strongly suggested by their specific localization close to exons encoding the membrane form of Ig molecules, and by their conservation during mammalian evolution.

Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

Exosomal PGE2 from M2 macrophages inhibits neutrophil recruitment and NET formation through lipid mediator class switching in sepsis.

BACKGROUND: Excess polymorphonuclear neutrophil (PMN) recruitment or excessive neutrophil extracellular trap (NET) formation can lead to the development of multiple organ dysfunction during sepsis. M2 macrophage-derived exosomes (M2-Exos) have exhibited anti-inflammatory activities in some inflammatory diseases to mediate organ functional protection, but their role in treating sepsis-related acute lung injury (ALI) remains unclear. In this study, we sought to investigate whether M2-Exos could prevent potentially deleterious inflammatory effects during sepsis-related ALI by modulating abnormal PMN behaviours. METHODS: C57BL/6 wild-type mice were subjected to a caecal ligation and puncture (CLP) mouse model to mimic sepsis in vivo, and M2-Exos were administered intraperitoneally 1 h after CLP. H&E staining, immunofluorescence and immunohistochemistry were conducted to investigate lung tissue injury, PMN infiltration and NET formation in the lung. We further demonstrated the role of M2-Exos on PMN function and explored the potential mechanisms through an in vitro coculture experiment using PMNs isolated from both healthy volunteers and septic patients. RESULTS: Here, we report that M2-Exos inhibited PMN migration and NET formation, alleviated lung injury and reduced mortality in a sepsis mouse model. In vitro, M2-Exos significantly decreased PMN migration and NET formation capacity, leading to lipid mediator class switching from proinflammatory leukotriene B4 (LTB4) to anti-inflammatory lipoxin A4 (LXA4) by upregulating 15-lipoxygenase (15-LO) expression in PMNs. Treatment with LXA4 receptor antagonist attenuated the effect of M2-Exos on PMNs and lung injury. Mechanistically, prostaglandin E2 (PGE2) enriched in M2-Exos was necessary to increase 15-LO expression in PMNs by functioning on the EP4 receptor, upregulate LXA4 production to downregulate chemokine (C-X-C motif) receptor 2 (CXCR2) and reactive oxygen species (ROS) expressions, and finally inhibit PMN function. CONCLUSIONS: Our findings reveal a previously unknown role of M2-Exos in regulating PMN migration and NET formation through lipid mediator class switching, thus highlighting the potential application of M2-Exos in controlling PMN-mediated tissue injury in patients with sepsis.

DNA repair and antibody diversification: the 53BP1 paradigm.

The DNA double-strand break (DSB) repair factor 53BP1 has long been implicated in V(D)J and class switch recombination (CSR) of mammalian lymphocyte receptors. However, the dissection of the underlying molecular activities is hampered by a paucity of studies [V(D)J] and plurality of phenotypes (CSR) associated with 53BP1 deficiency. Here, we revisit the currently accepted roles of 53BP1 in antibody diversification in view of the recent identification of its downstream effectors in DSB protection and latest advances in genome architecture. We propose that, in addition to end protection, 53BP1-mediated end-tethering stabilization is essential for CSR. Furthermore, we support a pre-DSB role during V(D)J recombination. Our perspective underscores the importance of evaluating repair of DSBs in relation to their dynamic architectural contexts.

PHRF1 promotes the class switch recombination of IgA in CH12F3-2A cells.

PHRF1 is an E3 ligase that promotes TGF-ß signaling by ubiquitinating a homeodomain repressor TG-interacting factor (TGIF). The suppression of PHRF1 activity by PML-RARα facilitates the progression of acute promyelocytic leukemia (APL). PHRF1 also contributes to non-homologous end-joining in response to DNA damage by linking H3K36me3 and NBS1 with DNA repair machinery. However, its role in class switch recombination (CSR) is not well understood. In this study, we report the importance of PHRF1 in IgA switching in CH12F3-2A cells and CD19-Cre mice. Our studies revealed that Crispr-Cas9 mediated PHRF1 knockout and shRNA-silenced CH12F3-2A cells reduced IgA production, as well as decreased the amounts of PARP1, NELF-A, and NELF-D. The introduction of PARP1 could partially restore IgA production in PHRF1 knockout cells. Intriguingly, IgA, as well as IgG1, IgG2a, and IgG3, switchings were not significantly decreased in PHRF1 deficient splenic B lymphocytes isolated from CD19-Cre mice. The levels of PARP1 and NELF-D were not decreased in PHRF1-depleted primary splenic B cells. Overall, our findings suggest that PHRF1 may modulate IgA switching in CH12F3-2A cells.

Interleukin-9 rescues class switching of Memory B cells derived from Common variable immunodeficiency patients.

Impaired class switch memory (CSM) B cell formation is the hallmark of common variable immunodeficiency (CVID). Various T cell abnormalities have been observed in CVID patients indicating inadequate T-cell help to B cells. A major setback in understanding its pathogenesis is due to diverse clinical presentation. Therefore, we performed extensive immunological investigation in a cohort of CVID patients with similar clinical findings in order to unravel the T cell dysfunction and its influence on the defective humoral immune response. All recruited CVID patients exhibited B cells in the normal range, but reduced CSM B cells. However, patients showed reduced T cell proliferation, reduced level of serum Interleukin-9 (IL-9) and frequency of IL-9 expressing CD4 (Th-9) cells. IL-9 supplementation along with CD40 engagement was effective in inducing in vitro CSM B cells formation in CVID patients. Thus, IL-9 supplementation has the potential to restore impaired CSM B cell formation in CVID.

Requirements of IL-4 during the Generation of B Cell Memory.

IL-4 has long been established as a key regulator of Th cells and for promoting effective B cell survival and isotype class switching. Yet, despite having been extensively studied, the specific role of IL-4 in generating humoral memory in vivo is unclear. In this review, we explore the recent studies that unravel the cellular sources and spatiotemporal production of IL-4, the relationship between IL-4 and IL-21 during germinal center responses and the formation of Ab-secreting cells, and the current understanding of whether IL-4 promotes or suppresses memory B cell generation in vitro and in vivo.

Attempts to evaluate locus suicide recombination and its potential role in B cell negative selection in the mouse.

Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain (IgH) locus under control of its 3' cis-regulatory region (3'RR). The 3'RR is itself transcribed and can undergo "locus suicide recombination" (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined. Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3'RR. Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies. Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro, this study suggests that LSR may contribute to the elimination of self-reactive B cells.

A liposomal carbohydrate vaccine, adjuvanted with an NKT cell agonist, induces rapid and enhanced immune responses and antibody class switching.

BACKGROUND: Congenital disorders of glycosylation (CDGs) are genetic diseases caused by gene defects in glycan biosynthesis pathways, and there is an increasing number of patients diagnosed with CDGs. Because CDGs show many different clinical symptoms, their accurate clinical diagnosis is challenging. Recently, we have shown that liposome nanoparticles bearing the ALG1-CDG and PMM2-CDG biomarkers (a tetrasaccharide: Neu5Ac-α2,6-Gal-ß1,4-GlcNAc-ß1,4-GlcNAc) stimulate a moderate immune response, while the generated antibodies show relatively weak affinity maturation. Thus, mature antibodies with class switching to IgG are desired to develop high-affinity antibodies that may be applied in medical applications. RESULTS: In the present study, a liposome-based vaccine platform carrying a chemoenzymatic synthesized phytanyl-linked tetrasaccharide biomarker was optimized. The liposome nanoparticles were constructed by dioleoylphosphatidylcholine (DOPC) to improve the stability and immunogenicity of the vaccine, and adjuvanted with the NKT cell agonist PBS57 to generate high level of IgG antibodies. The results indicated that the reformulated liposomal vaccine stimulated a stronger immune response, and PBS57 successfully induce an antibody class switch to IgG. Further analyses of IgG antibodies elicited by liposome vaccines suggested their specific binding to tetrasaccharide biomarkers, which were mainly IgG2b isotypes. CONCLUSIONS: Immunization with a liposome vaccine carrying a carbohydrate antigen and PBS57 stimulates high titers of CDG biomarker-specific IgG antibodies, thereby showing great potential as a platform to develop rapid diagnostic methods for ALG1-CDG and PMM2-CDG.

Optimal AID expression and efficient immunoglobulin class switch recombination are dependent on the hypoxia-inducible factor.

During immune responses, B cells engaging a cognate antigen are recruited to GCs in secondary lymphoid organs where they will diversify their BCR to generate highly specific and adapted humoral responses. They do so, by inducing the expression of activation-induced cytidine deaminase (AID), which initiates somatic hypermutation (SHM) and class switch recombination (CSR). AID deaminates cytosines in ss DNA, generating U:G mismatches that are processed to induce ds DNA break intermediates during CSR that result in the expression of a different antibody isotype. Interestingly, hypoxia regions have been reported in GCs and suggesting that hypoxia could modulate the humoral response. Furthermore, hypoxia inducible transcription factor (HIF) can bind to the AID promoter and induce AID expression in a non-B-cell setting, suggesting that it might be involved in the transcriptional induction of AID in B cells, hence, regulating SHM and CSR. We, thus, hypothesized that HIF could regulate the efficiency of CSR. Here, we show that the inactivation of both the HIF-1α and HIF-1ß subunits of the HIF transcription factor in murine CH12 B cells results in defective CSR and that this is due to the suboptimal induction of AID expression.