Zeliha Oruc

Polymeric IgA1 controls erythroblast proliferation and accelerates erythropoiesis recovery in anemia

Anemia because of insufficient production of and/or response to erythropoietin (Epo) is a major complication of chronic kidney disease and cancer. The mechanisms modulating the sensitivity of erythroblasts to Epo remain poorly understood. We show that, when cultured with Epo at suboptimal concentrations, the growth and clonogenic potential of erythroblasts was rescued by transferrin receptor 1 (TfR1)-bound polymeric IgA1 (pIgA1). Under homeostatic conditions, erythroblast numbers were increased in mice expressing human IgA1 compared to control mice. Hypoxic stress of these mice led to increased amounts of pIgA1 and erythroblast expansion. Expression of human IgA1 or treatment of wild-type mice with the TfR1 ligands pIgA1 or iron-loaded transferrin (Fe-Tf) accelerated recovery from acute anemia. TfR1 engagement by either pIgA1 or Fe-Tf increased cell sensitivity to Epo by inducing activation of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways. These cellular responses were mediated through the TfR1-internalization motif, YXXΦ. Our results show that pIgA1 and TfR1 are positive regulators of erythropoiesis in both physiological and pathological situations. Targeting this pathway may provide alternate approaches to the treatment of ineffective erythropoiesis and anemia.

B‐cell receptor signal strength influences terminal differentiation

B‐cell terminal differentiation into antibody secreting plasma cells (PCs) features a trans‐criptional shift driven by the activation of plasma cell lineage determinants such as Blimp‐1 and Xbp‐1, together with the extinction of Pax5. Little is known about the signals inducing this change in transcriptional networks and the role of the B‐cell receptor (BCR) in terminal differentiation remains especially controversial. Here, we show that tonic BCR signal strength influences PC commitment in vivo. Using immuno‐globulin light chain transgenic mice expressing suboptimal surface BCR levels and latent membrane protein 2A knock‐in animals with defined BCR‐like signal strengths, we show that weak, antigen‐independent constitutive BCR signaling facilitates spontaneous PC differentiation in vivo and in vitro in response to TLR agonists or CD40/IL‐4. Conversely, increasing tonic signaling completely prevents this process that is rescued by lowering surface BCR expression or through the inhibition of Syk phosphorylation. These findings provide new insights into the role of basal BCR signaling in PC differentiation and point to the need to resolve a strong BCR signal in order to guarantee terminal differentiation.

Replacement of Iγ3 germ-line promoter by Iγ1 inhibits class-switch recombination to IgG3

Class-switch recombination (CSR) enables IgM-producing B cells to switch to the production of IgG, IgE, and IgA. The process requires germ-line (GL) transcription that initiates from promoters upstream of switch (S) sequences and is regulated by the 3′ regulatory region (3′RR) located downstream of the Ig heavy chain (IgH) locus. How the 3′RR effect its long-range activation is presently unclear. We generated a mouse line in which Iγ3 GL promoter was replaced by Iγ1. We found that GL transcription could initiate from the inserted Iγ1 promoter and was induced by increased concentrations of IL-4 and that the transcripts were normally spliced. However, when compared with GL transcripts derived from the endogenous Iγ1 promoter in the same stimulation conditions, those from the inserted Iγ1 promoter were less abundant. CSR to Cγ3 was abrogated both in vivo and in vitro. The results strongly suggest that the endogenous Iγ1 promoter insulates the inserted Iγ1 from the long-range activating effect of the 3′RR. The implications of our findings are discussed in light of the prominent models of long-distance activation in complex loci.

Functional anatomy of the immunoglobulin heavy chain 3΄ super-enhancer needs not only core enhancer elements but also their unique DNA context

Abstract Cis-regulatory elements feature clustered sites for transcription factors, defining core enhancers and have inter-species homology. The mouse IgH 3΄ regulatory region (3’RR), a major B-cell super-enhancer, consists of four of such core enhancers, scattered throughout more than 25 kb of packaging ‘junk DNA’, the sequence of which is not conserved but follows a unique palindromic architecture which is conserved in all mammalian species. The 3’RR promotes long-range interactions and potential IgH loops with upstream promoters, controlling class switch recombination (CSR) and somatic hypermutation (SHM). It was thus of interest to determine whether this functional architecture also involves the specific functional structure of the super-enhancer itself, potentially promoted by its symmetric DNA shell. Since many transgenic 3’RR models simply linked core enhancers without this shell, it was also important to compare such a ‘core 3’RR’ (c3’RR) with the intact full-length super-enhancer in an actual endogenous IgH context. Packaging DNA between 3’RR core enhancers proved in fact to be necessary for optimal SHM, CSR and IgH locus expression in plasma cells. This reveals that packaging DNA can matter in the functional anatomy of a super-enhancer, and that precise evaluation of such elements requires full consideration of their global architecture.

Sequential activation and distinct functions for distal and proximal modules within the IgH 3' regulatory region.

Significance The immunoglobulin heavy chain (IgH) 3′regulatory region (3′RR) fine-tunes IgH gene expression during B cell development. One singularity of this region is its quasi-palindromic structure conserved in the 3′RR of other species. By comparing previous mouse knockout (KO) models (3′RR- and hs3b-4 KO) to a novel mutant devoid of the quasi-palindrome (3′PAL KO), we highlighted common features and differences that specify two distinct regulatory entities: (i) the distal module (hs4) is sufficient for normal IgH expression up to the naïve B cell stage; (ii) during B-cell activation, the proximal module (quasi-palindrome) is important for both class switch recombination and somatic hypermutation; and (iii) in plasma cells, the quasi-palindrome is required for robust transcription of the IgH locus. As a master regulator of functional Ig heavy chain (IgH) expression, the IgH 3′ regulatory region (3′RR) controls multiple transcription events at various stages of B-cell ontogeny, from newly formed B cells until the ultimate plasma cell stage. The IgH 3′RR plays a pivotal role in early B-cell receptor expression, germ-line transcription preceding class switch recombination, interactions between targeted switch (S) regions, variable region transcription before somatic hypermutation, and antibody heavy chain production, but the functional ranking of its different elements is still inaccurate, especially that of its evolutionarily conserved quasi-palindromic structure. By comparing relevant previous knockout (KO) mouse models (3′RR KO and hs3b-4 KO) to a novel mutant devoid of the 3′RR quasi-palindromic region (3′PAL KO), we pinpointed common features and differences that specify two distinct regulatory entities acting sequentially during B-cell ontogeny. Independently of exogenous antigens, the 3′RR distal part, including hs4, fine-tuned B-cell receptor expression in newly formed and naïve B-cell subsets. At mature stages, the 3′RR portion including the quasi-palindrome dictated antigen-dependent locus remodeling (global somatic hypermutation and class switch recombination to major isotypes) in activated B cells and antibody production in plasma cells.

IgA Structure Variations Associate with Immune Stimulations and IgA Mesangial Deposition

IgA1 mesangial deposition is the hallmark of IgA nephropathy and Henoch-Schönlein purpura, the onset of which often follows infections. Deposited IgA has been reported as polymeric, J chain associated, and often, hypogalactosylated but with no information concerning the influence of the IgA repertoire or the link between immune stimuli and IgA structure. We explored these issues in the α1KI mouse model, which produces polyclonal human IgA1 prone to mesangial deposition. Compared with mice challenged by a conventional environment, mice in a specific pathogen-free environment had less IgA deposition. However, serum IgA of specific pathogen-free mice showed more galactosylation and much lower polymerization. Notably, wild-type, α1KI, and even J chain-deficient mice showed increased polymeric serum IgA on exposure to pathogens. Strict germfree conditions delayed but did not completely prevent deposition; mice housed in these conditions had very low serum IgA levels and produced essentially monomeric IgA. Finally, comparing monoclonal IgA1 that had different variable regions and mesangial deposition patterns indicated that, independently of glycosylation and polymerization, deposition might also depend on IgA carrying specific variable domains. Together with IgA quantities and constant region post-translational modifications, repertoire changes during immune responses might, thus, modulate IgA propensity to deposition. These IgA features are not associated with circulating immune complexes and C3 deposition and are more pertinent to an initial IgA deposition step preceding overt clinical symptoms in patients.

Gammopathy with IgA mesangial deposition provides a monoclonal model of IgA nephritogenicity and offers new insights into its molecular mechanisms

BACKGROUND Henoch-Schönlein purpura (HSP) and IgA nephropathy (IgAN) are characterized by mesangial deposition of polyclonal IgA eventually showing aberrant glycosylation, affinity for mesangial cells and/or co-precipitation with antigen, bacterial peptides, autoantibodies or soluble receptors. IgA were also suggested to be negatively charged and predominantly of λ type but rarely in a monoclonal form. METHODS A gammopathy case with HSP provided us with a unique molecularly defined nephritogenic IgA1λ. Immunological analysis, biological activities, glycosylation analysis and finally IgA sequence were determined. RESULTS Compared to IgA1 from healthy subjects or IgAN patients, IgA1 CAT showed hyposialylation but no hypogalactosylation, in agreement with underexpression of sialyltransferase genes by the plasma cell clone. IgA variable domains had low pIs with negatively charged complementarity-determining regions. Weak reactivity appeared against the cationic autoantigen lactoferrin, which was, however, absent from kidney deposits. Deposition also occurred in mice upon injection of only the polymeric form of IgA1 CAT, despite whether or not co-injected with lactoferrin. CONCLUSIONS This monoclonal model of IgA nephritogenicity strongly suggests that beside hinge region glycosylation, V domains play a role in IgA stability and pathogenicity and supports the hypothesis that responses against cationic epitopes from pathogens or autoantigens may select negatively charged complementarity-determining regions prone either to bind charged structures of the mesangium or to promote by themselves IgA aggregation and deposition.

A plasma cell differentiation quality control ablates B cell clones with biallelic Ig rearrangements and truncated Ig production

Srour et al. identify a quality control, truncated Ig exclusion checkpoint dampening terminal plasma cell differentiation by eliminating cells expressing nonfunctionally rearranged Igκ alleles

First Membrane Proximal External Region–Specific Anti-HIV1 Broadly Neutralizing Monoclonal IgA1 Presenting Short CDRH3 and Low Somatic Mutations

Mucosal HIV-1–specific IgA have been described as being able to neutralize HIV-1 and to block viral transcytosis. In serum and saliva, the anti-HIV IgA response is predominantly raised against the envelope of HIV-1. In this work, we describe the in vivo generation of gp41-specific IgA1 in humanized α1KI mice to produce chimeric IgA1. Mice were immunized with a conformational immunogenic gp41-transfected cell line. Among 2300 clones screened by immunofluorescence microscopy, six different gp41-specific IgA with strong recognition of gp41 were identified. Two of them have strong neutralizing activity against primary HIV-1 tier 1, 2, and 3 strains and present a low rate of somatic mutations and autoreactivity, unlike what was described for classical gp41-specific IgG. Epitopes were identified and located in the hepted repeat 2/membrane proximal external region. These Abs could be of interest in prophylactic treatment to block HIV-1 penetration in mucosa or in chronically infected patients in combination with antiretroviral therapy to reduce viral load and reservoir.

Complete cis Exclusion upon Duplication of the Eμ Enhancer at the Immunoglobulin Heavy Chain Locus

ABSTRACT Developing lymphocytes somatically diversify their antigen-receptor loci through V(D)J recombination. The process is associated with allelic exclusion, which results in monoallelic expression of an antigen receptor locus. Various cis-regulatory elements control V(D)J recombination in a developmentally regulated manner, but their role in allelic exclusion is still unclear. At the immunoglobulin heavy chain locus (IgH), the Eμ enhancer plays a critical role in V(D)J recombination. We generated a mouse line with a replacement mutation in the constant region of the locus that duplicates the Eμ enhancer and allows premature expression of the γ3 heavy chain. Strikingly, IgM expression was completely and specifically excluded in cis from the mutant allele. This cis exclusion recapitulated the main features of allelic exclusion, including differential exclusion of variable genes. Notably, sense and antisense transcription within the distal variable domain and distal VH-DJH recombination were inhibited. cis exclusion was established and stably maintained despite an active endogenous Eμ enhancer. The data reveal the importance of the dynamic, developmental stage-dependent interplay between IgH locus enhancers and signaling in the induction and maintenance of allelic exclusion.