Michel Cogné

The Igκ 3′ Enhancer Influences the Ratio of Igκ versus Igλ B Lymphocytes

Abstract We generated mice harboring germline mutations in which the enhancer element located 9 kb 3′ of the immunoglobulin κ light chain gene (3′E κ ) was replaced either by a single loxP site (3′E κ Δ) or by a neomycin resistance gene (3′E κ N). Mice homozygous for the 3′E κ Δ mutation had substantially reduced numbers of κ-expressing B cells and increased numbers of λ-expressing B cells accompanied by decreased κ versus λ gene rearrangement. In these mutant mice, κ expression was reduced in resting B cells, but was normal in activated B cells. The homozygous 3′E κ N mutation resulted in a similar but more pronounced phenotype. Both mutations acted in cis . These studies show that the 3′E κ is critical for establishing the normal κ/λ ratio, but is not absolutely essential for κ gene rearrangement or, surprisingly, for normal κ expression in activated B cells. These studies also imply the existence of additional regulatory elements that have overlapping function with the 3′E κ element.

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.

Toward understanding renal Fanconi syndrome: step by step advances through experimental models.

Renal Fanconi syndrome (FS) is a generalized dysfunction of proximal tubular epithelial cells leading to the urinary leak of essential metabolites like phosphate, uric acid, glucose, amino acids and low molecular weight proteins. From inherited forms involving mutations on apparently unrelated genes to acquired forms induced by drugs, heavy metals or monoclonal immunoglobulin (Ig) light chains (LC), heterogeneous causalities of FS have complicated the understanding of this pathology for a long time. Experimental models of FS have allowed researchers to face the challenge and have helped unravel the main mechanisms disturbing proximal tubule reabsorption. Administration of cadmium to animals first demonstrated an inhibition of Na/K/ATPase activity, highlighting how a single toxic component could induce the general sodium-linked transport defect observed in FS. Today, genetically modified mice allow the development of reliable and reproducible experimental models for inherited or acquired forms of FS. One of the most exciting advances offered by these models is the unexpected major role of endocytosis in the function of the proximal tubule revealed by megalin and ClC-5 knockout mice. Using gene-targeted insertion, a transgenic mouse for LC-associated FS, the most frequent adult form of FS, has also been recently developed and represents a major step in the development of models of this pathology. Beyond deciphering molecular and cellular events at the origin of FS, these models also represent essential tools for the development of therapeutic strategies.

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.

Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells

Background While most antibody-based therapies use IgG because of their well-known biological properties, some functional limitations of these antibodies call for the development of derivatives with other therapeutic functions. Although less abundant than IgG in serum, IgA is the most abundantly produced Ig class in humans. Besides the specific targeting of its dimeric form to mucosal areas, IgA was shown to recruit polymorphonuclear neutrophils against certain targets more efficiently than does IgG1. Design and Methods In this study, we investigated the various pathways by which anti-tumor effects can be mediated by anti-CD20 IgA against lymphoma cells. Results We found that polymeric human IgA was significantly more effective than human IgG1 in mediating direct killing or growth inhibition of target cells in the absence of complement. We also demonstrated that this direct killing was able to indirectly induce the classical pathway of the complement cascade although to a lesser extent than direct recruitment of complement by IgG. Recruitment of the alternative complement pathway by specific IgA was also observed. In addition to activating complement for lysis of lymphoma cell lines or primary cells from patients with lymphoma, we showed that monomeric anti-CD20 IgA can effectively protect mice against tumor development in a passive immunization strategy and we demonstrated that this protective effect may be enhanced in mice expressing the human FcαRI receptor on their neutrophils. Conclusions We show that anti-CD20 IgA antibodies have original therapeutic properties against lymphoma cells, with strong direct effects, ability to recruit neutrophils for cell cytotoxicity and even recruitment of complement, although largely through an indirect way.

CHAPTER 19 – Regulation of Class Switch Recombination

This chapter focuses on the regulation of class-switch recombination (CSR) to all non-m isotypes, except for delta. The expression of immunoglobulin D (IgD) results from alternative splicing that regularly occurs as part of B-cell maturation, and only rarely involves Cμ deletion. In contrast, deoxyribonucleic acid (DNA) recombination is constantly needed for the other isotypes, and there are a number of elements that are now known to be essential. For example, a critical first step in CSR is germline transcription (GT), whose production for several isotypes is dependent on at least two of the 3´ immunoglobulin H (IgH) enhancers. GT of individual isotypes requires cell–cell interaction involving CD40 and CD40L, various B cell transcription factors, including NFkB and specific T cell cytokines. A predilection to switch to particular classes, especially immunoglobulin E (IgE) in allergic individuals can be harmful for human health, and most likely is fostered by the T cell cytokine profile associated with particular Th subsets. Mistakes in CSR have been associated with chromosomal translocations involving “c-myc” and are regularly detected in murine plasmacytomas, human myeloma, and Burkitts lymphoma. Current challenges are to identify the signals that trigger 3´ enhancer activity during B-cell development and the mechanisms that engage these enhancers with I promoters for GT. The formation of GT clearly involves both positive and negative regulation, providing an additional arena for critical investigation.

Membrane isoforms of human immunoglobulins of the A1 and A2 isotypes: structural and functional study

As for IgM, human IgA occurs either as soluble molecules in plasma and various other body fluids, or as membrane‐bound molecules on differentiated B cells, where they are part of the B‐cell receptor for antigen (BCR). We studied the structure of transcripts encoding the membrane‐anchored α‐chain of the human BCRα, which may be present in two different forms resulting from alternate splicing of the α‐chain mRNA (type I or type II). The ratio of type I versus type II did not vary upon stimulation of a B‐cell line with various cytokines. Rather, it differed strikingly in cells expressing either the IgA1 or IgA2 isotype of the BCRα, with virtually no type II α‐chain in the latter. Co‐modulation experiments also yielded different results for both isotypes, since they demonstrated a physical association of both membrane (m)IgA1 and mIgA2 with CD79b, the β component of the BCR Igα/Igβ heterodimer, but only of mIgA1 with CD19. Whatever the isotype, the BCR of the IgA class was able to carry out signal transduction upon cross‐linking by specific monoclonal antibodies but, in contrast to mIgM, it relied mainly on the entry of extracellular Ca2+ rather than on the release of intracellular stocks.

Production of Human or Humanized Antibodies in Mice

Mice are widely available laboratory animals that can easily be used for the production of antibodies against a broad range of antigens, using well-defined immunization protocols. Such an approach allows optimal in vivo affinity maturation of the humoral response. In addition, high-affinity antibodies arising in this context can readily be further characterized and produced as monoclonals after immortalizing and selecting specific antibody-producing cells through hybridoma derivation. Using such conventional strategies combined with mice that are either genetically engineered to carry humanized immunoglobulin (Ig) genes or engrafted with a human immune system, it is thus easy to obtain and immortalize clones that produce either fully human Ig or antibodies associating variable (V) domains with selected antigen specificities to customized human-like constant regions, with defined effector functions. In some instances, where there is a need for in vivo functional assays of a single antibody with a known specificity, it might be of interest to transiently express that gene in mice by in vivo gene transfer. This approach allows a rapid functional assay. More commonly, mice are used to obtain a diversified repertoire of antibody specificities after immunization by producing antibody molecules in the mouse B cell lineage from mouse strains with transgene Ig genes which are of human, humanized, or chimeric origin. After in vivo maturation of the immune response, this will lead to the secretion of antibodies with optimized antigen binding sites, associated to the desired human constant domains. This chapter focuses on two simple methods: (1) to obtain such humanized Ig mice and (2) to transiently express a human Ig gene in mice using hydrodynamics-based transfection.

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.

Impaired Lysosomal Function Underlies Monoclonal Light Chain–Associated Renal Fanconi Syndrome

Monoclonal gammopathies are frequently complicated by kidney lesions that increase the disease morbidity and mortality. In particular, abnormal Ig free light chains (LCs) may accumulate within epithelial cells, causing proximal tubule (PT) dysfunction and renal Fanconi syndrome (RFS). To investigate the mechanisms linking LC accumulation and PT dysfunction, we used transgenic mice overexpressing human control or RFS-associated κLCs (RFS-κLCs) and primary cultures of mouse PT cells exposed to low doses of corresponding human κLCs (25 μg/ml). Before the onset of renal failure, mice overexpressing RFS-κLCs showed PT dysfunction related to loss of apical transporters and receptors and increased PT cell proliferation rates associated with lysosomal accumulation of κLCs. Exposure of PT cells to RFS-κLCs resulted in κLC accumulation within enlarged and dysfunctional lysosomes, alteration of cellular dynamics, defective proteolysis and hydrolase maturation, and impaired lysosomal acidification. These changes were specific to the RFS-κLC variable (V) sequence, because they did not occur with control LCs or the same RFS-κLC carrying a single substitution (Ala30→Ser) in the V domain. The lysosomal alterations induced by RFS-κLCs were reflected in increased cell proliferation, decreased apical expression of endocytic receptors, and defective endocytosis. These results reveal that specific κLCs accumulate within lysosomes, altering lysosome dynamics and proteolytic function through defective acidification, thereby causing dedifferentiation and loss of reabsorptive capacity of PT cells. The characterization of these early events, which are similar to those encountered in congenital lysosomal disorders, provides a basis for the reported differential LC toxicity and new perspectives on LC-induced RFS.