Christelle Oblet

A mouse model recapitulating human monoclonal heavy chain deposition disease evidences the relevance of proteasome inhibitor therapy.

Randall-type heavy chain deposition disease (HCDD) is a rare disorder characterized by glomerular and peritubular amorphous deposits of a truncated monoclonal immunoglobulin heavy chain (HC) bearing a deletion of the first constant domain (CH1). We created a transgenic mouse model of HCDD using targeted insertion in the immunoglobulin κ locus of a human HC extracted from a HCDD patient. Our strategy allows the efficient expression of the human HC in mouse B and plasma cells, and conditional deletion of the CH1 domain reproduces the major event underlying HCDD. We show that the deletion of the CH1 domain dramatically reduced serum HC levels. Strikingly, even with very low serum level of truncated monoclonal HC, histologic studies revealed typical Randall-type renal lesions that were absent in mice expressing the complete human HC. Bortezomib-based treatment resulted in a strong decrease of renal deposits. We further demonstrated that this efficient response to proteasome inhibitors mostly relies on the presence of the isolated truncated HC that sensitizes plasma cells to bortezomib through an elevated unfolded protein response (UPR). This new transgenic model of HCDD efficiently recapitulates the pathophysiologic features of the disease and demonstrates that the renal damage in HCDD relies on the production of an isolated truncated HC, which, in the absence of a LC partner, displays a high propensity to aggregate even at very low concentration. It also brings new insights into the efficacy of proteasome inhibitor-based therapy in this pathology.

Specific impairment of proximal tubular cell proliferation by a monoclonal κ light chain responsible for Fanconi syndrome

BACKGROUNDnFanconi syndrome (FS) is a rare renal disorder featuring proximal tubule dysfunction that may occur following tubular reabsorption of a monoclonal light chain (LC), in patients with multiple myeloma. FS may precede the recognition of multiple myeloma by several years. In most cases, crystalline inclusions of monoclonal κ LCs are observed within the lysosomes of proximal tubular cells (PTCs) and probably participate in their functional alteration.nnnMETHODSnTo investigate the mechanism implicated in proximal tubule dysfunction, we compared the effects of κ LC-CHEB obtained from a patient with myeloma-associated FS to those of control κ LC-BON obtained from a patient without evidence of FS, on the viability and proliferation of two different PTC lines.nnnRESULTSnOur data suggest that the tubular atrophy in myeloma-associated FS does not result from increased apoptosis of PTCs, but from their impaired capacity to proliferate and renew. Indeed, in vitro incubation of cultured PTCs with physiological amounts of the nephrotoxic κ LC-CHEB was sufficient to cause a depression in DNA synthesis and in cell proliferation. This effect was observed neither with control κ LC-BON nor in the absence of κ LC.nnnCONCLUSIONSnThe reduced turnover of PTCs may affect tubular repair and regeneration. In addition, the reduced proliferation of myeloma cells producing the same monoclonal κ LC might explain the frequent association of FS with smoldering multiple myeloma.

The IgH 3′ regulatory region super-enhancer does not control IgA class switch recombination in the B1 lineage

The bone marrow-derived B2 population represents the vast majority of bone marrow, blood, lymph node and splenic B-cells. Mouse B1 B-cells mostly originate during embryonic life in the liver and represent the main B-cell population in the pleural and peritoneal cavities.1–5 B1 and B2 B-cells differ in their origin, antigen specificity, cell surface markers, tissue distribution and capacity for class switch recombination (CSR). Schematically, B1 B-cells appear earlier than B2 B-cells during fetal development and maintain their self-renewal ability throughout their life. Furthermore, they display a CSR biased toward IgA.1–5 IgH cis-regulatory regions, especially transcriptional super-enhancers, are major locus regulators.6 The IgH 3′ regulatory region (3′RR) super-enhancer promotes CSR in B2 B-cells.7,8 As B1 and B2 B-cells originate from different precursors and have clearly different development, function and regulation, we postulated that the 3′RR super-enhancer might differently regulate B1 and B2 B-cell IgA CSR. The 3′RR super-enhancer plays a key regulatory role in B2 B-cell CSR by poising the S acceptor region for efficient recombination toward all isotypes8 except IgD.9,10 Peritoneal cavity B1 B-cells switch preferentially to IgA.11,12 We thus investigated B1 B-cell IgA CSR in 3′RR-deficient mice.7 Our research was approved by our local ethics committee review board (Comité Régional dEthique sur lExpérimentation Animale du Limousin, Limoges, France) and carried out according to the European guidelines for animal experimentation. The 3′RR deletion was performed in a 129 ES cell line (IgH a allotype) and developed in a 129 background (IgH awt/awt). In this study, homozygous 3′RR-deficient mice (IgH aΔ3′RR/aΔ3′RR) were compared with 129 IgH awt/awt mice. Mouse B1 and B2 B-cells are distinguished on the basis of membrane cell surface markers. B1 B-cells are B220lowIgMhighIgDlowCD23−CD11b+/low, whereas B2 B-cells are B220highIgMhighIgDhighCD23+ CD11b−.2,4,13 As shown in Figure 1a, similar (P= 0.5) percentages of IgA+ B1 B-cells were found in the peritoneal cavity of 3′RR-deficient and wild type (wt) mice in response to a pristane-induced local inflammatory reaction. Almost all recruited inflammatory B-cells in response to pristane had a B1 B-cell phenotype. We next investigated their ability to switch in vitro toward IgA (LPS+BAFF+TGFβ stimulation). As shown in Figure 1b, no difference (P= 0.3) was found in B1 B-cell IgA CSR between 3′RR-deficient mice and wt mice. Despite similar in vivo and in vitro IgA CSR, lowered (P= 0.0003) levels of IgA were found in peritoneal ascites of 3′RR-deficient mice compared with wt mice (Figure 1c) and in IgA+ cell culture supernatants (P= 0.002) of 3′RR-deficient B1 B-cells compared with wt B1 B-cells (Figure 1d). q-PCR analysis indicated that the latter result originated from a significant (P= 0.01) decrease in Iμ-Cα transcripts in 3′RR-deficient mice compared with wt mice (Figure 1e). Taken together, these results suggest that in contrast to B2 B-cells, 3′RR deletion did not affect the B1 B-cell CSR toward IgA but only decreased Cα transcription after CSR. The IgA-producing B1 B-cells contribute substantially to mucosal IgA production and thus play a key role in regulating commensal microbiota.4,14 We thus investigated IgA production in the intestinal tract of 3′RR-deficient mice and wt mice. Mouse feces were recovered and tested for the presence of IgA. We found a significant (P= 0.002) and marked (up to 99%) decrease in secreted IgA levels in feces of 3′RR-deficient mice (0.08± 0.03 μg/g, six mice) compared with wt mice (11.34± 0.63 μg/g, six mice). Confirming that this result is related to an IgA secretion deficiency but not to an IgA CSR or a plasma cell maturation defect, immunohistochemistry experiments indicated similar IgA+ CNRS UMR 7276, CRIBL, Université de Limoges, Limoges, France

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

BACKGROUNDnHenoch-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.nnnMETHODSnA 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.nnnRESULTSnCompared 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.nnnCONCLUSIONSnThis 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.

The immunoglobulin heavy chain 3′ regulatory region superenhancer controls mouse B1 B-cell fate and late VDJ repertoire diversity

The immunoglobulin heavy chain (IgH) 3 regulatory region (3RR) superenhancer controls B2 B-cell IgH transcription and cell fate at the mature stage but not early repertoire diversity. B1 B cells represent a small percentage of total B cells differing from B2 B cells by several points such as precursors, development, functions, and regulation. B1 B cells act at the steady state to maintain homeostasis in the organism and during the earliest phases of an immune response, setting them at the interface between innate and acquired immunity. We investigated the role of the 3RR superenhancer on B1 B-cell fate. Similar to B2 B cells, the 3RR controls μ transcription and cell fate in B1 B cells. In contrast to B2 B cells, 3RR deletion affects B1 B-cell late repertoire diversity. Thus, differences exist for B1 and B2 B-cell 3RR control during B-cell maturation. For the first time, these results highlight the contribution of the 3RR superenhancer at this interface between innate and acquired immunity.