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International audience; Background: Primary central nervous system lymphoma (PCNSL) represents a particular entity within non-Hodgkin lymphomas and is associated with poor outcome. The present study addresses the potential clinical relevance of chimeric transcripts in PCNSL discovered by using RNA sequencing (RNA-seq).Methods: Seventy-two immunocompetent and newly diagnosed PCNSL cases were included in the present study. Among them, 6 were analyzed by RNA-seq to detect new potential fusion transcripts. We confirmed the results in the remaining 66 PCNSL. The gene fusion was validated by fluorescence in situ hybridization (FISH) using formalin-fixed paraffin-embedded (FFPE) samples. We assessed the biological and clinical impact of one new gene fusion.Results: We identified a novel recurrent gene fusion, E26 transformation-specific translocation variant 6-immunoglobulin heavy chain (ETV6-IgH). Overall, ETV6-IgH was found in 13 out of 72 PCNSL (18%). No fusion conserved an intact functional domain of ETV6, and ETV6 was significantly underexpressed at gene level, suggesting an ETV6 haploinsufficiency mechanism. The presence of the gene fusion was also validated by FISH in FFPE samples. Finally, PCNSL samples harboring ETV6-IgH showed a better prognosis in multivariate analysis, P = 0.03, hazard ratio = 0.33, 95% CI = 0.12-0.88. The overall survival at 5 years was 69% for PCNSL harboring ETV6-IgH versus 29% for samples without this gene fusion.Conclusions: ETV6-IgH is a new potential surrogate marker of PCNSL with favorable prognosis with ETV6 haploinsufficiency as a possible mechanism. The potential clinical impact of ETV6-IgH should be validated in larger prospective studies.

Somatic hypermutation in variable heavy chain rearranged regions is abrogated in the absence of the 3′ regulatory region enhancer, whereas transcription rate in the Ig heavy chain is only partially reduced., Interactions with cognate antigens recruit activated B cells into germinal centers where they undergo somatic hypermutation (SHM) in V(D)J exons for the generation of high-affinity antibodies. The contribution of IgH transcriptional enhancers in SHM is unclear. The Eμ enhancer upstream of Cμ has a marginal role, whereas the influence of the IgH 3′ regulatory region (3′RR) enhancers (hs3a, hs1,2, hs3b, and hs4) is controversial. To clarify the latter issue, we analyzed mice lacking the whole 30-kb extent of the IgH 3′RR. We show that SHM in VH rearranged regions is almost totally abrogated in 3′RR-deficient mice, whereas the simultaneous Ig heavy chain transcription rate is only partially reduced. In contrast, SHM in κ light chain genes remains unaltered, acquitting for any global SHM defect in our model. Beyond class switch recombination, the IgH 3′RR is a central element that controls heavy chain accessibility to activation-induced deaminase modifications including SHM.

International audience; V(D)J recombination occurs during the antigen-independent early steps of B-cell ontogeny. Multiple IgH cis-regulatory elements control B-cell ontogeny. IGCR1 (intergenic control region 1), the DQ52 promoter/enhancer, and the intronic Emu enhancer, all three located upstream of Cmu, have important roles during V(D)J recombination, whereas there is no clue about a role of the IgH regulatory region (RR) encompassing the four transcriptional enhancers hs3a, hs1,2, hs3b, and hs4 during these early stages. To clarify the role of the RR in V(D)J recombination, we totally deleted it in the mouse genome. Here, we show that V(D)J recombination is unaffected by the complete absence of the IgH RR, highlighting that this region only orchestrates IgH locus activity during the late stages of B-cell differentiation. In contrast, the earliest antigen-independent steps of B-cell ontogeny would be under the control of only the upstream Cmu elements of the locus.

Although c-myc is classically described as the driving oncogene in Burkitt’s lymphoma (BL), deregulation and mutations of c-myc have been reported in multiple solid tumors and in other mature B cell malignancies such as mantle cell lymphoma (MCL), myeloma, and plasma cell lymphoma (PCL). After translocation into the IgH locus, c-myc is constitutively expressed under the control of active IgH enhancers. Those located in the IgH 3′ regulatory region (3′RR) are master control elements of class switch recombination and of the transcriptional burst associated with plasma cell differentiation. c-myc-3′RR mice are prone to lymphomas with rather homogeneous, most often BL-like, phenotypes with incomplete penetrance (75% tumor incidence) and long latencies (10–12 mo). To reproduce c-myc–induced mature B cell lymphomagenesis in the context of an additional defect often observed in human lymphomas, we intercrossed c-myc-3′RR with p53+/− mice. Double transgenic c-myc-3′RR/p53+/− mice developed lymphoma with short latency (2–4 mo) and full penetrance (100% tumor incidence). The spectrum of B lymphomas occurring in c-myc-3′RR/p53+/− mice was widened, including nonactivated (CD43−) BL, activated (CD43+) BL, MCL-like lymphoma, and PCL, thus showing that 3′RR-mediated deregulation of c-myc can promote various types of B lymphoproliferation in cells that first acquired a p53 defect. c-myc/p53+/− mice closely reproduce many features of BL, MCL, and PCL and provide a novel and efficient model to dissect the molecular events leading to c-myc–induced lymphomagenesis and an important tool to test potential therapeutic agents on malignant B cells featuring various maturation stages.

International audience; Cyclin D1 overexpression is associated with mantle cell lymphoma and multiple myeloma. In myeloma, it often results from chromosomal translocations linking the CCND1 gene to the 3' part of the IgH locus constant region. This region includes a single and potent transcriptional regulatory region (RR) 3' of the Calpha gene mostly active in mature B-cells. To check whether this RR alone was sufficient to deregulate CCND1, we generated mice carrying a 3'IgH RR-driven human CCND1 transgene and specifically up-regulating cyclin D1 expression in B-cells. In transgenic B-cells, cyclin D1 enforced cell cycle entry in response to various stimuli (LPS, anti-IgM, anti-CD40) but also increased cell death, so that exaggerated proliferation did not result in peripheral lymphocytosis. Despite exaggerated B-cell entry into G(1) phase, malignant lymphoproliferation did not occur either. Crossing of CCND1-3'IgH RR mice with c-myc-3'IgH RR mice did not reveal accelerated tumorigenesis as compared with c-myc-3'IgH RR mice alone. The data presented here demonstrate that the 3'IgH RR-mediated deregulation of CCND1 in mature B-cells cannot by itself trigger the development of lymphomas and strengthen the concept that cyclin D1 per se is not an armful proto-oncogene. Rather its overexpression in several malignancies might be only a stigma of lymphomagenesis or represent a single hit within a multiple hit process.

Several studies have reported that regulatory elements located 3′ of the IgH locus (namely hs3a, hs1,2, hs3b, and hs4) might play a role during class switch recombination (CSR) and Ig synthesis. While individual deletion of hs3a or hs1,2 had no effect, pairwise deletion of hs3b (an inverted copy of hs3a) and hs4 markedly affected CSR and Ig expression. Among these two elements, hs4 was tentatively presented with the master role due to its unique status within the 3′ regulatory region: distal position outside repeated regions, early activation in pre-B cells, strong activity throughout B cell ontogeny. To clarify its role, we generated mice with a clean deletion of the hs4 after replacement with a floxed neoR cassette. Surprisingly, and as for previous deletion of hs3a or hs1,2, deletion of hs4 did not affect either in vivo CSR or the secretion level of any Ig isotype. In vitro CSR and Ig secretion in response to LPS and cytokines was not affected either. The only noticeable effects of the hs4 deletion were a decrease in the number of B splenocytes and a decreased membrane IgM expression. In conclusion, while dispensable for CSR and Ig transcription in plasma cells, hs4 mostly appears to contribute to Ig transcription in resting B lymphocytes.

International audience; MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. To examine the physiologic effects of an individual miRNA-mRNA interaction in vivo, we generated mice that carry a mutation in the putative microRNA-155 (miR-155) binding site in the 3'-untranslated region of activation-induced cytidine deaminase (AID), designated Aicda(155) mice. AID is required for immunoglobulin gene diversification in B lymphocytes, but it also promotes chromosomal translocations. Aicda(155) caused an increase in steady-state Aicda mRNA and protein amounts by increasing the half-life of the mRNA, resulting in a high degree of Myc-Igh translocations. A similar but more pronounced translocation phenotype was also found in miR-155-deficient mice. Our experiments indicate that miR-155 can act as a tumor suppressor by reducing potentially oncogenic translocations generated by AID.

International audience; BACKGROUND: Flow cytometry allows specific assessment of the expression of ZAP-70, a promising new prognostic factor in B-cell chronic lymphocytic leukemia (B-CLL), but suffers from a lack of multicenter standardization. DESIGN AND METHODS: An optimized method for direct detection of ZAP-70 in flow cytometry was tested in a multicenter fashion. Adapted for frozen cells, this method includes a normalization step by addition of B cells from a pool of peripheral blood mononuclear cells collected from normal donors. ZAP-70 expression levels were assessed for 153 patients with typical B-cell chronic lymphocytic leukemia chronic lymphocytic leukemia. Results were expressed as the ratio of ZAP-70 mean fluorescence intensity between B-CLL cells and normal B cells. RESULTS: The statistically optimized cut-off of ZAP-70 positivity was a ratio of 1.4. Concordance between ZAP-70 and CD38 expression was 67%. Concordance between the mutational status of IgVH genes and ZAP-70 or CD38 expression was 87% and 65%, respectively. ZAP-70 was significantly expressed in 28%, 54% and 61% of patients with Binet stages A, B and C B-cell chronic lymphocytic leukemia, respectively (p=0.008). The absence of ZAP-70 expression was associated with isolated del(13q14), a cytogenetic abnormality with a good prognosis, while most patients with the del(17p13) poor prognosis cytogenetic marker expressed ZAP-70 (p

Motivation: Antibodies are able to recognize a wide range of antigens through their complementary determining regions formed by six hypervariable loops. Predicting the 3D structure of these loops is essential for the analysis and reengineering of novel antibodies with enhanced affinity and specificity. The canonical structure model allows high accuracy prediction for five of the loops. The third loop of the heavy chain, H3, is the hardest to predict because of its diversity in structure, length and sequence composition. Results: We describe a method, based on the Random Forest automatic learning technique, to select structural templates for H3 loops among a dataset of candidates. These can be used to predict the structure of the loop with a higher accuracy than that achieved by any of the presently available methods. The method also has the advantage of being extremely fast and returning a reliable estimate of the model quality. Availability and implementation: The source code is freely available at http://www.biocomputing.it/H3Loopred/ Contact: anna.tramontano@uniroma1.it Supplementary Information: Supplementary data are available at Bioinformatics online.

A nonrestrictive method for identifying covariance in protein families is described and applied to human and mouse germline Vκ and VH sequence alignments. Amino acids that occur at each position in a sequence alignment are divided into two sets, called a word, by generating all possible combinations of alternative amino acids. Each word is associated with a pattern of changes. Words with identical patterns identify covariant positions. In antibody variable domains, the number of words generated ranged between 1103 and 2195 depending on the alignment, of which 4 to 12 % occurred in covariant pairs. Despite the nonrestrictive character of pattern generation, covariant residues did not reflect a random selection with respect to the nature of amino acid changes and/or their spatial proximity in a reference crystallographic structure. This approach allowed the identification of a covariance signal for positions with high variability, mostly located in the outer part of the common structural framework of antibody variable domains. Covariance in these regions may reflect the existence of alternative and mutually exclusive atomic arrangements that are compatible with antibody function. The method may be of general applicability to rationalize residue variability in protein families. Proteins 2000;41:475–484. © 2000 Wiley-Liss, Inc.

The surrogate light chain (SL) composed of the λ-like and VpreB polypeptides is organized as two Ig domains and an extra-loop structure. It associates to the μ-chain in preB cells. We have produced human VpreB, SL, two Fdμ (VH-CH1), and the two corresponding Fab-like (Fdμ-SL) recombinant proteins in baculovirus. The correctness of the general conformation of the proteins was assessed by epitope mapping and affinity measurements using a new batch of anti-VpreB mAbs. Plasmon resonance analysis showed that both VpreB and the entire SL associated with the Fdμ fragments, with Kd values of 3 × 10−8 M for VpreB-Fdμ and of 10−9 to 10−10 M, depending upon the VH, for SL-Fdμ. These results indicate that the λ-like chain, in addition to be covalently bound to the Cμ1 domain, also interacts with the VH domain. Therefore, a dual role of the SL emerges: 1) interaction of the C-domain of λ-like would release the μ-chain from its interaction with binding protein in the endoplasmic reticulum, and 2) interaction of a part of λ-like and most of VpreB would bind to VH, ensuring a “quality control” of the native heavy chain that represents the first step of selection of the B cell repertoire. We also demonstrated that two Fab-like fragments did not interact with each other, suggesting that activation of the cell surface preB receptor does not involve aggregation neither in cis nor in trans of the Fab-like structures.

Although the switch process is frequently associated with affin- ity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing iden- tical V H and V L sequences, but expressing different isotypes (IgA1 k PER and IgG1 k PER ), bind tubulin with significantly dif- ferent affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results sug- gest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mu- tations. Since the differences observed were found at the Fab level, they also suggest a role for the C H 1 domain in struc- turing the Ag-binding site into a more kinetically competent form. ( J. Clin. Invest. 1996. 98:2235-2243.) Key words: im- munoglobulinsisotypeswitchingC H 1 • affinity

The interface between the VL and VH domains of antibodies is highly conserved. To investigate the influence of conserved interface residues on Fab function, 13 interface residues were subjected to codon-based combinatorial alanine scanning mutagenesis in Fab 57P, specific for peptide 134 to 151 of the coat protein of tobacco mosaic virus. The 13 single mutants were analysed by Western blot to determine the effect of interface modifications on Fab expression. The kinetic rate constants of peptide-Fab mutant interactions were measured using the biosensor technology. Alanine replacements did not prevent assembly of the mutated Fabs and led to a modification of their binding properties in every case. Twelve of the 13 target residues correspond to homologous positions in the VL and VH domains, which have similar folds. Mutation at homologous positions mostly had different effects on antigen binding affinity. The replacement of bulky side-chains had the most drastic effect on binding. When smaller side-chains were replaced by alanine, the binding properties of Fab mutants differed slightly (by less than a factor of two), but significantly from that of Fab 57P. Modification of some of these residues, which are located 9 to 12 A away from the base of CDR loops, is unlikely to alter loop conformation. They may affect antigen binding indirectly by influencing the relative position of the VL and VH domains. Our results demonstrate that residues situated at the VL – VH interface and which are remote from the paratope are able to influence the antigen binding properties of antibodies.

International audience

International audience; Immunoglobulin (Ig) genes naturally acquire frequent premature termination codons during the error-prone V(D)J recombination process. Although B cell differentiation is linked to the expression of productive Ig alleles, the transcriptional status of nonfunctionally recombined alleles remains unclear. Here, we tracked transcription and posttranscriptional regulation for both Ig heavy-chain (IgH) alleles in mice carrying a nonfunctional knock-in allele. We show that productively and nonproductively VDJ-rearranged alleles are transcribed throughout B cell development, carry similar active chromatin marks, and even display equivalent RNA polymerase II (RNAPII) loading after B cell stimulation. Hence, these results challenge the idea that the repositioning of one allele to heterochromatin could promote the silencing of nonproductive alleles. Interestingly, the efficiency of downstream RNA surveillance mechanisms fluctuates according to B cell activation and terminal differentiation: unspliced nonfunctional transcripts accumulate in primary B cells, while B cell activation promotes IgH transcription, RNA splicing, and nonsense-mediated mRNA decay (NMD). Altogether, IgH transcription and RNA splicing rates determine by which RNA surveillance mechanisms a B cell can get rid of nonproductive IgH mRNAs.

International audience; Various renal disorders are associated with monoclonal gammopathies, secondary to tissue deposition or precipitation of a monoclonal immunoglobulin (Ig) or a fragment thereof (isolated Ig light chain or heavy chain). They are classified according to the localization of renal lesions, either glomerular or tubular and to the pattern of ultrastructural organization of Ig deposits. Renal disease in monoclonal gammopathies may be isolated, or associated with various systemic symptoms particularly in AL amyloidosis, Randall-type monoclonal Ig deposition disease and monoclonal cryoglobulinemias. Except for myeloma cast nephropathy, which occurs in the setting of high-mass myeloma and is recognized after electrophoretic analysis of proteinuria and AL amyloidosis, which diagnosis is usually made after pathological examination of non-invasive tissue specimens (i.e. abdominal fat or minor salivary glands), a kidney biopsy is required to identify the other types of renal disorders associated with monoclonal gammopathies and to estimate renal prognosis. Renal pathological diagnosis is difficult and relies on careful examination of kidney biopsy samples, by light microscopy, immunofluorescence studies using conjugates specific for Ig light and heavy chains, IgG sub-classes and heavy chain constant domains and by electron microscopy. In some cases, additional studies are required to identify the nature of deposits, such as immuno-electron microscopy or mass spectrometric-based proteomic analysis after laser dissection. In patients with renal disorders related to Ig light chain precipitation or deposition (myeloma cast nephropathy, AL amyloidosis, Randall-type light chain deposition disease), measurement of serum free light chains at baseline and throughout follow-up is mandatory to evaluate clonal response to chemotherapy. A more than or equal to 50% decrease in serum free light chain levels is associated with increased renal and patient survival. In AL amyloidosis, serum levels of markers of cardiac disease (NT-proBNP and troponin) are also closely associated with prognosis. Efficient chemotherapy, tailored to the underlying plasma cell or lymphoproliferative disorder and adapted to renal function, should be promptly introduced, even in the absence of overt malignant haematological disease. Renal prognosis and patient survival (particularly in AL amyloidosis and cast nephropathy) are closely associated with the rapid achievement of an haematological response. The combination of melphalan plus dexamethasone (MDex) is currently used as first-line chemotherapy in systemic AL amyloidosis. Bortezomib-based regimens are commonly employed as first-line treatment in myeloma cast nephropathy and Randall-type monoclonal Ig deposition disease and as second line therapy in AL amyloidosis patients with advanced cardiomyopathy or refractory to previous chemotherapy. Solid organ transplantation (heart and kidney) should be considered in patients with AL amyloidosis or Randall-type monoclonal Ig deposition disease and advanced cardiac or renal failure. Prolonged graft and patient survival may be obtained, providing that recipients do not have other severe organ involvement or symptomatic myeloma and that haematological remission has been achieved with chemotherapy before or after organ transplantation.

Chronic lymphocytic leukemia (CLL) is a heterogeneous disease characterized by defects in the DNA damage response and apoptosis. Among the factors involved in these pathways, we focused on the enzyme poly(ADP-ribose) polymerase 1 (PARP1) and on its substrate Che-1 by evaluating their basal expression and functional changes upon irradiation (IR). Microarray experiments were performed on 98 untreated CLL cases. Next, freshly isolated primary cells from 21 untreated patients were analyzed for invitro response to irradiation through Western blot, PARP activity assay, Annexin-V analysis, and PARP1 basal expression by quantitative polymerase chain reaction. Microarray analysis showed that PARP1 and CHE1 were constitutively expressed in CLL and had a high degree of correlation with each other and with TP53. PARP1 and TP53 downmodulation was associated with worse clinical outcomes, especially in TP53-mutated cases. Next, CLL samples from 21 untreated patients were classified as responders and nonresponders based on IR-induced PARP1 cleavage. Notably, while responder samples were characterized by Che-1 and p53 induction at 8 hours and reduction at 24 hours post-IR, nonresponders included both samples with p53 dysfunctions and cases with a normal IR-induced Che-1 and/or p53 response. Finally, we observed that PARP1 was downregulated in nonresponder vs responder samples and that its basal expression was positively correlated with PARP1 cleavage after IR. In conclusion, we showed that reduced expression of PARP1 is associated with an impairment of CLL responsiveness to cell death. © 2012 ISEH - Society for Hematology and Stem Cells.

The C-type lectin langerin/CD207 was originally discovered as a specific marker for epidermal Langerhans cells (LC). Recently, additional and distinct subsets of langerin+ dendritic cells (DC) have been identified in lymph nodes and peripheral tissues of mice. Although the role of LC for immune activation or modulation is now being discussed controversially, other langerin+ DC appear crucial for protective immunity in a growing set of infection and vaccination models. In knock-in mice that express the human diphtheria toxin receptor under control of the langerin promoter, injection of diphtheria toxin ablates LC for several weeks whereas other langerin+ DC subsets are replenished within just a few days. Thus, by careful timing of diphtheria toxin injections selective states of deficiency in either LC only or all langerin+ cells can be established. Taking advantage of this system, we found that, unlike selective LC deficiency, ablation of all langerin+ DC abrogated the activation of IFN-γ–producing and cytolytic CD8+ T cells after gene gun vaccination. Moreover, we identified migratory langerin+ dermal DC as the subset that directly activated CD8+ T cells in lymph nodes. Langerin+ DC were also critical for IgG1 but not IgG2a Ab induction, suggesting differential polarization of CD4+ T helper cells by langerin+ or langerin-negative DC, respectively. In contrast, protein vaccines administered with various adjuvants induced IgG1 independently of langerin+ DC. Taken together, these findings reflect a highly specialized division of labor between different DC subsets both with respect to Ag encounter as well as downstream processes of immune activation.

International audience; Antigen receptor gene loci are among the most complex in mammals. The IgH locus, encoding the immunoglobulin heavy chain (IgH) in B-lineage cells, undergoes major transcription-dependent DNA remodeling events, namely V(D)J recombination, Ig class-switch recombination (CSR), and somatic hypermutation (SHM). Various cis-regulatory elements (encompassing promoters, enhancers, and chromatin insulators) recruit multiple nuclear factors in order to ensure IgH locus regulation by tightly orchestrated physical and/or functional interactions. Among major IgH cis-acting regions, the large 3' regulatory region (3'RR) located at the 3' boundary of the locus includes several enhancers and harbors an intriguing quasi-palindromic structure. In this review, we report progress insights made over the past decade in order to describe in more details the structure and functions of IgH 3'RRs in mouse and human. Generation of multiple cellular, transgenic and knock-out models helped out to decipher the function of the IgH 3' regulatory elements in the context of normal and pathologic B cells. Beside its interest in physiology, the challenge of elucidating the locus-wide cross talk between distant cis-regulatory elements might provide useful insights into the mechanisms that mediate oncogene deregulation after chromosomal translocations onto the IgH locus.

In the mouse, the regulatory region located at the 3′ end of the IgH locus includes four transcriptional enhancers: HS3a, HS1-2, HS3b, and HS4; the first three lie in a quasi-palindromic structure. Although the upstream elements HS3a and HS1-2 proved dispensable for Ig expression and class switch recombination (CSR), the joint deletion of HS3b and HS4 led to a consistent decrease in IgH expression in resting B cells and to a major CSR defect. Within this pair of distal enhancers, it was questionable whether HS3b and HS4 could be considered individually as elements critical for IgH expression and/or CSR. Studies in HS4-deficient mice recently revealed the role of HS4 as restricted to Igμ-chain expression from the pre-B to the mature B cell stage and left HS3b as the last candidate for CSR regulation. Our present study finally invalidates the hypothesis that CSR could mostly rely on HS3b itself. B cells from HS3b-deficient animals undergo normal proliferation, germline transcription, and CSR upon in vitro stimulation with LPS; in vivo Ag-specific responses are not affected. In conclusion, our study highlights a major effect of the global ambiance of the IgH locus; enhancers demonstrated as being strongly synergistic in transgenes turn out to be redundant in their endogenous context.

International audience; The diversity of the human immune repertoire and how it relates to a functional immune response has not yet been studied in detail in humanized NOD.SCID.gammac(-/-) immunodeficient mice. Here, we used a multiplex PCR on genomic DNA to quantify the combinatorial diversity of all possible V-J rearrangements at the TCR-beta chain and heavy chain Ig locus. We first show that the combinatorial diversity of the TCR-beta chain generated in the thymus was well preserved in the periphery, suggesting that human T cells were not vastly activated in mice, in agreement with phenotypic studies. We then show that the combinatorial diversity in NOD.SCID.gammac(-/-) mice reached 100% of human reference samples for both the TCR and the heavy chain of Ig. To document the functionality of this repertoire, we show that a detectable but weak HLA-restricted cellular immune response could be elicited in reconstituted mice after immunization with an adenoviral vector expressing HCV envelope glycoproteins. Altogether, our results suggest that humanized mice express a diversified repertoire and are able to mount antigen-specific immune responses.

International audience; We have recently described a new form of light chain deposition disease (LCDD) presenting as a severe cystic lung disorder requiring lung transplantation. There was no bone marrow plasma cell proliferation. Because of the absence of disease recurrence after bilateral lung transplantation and of serum-free light chain ratio normalization after the procedure, we hypothesized that monoclonal light chain synthesis occurred within the lung. The aim of this study was to look for the monoclonal B-cell component in 3 patients with cystic lung LCDD. Histologic examination of the explanted lungs showed diffuse nonamyloid kappa light chain deposits associated with a mild lymphoid infiltrate composed of aggregates of small CD20(+), CD5(-), CD10(-) B lymphocytes reminiscent of bronchus-associated lymphoid tissue. Using polymerase chain reaction (PCR), we identified a dominant B-cell clone in the lung in the 3 studied patients. The clonal expansion of each patient shared an unmutated antigen receptor variable region sequence characterized by the use of IGHV4-34 and IGKV1 subgroups with heavy and light chain CDR3 sequences of more than 80% amino acid identity, a feature evocative of an antigen-driven process. Combined with clinical and biologic data, our results strongly argue for a new antigen-driven primary pulmonary lymphoproliferative disorder.

Objective: The aim of this study is the identification of HLA-A2 restricted T-cell epitopes in the conserved region of the immunoglobulin-G-heavy-chain (IgGH) that can be used for immunotherapy in multiple myeloma (MM) patients. Methods: After the IgGH gene sequence was scanned for HLA-A2 restricted T-cell epitopes with a high binding affinity to the MHC-I-complex, promising nona-peptides were synthesized. Peptide specific CD8 + T-cells were generated from peripheral blood mononuclear cells (PBMC) of healthy donors (HD) and patients with MM using peptide pulsed dendritic cells (DC) in vitro. The activation and cytotoxicity of CD8 + T-cells was analyzed by IFN-α ELISpot-assay and 51Chromium release-assay. HLA-A2 restriction was proven by blocking T-cell activation with anti-HLA-A2 antibodies. Results: Two HLA-A2 restricted T-cell epitopes–TLVTVSSAS derived from the IgGH-framework-region 4 (FR4) and LMISRTPEV from the constant region (CR)-induced expansion of specific CD8 + T-cells from PBMC in two of three (TLVTVSSAS) and one of three (LMISRTPEV) HD respectively. Specific T-cells were induced from PBMC in two of six (TLVTVSSAS) and eight of 19 (LMISRTPEV) patients with MM. Specific CD8 + T-cells also lysed peptide-pulsed target cells in 51Chromium release-assay. LMISRTPEV specific CD8 + T-cells from MM patients lysed specifically the HLA-A2 + IgG myeloma cell line XG-6. Conclusion: We identified two HLA-A2 restricted T-cell epitopes–TLVTVSSAS and LMISRTPEV–which can yield an expansion of CD8 + T-cells with the ability to kill peptide-loaded target cells and HLA-A2 + IgG+ myeloma cells. We conclude that TLVTVSSAS and LMISRTPEV could be T-cell epitopes for immunotherapy in MM patients.

International audience; We have recently described a new form of light chain deposition disease (LCDD) presenting as a severe cystic lung disorder requiring lung transplantation. There was no bone marrow plasma cell proliferation. Because of the absence of disease recurrence after bilateral lung transplantation and of serum-free light chain ratio normalization after the procedure, we hypothesized that monoclonal light chain synthesis occurred within the lung. The aim of this study was to look for the monoclonal B-cell component in 3 patients with cystic lung LCDD. Histologic examination of the explanted lungs showed diffuse nonamyloid kappa light chain deposits associated with a mild lymphoid infiltrate composed of aggregates of small CD20(+), CD5(-), CD10(-) B lymphocytes reminiscent of bronchus-associated lymphoid tissue. Using polymerase chain reaction (PCR), we identified a dominant B-cell clone in the lung in the 3 studied patients. The clonal expansion of each patient shared an unmutated antigen receptor variable region sequence characterized by the use of IGHV4-34 and IGKV1 subgroups with heavy and light chain CDR3 sequences of more than 80% amino acid identity, a feature evocative of an antigen-driven process. Combined with clinical and biologic data, our results strongly argue for a new antigen-driven primary pulmonary lymphoproliferative disorder.

International audience; The three-dimensional structure of the Fab fragment of an anti-2-phenyloxazolone monoclonal antibody (NQ10/12.5) in its native and complexed forms has been determined at 2.8 and 3.0 A resolution, respectively. Identification of hapten-contacting residues has allowed us to evaluate the contribution of individual somatic point mutations to maturation of the immune response. In particular, amino acid residues 34 and 36 of the light chain, which are frequently mutated in antibodies with increased affinity for 2-phenyloxazolone, are shown to interact directly with the hapten. We propose that the strict maintenance of certain amino acid sequences at the potentially highly variable VL-JL and VH-D-JH junctions observed among anti-2-phenyloxazolone antibodies is due largely to structural constraints related to antigen recognition. Finally, the three-dimensional model of NQ10/12.5, which uses the typical light chain of primary response anti-2-phenyloxazolone antibodies but a different heavy chain, allows an understanding of how, by preserving key contact residues, a given heavy chain may be replaced by another, apparently unrelated one, without loss of hapten binding activity and why the V kappa Ox1 germline gene is so frequently selected amongst the other known members of this family.

International audience; The immunoscope methodology has proven useful to analyze T-cell repertoires in mice and humans. We adapted it to the analysis of VH chains of human peripheral B cells by setting up a quantification of various VH and JH segments and the profiling of IgM-, IgG-, IgA- and IgE-expressing B cells. We then tested the hypothesis that the human B-cell and T-cell repertoires have a similar diversity of VH and V-beta rearrangements. We studied in more detail the VH5 family because it is not abundantly used, which facilitated the analysis. The data showed that the number of distinct VH5 rearrangements in all samples studied is close to the number of cells in the sample. This contrasts with T cells in which we previously showed that distinct V-beta rearrangements amount to a few percent of the number of T cells because each V-beta chain is on the average paired with approximately 25 alpha chains. Thus, in the VH5 family, the light chains add little quantitative diversity to that produced by the heavy chain alone. Whether this feature can be generalized to other VH chains is discussed.

Burkitt lymphoma (BL) features translocations linking c-myc to an Ig locus. Breakpoints in the H chain locus (IgH) stand either close to JH or within switch regions and always link c-myc to the 3′ IgH locus control region (3′ LCR). To test the hypothesis that the 3′ LCR alone was sufficient to deregulate c-myc, we generated mice carrying a 3′ LCR-driven c-myc transgene and specifically up-regulating c-myc in B cells. Splenic B cells from mice proliferated exaggeratedly in response to various signals had an elevated apoptosis rate but normal B220/IgM/IgD expression. Although all Ig levels were lowered in vivo, class switching and Ig secretion proved normal in vitro. Beginning at the age of 12 wk, transgenic mice developed clonal lymphoblastic lymphomas or diffuse anaplastic plasmacytomas with an overall incidence of 80% by 40 wk. Lymphoblastic lymphomas were B220+IgM+IgD+ with the BL “starry sky” appearance. Gene expression profiles revealed broad alterations in the proliferation program and the Ras-p21 pathway. Our study demonstrates that 3′ IgH enhancers alone can deregulate c-myc and initiate the development of BL-like lymphomas. The rapid and constant occurrence of lymphoma in this model makes it valuable for the understanding and the potential therapeutic manipulation of c-myc oncogenicity in vivo.

The predominant path of immunoglobulin class switch recombination follows the paradigm of intra-chromosomal deletion enabling expression of another heavy chain instead of micro and delta. This was, however, challenged by observations of inter-allelic class switch recombination in rabbit or mouse IgG3- or IgA-producing B cells. Assuming that the conditions of inter-chromosomal exchange are likely present at any target S regions in stimulated B cells, we explored trans-association of VH and C genes in a model allowing all C genes to be checked simultaneously. Heterozygous mutant mice are thus studied, which carry one non-functional IgH allele inactivated by a non-translatable mutation of VDJ-CH transcripts, while the functional allele is deficient for class switching due to a truncated 3'regulatory region. A fair level of switching to all Ig classes is restored in heterozygous mice despite the fact that cis-recombination is either non productive on one allele or deficient on the other. Molecular evidence at the DNA level of trans-CSR to IgG3 was demonstrated by cloning and sequencing Smu-Sgamma3 hybrid junctions. These data demonstrate that inter-allelic recombination may broadly rescue the production of various class-switched isotypes and allow complementation between mutations located at both ends of the IgH constant gene cluster.

In human, three transcriptional enhancers called hs1,2, hs3 and hs4 were identified downstream the 3′ Ig heavy (IgH) locus. We previously reported by PCR and Southern blotting the existence of various allelic forms for the hs1,2 enhancer, one allele being associated with a higher efficiency of switching to IgA in IgA nephropathy (IgAN) patients. Since it is strongly suggested in the mouse that the whole 3′ regulatory region is broadly involved in the regulation of class switch recombination (CSR), we wondered if the reported hs1,2 polymorphism was the sole difference possibly accounting for the varying ability to produce non-IgM antibodies in the human population. In this study, we report the absence of additional polymorphism of the hs3 and hs4 enhancers either by using a PCR method or by Southern blotting. DNA sequence analysis confirmed the existence of an invariant core sequence for human hs3 and hs4 enhancers, featuring multiple nuclear factor potential binding sites. In conclusion, human hs3 and hs4 enhancers are not polymorphic, a result that markedly contrasts with the hs1,2 enhancer for which the generation of multiple alleles in both rodents and humans has likely been favored by its central position within a large palindromic region.

Immunoglobulin heavy-chain class-switch recombination (CSR) occurs between highly repetitive switch sequences located upstream of the constant region genes. However, the role of these sequences remains unclear. Mutant mice were generated in which most of the Iμ-Cμ intron was deleted, including all the repeats. Late B-cell development was characterized by a severe impairment, but not a complete block, in class switching to all isotypes despite normal germ line transcription. Sequence analysis of the Iμ-Cμ intron in in vitro activated–mutant splenocytes did not reveal any significant increase in activation-induced cytidine deaminase (AID)–induced somatic mutations. Analysis of switch junctions showed that, in the absence of any Sμ repeat, the Iμ exon was readily used as a substrate for CSR. In contrast to the sequence alterations downstream of the switch junctions, very few, if any, mutations were found upstream of the junction sites. Our data suggest that the core Eμ enhancer could be the boundary for CSR-associated somatic mutations. We propose that the core Eμ enhancer plays a central role in the temporal dissociation of somatic hypermutation from class switching.

To ensure the B cell differentiation stage specificity of the intronic Emu element and of the locus control region (LCR) that lies downstream of the IgH chain locus, we generated transgenic mice harboring a V(H) promoter-GFP reporter gene linked to the 3'LCR region and the Emu element. By flow cytometry, GFP(+) lymphocytes were observed amongst pro-B cells (B220(+)CD43(+)CD117(+)) and at all stages of differentiation up to mature B cells (B220(+)IgM(+)IgD(+)). Expression was strictly confined to cells committed to the B lymphocyte lineage as judged by the lack of GFP(+)Thy1,2(+) cells (T lymphocytes) and GFP(+)B220(-)CD117(+)CD43(+) cells (uncommitted lymphohematopoietic progenitors). Therefore, the Emu-GFP-3'LCR transgene is not expressed by hematopoietic stem cells, begins its expression in pro-B cells and is specifically active at all stages of B cell maturation. The combination of 3' and 5' IgH regulatory elements thus appears as a potentially useful cassette in transgenes that require a stringent and early B lineage-specific expression.

Locus control regions (LCR) were first defined by their theoretical ability to enhance the expression of linked genes in a tissue-specific, position-independent and copy-number dependent manner. In fact, few of the so-called LCR identified completely fulfil this definition. For example, the regulatory elements located in 5' (Emu) and 3' (HS3a; HS1,2; HS3b; HS4) of the IgH locus display some properties of a LCR but lack a copy-number dependence and sometimes display position effects in transgenes. In order to study whether addition of insulators would allow to overcome such problems in transgenes, we studied constructs harboring a V(H) promoter-green fluorescent protein reporter gene linked to the 3' and/or 5' IgH elements, surrounded or not with the chicken beta-globin 5'HS4 insulator. When flanked with insulators it appeared that either 3' IgH and 5' IgH regulatory elements now behave as true LCR elements and noticeably display copy-number dependence in transfected pre-B or B cell lines.

The more distal enhancers of the immunoglobulin heavy-chain 3′ regulatory region, hs3b and hs4, were recently demonstrated as master control elements of germline transcription and class switch recombination to most immunoglobulin constant genes. In addition, they were shown to enhance the accumulation of somatic mutations on linked transgenes. Since somatic hypermutation and class switch recombination are tightly linked processes, their common dependency on the endogenous locus 3′ enhancers could be an attractive hypothesis. VDJ structure and somatic hypermutation were analyzed in B cells from mice carrying either a heterozygous or a homozygous deletion of these enhancers. We find that hs3b and hs4 are dispensable both for VDJ assembly and for the occurrence of mutations at a physiologic frequency in the endogenous locus. In addition, we show that cells functionally expressing the immunoglobulin M (IgM) class B-cell receptor encoded by an hs3b/hs4-deficient locus were fully able to enter germinal centers, undergo affinity maturation, and yield specific antibody responses in homozygous mutant mice, where IgG1 antibodies compensated for the defect in other IgG isotypes. By contrast, analysis of Peyer patches from heterozygous animals showed that peanut agglutinin (PNAhigh) B cells functionally expressing the hs3b/hs4-deficient allele were dramatically outclassed by B cells expressing the wild-type locus and normally switching to IgA. This study thus also highlights the role of germinal centers in the competition between B cells for affinity maturation and suggests that membrane IgA may promote recruitment in an activated B-cell compartment, or proliferation of activated B cells, more efficiently than IgM in Peyer patches.

A locus control region (LCR) containing four transcriptional enhancers lies downstream of the IgH chain locus. We studied transgenes carrying a 3(')IgH LCR-driven GFP reporter gene for expression and B cell differentiation stage specificity. We also compared transgenes that were or were not flanked by two copies of the beta-globin HS4 insulator, an element defined by its ability to protect transgenes from the influences of surrounding genes at the insertion site. Results indicate that insulators are instrumental in sustaining GFP expression in GFP-3(')LCR transgenic mice when they were included. Flow cytometry experiments reported a strictly B cell specific GFP expression from pre-B cells in bone marrow to mature B cells in spleen. Despite addition of 5(')HS4 insulators to the GFP-3(')LCR construct, complete transgene silencing occurred in some transgenic lines and was systematically observed in ageing animals from all lines.

In B cells undergoing class switching, recombination is usually directed to the same switch sequences on both homologous chromosomes and is generally preceded by germ-line transcription initiated from I promoters upstream of immunoglobulin constant heavy chain genes. Whether germ-line transcription occurs on both alleles or is restricted to the productive one has not been directly addressed before. To clearly distinguish between these two possibilities, a system is needed in which one might detect allele-specific germ-line transcripts. We used homologous recombination to set up two in vivo systems in which one gamma3 allele was marked by a loxP sequence whereas the other allele was silenced by inhibiting mu heavy chain production. Here, we provide evidence that germ-line transcription occurs on both the functional and the non-functional alleles.

Previous targeting experiments within the IgH locus have shown that V(D)J recombination was affected by an insertion of a neo gene within Eμ upstream of the core enhancer, but not by insertions downstream of the enhancer. Similarly, class switch recombination to a given (C) gene was affected only by interposition of neo in between that gene and the 3′ IgH enhancers. Here we show that insertion of neo upstream Eμ only marginally impairs V(D)J recombination, but results in an altered D and JH gene usage and completely blocks transcription of the germline JH region and the rearranged VDJ segments. Although transcriptional silencing of JH occurs upstream of the insertion and results in the lack of mature B cells in homozygous mutant animals, IgH transcription is maintained downstream of the insertion together with neo transcription and can be up-regulated by LPS stimulation or upon fusion with plasmacytoma cells. Altogether these data argue for a polarized “neo effect” involving promoter competition and further show that V(D)J rearrangement can be uncoupled from transcription.

SUMMARYGlomerulonephritis with organized microtubular monoclonal immunoglobulin deposits (GOMMID) and glomerulonephritis related to type I cryoglobulin are well-known but rare complications of B cell derived chronic lymphocytic leukaemia. In these disorders, monoclonal Ig have never been studied at the molecular level. We conducted a pathological and molecular analysis in a patient with chronic lymphocytic leukaemia, glomerulonephritis and a single circulating monoclonal Ig. Unusual IgG1κ kidney deposits were observed. The heavy and light chain variable region sequences of that cryoprecipitating monoclonal Ig were characterized. Light microscopy revealed glomerulonephritis typical of cryoglobulinaemia, with neutrophil and macrophage infiltration, endocapillary hyperplasia and few protein thrombi. Electron microscopic study clearly evidenced numerous subepithelial mixed granular and organized deposits with a unique microtubular organization, reminiscent of the GOMMID. The Ig molecule sequence revealed alterations of charge and hydrophobicity potentially promoting a crystal-like aggregation and the aggregation of microtubules. This description suggests that common mechanisms are involved in various forms of precipitation and/or deposition of complete Ig molecules, with a variable extent of organization and with a possible overlap between pathological patterns of either glomerulonephritis with microtubular deposits or type I cryoglobulinic glomerulonephritis.

Surface-exposed charges can affect protein structure, stability and solubility as well as the kinetics of both the folding process and interaction with binding partners. We have investigated the influence on kinetic interaction parameters of 14 conserved, surface-exposed charges located away from the paratope in the variable domains of two antibodies of different specificity. We found that conserved, surface-exposed, charged framework residues are asymmetrically distributed on opposite faces of both VH and VL domains. Some of the charges play a critical role in protein folding and stability. While electrostatic forces within or close to the binding interface can be used to optimize the association rate, we confirmed the predicted minor effects of charge modifications remote from the binding site. They had no effect on the dissociation rate parameter. Our study demonstrates the role of residues remote from the interaction site in the recognition function as well as the limited effect of surface charge modifications in antibody fragments on kinetic interaction parameters.

International audience; Four different monoclonal Ig (MIg) (IgA1kappa, IgG1kappa, IgG2kappa and IgG4kappa) displaying anti-tubulin activity were detected in the serum from a lymphoma patient. The complete sequence of three of these MIg showed identical V(H) and V(L) domains and the presence of mutations compatible with an antigen-driven process. Surprisingly, despite complete homology in their variable domains, IgA1kappa, IgG1kappa, or their Fab fragments bound to a common motif recognized in beta tubulin, with significant differences in affinity (IgA1kappa 1.52x10(-8) M, and IgG1kappa 2.09x10(-7) M). To substantiate these results, the V(H) and V(L) domains from IgA1kappa were cloned and introduced into expression vectors containing the constant kappa exon and either the mu or the gamma1 constant exon, and complete recombinant IgMkappa and IgG1kappa were obtained. Like the IgA1kappa, the IgMkappa construction bound to the tubulin epitope with consistent affinity (7.7x10(-9) M), whereas the IgG1kappa construction displayed a significantly lower affinity (3.28x10(-7) M). These results provide definitive evidence that isotype can influence binding affinity to antigen and suggest that malignant transformation occurred at the germinal center once the mutational process was achieved and the switch process was still active.

The influence of framework residues belonging to VH and VL modules of antibody molecules on antigen binding remains poorly understood. To investigate the functional role of such residues, we have performed semi-conservative amino acid replacements at the VH–VL interface. This work was carried out with (i) variants of the same antibody and (ii) with antibodies of different specificities (Fab fragments 145P and 1F1h), in order to check if functional effects are additive and/or similar for the two antibodies. Interaction kinetics of Fab mutants with peptide and protein antigens were measured using a BIACORE® instrument. The substitutions introduced at the VH–VL interface had no significant effects on ka but showed small, significant effects on kd. Mutations in the VH module affected kd not only for the two different antibodies but also for variants of the same antibody. These effects varied both in direction and in magnitude. In the VL module, the double mutation FL37L–QL38L, alone or in combination with other mutations, consistently decreased kd about two-fold in Fab 145P. Other mutations in the VL module had no effect on kd in 145P, but always decreased kd in 1F1h. Moreover, in both systems, small-magnitude non-additive effects on kd were observed, but affinity variations seemed to be limited by a threshold. When comparing functional effects in antibodies of different specificity, no general rules could be established. In addition, no clear relationship could be pointed out between the nature of the amino acid change and the observed functional effect. Our results show that binding kinetics are affected by alteration of framework residues remote from the binding site, although these effects are unpredictable for most of the studied changes. Copyright © 2000 John Wiley & Sons, Ltd.

International audience; In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Cellular competition for survival signals offers a cogent and appealing mechanism for the maintenance of cellular homeostasis [Raff, M. C. (1992)Nature (London)356, 397–400]. We present a theoretical and experimental investigation of the role of competition for resources in the regulation of peripheral B cell numbers. We use formal ecological competition theory, mathematical models of interspecific competition, and competitive repopulation experiments to show that B cells must compete to persist in the periphery and that antigen forms a part of the resources over which B cells compete.

SMI is a previously characterized IgM kappa polyreactive (natural) autoantibody. The variable regions of the heavy and light chains of SMI are respectively encoded by a nonmutated VH1 gene, designated 51p1, and a conserved nonmutated V kappa gene, designated Humkv325. These V genes seem to be over-represented in the autoimmune and fetal B cell repertoires, and to be frequently expressed in malignant B cells during certain lymphoid proliferations such as chronic lymphocytic leukemia. Polyreactive natural autoantibodies are thought to rely mainly on the use of such V genes in germ-line configuration. However, this model underestimates the contribution of the somatically generated heavy chain third complementarity-determining region (HCDR3) to autoantibody specificity. We used oligonucleotide site-directed mutagenesis to permute the sequence of the SMI-HCDR3 to generate a family of mutant proteins, each of which differed from the original SMI-IgM kappa by one amino acid residue. This allowed us to examine the relative contribution of selected amino acid residues in this region to the binding affinity of SMI against a panel of self-Ags. We found that a single amino acid substitution within the HCDR3 could dramatically alter the specificity of this autoantibody. Some substitutions abrogated the reactivity with all the tested Ags, whereas others changed the affinity or spectrum of reactivity for certain self-Ags. These results demonstrate that the autoantibody-binding activity of these conserved autoantibody-associated germ-line V genes is dependent upon heavy chain junctional sequences that are generated somatically during Ig gene rearrangement.

Chromosome translocations between oncogenes and the region spanning the immunoglobulin (Ig) heavy chain (IgH) variable (V), diversity (D), and joining (J) gene segments (Ig V-JH region) are found in several mature B cell lymphomas in humans and mice. The breakpoints are frequently adjacent to the recombination signal sequences targeted by recombination activating genes 1 and 2 during antigen receptor assembly in pre–B cells, suggesting that these translocations might be the result of aberrant V(D)J recombination. However, in mature B cells undergoing activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM), duplications or deletions that would necessitate a double-strand break make up 6% of all the Ig V-JH region–associated somatic mutations. Furthermore, DNA breaks can be detected at this locus in B cells undergoing SHM. To determine whether SHM might induce c-myc to Ig V-JH translocations, we searched for such events in both interleukin (IL) 6 transgenic (IL-6 tg) and AID−/− IL-6 tg mice. Here, we report that AID is required for c-myc to Ig V-JH translocations induced by IL-6.

Natural polyreactive autoantibodies (NAA) are an important component of the normal B cell repertoire. One intriguing characteristic of these Abs is their binding to various dissimilar Ags. It has been generally assumed that these Abs bind the Ags with low affinity, and are encoded by germline genes. We have used surface plasmon resonance to determine binding of avidities, and conducted a structural analysis of five murine monoclonal natural autoantibodies displaying a typical polyreactive binding pattern against cytoskeleton Ags and DNA. We show that 1) all the five Abs bind the different Ags with kinetic constants similar to those observed for immune Abs; 2) they express a restricted set of V(H) and V(L) genes, since the same V(H) gene is expressed by three out of the five, and one particular Vkappa gene was expressed twice. In addition, a single D gene segment was used by three of the five Abs; and 3) they express, in most cases, genes in a close germline configuration. Our amino acid sequence and modeling studies show that the distribution of exposed side chains in the NAA paratopes is close to the general pattern observed in the complementarity-determining regions (CDRs) of variable domains from immune Abs. Although CDR3 regions of the heavy chain have been postulated to play a major role in determining polyreactivity on the basis of recombinatorial experiments, our results failed to show any distinctive particularity of this region in terms of length or charge when compared with classical immune Abs.

Allelic exclusion of IgH chain expression is stringently established before or during early B cell maturation. It likely relies both on cellular mechanisms, selecting those cells in which a single receptor allows the best possible Ag response, and on molecular restrictions of gene accessibility to rearrangement. The extent to which transcriptional control may be involved is unclear. Transcripts arising from nonfunctional alleles would undergo nonsense-mediated degradation and their virtual absence in mature cells cannot ensure that transcription per se is down-regulated. By contrast, somatic hypermutation may provide an estimate of primary transcription in Ag-activated cells since both processes are directly correlated. For coding regions, the rate and nature of mutations also depend upon Ag binding constraints. By sequencing intronic sequence downstream mouse VDJ genes, we could show in the absence of such constraints that somatic hypermutation intrinsically targets nonfunctional rearranged alleles at a frequency approaching that of functional alleles, suggesting that transcription also proceeds on both alleles at a similar rate. By contrast and confirming the strong dependency of somatic hypermutation upon transcription, we show that artificial blockade of transcription on the nonfunctional allele by a knock-in neomycin resistance cassette keeps the VDJ region unmutated even when its promoter is intact and when it is fully rearranged.