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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.

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; 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.

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.

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.

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; 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.

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.

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.

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; 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.

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.