Glycotranscriptome study reveals an enzymatic switch modulating glycosaminoglycan synthesis during B-cell development and activation.

B-cell fate and responses are modulated by soluble mediators and direct cellular interactions. Migration properties also vary during differentiation, commitment and activation. In many cells, modulation of responses to stimuli involves cell surface glycans, whose architecture depends on the simultaneous expression of multiple enzymes. By looking at the glycosylation-related gene expression patterns among B-cell populations, we determined in this study that the strongest variations were observed for CSGalNAcT-1 and EXTL1. These are enzymes involved in the biosynthesis of alternative forms of glycosaminoglycans (GAGs), namely chondroitin sulfate and heparan sulfate, respectively. These two enzymes showed inverse fluctuations in progenitors, resting B cells and activated B cells, suggesting a developmentally regulated switch between chondroitin and heparan sulfate synthesis. To explore whether these variations contributed to optimal B-cell differentiation, we overexpressed EXTL1 in the B-cell lineage of transgenic mice, yielding a partial differentiation blockade at the pro-B to pre-B transition. In the periphery, this defect was almost fully compensated for in vivo, with normal-size B-cell compartments and normal serum immunoglobulin levels in the transgenic EXTL1 mice. The peripheral B cells from EXTL1 transgenics were only affected with regard to their in vitro responses to polyclonal activation, showing reduced proliferation. Together the data suggest that despite their low amounts in lymphocytes, the heparan sulfate chains decorating the endogenous GAGs appear to be regulators of B-cell physiology.

Genomic deletion of the whole IgH 3' regulatory region (hs3a, hs1,2, hs3b, and hs4) dramatically affects class switch recombination and Ig secretion to all isotypes.

The immunoglobulin heavy chain locus (IgH) undergoes multiple changes along B-cell differentiation. In progenitor B cells, V(D)J assembly allows expression of µ heavy chains. In mature B cells, class switch recombination may replace the expressed constant (C)µ gene with a downstream C(H) gene. Finally, plasma cell differentiation strongly boosts IgH transcription. How the multiple IgH transcriptional enhancers tune these changes is unclear. Here we demonstrate that deletion of the whole IgH 3' regulatory region (3'RR) allows normal maturation until the stage of IgM/IgD expressing lymphocytes, but nearly abrogates class switch recombination to all C(H) genes. Although plasma cell numbers are unaffected, we reveal the role of the 3'RR into the transcriptional burst normally associated with plasma cell differentiation. Our study shows that transcriptional changes and recombinations occurring after antigen-encounter appear mainly controlled by the 3'RR working as a single functional unit.

Premature replacement of mu with alpha immunoglobulin chains impairs lymphopoiesis and mucosal homing but promotes plasma cell maturation.

Sequentially along B cell differentiation, the different classes of membrane Ig heavy chains associate with the Ig alpha/Ig beta heterodimer within the B cell receptor (BCR). Whether each Ig class conveys specific signals adapted to the corresponding differentiation stage remains debated. We investigated the impact of the forced expression of an IgA-class receptor throughout murine B cell differentiation by knocking in the human C alpha Ig gene in place of the S mu region. Despite expression of a functional BCR, homozygous mutant mice showed a partial developmental blockade at the pro-B/pre-BI and large pre-BII cell stages, with decreased numbers of small pre-BII cells. Beyond this stage, peripheral B cell compartments of reduced size developed and allowed specific antibody responses, whereas mature cells showed constitutive activation and a strong commitment to plasma cell differentiation. Secreted IgA correctly assembled into polymers, associated with the murine J chain, and was transported into secretions. In heterozygous mutants, cells expressing the IgA allele competed poorly with those expressing IgM from the wild-type allele and were almost undetectable among peripheral B lymphocytes, notably in gut-associated lymphoid tissues. Our data indicate that the IgM BCR is more efficient in driving early B cell education and in mucosal site targeting, whereas the IgA BCR appears particularly suited to promoting activation and differentiation of effector plasma cells.

In vivo redundant function of the 3' IgH regulatory element HS3b in the mouse.

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 Igmicro-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.

A myeloma translocation-like model associating CCND1 with the immunoglobulin heavy-chain locus 3' enhancers does not promote by itself B-cell malignancies.

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.

Ig synthesis and class switching do not require the presence of the hs4 enhancer in the 3' IgH regulatory region.

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 neo(R) 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.

Comparative analysis of oncogenic properties and nuclear factor-kappaB activity of latent membrane protein 1 natural variants from Hodgkin's lymphoma's Reed-Sternberg cells and normal B-lymphocytes.

BACKGROUND: In Epstein-Barr virus-associated Hodgkin's lymphomas, neoplastic Reed-Sternberg cells and surrounding non-tumor B-cells contain different variants of the LMP1-BNLF1 oncogene. In this study, we raised the question of functional properties of latent membrane protein 1 (LMP1) natural variants from both Reed-Sternberg and non-tumor B-cells. DESIGN AND METHODS: Twelve LMP1 natural variants from Reed-Sternberg cells, non-tumor B-cells of Hodgkin's lymphomas and from B-cells of benign reactive lymph nodes were cloned, sequenced and stably transfected in murine recombinant interleukin-3-dependent Ba/F3 cells to search for relationships between LMP1 cellular origin and oncogenic properties as well as nuclear factor-kappaB activation, and apoptosis protection. RESULTS: LMP1 variants of Reed-Sternberg cell origin were often associated with increased mutation rate and with recurrent genetic events, such as del15bp associated with S to N replacement at codon 309, and four substitutions I85L, F106Y, I122L, and M129I. Oncogenic potential (growth factor-independence plus clonogenicity) was consistently associated with LMP1 variants from Reed-Sternberg cells, but inconstantly for LMP1-variants from non-tumor B-cells. Analysis of LMP1 variants from both normal B-cells and Reed-Sternberg cells indicates that protection against apoptosis through activation of nuclear factor-kappaB - whatever the cellular origin of LMP1 - was maintained intact, regardless of the mutational pattern. CONCLUSIONS: Taken together, our results demonstrate that preserved nuclear factor-kappaB activity and protection against apoptosis would be the minimal prerequisites for all LMP1 natural variants from both normal and tumor cells in Hodgkin's lymphomas, and that oncogenic potential would constitute an additional feature for LMP1 natural variants in Reed-Sternberg cells.

Uncoupling between Ig somatic hypermutation and oncogene mutation in mouse lymphoma.

Burkitt lymphoma (BL) features translocations linking c-myc to the immunoglobulin heavy chain (IgH) locus. By inserting a c-myc gene under the control of the 3'IgH locus control region (LCR) into the mouse genome, we generated c-myc-3'LCR mice that develop clonal BL or diffuse anaplastic lymphoma. We show in the present study that while BL from c-myc-3'LCR mice would be classified as pre-germinal center (GC) cells due to the absence of both BCL-6 expression and somatic hypermutation (SHM) in V(H) sequences, they show a high level of SHM focused on the c-myc oncogene itself. This observation suggests that the c-myc-3'IgH LCR tandem association drives development of lymphoma from naïve B cells by specifically recruiting AID activity on c-myc in a process that early becomes independent from antigen selection and where the successive rounds of SHM rather rely on the selection of the most efficient mutations for oncogene deregulation. Similar to the translocated c-myc gene in human BL, mutations were found in first exon and 5' flanking sequences of transgenic c-myc and specially focused on negative regulatory elements, thus leading to high and constitutive oncogene expression. In conclusion while 3'IgH transcriptional enhancers in c-myc-3'LCR mice first simply act in cis to slightly stimulate c-myc transcription in untransformed B cells, the occurrence of lymphoma appears to result from an additional mechanism necessitating AID-driven mutations within the first exon and 5' flanking sequences which does not occur in parallel but rather circumvents antigen-driven selection.

The 3' IgH locus control region is sufficient to deregulate a c-myc transgene and promote mature B cell malignancies with a predominant Burkitt-like phenotype.

Burkitt lymphoma (BL) features translocations linking c-myc to an Ig locus. Breakpoints in the H chain locus (IgH) stand either close to J(H) 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.

Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis, and correlation with clinical findings.

To study the influence of Toxoplasma gondii genotypes on the severity of human congenital toxoplasmosis (asymptomatic, benign, or severe infection or newborn or fetal death), 8 microsatellite markers were used to analyze 86 T. gondii isolates collected from patients with congenital toxoplasmosis. Seventy-four different genotypes were detected, some identical genotypes originating probably from the same source of contamination. The 3 less polymorphic microsatellite markers associated with 6 isoenzymatic markers allowed a classification of isolates into the 3 classical types and detected atypical genotypes. Whatever the clinical findings, type II isolates were largely predominant (84.88% in the whole collection and 96.49% in 57 consecutive cases). Type I and atypical isolates were not found in asymptomatic or benign congenital toxoplasmosis. However, in 4 cases in which children were not infected despite isolation of T. gondii from placenta, only type I isolates were found.