Béatrice Payelle-Brogard

Predictive value of serum thymidine kinase level for Ig-V mutational status in B-CLL.

In B-CLL IgVH genes mutational status is a major prognostic factor. Since sequencing of IgVH genes is not available in most laboratories, an easily performed surrogate assay is desirable. To identify the best surrogate assay, and to better discriminate prognostic subgroups we analyzed clinical and biological data from 58 typical CLL cases. A higher serum thymidine kinase level (>15 U/l) proved to be a strong predictor of mutational status, and the only independent one among the studied parameters. To further identify prognostic subgroups, cluster analysis was employed on 38 cases on which all data were available, which segregated two groups including 25 and 13 patients, respectively. These two clusters differed by their proliferative potential and appeared to discriminate patients with very different clinical course and outcome. s-TK was strikingly different among these two clusters, suggesting that s-TK level could be used routinely to identify patients at risk of progression.

VH gene usage by family members affected with chronic lymphocytic leukaemia

The excess risk of chronic lymphocytic leukaemia (CLL) in the first‐degree relatives of affected patients suggests that familial CLL might constitute a useful model to study the pathogenesis of this disease, as has been demonstrated in numerous other neoplastic disorders. Previous studies have shown non‐random utilization of immunoglobulin genes in CLL, some germline in sequence and others containing numerous somatic mutations. To investigate whether familial cases of CLL exhibit similarities in the composition of the B‐cell receptor repertoire to the pattern expressed by CLL patients as a whole, we have studied 25 CLL patients belonging to 12 different families (four French and eight Italian), each of which contained at least two affected members.

Defective assembly of the B-cell receptor chains accounts for its low expression in B-chronic lymphocytic leukaemia

Summary. B‐cell chronic lymphocytic leukaemia (B‐CLL) characteristically displays low amounts of B‐cell receptor (BCR), which mainly consists of the heterodimer CD79a/CD79b bound non‐covalently with the surface immunoglobulin (SIg). This heterodimer is required for SIg expression and BCR signalling. To better define the mechanisms related to low BCR expression, we have investigated transcription, protein synthesis, assembly and transport of the BCR in B‐CLL cells. Our results demonstrated that: (1) there was no major defect in transcriptional expression of the B29 (CD79b) gene; (2) the BCR components were intracellularly detected, thus adequately synthesized, in almost all patients; (3) neither a genetic defect in the transmembrane region of SIg, which associated with CD79a/CD79b, nor a genetic abnormality in the chaperone protein calnexin that is involved in folding and assembly of the BCR were found; (4) a constant defect in the assembly of IgM and CD79b chains occurred leading to abnormal accumulation of both chains in different intracellular compartments; (5) in a majority of CLL patients all of the nascent IgM failed to be processed into mature chains and remained unsuitable for transport. These findings demonstrated that a post‐transcriptional defect located at the BCR intracellular assembly and/or trafficking levels could be involved in its low surface expression in B‐CLL.

Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells

Recent work suggests that chronic lymphocytic leukemia (B-CLL) expressing unmutated immunoglobulin V genes could correspond to the proliferation of naive B cells whereas those expressing mutated genes, may correspond to the proliferation of post-germinal center B cells. Current data from gene profiling expression have failed to demonstrate a clear-cut distinction between these two forms of B-CLL disease. In the present study, we have investigated the complete VH nucleotide sequence and the presence of RNA transcripts from different CH domains in 25 B-CLL patients. Our results demonstrate that: (1) expression of IgD is not related to the mutational frequency and activation of the isotype switch pathway; (2) isotype switch, leading to simultaneous expression at the transcriptional and protein level of IgM, IgD, IgG and IgA, occurs in a small percentage of patients, and (3) different mechanisms such as VDJ duplication and trans-splicing or RNA splicing of long nuclear transcript, could be involved in isotype switch. Our results highlight the difficulty in assigning a normal counterpart to B-CLL cells and raise the possibility that a different B cell development pathway, independent from classical germinal centers, might exist in B-CLL.

USP18 establishes the transcriptional and anti-proliferative interferon α/β differential

Type I IFNs (interferons) are pathogen-induced immunoregulatory cytokines that exert anti-viral and anti-proliferative activities through binding to a common cell-surface receptor. Among the 17 human IFN subtypes, IFNβ binds the IFNAR (IFNα receptor) 1/IFNAR2 receptor chains with particularly high affinity and is especially potent in select bioactivities (e.g. anti-proliferative and pro-apoptotic) when compared with IFNα2. However, no molecular basis has been ascribed to this differential action, since the two ligands are equipotent in immediate early signalling events. In the present study we report that IFNβ induces Stat (signal transducer and activator of transcription) phosphorylation and transcriptional activation of ISGs (interferon-stimulated genes), including two genes with pro-apoptotic functions, for a considerably longer time frame than does IFNα2. We show that the diversification of α2/β responses progressively builds up at the receptor level as a result of accumulating USP18 (ubiquitin specific protease 18), itself an ISG, which exerts its negative feedback action by taking advantage of the weakness of IFNα2 binding to the receptor. This represents a novel type of signalling regulation that diversifies the biological potential of IFNs α and β.

Comparison of natural antibodies to autoantibodies arising during lupus in (NZB × NZW)F1 mice

Autoantibodies arising in (NZB x NZW)F1 (B/W) mice during the lupus-like syndrome were studied and compared to natural antibodies present in normal mice. The antibody activities were tested in sera, circulating immune complexes (CIC) and kidney eluates, using an enzyme immunoassay against a panel of self and non-self antigens: actin, myosin, tubulin, DNA, myoglobin, spectrin and trinitrophenylated bovine serum albumin (TNP/BSA). In the B/M mouse sera, IgM antibodies reacting with all the panel of antigens (PAg) and comparable to those of normal mice, increased moderately from 5 to 9 months and markedly during the last stage preceding death (10 months), when particularly high levels of anti-DNA, anti-tubulin and anti-myoglobin antibodies were noted. Polyreactive IgM antibodies present in CIC were moderately increased while those present in complexes deposited in kidneys were strongly enhanced after the 8th month. IgG antibodies showed an early increase (2 months) in B/W sera for anti-TNP activity, which remained more or less constant until death, while a later (5-6 months) and greater increase of activity, mainly directed against DNA but also against the other antigens of the panel, was observed. In CIC, IgG, mainly anti-DNA but also anti-TNP, were enhanced at the end of the disease while at the same time IgG reacting with all the PAg were found in kidney deposits. Isolation of antibodies from sera on a DNA-immunoadsorbent demonstrated that eluted IgM reacted with all the PAg but mainly with DNA, while IgG reactivity was more restricted to DNA and to a lesser degree to TNP. The D23 idiotype, characteristics of natural polyspecific antibodies, was expressed on IgM and IgG autoantibodies from B/W mice and was enhanced, particularly in kidneys, at the end of the disease. These results demonstrate that natural antibodies are a part of the population of increased autoantibodies in this disease and could participate with IgG anti-DNA antibodies in lupus.

Idiotype-pulsed dendritic cells are able to induce antitumoral cytotoxic CD8 cells in chronic lymphocytic leukaemia

Summary. Idiotypic structures of immunoglobulins from malignant B cells constitute tumour‐specific antigens, though the function of immunoglobulin‐specific CD8+ T cells in disease control and rejection remains unclear. We have studied five cases of B chronic lymphocytic leukaemia patients affected with indolent (three patients) or aggressive (two patients) disease. We showed that CD8+ T cells with major histocompatibility complex class I‐restricted cytotoxicity against autologous tumour B cells could be generated following repeated stimulations with idiotype‐pulsed dendritic cells in vitro. CD8+ T‐cell lines were able to upregulate CD69 expression and to release interferon (IFN)‐γ upon contact with the autologous B cells, though cytolytic activity was only substantiated for patients with indolent disease. The failure of cytolytic activity in patients with aggressive disease may be explained by a skewed maturation of memory CD8 cells.

Biochemical Monitoring of the Early Endocytic Traffic of the Type I Interferon Receptor

The type I interferon (IFN) receptor consists of two transmembrane chains IFNAR1 and IFNAR2, associated with the tyrosine kinases Tyk2 and Jak1, respectively. Binding of IFN to this receptor complex induces activation of Jak/Stat and non-Stat signaling pathways. Ligand binding also drives receptor internalization and sorting toward degradation or recycling. To gain insights into receptor trafficking and its relation to signaling, we performed subcellular organelle fractionation from IFN-stimulated Daudi cells and defined biochemically an early endosomal antigen-1 (EEA1)-positive compartment bearing the activated IFN receptor. Endosomes were thus purified by immunoaffinity isolation on anti-EEA1 antibodies-coated beads. The content of these purified endosomal fractions was analyzed by Western blot and proteomics. Shortly after IFN stimulation, robustly ubiquitinated IFNAR1 and a small amount of IFNAR2 were found in this endosomal compartment, which also contained tyrosine-phosphorylated Tyk2 and Jak1. These data strongly point to the prolonged interaction during traffic of the tyrosine kinases, still in an activated configuration, with the receptors. Among the major constituents of this EEA1-positive compartment, some proteins that have been implicated in IFN signaling were identified. Altogether, these observations suggest that trafficking of the IFN receptor through endosomes may regulate signaling pathways.

High occurence of DRB1 11 in chronic lymphocytic leukaemia families

Summary. Recently, linkage analysis of a series of familial chronic lymphocytic leukaemia (CLL) showed that affected sibling pairs did not share common major histocompatibilty complex haplotypes. We analysed Class I and II antigens in 11 Italian families with familial CLL. Although there was no association of disease status with any particular human leucocyte antigen, there was an overrepresentation of DRB1 11 alleles in these families (P = 0·009). A similar trend was also observed in a second series of nine French families (P = 0·002). Larger studies are needed to determine whether non‐inherited paternal or maternal DRB1 antigens play a role in familial CLL development.

Anti-tubulin antibodies in rabbits before and after immunization with pig tubulin

Sera from rabbits before and after repeated injections of pig tubulin in complete Freunds adjuvant were examined for antibody activity against pig and rabbit tubulins and against a panel of antigens: actin, myosin, DNA, TNP/BSA. Antibody activity against all the antigens of the panel (PAg) increased moderately after the first but not after subsequent injections. Antibody activity against pig and rabbit tubulins strongly increased after the second immunization when the maximum was reached. Isolation of anti-tubulin antibodies from normal or immune sera on tubulin-immunoadsorbent demonstrated the presence of three different antibody populations: (1) polyspecific IgM reacting with the PAg and the tubulins, present in substantial amounts in normal sera and moderately increased in immune sera; (2) small amounts of polyspecific IgG detected only in immune sera; (3) high amounts of specific IgG reacting with pig and rabbit tubulins, present in immune but not normal sera. Western blot analysis of the specific IgG population showed that it contained antibodies reacting with both native pig and rabbit tubulins, as well as antibodies recognizing only the 30,000 proteolytic fragment of pig, but not that of rabbit tubulin. The results indicate that the immunization of rabbits with heterologous tubulin induced specific IgG anti-tubulin antibodies which recognize the self and non-self antigens differently.