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  Director : Guillaume DIGHIERO (dighiero@pasteur.fr)


  abstract

 

The work from our laboratory was devoted to: 1) the study of the mechanisms involved in the low expression of the B cell receptor (BCR) in this disease; 2) the study of AID (activation induced cytidine deaminase) expression in this disease; 3) the study of gene expression profiles in CLL and 4) the " in vitro " feasability study of an anti-idiotypic vaccine by using dendritic cells from CLL patients pulsed with clonal idiotypic determinants.



  report

cale

Introduction

Chronic lymphocytic leukemia (CLL), the most frequent form of leukemia in Western Countries, is characterized by a progressive accumulation of functionally incompetent, long-lived small mature monoclonal B lymphocytes, with a characteristic phenotype:CD5+,CD23+, low BCR expression resulting from low surface immunoglogulin and CD79b expression. It is far from uniform in presentation and clinical course. About one-third of patients never require treatment and have a long survival; in another third an initial indolent phase is followed by progression of the disease; the remaining third of patients have aggressive disease at the outset and need immediate treatment. The development of the Rai and Binet staging systems has allowed the division of patients with chronic lymphocytic leukemia into three prognostic groups: good, intermediate and poor prognosis. However, neither of these staging systems is able to accurately predict evolution of these patients. Recently, it has been demonstrated that the absence of mutations inV genes is strongly linked to advanced stages of the disease even within the good prognosis group and mutational status appears as the most relevant prognostic parameter in CLL. Although classical chemotherapeutic treatments are able to induce prolonged remission in this disease, relapse invariably occurs and CLL is presently considered as an incurable disease. Since the only treatment presently available able to cure disease is allogenic bone marrow transplantation, which acts through the graft versus leukemia effect, this indicates that immunotherapy may play an important role in the treatment of this disease.

1) What are the mechanisms underlying underexpression of the BCR and impaired functional response in CLL B cells? (Responsible: B. Payelle-Brogard in collaboration with C. Magnac and F. Vuillier).

The BCR consists of two heterodimers CD79a/CD79b bound non-covalently with the surface membrane Ig (SIg). In CLL, both SIg and CD79b have been reported to be consistently under-expressed, though the mechanisms accounting for this phenomenon have not been elucidated. In addition, CLL B cells display very poor signaling upon stimulation through the BCR. Since anergic B cells have been demonstrated to express low levels of the BCR and to poorly signal upon BCR stimulation, CLL B cells may correspond to malignant transformation of an anergic B cell.

In a previous work, we have studied the complete sequence of the B29 gene, encoding for CD79b, in 20 different familial CLL patients and demonstrated the absence of genetic abnormalities underlying the low surface expression of the CD79b. Since genetic alterations could not account for under-expression of the BCR in CLL, we have undertaken transcriptional and post-transcriptional analysis of this gene. Our results demonstrate that: 1) there is no major defect in transcriptional expression of the BCR. 2) Analysis of intracellular expression of the BCR components showed adequate synthesis, despite consistent low membrane expression of the receptor. 3) Neither a genetic defect in the transmembrane domain of SIg which it associates with CD79a/CD79b nor a genetic abnormality in the chaperone protein calnexin involved in folding and assembly of the BCR were found. 4) In contrast a defect in the processing of nascent IgM, demonstrated by its failure to become resistant to endoglycosidase H treatment was found. This defect indicates that they are retained in the endoplasmic reticulum. 5) In addition, a constant defect in assembly of Ig M and CD79b chains of the BCR could be demonstrated in CLL B cells, by using confocal microscopy techniques, which allowed to demonstrate its retention in the Reticulum Endoplasmic compartment and its inability to progress to the Golgi cell surface. These findings demonstrate that a post-transcriptional defect located at the BCR intracellular assembly and/or trafficking levels could be involved in the low expression of the BCR in B-CLL and are reminiscent to those previously reported in a transgenic murine model of anergic B cells. This project is supported by a grant of the Fondation contre la Leucémie.

Figure illustrate this defect: whereas normal and Daudi Burkitt B cell line cells, succeed to merge the µ (red) and the CD79b (green) molecules at the membrane (yellow color), this is very poorly achieved by CLL B cells.

2) AID expression in CLL (Responsibles: G. Dighiero with P. Oppezzo and G. Dumas).

After rearrangement, immunoglobulin (Ig) variable genes are diversified by somatic hypermutation (SHM), while the effector function of the constant domain is modified by class switch recombination (CSR). These processes depend on activation induced cytidine deaminase (AID), a putative RNA-editing enzyme, expressed in B-cells from secondary lymphoid organs upon CD40 ligand (CD40L) stimulation. Since the absence of AID expression in one form of the hyper-IgM syndrome in humans and in AID targeted mice, abolishes both CSR and SHM, this protein is supposed to play a major role in both processes. Honjo and colleagues postulated that this enzyme might be involved in them, through the production of similar or identical molecular substrates for RNA editing. On the other hand, recent reports suggest that SHM and CSR could all be initiated by the direct action of AID on DNA through a DNA-deamination mechanism of antibody diversification. However, at present its precise mode of action remains unknown.

Since some CLL B-cells can undergo CSR without somatic mutations, they may constitute a useful model to dissect AID function. In this work, we have studied AID expression, the presence of mutations in pre-switch µ DNA region, CSR and SHM in 65 CLL patients. Our results demonstrate that: 1) CLL B-cells can constitutively express AID, which expression is associated with mutations in the pre-switch region and clonally related isotype-switched transcripts. Interestingly, this phenomenon is almostly exclusively restricted to CLL B cells exhibiting Ig V genes in unmutated form and 2) in CLL without constitutive AID expression, its induction upon stimulation results in pre-switch mutations and CSR process. However, despite that either constitutive or induced AID expression were able to induce somatic mutations in the pre-switch µ region and to induce active CSR, somatic hypermutations in the variable domain could never be obtained. Overall, our results show a dissociation between SHM and CSR in CLL and suggest that, at least in this disease, AID would require additional help to carry out SHM process.

3) Micro-array studies of gene profile expression in different CLL prognostic groups (Responsibles: G. Dighiero with Yuri Vasconcelos and Frédéric Davi (Hôpital Pitié-Salpêtrière), Florence Cymbalista (Hôtel-Dieu de Paris) and Xavier Troussard (CHU de Caen) on behalf of the French Cooperative Group on CLL.

Chronic lymphocytic leukemia (CLL) is a disease with a highly variable clinical course. During recent years the development of biologic markers has allowed a better definition of prognosis in CLL. Different serological, phenotypic and cytogenetic markers have been proposed to help better prediction of survival and progression of this disease. So far, the best biological parameter capable of predicting its evolution is the mutational status of the immunoglobulin (Ig) genes, with Ig mutated cases having a far better prognostic than those with unmutated genes. This may suggest that there are two types of CLL: one arises from relatively less differentiated (immunologically naive) B cells with unmutated heavy chain genes, and displays poor prognosis; the other evolves from more differentiated B cells (memory B cells) with somatically mutated heavy chain genes, and has a good prognosis. However, recent data derived from gene expression profiling analysis failed to clearly distinguish unmutated and mutated cases and favor the view that all cases of CLL have a common cell origin and/or a common mechanism of malignant transformation. We have used the microarray technology to determine whether gene expression profile could distinguish these two groups of CLL.

Eighteen cases of CLL, including 9 indolent forms of the disease ( Binet stage A; i.e. at least 5 years without treatment and any evolution) and mutated Ig genes and 9 cases with stage B or C aggressive disease and unmutated Ig genes, were studied. For these latter cases, only cells collected before therapy were analyzed. Leukemic cells were purified by negative selection, using anti-CD3, -CD14, -CD16, -CD56 antibodies, yielding > 98% pure CLL cell populations. RNAs were extracted and gene expression analyzed using the Affymetrix human U133 Genechip with 22283 probe sets.

In agreement with previous reports indicating that Ig mutated and unmutated cases have globally the same gene expression pattern, a supervised statistical analysis showed that only 85 genes were differentially expressed by a factor >2 between the two groups of CLL. Among these 85 genes, our results showed homogeneous overexpression of ZAP-70, lipoprotein lipase (LPL), BCL-7A, VLA-4, dystrophin (DMD) and gravin (AKAP12) in the aggressive unmutated cases, while stable mutated cases overexpressed T cell function-related genes HLA-DQB1, CTLA-4, TCF7, ADAMDEC1, as well as Wnt-3 and JUN oncogene. However, in contrast to previous reports, a non supervised hierarchical clustering analysis could clearly separate the stable mutated group from the aggressive unmutated one, except for one case. Microarray results could subsequently be confirmed by using Real Time PCR.

These results show that gene expression profiling can distinguish CLL cases with a stable evolution and mutated Ig genes from those with unmutated Ig genes and progressive disease. Thus, monitoring the expression of a very limited number of genes might suffice to identify patients displaying an indolent disease from patients exhibiting an aggressive one.

4) Is there a role for anti-idiotypic vaccines in CLL? (Responsible: F. Vuillier).

Since all major chemotherapeutic treatments in CLL succeed to induce good responses but are unable to cure this disease, one of the major questions that needs to be answered is whether there is a place for immunotherapeutic approaches like anti-idiotypic vaccines. Since each B cell undergoes a unique rearrangement that is characteristic for each individual B cell and since malignant B cell hemopathies are characterized by clonal expansion of a clone displaying a unique rearrangement, idiotype constitutes a privileged tumor antigen.

One of the aims of tumor immunotherapy is the induction of CD8 cytotoxic T lymphocytes (CTL). It is established that various tumor cells share tumor associated antigens, though whether such antigens can elicit CTL response remains elusive.

The French Cooperative Group on CLL has committed our group to initiate a feasibility "in vitro" study on the possibility to generate specific cytotoxic anti-idiotypic CD8 cells, through incubation of these T cells with dendritic cells (DC) previously pulsed with the tumoral idiotype. Following the complete sequence of VH and VL genes from the CLL clone, the Ig is expressed by transfecting X-63 myeloma with the same expression vectors used to construct transgenic mice. In these conditions, DCs obtained from patient's monocytes are pulsed with the complete Ig or different peptides corresponding to CDR3 hypervariable Ig region.

In a first step, freshly purified monocytes from CLL patients and normal donors were induced to differentiate in GM-CSF and IL-4 in serum-free medium and compared for their morphological, phenotypic and functional characteristics. Our results demonstrate that: 1) functional DCs can be generated from CLL patients with similar phenotype and function to that observed with normal donors, 2) in contrast to normal controls Mono-derived DCs from CLL patients spontaneously secrete endogenous IL-10 and 3) IFN-g in combination with CD40L plays a major role in priming DC from CLL patients for IL-12 and IL-15 production. These results indicate that it is possible to derive functionally competent dendritic cells from circulating monocytes in CLL patients. In a second step, VH and VL genes from CLL B lymphocytes from different patients have been isolated and cloned. This allowed us to produce patient's respective idiotype in different forms: complete IgM by recombinant technology and CDR3 peptides displaying class I high affinity. Next, monocyte derived dendritic cells from CLL patients were pulsed with these idiotypic reagents and evaluated for their capacity to induce specific CD4 and CD8 cell lines with anti-tumoral activity. In these conditions, CD8 cells lines with a moderate cytotoxic activity have been obtained (about 20% of cytotoxic activity in a 25/1 ratio; manuscript in preparation). To improve the efficiency of this system, pulsing of CLL monocyte-derived dendritic cells with exosomes derived from tumoral B cells is attempted.

STUDY OF RESTORATION OF IMMUNE FUNCTION REGARDING TWO PATHOGENS: CMV AND T. GONDII. (Responsible: D. Scott-Algara, M. Alfonzo, M. Bedora).

We conducted a prospective study of the immune restoration regarding two pathogens after the start of triple antiretroviral in chronically HIV-1 infected patients. We showed that SCID mice humanized with lymphoid cells from HIV-1 subjects after beginning drug therapy but infected with T. gondii survive longer than control mice. Untreated patients expressed a CMV-specific CTL activity despite of an absence of CMV-specific proliferation, secretion of IFN-g and IL-2. The reconstitution of these CMV-specific responses was observed after 6 months of HAART simultaneously with an increase of CMV-specific CTL activity. However, IL-2 response was never restored during the follow-up. In conclusion, partially immune restoration was observed during anti-retroviral therapy.

Keywords: Autoimmunity, Chronic Lymphocytic Leukemia, Immunopathology



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  DIGHIERO Guillaume, Head of the Unit,dighiero@pasteur.fr BROGARD Béatrice, CNRS,bbrogard@pasteur.fr

SCOTT-ALGARA Daniel, IP,scott@pasteur.fr

ALFONZO DIAZ Miguel

GIORDANO Mirta

OPPEZZO Pablo,popezzzo@pasteur.fr

TISCORNIA Adriana

VASCONCELOS Yuri,yvasconc@pasteur.fr

BEDORA Marie, Technician IP,mbedora@pasteur.fr

BOUYSSIE Reine, Secretary,bouyssie@pasteur.fr

DUMAS Gérard, Technician IP,dumas@pasteur.fr

LAURENT Nathalie, Technical personnel

MAGNAC Christian, Engineer IP,

VUILLIER Françoise, Engineer IP,vuillier@pasteur.fr


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