Unit: Immuno-Hematology and Immunopathology

Director: Guillaume DIGHIERO

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) and of the functional impairment of BCR signaling in this disease; 2) the study of AID (activation induced cytidine deaminase) expression and mechanisms of its regulation in this disease; 3) the study of gene expression profiles in CLL and of their application to prognosis of this disease and 4) the search of a viral origin for CLL.



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. 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. In 1994, we reported for the first time (Schroeder and Dighiero) that about half of CLL patients were displaying somatic mutations in their immunoglobulin (Ig) genes, whereas the other half expressed Ig genes without somatic mutations. Since then, the mutational status of Ig heavy chain variable (IgVH) genes has been considered as the best prognostic marker in CLL. Mutated patients usually demonstrate a favorable evolution when compared to unmutated cases, which are characterized by progressive disease, continuing treatment needs and a high proportion of CLL-related deaths.

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

The BCR consists of an heterodimer CD79a/CD79b bound non-covalently with the surface Ig (SIg). In CLL, both SIg and CD79b are consistently under-expressed. 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 demonstrated in CLL the absence of genetic abnormalities of the B29 gene, encoding for CD79b, underlying the low surface expression (Payelle-Brogard et al Blood 1999). Furthermore there is no major defect in transcriptional expression or in synthesis of the BCR components. In contrast a defect in the processing of IgM, in assembly with CD79b chain, and an IgM retention in the Endoplasmic Reticulum (ER) have been demonstrated (Payelle-Brogard et al Br J Haematol 2002, Leukemia 2003).

We thus investigated the maturation processes and cellular localization of the BCR components in CLL B-cells. These processes take place in the ER where the proteins are modified (cleavage of signal peptide, N-glycosylation, formation of disulfide bonds) and then properly folded before exit to the Golgi. If the folding and maturation process fails, the proteins are retained in the ER and exposed to ER resident chaperones to attempt adequate folding. Improperly folded or assembled proteins may be degraded by the ubiquitin proteasome pathway or if they fail to be degraded they may accumulate as aggregates.

Our results obtained in CLL patients demonstrate a severe impairment in µ and CD79a glycosylation and folding, as well as a constant retention in the ER, as shown by their colocalization with calnexin (Figure 1). In the contrary to the CD79a chains, no clear impairment in the glycosylation and folding status of the CD79b, which colocalize in the Golgi, detected by TGN46 Ab (Figure 1), was found. No structural abnormalities of the BCR components and of the chaperone proteins involved in the BCR folding processes could be substantiated. Altogether, these results show for the first time that lower BCR surface expression in CLL is accounted for by an impaired glycosylation and folding of the μ and CD79a chains (Vuillier et al Blood 2004).

To better define the functional abnormality of CLL BCR, we have decided to study the traffic of the BCR molecules in CLL B cells, upon stimulation by the BCR pathway. Indeed, recent advances in membrane biology have led to the identification of glycosphingolipid and cholesterol-rich plasma membrane microdomains, or lipid rafts, that have been proposed to function as platforms for both signal transduction and membrane trafficking. Following the proteomic characterization of lipid rafts in CLL previous to any stimulation, we will proceed in a second step, to the study of the traffic of the different components of BCR following stimulation with anti-µ antibodies. This project led by F. Vuillier, has received the scientific and financial support of the Ligue contre le Cancer.

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

After rearrangement, 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). Since some CLL B-cells can undergo CSR without somatic mutations, they may constitute a useful model to dissect AID function (Oppezzo et al Leukemia, 2002). We have studied AID expression, 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 and almost 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, without somatic hypermutations in the variable domain. Overall, our results show a dissociation between SHM and CSR in CLL and suggest that AID may act differentially on CSR and SHM pathways, and at least in this disease, would require additional help to carry out SHM process (Oppezzo et al Blood, 2003).

Inhibitor of Differentiation (Id) proteins family also appears to be involved in the process of CSR, particularly Id-2 and Pax-5. The balance between Pax-5 and Id-2 activities is key to the regulation of AID gene expression and CSR (Gonda et al J Exp Med 2003). BSAP, the protein encoded by Pax-5, up-regulates AID expression and this effect can be down regulated by over-expression of Id-2. In addition, expression of Pax-5 and AID genes is regulated by the transcriptional repressor Blimp-1 (B-cell lineage-specific activator protein). We have analyzed the role of Pax-5, Id-2 and prdm-1 genes in regulation of AID expression and CSR in normal and CLL B-cells. Our results show that the presence of AID protein and CSR is associated with high expression of the complete form of Pax-5 gene (Pax-5a). In contrast, an absence of AID and CSR is consistently associated to a reduction of Pax-5a transcripts and the appearance of a spliced form displaying a deletion in the C-terminal domain (Pax-5/Δ Ex8) (Figure 2). Both isoforms of BSAP are able to bind to the AID-promoter region and AID expression is correlated with a decrease in Id-2 and prdm-1 transcripts. These results propose that Pax-5/Δ Ex8, an alternative splicing product of Pax-5 gene in human B-cells, could play an important role in the control of its own transcription and indirectly in AID expression of CSR regulation (Oppezzo et al Blood, 2004).

The important question addressed is whether "ectopic" expression of AID in these CLL patients, is recruiting the same AID partners as in normal germinal center B cells. To assess these questions we propose to: a) Identify putative protein/s factors partners of AID protein by using the "Two Hybrid system" and b) Characterize structurally and functionally AID and its putative partners through a collaboration with the Structural Biochemistry Laboratory of the Pasteur Institute. For this we are attempting to produce AID in its native form and to solve the tri-dimensional structure of AID alone and in complex with its putative partners. This project has received the support from ARC.

3) Micro-array studies of gene profile expression in CLL and their application to prognosis (Responsibles: G. Dighiero with Yuri Vasconcelos, P. Oppezzo and G. Dumas in collaboration with F. Davi, H. Merle-Béral and C. Settegrana (Hôpital Pitié-Salpêtrière).

The development of the Rai and Binet staging systems has allowed the division of CLL patients into three prognostic groups: good, intermediate and poor prognosis. However, neither of these staging systems is able to accurately predict evolution of these patients. An important long term study from our group (Dighiero et al N Eng J Med 1998) demonstrated that about half of patients included in the good prognosis group never evolve, do not require treatment and die of causes unrelated to CLL. The other half however, will evolve, require treatment and die of CLL related causes. Given that the absence of mutations in V genes is strongly linked to poor prognosis, we have undertaken a study on 146 patients aiming at defining whether the mutational profile of Ig was able to better predict prognosis in CLL patients when associated to the Binet's clinical staging. Our results demonstrated that both the Binet's clinical staging and the mutational pattern of Ig genes result in a high improvement of prognosis assessment in this disease (Vasconcelos et al J Clin Oncol 2003).

The best biological parameter capable of predicting the evolution of this disease is the mutational status of the 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 Ig genes, and displays poor prognosis; the other evolves from more differentiated B cells (memory B cells) with somatically mutated Ig genes, and has a good prognosis. However, recent data derived from gene expression profiling analysis 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 unmutated and mutated two groups of CLL. Gene expression was analyzed using the Affymetrix human U133 Genechip with 22283 probe sets.

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, overexpression of ZAP-70 and lipoprotein lipase (LPL) genes was observed in the aggressive unmutated cases, while stable mutated cases overexpressed the ADAM29 gene encoding for metallo-proteinase. 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 (Vasconcelos et al, Leukemia, resubmitted after revision).

We then investigated which of these genes (individually or in combination) could better predict the IgVH mutational status in 127 CLL patients. Simultaneaous usage of the LPL/ADAM29 (L/A) ratio and ZAP-70 expression allowed an almost perfect (99%) assessment of the IgVH status in the 80% of patients with concordant results (L/A+, ZAP-70+ or L/A-, ZAP-70-). IgVH mutational status, ZAP-70 and the L/A ratio were predictive of event-free survival for the whole cohort and for stage A patients. In addition the L/A ratio was an independent pronostic factor for stage B and C patients (Oppezzo et al Blood, resubmitted after revision).

4) Is there a viral origin in the CLL? (Responsible: Evie Melanitou in collaboration with O. Pritsch, G. Dumas and F. Vuillier).

Several observations suggest a viral origin of the CLL. The presence of a viral etiology as demonstrated in the Bovin Chronic Lymphocytic Leukaemia (Burny et al Retr Biol Hum Dis 1990) that shares phenotypic and clinical characteristics with the human CLL, is one of the arguments in favour of a viral origin, also, in the aetiology of CLL. The BLV is a retrovirus similar to HTLV1. Another concurring observation is that in human CLL patients, a constitutive IL-10 expression has been observed in the B cells and in monocytes deriving from dendritic cells. Finally, a defect observed in the BCR expression in CLL B-cells, has been also observed in a B cell line after transfection with a viral protein, which is responsible for the retention of the BCR in the endoplasmic reticulum. .

To explore the possibility of a viral origin for CLL, we used a subtractive hybridization procedure derived from the Representational Difference analysis (RDA). This is a powerful method that gives the possibility to detect eventually the existence of a viral exogenous sequence, known or unknown, in the B cells of CLL. It has been successfully applied in the identification of the Human Herpes Virus 8 (HHV8) responsible for the Kaposi's sarcoma. Although our first data did not identify yet the presence of a viral sequence, revealed however interesting results, as several genes have been found to be differentially expressed between CLL B cells non activated and activated with CD40 ligand. This is an ongoing project.

Deregulation of the immune system contributes to several diseases such as cancer, infection and autoimmunity. The majority of such diseases have a genetic component with a multifactorial inheritance. Evie Melanitou, with the participation of Sadrina Benabla, has been interested in a functional genomics approach applied in deciphering the immune deregulation of early inflammatory responses, initially applied in autoimmune diseases. The aim of these studies is to be able to unravel the early molecular mechanisms taking place in the immune surveillance of the self-recognition, as well as controlling infection and malignancy. Early sub phenotypic markers have been established and we have demonstrated that they correlate with the final disease onset (Melanitou et al J Immunol 2004). Subsequently, we used these markers and proceeded in the identification of genes implicated in the early stages of immune deregulation, before disease appears by high throughput differential gene expression analysis. We have thus identified several genes playing a role in the immune system as well as genes implicated in the cellular cycle. Functional analysis of these genes allowed their classification in specific networks. Further analysis includes undertaking experimental approaches aiming to study the pre inflammatory processes occurring in pathologies as different as cancer, autoimmunity and infection. Integrative approaches of candidate genes identified by genomics are under development to gain insights into the molecular processes taking place in pre inflammatory conditions. Finally, the implication of environmental factors, such as viral infections, will be assessed as environmental triggers of the immune system, in relation to disease.

Keywords: Autoimmunity, Chronic Lymphocytic Leukemia, Immunopathology

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