No abstract

IntroductionAfter gene rearrangement, immunoglobulin (Ig) variable genes are diversified by somatic hypermutation (SHM), whereas 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 on CD40 ligand (CD40L) stimulation. 1 Given that the absence of AID expression in one form of the hyper-IgM syndrome in humans 2 and in AID-targeted mice abolishes CSR and SHM, this protein is thought to play a major role in both processes. 3 Fifty percent of patients with chronic lymphocytic leukemia (CLL) display mutated V H genes. 4 The mutational profile of immunoglobulin genes represents an important prognostic factor 5,6 because patients expressing unmutated V H genes exhibit poor prognoses. In addition, previous reports 7-9 have demonstrated that CSR frequently occurs in CLL and predominates in unmutated B-cell CLLs (B-CLLs).In this work, we have examined the expression of AID transcripts, SHM, and CSR in 65 patients with CLL expressing either unmutated (33 of 65) or mutated (32 of 65) V H genes. Our results show that patients with unmutated B-CLL can constitutively express AID transcripts. This fact is associated with the presence of mutations in the preswitch DNA region and with an active CSR, but it is not related to the SHM process. Additionally, in patients with mutated CLL without constitutive AID expression, AID induction on stimulation results in preswitch mutations and the CSR process. Study design Healthy and CLL samplesBlood was collected from 4 healthy controls and 65 typical CLL patients from Hôtel-Dieu, Pitié-Salpêtrière, and Pasteur hospitals (Paris, France), Sao Paulo and Servidor Publico Estadual hospitals (Sao Paulo, Brazil), and Hospital Maciel (Montevideo, Uruguay). B cells were purified through negative depletion by using RosetteSep antibody cocktail (StemCell Technologies, Vancouver, BC, Canada) and were stimulated in vitro for 5 days with 1 g/mL recombinant soluble CD40L (Immunex, Seattle, WA) and 1000 U/mL interleukin (IL)-4 (PharMingen, San Diego, CA). Analysis of AID transcriptsPolymerase chain reaction (PCR) amplification of CLL cDNA was carried out as described previously, 2 and a semiquantitative protocol for AID expression was performed. Briefly, cDNA obtained from 5 ϫ 10 6 B cells was amplified by 20 cycles of PCR using AID 2 and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) 10 primers. The fragments obtained were transferred and hybridized with specific AID and GAPDH ␣-[ 32 P] dCTP (deoxycytidine-5Ј-triphosphate)-labeled probes. Analysis of CSR processPCR amplification of different isotype transcripts was performed as described by Oppezzo et al. 9 Circle isotype-specific (I-C) transcripts, termed circle transcripts (CTs), were analyzed by PCR with primers I-␥ (forward) and C-(reverse), as described by Kinoshita et al. 11 Mutation analysis of V H and preswitch regions Sequences of V H genes were det...

Although the zeta-associated protein of 70 kDa (ZAP-70) is overexpressed in patients with chronic lymphocytic leukemia (CLL) displaying unmutated IGVH genes and poor prognosis, a previous microarray study from our group identified overexpression of LPL and ADAM29 genes among unmutated and mutated CLL, respectively. To assess the prognostic value of these genes, we quantified their expression by real-time quantitative polymerase chain reaction (

Base substitutions, deletions, and duplications are observed at the immunoglobulin locus in DNA sequences involved in class switch recombination (CSR). These mutations are dependent upon activation-induced cytidine deaminase (AID) and present all the characteristics of the ones observed during V gene somatic hypermutation, implying that they could be generated by the same mutational complex. It has been proposed, based on the V gene mutation pattern of patients with the cancer-prone xeroderma pigmentosum variant (XP-V) syndrome who are deficient in DNA polymerase η (pol η), that this enzyme could be responsible for a large part of the mutations occurring on A/T bases. Here we show, by analyzing switched memory B cells from two XP-V patients, that pol η is also an A/T mutator during CSR, in both the switch region of tandem repeats as well as upstream of it, thus suggesting that the same error-prone translesional polymerases are involved, together with AID, in both processes.

Superantigens bind class II major histocompatibility proteins and stimulate powerful proliferative responses of T lymphocytes bearing particular V beta sequences as part of their alpha beta antigen receptor. Exogenous bacterial superantigens are responsible for food poisoning and toxic shock syndrome. Murine virus-encoded self-superantigens induce clonal deletion of T lymphocytes. Although superantigen-like properties have been suggested for human immunodeficiency virus-1, no viral superantigen has been identified in humans. Here we report that the nucleocapsid of the rabies virus is an exogenous superantigen specific for V beta 8 human T lymphocytes which binds to HLA class II alpha-chains.