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...

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Four different monoclonal Ig (MIg) (IgA1κ, IgG1κ, IgG2κ and IgG4κ) displaying anti‐tubulin activity were detected in the serum from a lymphoma patient. The complete sequence of three of these MIg showed identical VH and VL domains and the presence of mutations compatible with an antigen‐driven process. Surprisingly, despite complete homology in their variable domains, IgA1κ, IgG1κ, or their Fab fragments bound to a common motif recognized in β tubulin, with significant differences in affinity (IgA1κ 1.52×10–8 M, and IgG1κ 2.09×10–7 M). To substantiate these results, the VH and VL domains from IgA1κ were cloned and introduced into expression vectors containing the constant κ exon and either the μ or the γ1 constant exon, and complete recombinant IgMκ and IgG1κ were obtained. Like the IgA1κ, the IgMκ construction bound to the tubulin epitope with consistent affinity (7.7×10–9 M), whereas the IgG1κ construction displayed a significantly lower affinity (3.28×10–7 M). These results provide definitive evidence that isotype can influence binding affinity to antigen and suggest that malignant transformation occurred at the germinal center once the mutational process was achieved and the switch process was still active.

The significant survival differences observed between the VH mutational groups, among stage A and stage B and C patients, indicate that Binet's classification and VH genes are independent prognostic variables and are most likely complementary.