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 (

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Antibody-dependent cell cytotoxicity (ADCC) plays a critical role in monoclonal antibody (mAb)-mediated cancer therapy. ADCC, however, has not been directly shown in vivo but inferred from the requirement for IgG Fc receptors (FcgR) in tumor rejection in mice. Here, we investigated the mechanism of action of a Tn antigenspecific chimeric mAb (Chi-Tn), which binds selectively to a wide variety of carcinomas, but not to normal tissues, in both humans and mice. Chi-Tn mAb showed no direct toxicity against carcinomas cell lines in vitro but induced the rejection of a murine breast tumor in 80% to 100% of immunocompetent mice, when associated with cyclophosphamide. Tumor rejection was abolished in Fc receptors-associated g chain (FcR-g)-deficient mice, suggesting a role for ADCC. Indeed, tumor cells formed stable conjugates in vivo with FcR-g chainexpressing macrophages and neutrophils in Chi-Tn mAb-treated but not in control mAb-treated mice. The contact zone between tumor cells and ADCC effectors accumulated actin, FcgR and phospho-tyrosines. The in vivo formed ADCC synapses were organized in multifocal supra-molecular activation clusters. These results show that in vivo ADCC mediated by macrophages and neutrophils during tumor rejection by Chi-Tn mAb involves a novel type of multifocal immune synapse between effectors of innate immunity and tumor cells. Cancer Res; 71(15); 5134-43. Ó2011 AACR.