ORIGINAL ARTICLE AID-induced T-lymphoma or B-leukemia/lymphoma in a mouse BMT model Y Komeno 1,2 , J Kitaura 1,2 , N Watanabe-Okochi 3,4 , N Kato 1,2 , T Oki 1,2 , F Nakahara 1,2 , Y Harada 5 , H Harada 6 , R Shinkura 7 , H Nagaoka 7 , Y Hayashi 8 , T Honjo 7 and T Kitamura 1,2 1 Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan; 2 Division of Stem Cell Signaling, Center for Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan; 3 Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan; 4 Department of Transfusion Medicine, Graduate School of Medicine, University of Tokyo, Bunkyo-ku, Tokyo, Japan; 5 International Radiation Information Center, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima, Japan; 6 Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima, Japan; 7 Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto, Japan and 8 Department of Hematology/Oncology, Gunma Children’s Medical Center, Shibukawa, Gunma, Japan Activation-induced cytidine deaminase (AID) diversifies immunoglobulin through somatic hypermutation (SHM) and class-switch recombination (CSR). AID-transgenic mice develop T-lymphoma, indicating that constitutive expression of AID leads to tumorigenesis. Here, we transplanted mouse bone marrow cells transduced with AID. Twenty-four of the 32 recipient mice developed T-lymphoma 2–4 months after the transplantation. Surprisingly, unlike AID-transgenic mice, seven recipients developed B-leukemia/lymphoma with longer laten- cies. None of the mice suffered from myeloid leukemia. When we used nude mice as recipients, they developed only B-leukemia/ lymphoma, presumably due to lack of thymus. Analysis of AID mutants suggested that an intact form with SHM activity is required for maximum ability of AID to induce lymphoma. Except for a K-ras active mutant in one case, specific mutations could not be identified in T-lymphoma; however, Notch1 was constitutively activated in most cases. Importantly, truncations of Ebf1 or Pax5 were observed in B-leukemia/lymphoma. In conclusion, this is the first report on the potential of AID overexpression to promote B-cell lymphomagenesis in a mouse model. Aberrant expression of AID in bone marrow cells induced leukemia/lymphoma in a cell-lineage–dependent manner, mainly through its function as a mutator. Leukemia (2010) 24, 1018–1024; doi:10.1038/leu.2010.40; published online 1 April 2010 Keywords: AID; BMT; T-lymphoma; B-leukemia/lymphoma Introduction Under physiological conditions, activation-induced cytidine deaminase (AID) is expressed in germinal center (GC) B-cells and initiates somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating a cytosine to create a uracil. 1,2 Structurally, the N-terminal or C-terminal domain of AID is indispensable for SHM or CSR, respectively. 3–5 Interest- ingly, expression of AID is increased in B-lymphoid leukemia or GC-derived B-lymphoma, with frequent hypermutation of proto-oncogenes and reciprocal chromosomal translocation. 6–9 In fact, recent studies have shown that AID is required for GC-derived lymphomagenesis and c-Myc/IgH chromosomal translocations. 10,11 In addition, elevated expression of endo- genous AID and aberrant somatic mutations in tumor-related genes have also been observed in cancerous tissues related to inflammation. 12 Analysis of AID-transgenic (Tg) mice has revealed that constitutive expression of AID leads to tumori- genesis; ubiquitous and constitutive expression of AID induced lethal T-lymphoma with no apparent chromosomal trans- location, occasionally accompanied by lung, liver, and gastric cancers, 13,14 and specific expression of AID in double-positive thymocytes also induced T-lymphoma. 15 However, neither AID-Tg mice specifically expressing AID in single-positive thymocytes and mature T-cells nor AID-Tg mice with CD19 þ B-cell-specific expression of AID developed lymphoma/ leukemia. 15,16 These results suggest that susceptibility to AID- induced tumorigenesis depends on tissue or cell lineage, but the underlying mechanism remains obscure. Importantly, sequencing analysis in AID-Tg mice indicated that AID is an organ-specific mutator of non-Ig genes. 14 To prevent accumula- tion of unfavorable mutations induced by AID, its activity is tightly regulated by several mechanisms. 17 In this study, we focused on AID-mediated leukemogenesis and created a mouse bone marrow transplantation (BMT) model, using BM cells retrovirally transduced with AID. Notably, recipient mice developed B-leukemia/lymphoma, albeit less frequently as compared with thymic T-lymphoma. Materials and methods Retroviral constructs, transfection, and retrovirus production Murine AID (mAID), mAID mutants (G23S 4 and D189–198 5 ), human AID (hAID), and hAID mutants (P20 3 and JP8B 3 ) were subcloned into the pMYsIG vector as described in ‘Supplemen- tary Materials and methods’. All constructs were verified by DNA sequencing. Expression of wild-type or mutant AID was recognized in 293T cells transiently transfected with each construct. Retroviruses were generated by transient transfection of Plat-E packaging cells with FuGene 6 (Roche Diagnostics, Mannheim, Germany), as described earlier. 18–20 Mouse BMT Mouse BMT was performed as described earlier. 20 C57BL/6 CD45.1 or CD45.2 mice were used as donors or recipients, Received 23 November 2009; revised 8 January 2010; accepted 9 February 2010; published online 1 April 2010 Correspondence: Dr T Kitamura, Division of Cellular Therapy, Advanced Clinical Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. E-mail: kitamura@ims.u-tokyo.ac.jp Leukemia (2010) 24, 1018–1024 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu