Eur. J. Immunol. 1987.17: 1593-1597 T cell receptor zyxwv y chain variable gene rearrangements in zyx ALL 1593 Thomas L. J. Boehm", Andreas Werle', Arnold Gamern, Bernhard Kornhuber' and Dusan Drahovsky' Zentrum der Kinderheilkunde', Zentrum der Biologischen Chemie', Zentrum der Inneren Medizina, Klinikum der Johann Wolfgang Goethe-Universitat, Frankfurt T cell receptor y chain variable gene rearrangements in acute lymphoblastic leukemias of T and B lineage* The status of immunoglobulin and T cell receptor genes in acute lymphoblastic leukemia (ALL) of T and B lineage has been studied. Our data indicate that illegiti- mate gene rearrangements at immunoglobulin heavy chain (in T cell ALL), and T cell receptor P chain (in pre-B ALL) genes are only rarely found (2 out of 30 patients). In contrast, T cell receptor y chain gene rearrangements, characteristically found in T- ALL, are also present in 7 of 18 patients with pre-B ALL. Several features distinguish these illegitimate T cell receptor y chain gene rearrangements from those in normal and leukemic T cells. V, genes located far upstream of the JdC, complexes (V,2, V,3, V,4, V,5) appear to be preferentially used in normal adult peripheral blood T cells. In contrast, V, genes located immediately 5' to the J,/C, complexes (V,8, V,9, V,lO, V,11) predominate in V,-J, recombinations observed in T-ALL and pre-B ALL. Whereas the J,2 region is primarily used in T cell receptor y gene rearrangements observed in T-ALL, those in pre-B ALL are confined mostly to the J,1 region. These data suggest a limited accessibility of the T cell receptor y chain gene locus for recombination processes in early stages of B cell differentiation. 1 Introduction 2 Materials and methods The T cell receptor (TCR) y chain, together with a fourth 2.1 Patients polypeptide chain, designated 6 is expressed in association with the CD3 complex presumably as a receptor complex on a Acute leukemia (AL) patients were typed according to estab- distinct set of T cells [l-31. The function of this putative recep- lished morphological, cytochemical and immunological tor complex, however, is only just beginning to be clarified. methods [13-161 as described [17-191. Only patients with an The genomic locus for the y chain has recently been character- unequivocal phenotype were included in the present study. ized in both mouse and man, and shown to be much less diverse than the genes encoding the P and zyxwvutsrq a chains of the classical TCR molecule [4-121. 2.2 Strategy of analysis Recent studies have indicated that the human V, locus con- tains at least 13 V, genes, belonging to four subgroups; five genes in this locus are considered to be pseudogenes [7, 121. The genomic organization of the TCR V, locus in man is: 5' V,1O-Vyll-3' [7, 121. Previous studies have also shown that each V,-J,-recombination results in a rearranged fragment of distinct size [7, 121, thereby allowing the assingment of a V-J recombination to a specific V, gene by rearrangement analy- sis. We have exploited this information to analyze V, gene usage in V,-J, recombinations observed in acute lymphoblastic leukemia (ALL) of T and B lineage. QV,l -V,2-V,3-V,4-V,5-~V,5-wV,6-~V,7-V,8-.V~A-V,~- [I 62111 Hybridization analysis of immunoglobulin heavy chain (IgH) gene arrangement was performed using a 3.0-kb Eco RI-Hind I11 fragment encompassing most the the JH gene [20]. The unrearranged C, locus is comprised of a 12-kb BamHI germ- line fragment detectable by hybridization with a 2.5-kb Eco RI fragment, which encompasses the C, gene [21]. The analysis of h light chain gene rearrangement was accomplished by use of a combined probe encompassing both Ke-Oz-h and Mcgh genes (2.5-kb Eco RI-Hind 111 and 3.5-kb Eco RI-Hind I11 frag- ments) [22], capable of cross-hybridizing with all known Ch genes. Analysis of TCRy gene status used a 0.7-kb EcoRI- HindIII genomic J,1 fragment [6]. The J,1 complex is con- tained in a 1.4-kb Eco RI, the J,2 complex is situated in a 3.2- kb EcoRI fragment. TCRP analysis was accomplished by hybridization of YT35 cDNA [23] to BamHI, HindIII and Eco RI digests, and rearrangements were evaluated as described [24]. * Supported by grants from the Deutsche Stiftung fur Krebsforschung (to TLJB), and the Deutsche Forschungsgemeinschaft (Ga33311-1). 2.3 DNA biochemistry and hybridization analysis Correspondence: Thomas L. J. Boehm, Medical Research Council Leukemic blasts were purified through Ficoll (Pharmacia, Uppsala, Sweden) gradients and Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, the DNA was extracted from purified nuclei [18]. After com- GB Abbreviations: zyxwvutsrq TCR: T cell receptor I@: Immunoglobulin heavy pkte digestion with restriction endonucleases, DNAS Were chain V: Variable J: Joining AL: Acute leukemias ALL: Acute prepared for Southern blot hybridization analysis as described 9570) by lymphoblastic leukemias PBL: Peripheral blood lymphocytes [181. zyxwvu 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1987 0014-2980/87/1111-1593$02.50/0