TEL-JAK2 transgenic mice develop T-cell leukemia by Clémence Carron, Françoise Cormier, Anne Janin, Virginie Lacronique, Marco Giovannini, Marie-Thérèse Daniel, Olivier Bernard, and Jacques Ghysdael Blood Volume 95(12):3891-3899 June 15, 2000 ©2000 by American Society of Hematology

Schematic representation of HA-TEL-JAK2 transgene Schematic representation of HA-TEL-JAK2 transgene.The HA-TEL-JAK2 cDNA13 was inserted 3′ of the Eμ/SRα enhancer/promoter (thick hatched and dark boxes), which allows high-level specific expression in lymphoid cells but only low to undetectable expression in... Schematic representation of HA-TEL-JAK2 transgene.The HA-TEL-JAK2 cDNA13 was inserted 3′ of the Eμ/SRα enhancer/promoter (thick hatched and dark boxes), which allows high-level specific expression in lymphoid cells but only low to undetectable expression in nonlymphoid tissues.27 The 3′ end of the transgene contains the rabbit globin poly(A) addition site. The self-association domain of TEL is represented by a black box. The JH1 domain (catalytic domain) of JAK2 is represented by a thin hatched box. Clémence Carron et al. Blood 2000;95:3891-3899 ©2000 by American Society of Hematology

: Histopathological and cytologic analysis of TEL-JAK2 transgenic mice : Histopathological and cytologic analysis of TEL-JAK2 transgenic mice.(A) Macroscopic appearance of an F1 transgenic mouse showing hyperplasia of spleen (Sp); thymus (Th); and cervical (C), axillary (A), and femoral (F) lymph nodes. : Histopathological and cytologic analysis of TEL-JAK2 transgenic mice.(A) Macroscopic appearance of an F1 transgenic mouse showing hyperplasia of spleen (Sp); thymus (Th); and cervical (C), axillary (A), and femoral (F) lymph nodes. (B) May-Grünwald-Giemsa (MGG) staining of blood smears of transgenic mice. Note the morphology resembling that of prolymphocytes, the size heterogeneity, and the presence of a mitotic cell. (C-E) Histopathology of hematopoietic organs. (C) Thymus showing invasion of the perilobular capsule (white arrows) (HE). (D) Spleen; note the loss of its normal architecture (HE). (E) Bone marrow; note infiltration by leukemic cells (MGG). (F and G) Histopathology of nonhematopoietic organs (HE) showing infiltration of leukemic cells in the (F) pericentrolobular area and sinusoids of the liver (HE) and (G) brain; subarachnoid area (HE). (H) Kidney; infiltration of leukemic cells around peritubular capillaries (HE). Clémence Carron et al. Blood 2000;95:3891-3899 ©2000 by American Society of Hematology

Infiltrating lymphoid cells in TEL-JAK2 transgenic mice are T cells Infiltrating lymphoid cells in TEL-JAK2 transgenic mice are T cells.A lymph node section from a diseased mouse was double stained with anti-Thy1.2–FITC antibody and anti-B220–PE antibody and examined for expression of Thy1.2 (A) or B220 (B). Infiltrating lymphoid cells in TEL-JAK2 transgenic mice are T cells.A lymph node section from a diseased mouse was double stained with anti-Thy1.2–FITC antibody and anti-B220–PE antibody and examined for expression of Thy1.2 (A) or B220 (B). A nontransgenic liver section (C) and the liver section from a diseased mouse (D) were stained with anti-Thy1.2 antibody. Clémence Carron et al. Blood 2000;95:3891-3899 ©2000 by American Society of Hematology

: CD4 and CD8 expression in TEL-JAK2 leukemic cells : CD4 and CD8 expression in TEL-JAK2 leukemic cells.(A) Single-cell suspensions from lymph nodes (LN), thymus (Th), spleen (Sp), and bone marrow (BM) were obtained from a transgenic leukemic animal (71-4) and its sibling control (71-2). : CD4 and CD8 expression in TEL-JAK2 leukemic cells.(A) Single-cell suspensions from lymph nodes (LN), thymus (Th), spleen (Sp), and bone marrow (BM) were obtained from a transgenic leukemic animal (71-4) and its sibling control (71-2). (B) Single-cell suspensions obtained from a tumor generated in nude mice (Nu18; Table3) were sorted as CD8+ SP and CD4+CD8+ DP as described in “Materials and methods.” The CD4 and CD8 expression pattern of unsorted cells (left), CD8+ SP cells (middle), and DP cells (right) was determined by flow cytometry. For each experiment, 106cells were incubated with anti-CD4–PE (Y-axis) and anti-CD8–FITC (X-axis) and analyzed by flow cytometry. The results were computed from the acquisition of 10 000 events. The relative percentage of each subpopulation (CD4+CD8−; CD4−CD8+; and CD4+CD8+) is indicated in the corresponding area of the diagrams. Clémence Carron et al. Blood 2000;95:3891-3899 ©2000 by American Society of Hematology

Expression of HA-TEL-JAK2 transgene.(A) Northern blot analysis. Expression of HA-TEL-JAK2 transgene.(A) Northern blot analysis. Total mRNA from diseased spleen (lane 1), thymus (lane 2), lymph nodes (lane 3), and from a control spleen (lane 4) were hybridized as indicated with probes specific for TEL or JAK2. The TEL-JAK2 transgene mRNA is indicated by a white arrowhead. Bands corresponding to the endogenous TEL and JAK2 transcripts are indicated by gray and black arrowheads, respectively. (B) Flow cytometric detection of HA-TEL-JAK2 protein expression in diseased thymus and lymph nodes. Double labeling with mouse anti-Thy1.2–PE and rabbit anti-HA plus antirabbit Ig-FITC antibodies revealed that Thy1.2+ cells express the HA epitope. For the thymus, FITC-fluorescence intensity in gated Thy1.2+ cells is represented for a transgenic animal (71-10, dark surface) and for a control animal (71-6, open surface). For diseased lymph nodes, labeling is with the anti-HA rabbit antibody plus antirabbit Ig-FITC (dark surface) or with antirabbit Ig-FITC alone (open surface). (C) Western blot analysis of cellular extracts from lymph nodes, thymus, and spleen from diseased and control animals. Extracts corresponding to the same number of cells from each organ were immunoprecipitated by anti-JAK2 antibody, separated by SDS-PAGE, and blotted on nitrocellulose. Blots were analyzed with either the anti-HA (12CA5, Boehringer) or anti-TEL23 antibodies (upper panel) or with the 4G10 phosphotyrosine-specific antibody (lower panel). Lanes 1 and 2: lymph nodes from transgenic animals 71-4 and 71-18. Lanes 3 and 5: thymus from transgenic mice 65 and 71-18. Lane 6: spleen from transgenic mouse 71-18. Lanes 4 and 7: control thymus and spleen, respectively. The HA-TEL-JAK2 protein is indicated by a white arrowhead. Clémence Carron et al. Blood 2000;95:3891-3899 ©2000 by American Society of Hematology

TEL-JAK2 leukemic cells express activated STAT1 and STAT5 TEL-JAK2 leukemic cells express activated STAT1 and STAT5.Electrophoretic mobility shift assays were carried out with either the m67SIE probe (upper panel) or the β casein probe (lower panel) and whole-cell extracts from the spleen and lymph nodes of a dise... TEL-JAK2 leukemic cells express activated STAT1 and STAT5.Electrophoretic mobility shift assays were carried out with either the m67SIE probe (upper panel) or the β casein probe (lower panel) and whole-cell extracts from the spleen and lymph nodes of a diseased transgenic animal (71-45) or from a control spleen. Positions of the STAT/DNA complexes are indicated by open arrowheads. Specific and nonspecific competitors were added as indicated. For supershift experiments, anti-STAT1 or anti-STAT5 antibodies were added as indicated. Supershifted complexes are indicated by black arrowheads. Clémence Carron et al. Blood 2000;95:3891-3899 ©2000 by American Society of Hematology

TCRβ gene rearrangement in TEL-JAK2 leukemic cells TCRβ gene rearrangement in TEL-JAK2 leukemic cells.(A) DNA was extracted from the thymus (T) from founder mouse 65, from 4 F1 animals of transgenic line 71 (71-11, 71-24, 71-10, and 71-4), and from a control thymus. TCRβ gene rearrangement in TEL-JAK2 leukemic cells.(A) DNA was extracted from the thymus (T) from founder mouse 65, from 4 F1 animals of transgenic line 71 (71-11, 71-24, 71-10, and 71-4), and from a control thymus. Lymph nodes (LN) from transgenic mice 30 and 71-4 and the liver (L) from mouse 65 were also analyzed. DNA were digested by HindIII and analyzed by Southern blot using a jβ2-specific probe. An arrow points to germline configuration. TCRβ-rearranged bands (indicated by stars) are prominant in all leukemic samples. (B) DNA was extracted from a control thymus (lane 1), from the thymus of a diseased transgenic mouse 71-18 (lane 2), from nude mice tumors obtained following transplantation either of the leukemic cells of animal 71-18 (Nu18, lane 3) or following secondary transplantation of Nu18 tumor cells (lanes 4 and 5), from nude mice tumors obtained from the transplantation of sorted SP (lanes 6-8) or DP cells (lane 11), and from the sorted CD8+SP and DP populations derived from transplanted tumors Nu18/SP4 and Nu18/DP1 (lanes 9-10 and 12-13) (see also Table 3). DNA were digested by HindIII and analyzed by Southern blot using a jβ2-specific probe. Germline configuration is indicated by an arrow. TCRβ-rearranged bands (indicated by stars) are prominant in all leukemic samples. (C) DNA was extracted from a control thymus (lane 1), from the thymus of diseased transgenic mouse 71-24 (lane 2), from nude mice tumors obtained either following transplantation of the leukemic cells of animal 71-24 (Nu24, lane 3) or following secondary transplantation of Nu24 tumor cells (Nu24/1, lane 4), from nude mice tumors obtained from the transplantation of sorted SP (Nu24/SP1, lane 7), and from the sorted DP and CD8+ SP populations derived from either transplanted tumors Nu24/1 (lanes 5 and 6) or Nu24/SP1 (lanes 8 and 9) (see also Table 3). DNA were digested byHindIII and analyzed by Southern blot using a jβ2-specific probe. Germline configuration is indicated by an arrow. TCRβ-rearranged bands (indicated by stars) are prominant in all leukemic samples. Clémence Carron et al. Blood 2000;95:3891-3899 ©2000 by American Society of Hematology