Tumor formation in nude mice. (A) Kaplan-Meier plot of nude mice after tail vein injection with 10⁶ transformed cells. No difference in the course of the disease was detected, irrespective of whether TYK2^+/– or TYK2^–/– transformed cells were injected (n = 8 mice for each genotype). (B) Mean spleen weights of nude mice injected with TYK2^+/– or TYK2^–/– transformed cells. Data show means ± SE (n = 8 for each genotype). (C) Infiltration of CD19⁺ cells in bone marrow was determined by FACS. Data show means ± SE (n = 8 for each genotype).

Effect of TYK2 deficiency on tumor surveillance in other lymphoid malignancies. (A) Kaplan-Meier plot of TEL-JAK2 transgenic TYK2^+/– and TEL-JAK2 transgenic TYK2^–/– mice (n = 10 for TEL-JAK2/TYK2^+/– and n = 6 for TEL-JAK2/TYK2^–/–). TEL-JAK2 transgenic TYK2^–/– animals developed disease significantly earlier when compared with their TEL-JAK2 transgenic TYK2^+/– littermate controls as determined by a log rank test (P = 0.0273). (B) Mean tumor weights of TYK2^+/+ and TYK2^–/– animals injected with 10⁵ EL-4 cells. Data show means ± SE (n = 6 for each genotype; weight, 0.13 ± 0.079 g and 0.476 ± 0.076 g for TYK2^+/+ and TYK2^–/– mice, respectively; P = 0.0099).

Effect of in vivo oncogenic challenge with the Abelson oncogene. (A) Kaplan-Meier plot of TYK2^+/– and TYK2^–/– mice after injection of a replication-incompetent A-MuLV retrovirus (n = 20 for each group). All 20 TYK2^–/– mice died of leukemia/lymphoma within 10 weeks, whereas 6 TYK2^+/– mice remained disease free for more than 6 months. (B) White blood cell count of the diseased TYK2^+/– and TYK2^–/– animals (n = 8 for TYK2^–/– and n = 15 for TYK2^+/–). (C) H&E-stained blood smears of TYK2^+/– and TYK2^–/– mice (magnification, ×200). Typical examples of each genotype are shown. (D) H&E-stained histological sections (magnification, ×200) of liver (upper panels) and spleen (lower panels) of TYK2^+/– and TYK2^–/– animals. One representative example is depicted.

Analysis of lymph nodes and primary lymphoma. (A) Lymph nodes of TYK2^+/+, TYK2^+/–, and TYK2^–/– mice (n = 4 each) were excised and analyzed for the composition of B, T, NK, and NKT cells by FACS. One representative example of each genotype is depicted. Numbers of CD4⁺ T cells and CD8⁺ T cells in the lymph nodes are shown in the upper panels and numbers of NK cells (NK1.1⁺CD3^–), NKT cells (NK1.1⁺CD3⁺), and CTLs (NK1.1^–CD3⁺) in the lower panels. (B) MHC class I expression of TYK2^+/– and TYK2^–/– tumor-derived cell lines was analyzed by FACS (n = 4 for each genotype). One representative example is depicted; no differences were observed in any of the samples investigated. (C) Lymphomas of TYK2^+/– and TYK2^–/– mice were excised and analyzed for infiltrated NK cells (left panel) and NKT cells (right panel) by FACS (n = 7 individual TYK2^–/– mice and n = 10 individual TYK2^+/– mice).

Effect of TYK2^–/– NK/NKT cells on tumor target cells. (A and B) Cytotoxicity assay using in vitro_expanded NK/NKT cells of WT (diamonds) and TYK2^–/– animals (squares) as effector cells and the TYK2^–/– tumor-derived cell line (A) or the TYK2^+/– cell line (B) as target cells. Irrespective of the target cells, TYK2^–/–-derived NK/NKT cells had a reduced cytolytic ability. Each assay combination was tested at least 3 times. One representative experiment is depicted. (C) Kaplan-Meier plot of TYK2^+/– and TYK2^–/– mice on a RAG2^–/– background after injection of a replication-incompetent A-MuLV retrovirus (n = 14 for TYK2^+/– and n = 18 for TYK2^–/–). RAG2^–/–TYK2^–/– animals succumbed significantly earlier to Abelson-induced disease than their RAG2^–/–TYK2^+/– littermates as determined by a log rank test (P = 0.0027).

In vitro characterization of TYK2^+/– and TYK2^–/– NK/NKT cells. (A) An MTT assay did not reveal any differences in growth characteristics of TYK2^+/+-derived and TYK2^–/–-derived NK/NKT cells. Each data point represents mean ± SD from 5 individual wells. One representative experiment is depicted (n = 3). (B) Similarly, FACS analysis of the in vitro_derived cells showed a comparable composition of NK (NK1.1⁺CD3^–) and NKT (NK1.1⁺CD3⁺) cells, with an estimate of 20% NKT cells. One representative experiment is depicted (n = 6 for each genotype). (C) Production of IFN-γ in NK/NKT cells after expansion with IL-2. NK/NKT cells were either left untreated or incubated with IL-12 or IL-2 for 48 hours. Culture supernatants were analyzed for IFN-γ production by ELISA. One representative experiment is shown (n = 4). (D) RT-PCR and subsequent oligohybridization for IFN-γ from TYK2^+/–-derived and TYK2^–/–-derived tumors. Representative examples of the analysis are shown in the upper panel. Activated (IL-2 and anti-CD3) primary T lymphocytes were used as positive control (Co). An RT-PCR reaction for β-actin was performed to control for the prepared cDNA. IFN-γ was only detected in tumors derived from TYK2^+/– animals. (E) Cytotoxicity assay using TYK2^+/+ and TYK2^–/– NK/NKT cells as effector and YAC-1 cells as target cells. One representative experiment is shown (n = 4). TYK2^–/– NK/NKT cells were consistently less efficient in lysing YAC-1 cells. E/T, effector/target; conc., concentration.

In vitro characterization of B lymphoid transformation. (A) CD19⁺CD43⁺ B lymphoid precursor cells of TYK2^+/+ and TYK2^–/– bone marrow were identified by FACS analysis. One typical example is depicted (n = 10 for each genotype). (B) Bone marrow of TYK2^+/– and TYK2^–/– mice was kept in IL-7_containing medium for 7 days and subsequently applied to a [³H]thymidine incorporation assay. (C and D) Single-cell suspensions derived from bone marrow were infected with A-MuLV and subsequently cloned in growth factor_free (no GF) (C) or IL-7_containing methylcellulose (D). Mock-infected cell preparations are included as controls (C). No significant differences were observed. The initial number of B cells in each individual bone marrow preparation was determined by FACS. Results show the average numbers ± SD of colonies obtained (n = 6 for TYK2^+/– and n = 6 for TYK2^–/– bone marrow). (E) STAT activation pattern was analyzed by electrophoretic mobility shift assay and supershift analysis in 3 individual TYK2^+/– and TYK2^–/– primary tumor samples. One representative example of each genotype is shown. Antibodies directed against STAT1 (S1) and STAT3 (S3) reveal an activation of STAT1 and of STAT3 as indicated by supershifts (SS).