The fusion kinase ITK-SYK mimics a T cell receptor signal and drives oncogenesis in conditional mouse models of peripheral T cell lymphoma

Abstract

Peripheral T cell lymphomas (PTCLs) are highly aggressive malignancies with poor prognosis. Their molecular pathogenesis is not well understood and small animal models for the disease are lacking. Recently, the chromosomal translocation t(5;9)(q33;q22) generating the interleukin-2 (IL-2)-inducible T cell kinase (ITK)-spleen tyrosine kinase (SYK) fusion tyrosine kinase was identified as a recurrent event in PTCL. We show that ITK-SYK associates constitutively with lipid rafts in T cells and triggers antigen-independent phosphorylation of T cell receptor (TCR)-proximal proteins. These events lead to activation of downstream pathways and acute cellular outcomes that correspond to regular TCR ligation, including up-regulation of CD69 or production of IL-2 in vitro or deletion of thymocytes and activation of peripheral T cells in vivo. Ultimately, conditional expression of patient-derived ITK-SYK in mice induces highly malignant PTCLs with 100% penetrance that resemble the human disease. Our work demonstrates that constitutively enforced antigen receptor signaling can, in principle, act as a powerful oncogenic driver. Moreover, we establish a robust clinically relevant and genetically tractable model of human PTCL.

Figure 1.

ITK-SYK mimics a TCR signal in vitro. (A) Schematic representations of ITK, SYK, and ITK-SYK. Kinase, tyrosine kinase domain. See text for details. (B) Constitutive ITK-SYK signaling in T cell lipid rafts. Jurkat cells were infected with retroviruses carrying ITK-SYK, ITK-SYK^KD, ITK-SYK^PHmut together with GFP (ITK-SYK, ITK-SYK^KD, or ITK-SYK^PHmut), or GFP only as a control (GFP). Lipid raft fractions from unstimulated or anti-CD3– (5 µg/ml) and anti-CD28 (1 µg/ml)–stimulated cells were prepared and subjected to Western blot analysis with antibodies against ITK (top), phospho-tyrosine (p-Tyr), or phosphorylated PLCγ1 (p-PLCγ1). Dot blots with the lipid raft marker GM1 confirm equal loading. Data shown are representative of at least two independent experiments. (C) Phosflow analysis of ITK-SYK signaling. Jurkat cells were infected with ITK-SYK together with GFP or GFP only expressing retrovirus and intracellularly stained with activation-specific anti-phospho antibodies directed against the indicated T cell signaling molecules. Signaling was analyzed within the infected viable GFP⁺ populations. Data shown are representative of four independent experiments. (D) ITK-SYK triggers kinase and PH domain–dependent T cell activation. Jurkat cells were infected as in B, left unstimulated, stimulated with 10 µg/ml anti-CD3 and 2 µg/ml anti-CD28, or treated with 2 µM R406 as indicated and analyzed by FACS. The percentage of infected GFP⁺ CD69-expressing cells from unstimulated, stimulated, or R406-treated cells is indicated. Data shown are representative of five independent experiments. (E) ITK-SYK induces kinase and PH domain–dependent IL-2 production. Jurkat cells were infected as in B. Cells were left unstimulated, stimulated with 10 µg/ml anti-CD3 and 2 µg/ml anti-CD28, or treated with 2 µM R406 as indicated. IL-2 concentrations in the cell supernatants were determined by ELISA. Shown are the mean ± SD from triplicate samples. Data shown are representative of four independent experiments.

Figure 2.

A mouse model for conditional ITK-SYK expression. (A) Schematic representation of the gene-targeting strategy. A targeting vector that carries the human ITK-SYK cDNA together with an IRES eGFP sequence preceded by a loxP-flanked NEO-STOP cassette was constructed and used to generate Rosa26^{loxSTOPlox-ITK-SYK} mice as described in Materials and methods. The recombinant Rosa26 locus and the ITK-SYK–expressing locus upon Cre-mediated deletion of the NEO-STOP cassette are indicated. SA, splice acceptor site; pA, polyA sequence; 1–3, Rosa26 exon 1–3; probe, flanking probe for Southern blot analysis. (B) Southern blot analysis. Genomic DNA from a wild-type (WT) and a successfully targeted Rosa26^{loxSTOPlox-ITK-SYK} embryonic stem cell clone was digested with XbaI and Southern blotted with the flanking probe indicated in A. Sizes of the wild-type and recombinant (Rec) fragments are indicated. (C) Conditional expression of ITK-SYK in T and B cells in vivo. ROSA26^{loxSTOPlox-ITK-SYK} mice were crossed to CD4-Cre or CD19-Cre transgenic mice for T or B cell–specific ITK-SYK expression. Peripheral lymphocyte suspensions of double transgenic 5-wk-old ITK-SYK^CD4-Cre or ITK-SYK^CD19-Cre mice were stained against TCR-β or B220. eGFP fluorescence indicative of ITK-SYK expression in the TCR-β⁺ T cells or B220⁺ B cells of the respective animals was analyzed using FACS. Data shown are representative of >40 mice per genotype analyzed. (D) Western blot analysis of conditional ITK-SYK expression. Individual mature T and B cell populations from 5-wk-old Rosa26^{loxSTOPlox-ITK-SYK}, ITK-SYK^CD4-Cre, and ITK-SYK^CD19-Cre mice were sorted with magnetic beads and subjected to Western blot analysis with an anti-ITK antibody. Bands of wild-type ITK and ITK-SYK are indicated. Western blotting for β-actin demonstrated equal protein loading. Data shown are representative of five independent experiments.

Figure 3.

ITK-SYK mimics a TCR signal in vivo. (A) ITK-SYK expression in the thymus of ITK-SYK^CD4-Cre animals. Thymocytes were stained against CD4 and CD8 and analyzed by FACS. eGFP fluorescence indicative of ITK-SYK expression was measured in the CD4⁺CD8⁺ DP and in the CD4⁺ or CD8⁺ SP populations of 4–5-wk-old ITK-SYK^CD4-Cre or control (CD4-Cre transgenic) mice. (B and C) ITK-SYK induces DP thymocyte deletion. (B) Thymocytes were stained as in A and analyzed for the expression of CD4 or CD8. The frequencies of individual thymocyte subsets are indicated. (C) The total DP or SP thymocyte cell numbers from ITK-SYK^CD4-Cre (n = 9) or control (n = 6) mice are shown. (D) Total splenic B or T cell numbers from ITK-SYK^CD4-Cre (n = 10) or control (n = 9) mice are indicated. Each symbol in C and D represents an individual mouse. Statistical significance was analyzed using the unpaired two-tailed Student’s t test. ***, statistically significant (P < 0.0001); ns, not statistically significant (P ≥ 0.05). Horizontal bars indicate the means. (E and F) Single cell suspensions from lymph node (LN) or spleen (SPL) from 4–5-wk-old ITK-SYK^CD4-Cre or control mice were stained against CD4 and CD8. (E) CD4 and CD8 expression was analyzed by FACS. The frequencies of individual T cell subsets are indicated. (F) eGFP fluorescence indicative of ITK-SYK expression was determined in peripheral CD4⁺ or CD8⁺ T cells of ITK-SYK^CD4-Cre or control mice. Data from spleen are shown. (G) ITK-SYK expressing T cells exhibit an activated phenotype in vivo. Peripheral lymphocytes from 4–5-wk-old ITK-SYK^CD4-Cre or control mice were stained against CD4, CD8, CD44, CD62L, and TCR-β. Forward scattering (FCS) as a parameter for cell size and expression of CD44, CD62L, and TCR-β was analyzed in the CD4⁺ and CD8⁺ T cell compartments using FACS. Data shown in A, B, and E–G are representative of five independent experiments with a total number of at least 10 mice analyzed per genotype. (H) ITK-SYK induces PLCγ1 phosphorylation in primary T cells in vivo. Splenic T cells were isolated from control or ITK-SYK^CD4-Cre mice that were >12 wk old. Cells were left unstimulated and control cells additionally stimulated with 5 µg/ml of anti-CD3 and 1 µg/ml of anti-CD28. Lipid raft fractions were prepared and subjected to Western blot analysis with an anti–phospho-PLCγ1 antibody. Dot blots for GM1 show successful raft preparation and equal loading. Data shown are representative of three independent experiments. Black lines indicate that intervening lanes have been spliced out.

Figure 4.

B cell–intrinsic ITK-SYK expression does not affect B cell development or activation. (A) Single cell suspensions from the spleens or peritoneal cavities of ITK-SYK^CD19-Cre or control (CD19-Cre) mice were stained against B220, CD21, CD23, and CD5. eGFP fluorescence indicative of ITK-SYK expression was determined in the splenic follicular (FO), splenic marginal zone (MZ), or peritoneal B1 B cell compartments using FACS. (B) Cytoplasmic extracts of purified mature B cells from 5-wk-old control or ITK-SYK^CD19-Cre mice were subjected to Western blot analysis with an antibody against phospho–tyrosine (p-Tyr). Western blotting for β-actin demonstrated equal protein loading. Data shown are representative of three independent experiments. (C) Total splenic B or T cell numbers from ITK-SYK^CD19-Cre (n = 12) or control (n = 6) mice are shown. Each symbol represents an individual mouse. Statistical significance was analyzed using the unpaired two-tailed Student’s t test. ns, non–statistically significant differences (P ≥ 0.05). Horizontal bars indicate the means. (D) Lymphocyte preparations from bone marrow (BM) or spleen (SPL) of ITK-SYK^CD19-Cre or control mice were stained against B220, IgM, CD21, or CD23 and analyzed by FACS. The percentages of individual B cell populations are indicated. FO, follicular B cells; MZ, marginal zone B cells. (E) Forward scattering (FCS) and expression of CD86 was analyzed on B220⁺ mature B cells from ITK-SYK^CD19-Cre or control mice using FACS. Data shown in A and C–E are representative of four independent experiments with total numbers of at least six 5–7-wk-old mice per genotype analyzed. (F) ITK-SYK is constitutively associated with T but not with B cell lipid rafts. Lipid raft and nonraft fractions were prepared from purified splenic T cells of control or ITK-SYK^CD4-Cre mice or from mature B cells of ITK-SYK^CD19-Cre mice that were >12 wk old. The fractions were subsequently analyzed for the presence of ITK or ITK-SYK by Western blot analysis using an antibody against ITK. Dot blots for GM1 show successful raft preparation and equal loading. Data shown are representative of three independent experiments.

Figure 5.

Conditional expression of ITK-SYK in T cells induces a lymphoproliferative disease. (A) Peripheral blood samples from ITK-SYK^CD4-Cre (starting with n = 19) and ITK-SYK^CD19-Cre mice (n = 8) were obtained at the indicated time points. The frequencies of eGFP fluorescent lymphocytes in individual animals were determined by FACS analysis. The total number of ITK-SYK^CD4-Cre mice declined over time as a result of disease-related mortality. Horizontal bars indicate the means. (B) Kaplan-Meier curve of ITK-SYK^CD4-Cre (n = 73) and control (CD4-Cre) mice (n = 15). (C) Macroscopic appearance of representative spleens from 20-wk-old control and ITK-SYK^CD4-Cre mice are shown (in centimeters). (D) Splenocytes from 20-wk-old control and ITK-SYK^CD4-Cre mice were stained with antibodies against TCR-β. The frequency of eGFP–expressing T cells in a diseased ITK-SYK^CD4-Cre mouse is indicated. Data shown in C and D are representative of a total number of 20 mice per genotype analyzed.

Figure 6.

PTCL in ITK-SYK^CD4-Cre mice. (A) Disruption of the splenic architecture with highly proliferative cells in ITK-SYK^CD4-Cre mice was revealed by hematoxylin and eosin (H&E) staining and immunohistochemistry with anti–Ki-67 antibodies. Bars: (black) 1 mm; (white) 50 µm. Data shown are representative of five diseased ITK-SYK^CD4-Cre mice analyzed. (B) Splenic cells from diseased ITK-SYK^CD4-Cre mice were stained against CD4 and CD8 and analyzed by FACS. Selected examples of each type of T cell expansion from a total number of 40 mice analyzed are shown. (C) Bone marrow cell preparations from diseased ITK-SYK^CD4-Cre or control (CD4-Cre) mice were stained with antibodies against TCR-β. The frequency of eGFP⁺ T cells is indicated. Data are representative of five independent experiments with a total number of 15 mice per genotype analyzed. (D) Solid organ infiltration of abnormal CD3⁺ T cells. Tissue sections from kidney (KID), liver (LIV), and lung (LNG) of affected ITK-SYK^CD4-Cre animals were stained with H&E. Immunohistochemistry with anti-CD3 antibodies was additionally performed. Bars: (black) 200 µm; (white), 1 mm. Data shown are representative of five diseased ITK-SYK^CD4-Cre mice analyzed. (E) Selective expansion of distinct T cell clones. Single cell suspensions from spleen (SPL), kidney (KID), and liver (LIV) of three individual mice were stained with antibodies against CD4, CD8, and a panel of TCR-Vβ chain–specific antibodies (see Materials and methods). Frequencies of CD4⁺ or CD8⁺ cells expressing the indicated TCR-Vβ chains in the spleen of control mice (open histograms) or frequencies of cells expressing the indicated TCR-Vβ chains in spleen, kidney, and liver from diseased ITK-SYK^CD4-Cre mice (gray histograms) are shown. (F) Genescan analysis for TCR gene rearrangements. Representative fragment size distributions of fluorochrome-labeled PCR products of the Dβ2/Jβ2 junction of a control and a diseased ITK-SYK^CD4-Cre mouse are shown. Data shown are representative of three mice per genotype analyzed. Data in A–F are from ITK-SYK^CD4-Cre mice that were older than 12 wk and showed disease symptoms.

Figure 7.

Proliferation and infiltration of ITK-SYK–expressing T cells upon transplantation. (A) Splenic cells from diseased ITK-SYK^CD4-Cre animals older than 12 wk of age were intravenously injected into nude recipient mice for transplantation. The frequencies of ITK-SYK–expressing eGFP⁺ peripheral blood cells in the recipients were monitored over time. Recipients that succumbed to the disease are indicated (†). Representative examples are shown. 9 out of 10 donor mice transplanted the disease. Five independent transplantations were performed. (B) Spleens from a control and a representative recipient mouse from A are shown (in centimeters). (C) Recipients were sacrificed upon signs of disease, and bone marrow (BM) and spleen (SPL) suspensions were stained against TCR-β. Representative frequencies of eGFP fluorescent T cells in spleen and bone marrow of 10 analyzed diseased recipients are indicated. (D) Tissue sections from spleen (SPL), liver (LIV), and lung (LNG) were analyzed after H&E staining or immunohistochemistry with anti-CD3 or anti-Ki-67 antibodies. Bars, 1 mm. Representative examples of four analyzed diseased recipients are shown.

Figure 8.

ITK-SYK^CD19-Cre mice develop clonal PTCLs. (A) ITK-SYK^CD19-Cre mice succumb to disease. Kaplan-Meier curve of ITK-SYK^CD19-Cre (n = 18) and control (CD19-Cre) mice (n = 10). (B) Splenomegaly in ITK-SYK^CD19-Cre mice. Representative spleens from 50-wk-old control and ITK-SYK^CD19-Cre mice are shown (in centimeters). (C) Solid organ infiltration with abnormally proliferating T cells in ITK-SYK^CD19-Cre mice. H&E staining and immunohistochemistry with anti-CD3 and anti–Ki-67 antibodies were performed on liver (LIV) and lung (LNG) sections. Bars, 1 mm. Data representative of six diseased ITK-SYK^CD19-Cre mice are shown. (D) Bone marrow (BM) or spleen (SPL) cell suspensions of 50-wk-old control and ITK-SYK^CD19-Cre mice were stained against TCR-β. The frequency of eGFP⁺ T cells in a representative ITK-SYK^CD19-Cre mouse is indicated. (E) Expanded T cells in ITK-SYK^CD19-Cre mice display an activated phenotype. CD44 and CD62L surface expression on affected T cells or controls was assessed using FACS. One representative example of CD4⁺ T cells is shown. (F) Spleen cells from diseased ITK-SYK^CD19-Cre mice (n = 16) were stained against CD4 and CD8. Examples of preferential CD4⁺ T cell (Type I) or CD8⁺ T cell (Type II) expansions are shown. (G) T cell populations in ITK-SYK^CD19-Cre mice are clonal. Single cell suspensions from spleens of individual mice were stained with antibodies against CD4, CD8, and a panel of antibodies against TCR-Vβ chains (see Materials and methods). Frequencies of CD4⁺ or CD8⁺ cells expressing the indicated TCR-Vβ chains in control (open histograms) or diseased ITK-SYK^CD19-Cre (gray histograms) mice are shown. Data in B and D–G are representative of six independent experiments with a total number of at least 10 mice analyzed per genotype are shown. If not indicated otherwise, all panels show data from ITK-SYK^CD19-Cre mice that were older than 40 wk and showed disease symptoms.