Premature Terminal Differentiation Protects from Deregulated Lymphocyte Activation by ITK-Syk

This information is current as Martina P. Bach, Eva Hug, Markus Werner, Julian Holch, of September 28, 2021. Clara Sprissler, Konstanze Pechloff, Katja Zirlik, Robert Zeiser, Christine Dierks, Jürgen Ruland and Hassan Jumaa J Immunol 2014; 192:1024-1033; Prepublished online 27 December 2013;

doi: 10.4049/jimmunol.1300420 Downloaded from http://www.jimmunol.org/content/192/3/1024

Supplementary http://www.jimmunol.org/content/suppl/2013/12/26/jimmunol.130042 Material 0.DCSupplemental http://www.jimmunol.org/ References This article cites 55 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/192/3/1024.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Premature Terminal Differentiation Protects from Deregulated Lymphocyte Activation by ITK-Syk

Martina P. Bach,*,† Eva Hug,† Markus Werner,*,† Julian Holch,‡ Clara Sprissler,x Konstanze Pechloff,‡,{ Katja Zirlik,x Robert Zeiser,x Christine Dierks,x Ju¨rgen Ruland,‡,{ and Hassan Jumaa*,†

The development of hematopoietic neoplasms is often associated with mutations, altered expression or chromosomal trans- locations. Recently, the t(5, 9)(q33;q22) translocation was found in a subset of peripheral lymphomas and was shown to result in an IL-2–inducible –spleen (ITK-Syk) fusion transcript. In this study, we show that T cell–specific expression of the ITK-Syk oncogene in mice leads to an early onset and aggressive polyclonal T cell lymphoproliferation with concomitant B cell expansion and systemic inflammation by 7–9 wk of age. Because this phenotype is strikingly different from previous work showing that ITK-Syk expression causes clonal T cell lymphoma by 20–27 wk of age, we investigated the underlying Downloaded from molecular mechanism in more detail. We show that the reason for the severe phenotype is the lack of B-lymphocyte–induced maturation -1 (Blimp-1) induction by low ITK-Syk expression. In contrast, high ITK-Syk oncogene expression induces terminal T cell differentiation in the thymus by activating Blimp-1, thereby leading to elimination of oncogene-expressing cells early in development. Our data suggest that terminal differentiation is an important mechanism to prevent oncogene-expressing cells from malignant transformation, as high ITK-Syk oncogene activity induces cell elimination. Accordingly, for transformation, a specific amount of oncogene is required, or alternatively, the induction of terminal differentiation is defective. The Journal of http://www.jimmunol.org/ Immunology, 2014, 192: 1024–1033.

pleen tyrosine kinase (Syk) is a nonreceptor tyrosine kinase phocytic leukemia and diffuse large B cell lymphoma cells, in- that possesses two N-terminal Src homology 2 domains and dicating that Syk is crucial for the survival of these cells (7, 8). S a C-terminal kinase domain. Syk is widely expressed in Syk has also been linked to malignancies of other hematopoietic all hematopoietic cells, mediates various biological functions, and lineages such as peripheral T cell lymphomas (PTCLs), in which plays a central role in B cell development and signal transduction Syk is overexpressed in .90% of all PTCLs (9).

downstream of the BCR (1–4). In T cells, Syk is mainly expressed ThegeneencodingSykhasalsobeenshowntobeaffected by guest on September 28, 2021 at the double-negative and double-positive (DP) developmental by chromosomal translocations. Chromosomal translocations are stages, but its expression is markedly reduced in later stages of thought to play a major role in the development and pathogenesis of T cell development (5, 6). Deregulated Syk expression or activity various malignancies. For example, the chromosomal translocation has been frequently linked to distinct hematological and non- t (9, 12)(q22;p12) was identified in a patient with myelodysplastic hematological malignancies and seems to play an important role syndrome and results in the expression of the TEL-Syk fusion in the development and maintenance of B cell lineage neoplasms. protein, leading to constitutive activation of Syk and the ability to For instance, inhibition of Syk induces apoptosis of chronic lym- transform various cell lines and induce leukemia in mice (10–12). Recently, an additional chromosomal translocation involving the Syk gene, the t (5, 9)(q33;q22), was found in ∼17% of unspecified † *Institute of Immunology, University Clinics Ulm, 89081 Ulm, Germany; Department PTCLs, which represent a heterogeneous and aggressive group of of Molecular Immunology, Faculty of Biology, Albert-Ludwigs University of Freiburg and Max Planck Institute of Immunobiology and Epigenetics, 79108 non-Hodgkin lymphomas (13). This translocation fuses Syk to the Freiburg, Germany; ‡Institut fu¨r Klinische Chemie und Pathobiochemie, Klini- IL-2–inducible kinase (ITK) and results in an ITK-Syk fusion kum rechts der Isar, Technische Universita¨tMu¨nchen, 81675 Munich, Germany; xDepartment of Hematology/Oncology, University Medical Center Freiburg, transcript. ITK is a member of the Tec kinase family, predomi- 79106 Freiburg, Germany; and {German Cancer Consortium, German Cancer nantly expressed in T cells and involved in normal T cell devel- Research Center, 69120 Heidelberg, Germany opment and activation. Mice deficient for ITK show a partial block Received for publication February 11, 2013. Accepted for publication November 25, in T cell development and impaired signaling downstream of the 2013. TCR (14). Upon TCR activation, ITK is recruited to the mem- This work was supported by the Deutsche Krebshilfe (Project 108935) and the Deutsche Forschungsgemeinschaft (SFB746). brane in proximity of the TCR, where it is phosphorylated and Address correspondence and reprint requests to Prof. Hassan Jumaa, Institute of interacts with various substrates including linker for activation of Immunology, University Clinics Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany. T cells, SLP-76, Grb2, and phospholipase Cg1 (PLCg1) (15). ITK E-mail address: [email protected] localization to the plasma membrane is mediated by the pleckstrin The online version of this article contains supplemental material. homology (PH) domain of ITK and is dependent on PI3K activity Abbreviations used in this article: Bcl-6, B cell lymphoma-6; Blimp-1, B-lympho- that converts phosphatidylinositol-(4, 5)-bisphosphate to phos- cyte–induced maturation protein-1; CBA, cytometric bead array; DP, double-positive; hSyk, human spleen tyrosine kinase; IRF-4, IFN regulatory factor 4; ITK, IL-2– phatidylinositol-(3,4,5)-trisphosphate, the ligand for the PH do- inducible kinase; ITK-Syk, IL-2-inducible kinase–spleen tyrosine kinase; PH, pleck- main. Once activated, ITK phosphorylates PLCg1, which induces strin homology; PLCg1, phospholipase Cg1; PTCL, peripheral T cell lymphoma; SP, calcium mobilization and the activation of downstream pathways single-positive; Syk, spleen tyrosine kinase; wt, wild-type. regulating the activation and effector function of T cells (15). The Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 ITK-Syk fusion transcript contains the PH domain, the Tec ho- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300420 The Journal of Immunology 1025 mology domain of ITK, and the kinase domain of Syk. It was (Thr202/Tyr204; Cell Signaling Technology), anti–p-PKB (Ser473; Cell Sig- naling Technology), anti–p-ZAP70 (Tyr493; Cell Signaling Technology), demonstrated that this protein exhibits kinase activity and is 783 able to phosphorylate various important signaling such anti–p-PLCg1(Tyr ; Cell Signaling Technology), and anti–Blimp-1 (C-21/ N-20; Santa Cruz Biotechnology). An APC labeling (AbD Serotec) or as PLCg1, Akt, and Erk1/2 (16, 17). secondary Ab (anti-rabbit DyLight-649; AbD Serotec) was used in case of To study the role of the ITK-Syk oncogene in disease development, unlabeled primary Abs. If not stated otherwise, Abs were obtained from BD we generated a mouse model for conditional ITK-Syk expression. Biosciences and eBioscience. Intracellular staining was performed using We found that T cell–specific ITK-Syk expression leads to a severe FIX&PERM Cell PermeabilizationKit(AnDerGrubBioResearch, Kaumberg, Austria). FACS analysis was performed with FACSCalibur or disease development with an early onset and aggressive polyclonal LSR II (BD Biosciences). Data were analyzed using FlowJo software lymphoproliferation by 7–9 wk of age, which is strikingly different (Tree Star). from available data showing that T cell–specific ITK-Syk expression Western blot analysis results in clonal T cell proliferation by 20–27 wk of age (17). In this study, we characterize the underlying mechanism for the early onset Transduced pre-T cells were sorted with a MoFlo (DakoCytomation) cell of disease and show that reduced oncogene expression prevents cell sorter. For isolation of splenic T cells, splenocytes were stained with anti– Thy1.2-biotin (BD Biosciences) before incubation with streptavidin- elimination by a cell-autonomous mechanism. labeled magnetic beads (Miltenyi Biotec). Purification was performed with AutoMACS (Miltenyi Biotec). Cells were lysed in RIPA lysis buffer Materials and Methods containing 50 mM Tris-HCl, 1% Nonidet P-40, 0.25% deoxycholate, 150 mM Mice NaCl, 1 mM EDTA, 0.5 mM Na3VO4, 5 mM NaF, and protease inhibi- tor mixture (Sigma-Aldrich). Samples were separated on 10% SDS- To generate the ITK-Syk mice, the human ITK-Syk cDNA (Supplemental polyacrylamide gels and transferred onto polyvinylidene difluoride mem- Fig. 1A) (13) was cloned into a ROSA26 targeting vector (18), linearized, branes (Millipore). Membranes were blocked in 5% milk powder in PBS Downloaded from and electroporated into W4 embryonic stem cells obtained from 129S6/ with 0.1% Tween 20. Primary Abs were diluted in PBS with 0.1% Tween SvEv mice. Homologous recombination at the ROSA26 was con- 20 supplemented with 2% BSA fraction V (Biomol) and 0.1% sodium firmed by PCR screening (forward, 59-CCTCAGAGAGCCTCGGCTA-39; azide (Sigma-Aldrich). Immunoreactive proteins were detected using reverse, 59-GGACAGGATAAGTATGACATCA-39) and Southern blot a chemoluminescence detection system (ECL). Anti-hSyk (4D10; Santa analysis. Southern blots from HindIII-digested embryonic stem cell ge- Cruz Biotechnology), anti–Blimp-1 (6D3; Santa Cruz Biotechnology), nomic DNA were hybridized with a 59-probe (19) and analyzed by phos- anti-actin (l-19; Santa Cruz Biotechnology), and anti-BAP31 (22) Abs

phoimaging. Two clones were injected into blastocysts obtained from were used for immunoblotting. http://www.jimmunol.org/ C57BL/6 mice and transferred back into foster females to obtain chimeric mice. The resulting ROSA26-ITK-Syk mice were crossed to either CD4-Cre ELISA and cytometric bead array analysis mice (20) or -Cre mice (21). Mice were checked daily and subsequently Serum of mice was collected using serum collection tubes (Microtainer; sacrificed and analyzed once the first prognostic symptoms were observed. All BD Biosciences), and Ig titers were determined by ELISA. All Abs were mice were bred at the animal facility of the Max Planck Institute of Immu- obtained from Southern Biotechnology Associates. Secondary Abs were nobiology and Epigenetics. Animal experiments were done in compliance with AP-labeled using 4-nitrophenyl phosphate disodium salt hexahydrate the guidelines of German law. (Sigma-Aldrich) as detection reagent. Human subjects Cytokine levels were analyzed using a cytometric bead array (CBA) mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences). For in vitro cytokine All human tissue samples were collected after approval by the ethics detection, splenic T cells were isolated by negative selection with magnetic by guest on September 28, 2021 committee of the Albert-Ludwigs University Freiburg (protocol number: beads (Miltenyi Biotec). Cells were either left untreated or stimulated with 267/11) and after written informed consent. Defined as positive for ITK-Syk 3 mg/ml anti-CD3ε and 10 mg/ml anti-CD28 (eBioscience). Supernatants were those samples showing a Syk-specific protein band at the expected size. were harvested after 30 h and subjected to CBA analysis. Measurements were performed with FACSCalibur or LSR II (BD Biosciences). Data were Plasmids and retroviral transduction analyzed using FCAP Software (Soft Flow). Retroviral constructs were generated by cloning the human ITK-Syk cDNA Real-time PCR analysis together with flanking sequences leading to low and high ITK-Syk ex- pression into a retroviral vector backbone containing an internal ribosome Total RNA was isolated using Quick-RNA Mini Kit (Zymo Research) or entry site–GFP sequence (pMIG). For retroviral transductions, viral RNeasy Plus Mini KIT MiniPrep (Qiagen). The synthesis of cDNA was supernatants were produced using the Phoenix retroviral producer cell line performed as previously described (23). was determined and GeneJuice (EMD Millipore) transfection reagent. Retroviral super- with gene-specific primers using the SYBR Green detection method (Applied natants were harvested after 48 h, subsequently mixed with Beko pre- Biosystems) and a 7500 Fast Real-Time PCR machine (Applied Biosystems). T cells or freshly isolated wild-type (wt) thymocytes, and centrifuged at Results were calculated by the D threshold cycle method. 300 3 g at 37˚C for 2 to 3 h. Histology and immunofluorescence Culture conditions For immunohistochemistry, mouse tissues were fixed in formalin, and Primary cells were grown in Iscove’s medium (Biochrom) containing 10% paraffin-embedded sections were stained with H&E or overnight with anti- heat-inactivated FCS (PAN-Biotech), 10 mM L-glutamine (Life Technolo- CD3 (Abcam) primary Ab followed by a 30-min incubation with HRP- gies), 100 U/ml penicillin, 100 U/ml streptomycin (Life Technologies), coupled secondary Abs. Immunodetection was performed using 3,39- 50 mM 2-ME, and 9 ml (per 500 ml medium) culture supernatant from J558L diaminobenzidine (EnVision+System-HRP; DakoCytomation). Photographs cells stably transfected with murine IL-7 expression vector. Phoenix cell were were taken with Axioplan 2 (Zeiss). For immunofluorescence, cells (5 3 103 grown in Iscove’s medium (Biochrom) containing 10% of heat- cells/chamber) were seeded into eight-chamber slides and fixed with inactivated FCS (PAN-Biotech), 10 mM L-glutamine (Life Technolo- 2% paraformaldehyde for 15 min at room temperature, followed by gies), 100 U/ml penicillin, 100 U/ml streptomycin (Life Technologies), permeabilization with 0.5% saponin and 1% BSA in PBS for 30 min. and 50 mM 2-ME. Beko pre-T cells were grown in Life Technologies After blocking, cells were subjected to immunofluorescence staining with IMDM complemented with 25 mM HEPES, 4 mM L-glutamine, 10% of anti-hSyk (4D10; Santa Cruz Biotechnology) and anti-mouse Cy3 (Invi- heat-inactivated FCS (PAN-Biotech), 1 mM MEM nonessential amino trogen). Cells were examined by fluorescence microscopy with the LSM acids (Life Technologies), 2.5 mg insulin (Sigma-Aldrich), 100 U/ml 780 confocal microscope (Zeiss). penicillin, 100 U/ml streptomycin (Life Technologies), and 150 mM Peptone Primatone (Sigma-Aldrich). For inhibition of Syk, cells were Genscan analysis m treated with 2 M R406 for 24 h. Genomic DNA from purified primary total lymphocytes was analyzed for Flow cytometry clonal TCR gene rearrangements on the TCR-b locus. TCR-b gene clonality was assayed at V-D-J and incomplete D-J rearrangements in five Primary cells were stained with Abs including anti-CD4, anti-CD8, anti- different PCR reactions: Vb (1–20)-Jb1, Vb (1–20)-Jb2, Db1-Jb1, Db2- Thy1.2, anti-B220, anti-CD19, anti-IgM, anti-IgD, anti-CD44, anti-CD62L, Jb2, and Db1-Jb2. The primers used for detection of gene recombination anti–human Syk (hSyk; 4D10; Santa Cruz Biotechnology), anti–p-ERK1/2 consist of a mixture of 20 family-specific upstream primers located within 1026 LOW ITK-Syk EXPRESSION LEADS TO SEVERE DISEASE

Vb gene segments, consensus primers immediately located 59 of Db1/Db2, STOP cassette allows ITK-Syk expression under the control of the and 59 FAM-labeled consensus downstream primers immediately located ROSA26 promoter (Supplemental Fig. 1B). To express the ITK- 39 of Jb1/Jb2 as described previously (24, 25). 59-FAM–labeled PCR products Syk fusion protein specifically in T cells, the mice were crossed to were size separated by capillary POP-7-Polymer electrophoresis and visual- ized via automated scanning using a 3700 Genetic Analyzer (Applied Bio- either CD4-Cre (20) or Lck-Cre (21) mice. Because both mouse systems). All reagents were obtained from Applied Biosystems. Fragment size strains showed the same phenotype, we summarized both as ITK- distribution was determined by GeneMapper software (Applied Biosystems) Syk mice. T cell–specific ITK-Syk expression was confirmed by and analyzed as previously described (26, 27). immunoblot and FACS analysis of splenic T cells (Fig. 1A, 1B). Statistical analysis We first analyzed the T cell compartment in 4- to 5-wk-old ITK- Syk mice. No differences in percentages or total cell numbers were Results were analyzed for statistical significance with GraphPad Prism 4 + software (GraphPad) using Student t test or Mann–Whitney U test. detected for DP or single-positive (SP) thymocytes as well as CD4 or CD8+ peripheral T cells in the spleen (Fig. 1C–F), suggesting Results that expression of the ITK-Syk oncogene does not interfere with Generation of mice with T cell–specific ITK-Syk oncogene T cell development in the thymus. expression ITK-Syk mice develop early lymphoproliferation and systemic To study the effect of the ITK-Syk oncogene in vivo, we generated inflammation a mouse model for conditional expression of the ITK-Syk fusion By 7–9 wk of age, ITK-Syk mice showed first signs of malaise. protein in T cells. We introduced the cDNA for the human ITK-Syk Diseased ITK-Syk mice have enlarged spleens comprising an + fusion protein isolated from a PTCL patient harboring the t(5, 9) expanded T cell pool with mainly CD4 T cell numbers being Downloaded from (q33;q22) translocation together with a loxP-flanked transcrip- increased (Fig. 2A, 2B). Histological analysis of spleen and liver tional STOP cassette into the ubiquitously expressed ROSA26 demonstrated a severe disruption of their normal architecture with locus (Supplemental Fig. 1) (13). Cre-mediated excision of the altered follicular structures and widely scattered T cells in the http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Normal T cell development in mice expressing the ITK-Syk oncogene in T cells. (A) Immunoblot analysis of T cell–specific ITK-Syk expression. Splenocytes were isolated from an ITK-Syk mouse and a control littermate, sorted with magnetic beads for Thy1.2+ and Thy1.22 cells, and subjected to immunoblot analysis. ITK-Syk expression was detected using an anti-hSyk Ab. Actin served as loading control. Data are representative of two independent experiments. (B) Specific ITK-Syk expression in CD4+ and CD8+ T cells. FACS analysis of splenocytes (gated on living cells) from an ITK- Syk mouse (red line) and a control littermate (gray filled) stained for CD4, CD8, CD19, and intracellular hSyk. Data are representative of two independent experiments. FACS analysis of thymocytes (C) and splenic T cells (D) from 4- to 5-wk-old control and ITK-Syk mice (gated on living cells) stained for the expression of CD4 and CD8. Numbers in quadrants indicate percentage of individual thymocytes subsets. Data are representative of more than three independent experiments. (E) Cell numbers from total, DP, and SP thymocytes of 4- to 5-wk-old control and ITK-Syk mice (n = 7/group) are shown. Statistical significance was analyzed using Student t test (p = 0.19 [total]; p = 0.23 [DP]; p = 0.39 [SP]). (F) Total splenic T cell numbers (Thy1.2+) from 4- to 5-wk-old control and ITK-Syk mice (n = 7/group) are indicated. Statistical significance was analyzed using Student t test (p = 0.82). The Journal of Immunology 1027 spleen (Fig. 2C, Supplemental Fig. 2A). To assess whether the cytokines, similar to control T cells or even significantly higher in expanded T cells arose from one or several T cell clones, we case of IL-4 and IL-10 (Fig. 3B). This suggests that the activated characterized T cell clonality by performing a PCR-based and ITK-Syk–expressing T cells are not anergic and are still able to computer-assisted fragment length analysis (Genscan) for TCR-b react to extrinsic stimuli comparable to or even more sensitive locus gene rearrangement (26, 27). In a clonal disease, only few than wt T cells. Together, these data show that peripheral ITK- rearrangements representing one or more cell clones would be Syk–expressing T cells are activated and secrete both Th1 and Th2 predominant over a polyclonal background, whereas in polyclonal inflammatory cytokines that might lead to severe systemic in- samples, many different amplification products will be detected. flammation in these mice. Diseased ITK-Syk mice showed a polyclonal TCR-b repertoire comparable to healthy control littermates (Fig. 2D). To determine Elevated B cell numbers and Ig titers in ITK-Syk mice whether the polyclonal expanded T cells are activated, we per- T cell–derived cytokines play a crucial role in promoting B cell formed FACS analysis of splenic T cells from control and ITK-Syk activation and Ig class switch (30). Hence, the secretion of various mice. T cell activation is associated with an increased expression cytokines by ITK-Syk–expressing T cells raised the question of the membrane Ag CD44 and a decreased expression of the cell whether B cells are also affected in these mice. Interestingly, ITK- adhesion molecule CD62L (28). Indeed, both CD4+ and CD8+ Syk mice showed increased numbers of mature B cells in the T cells in the spleen of ITK-Syk mice upregulated CD44 and spleen possessing similar expression levels of CD19, IgM, and downregulated the expression of CD62L (Fig. 2E). IgD BCRs compared with splenic B cells from control littermates Because activated T cells are an important source of cytokines (Fig. 4A, 4B). Further, we could detect an increased amount of during immune responses (29), we determined cytokine levels in plasma cells in the spleen of ITK-Syk mice (Fig. 4C). Thus, we Downloaded from the serum of diseased mice. We detected elevated levels of in- determined Ig titers in the serum of diseased ITK-Syk mice and flammatory cytokines such as IFN-g, TNF, and IL-6 and slightly could detect elevated levels of IgM, IgG, and IgA Abs (Fig. 4D). increased levels of IL-2, IL-4, and IL-10 in the serum (Fig. 3A), These data suggest that the increased secretion of various cyto- suggesting that ITK-Syk mice developed systemic inflammation. kines by ITK-Syk–expressing T cells affects B cell numbers and To examine whether the ITK-Syk–expressing T cells secrete these Ig titers observed in ITK-Syk mice.

inflammatory cytokines, we isolated splenic T cells from ITK-Syk http://www.jimmunol.org/ and control mice and measured cytokine levels in the supernatants Role of ITK-Syk protein expression levels of in vitro cultured T cells. As in vivo, we detected significantly Recently, we reported that mice with T cell–specific ITK-Syk elevated amounts of inflammatory cytokines in the supernatants expression show a severe reduction of total thymocytes and pe- of T cells from ITK-Syk mice such as IFN-g, TNF, IL-6, IL-2, and ripheral T cells by 4 to 5 wk, but later developed a lymphoproli- IL-10, whereas splenic T cells from control mice barely secreted ferative disease by 20–27 wk with clonal T cells infiltrating all cytokines (Fig. 3B). Upon in vitro stimulation, ITK-Syk– organs (17). Although the targeted locus, the targeting strategy for expressing T cells were able to secrete higher amounts of these conditional ITK-Syk expression, and the induction of ITK-Syk by guest on September 28, 2021

FIGURE 2. ITK-Syk mice early develop polyclonal T lymphoproliferation. (A) Macroscopic appearance of representative spleens from a control and diseased ITK-Syk mouse (n . 10/group). (B) Total splenic T cell (Thy1.2+), CD4+,andCD8+ Tcell(p = 0.64) numbers from control and ITK-Syk mice (n = 12/group) are shown. Statistical significance was analyzed using Student t test or Mann–Whitney U test. (C) Disruption of the splenic architecture was demonstrated by H&E (HE) staining and immunohistochemistry for CD3 in paraffin-embedded spleens from control and ITK-Syk mice (n = 4/group). Original magnification 3100 (left and middle images)and3400 (right images). (D) Genscan analysis for TCR-b gene rearrangements of control (n = 2) and diseased ITK-Syk mice (n = 5). Shown are representative fragment size distributions of fluorochrome-labeled PCR products of the Db1/Jb2 junction from one control and one diseased ITK-Syk mouse. (E) ITK-Syk–expressing peripheral T cells show an activated phenotype. FACS analysis of splenic CD4+ and CD8+ Tcellsof control (gray filled) and ITK-Syk mice (red line) stained for the expression of CD44 and CD62L. Living cells were gated on CD4 or CD8 SP cells, which were depicted as histogram for expression of CD44 and CD62L. Data are representative of three independent experiments. ***p , 0.001. 1028 LOW ITK-Syk EXPRESSION LEADS TO SEVERE DISEASE

FIGURE 3. ITK-Syk–expressing T cells secrete inflammatory cytokines. (A)CBA analysis for Th1/Th2 cytokines in the serum of diseased ITK-Syk mice and control lit- termates. Total levels of IFN-g,TNF,IL-6, IL-2, IL-4 (p = 0.18), and IL-10 (p = 0.43) were determined for control (n =6)and diseased ITK-Syk mice (n = 10). Statistical significance was analyzed using Mann– Whitney U test. (B) Splenic T cells from control (n =5)andITK-Sykmice(n =6) were isolated using magnetic beads and cultured for 30 h in vitro either unstimu- lated (unstim; left bars)orstimulatedwith anti-CD3ε (3 mg/ml) and anti-CD28 (10 mg/

ml) (right bars; n = 3/genotype). Total levels Downloaded from of IFN-g,TNF,IL-6,IL-2,IL-4,andIL-10 were determined in the cell-culture super- natants by CBA analysis. Samples were measured in doublets. Statistical signifi- cance was analyzed using Student t test or Mann–Whitney U test (stimulated: p = 0.2 [IFN-g], p =0.29[TNF],p =0.76 http://www.jimmunol.org/ [IL-6], and p = 0.55 [IL-2]; unstimu- lated: p = 0.43 [IL-4]). *p , 0.05, **p , 0.01, ***p , 0.001.

expression by CD4-Cre transgenic mice were the same in both wt thymocytes with the expression constructs for IS(hi), IS(lo), or by guest on September 28, 2021 studies, the phenotypes are strikingly different. The expected ITK- an empty control vector and detected an upregulation of CD69 and Syk protein sequence was identical, but the sequence preceding the phosphorylation of important TCR signaling molecules such the translational initiation site of the ITK-Syk cDNA was different as ZAP70, PLCg1, Akt, and Erk1/2, when ITK-Syk was highly in the two models. This sequence is important for the initiation of expressed (Fig. 5D, 5E). We also investigated the subcellular lo- translation and thus protein production (31). Therefore, we com- calization of IS(lo) and IS(hi) and found that the majority of the pared the ITK-Syk expression levels in thymocytes from both ITK-Syk protein was localized at the plasma membrane in both ITK-Syk mouse models and found striking differences in ITK-Syk cases (Supplemental Fig. 3). However, we also found that the protein levels, whereas ITK-Syk RNA levels were similar (Fig. increased expression of IS(hi) resulted in higher ITK-Syk protein 5A, Supplemental Fig. 2B). amounts at the plasma membrane, and it is not clear whether and To investigate whether also in human PTCLs ITK-Syk protein how this interferes with the function of other signaling molecules. expression levels vary, we examined the presence of ITK-Syk in 22 High ITK-Syk expression induces premature T cell PTCL samples, out of which we identified 2 as positive for ITK- differentiation in the thymus Syk. Interestingly, the ITK-Syk protein levels differed in these two patients (Supplemental Fig. 2C), indicating that the ITK-Syk ex- Developing T cells that receive strong signals via their TCR are pression level may vary among humans carrying the t(5, 9)(q33; eliminated from the repertoire by negative selection (32). Because q22) translocation. However, to more conclusively assess the de- high ITK-Syk expression in wt thymocytes led to their activation, gree of variation of ITK-Syk protein expression in patients, a we investigated whether associated with negative selection larger sample size needs to be analyzed. are upregulated in thymocytes from young (4 to 5 wk of age) mice with high ITK-Syk oncogene levels [IS(hi) mice]. Compared with Different cellular activation by low and high ITK-Syk thymocytes from control and low ITK-Syk–expressing mice [IS expression (lo) mice], thymocytes from IS(hi) mice showed only a slight, but To explain the finding that low and high ITK-Syk oncogene ex- not significant, increase of BIM and NUR77 transcripts (Supple- pression in T cells lead to different disease development in mice, mental Fig. 4A), genes known to be key mediators of thymocyte we investigated the molecular mechanisms of how low and high negative selection (33, 34). This suggests that high ITK-Syk ex- ITK-Syk expression influence thymocyte development. We gen- pression may trigger other signaling cascades than those activated erated retroviral expression constructs for ITK-Syk carrying dif- during negative selection. Activated, Ag-experienced T cell sub- ferent flanking sequences resulting in low and high ITK-Syk sets, which have undergone effector differentiation, show elevated protein levels when expressed in different cell lines (ITK-Syklow expression of Blimp-1, a protein important for terminal differen- [IS(lo)] and ITK-Sykhigh [IS(hi)]), similar to the levels found in tiation of both effector T and B cells and tumor suppression in the corresponding mice (Fig. 5B, 5C). We retrovirally transduced B lymphomas (35–38). Thus, we tested whether high ITK-Syk The Journal of Immunology 1029 Downloaded from

FIGURE 4. Diseased ITK-Syk mice have elevated B cell numbers and Ig titers. (A) Total splenic B cell (B220+) numbers from control and ITK-Syk mice (n = 12/ group) are shown. (B) FACS analysis of splenocytes (gated on living cells) from ITK-Syk and control mice stained for CD19 versus CD3ε (left panel). CD19+ B cells from ITK-Syk mice show expression of IgM and IgD similar to B cells from control mice (right panel). Data are representative of more than three independent experiments. (C) ITK-Syk mice have an increased amount of plasma cells as shown by FACS analysis of splenocytes, gated on living cells, from ITK-Syk and control http://www.jimmunol.org/ mice stained for B220 and CD138. Data are representative of three independent experiments. (D) ELISA analysis of serum IgM, IgG, and IgA Ig titers of control and diseased ITK-Syk mice (n = 5/group). (A and D) Statistical significance was analyzed using Student t test or Mann–Whitney U test. **p , 0.01, ***p , 0.001. expression induced the expression of Blimp-1. Indeed, we found (Fig. 6A). Interestingly, this could be prevented by treating IS(hi)- that wt thymocytes transduced with the construct for IS(hi), expressing thymocytes with the Syk inhibitor R406 (Supplemental but not IS(lo), showed a clear induction of Blimp-1 expression Fig. 4B). by guest on September 28, 2021

FIGURE 5. Role of different ITK-Syk protein ex- pression levels. (A) ITK-Syk protein levels were detected by immunoblot analysis in total lysates from thymocytes isolated from a control and an ITK-Syk mouse as well as from an ITK-Syk mouse from our previous work (17). ITK-Syk expression was detected using an anti-hSyk Ab. BAP31 served as loading con- trol. Data are representative of three independent experiments. (B) Schematic illustration of the human ITK-Syk cDNA together with the sequence context preceding the translational initiation site accounting for low [IS(lo)] and high [IS(hi)] ITK-Syk expression. (C) Beko pre-T cells were retrovirally transduced with an empty vector control (EV) and the IS(lo) and IS(hi) constructs shown in (B), sorted, and subjected for im- munoblot analysis. ITK-Syk expression was detected using an anti-hSyk Ab. BAP31 served as loading con- trol. Data are representative of three independent experiments. (D and E)High,butnotlow,ITK-Syk expression immediately activates wt thymocytes. FACS analysis of wt thymocytes retrovirally transduced with an empty vector control (EV; gray filled), the IS(lo) construct (red line), and the IS(hi) construct (blue line) and stained for the expression of CD69 (D) and p- ZAP70, p-PLCg1, and p-Akt, as well as p-Erk1/2 (E). Data are representative of two independent experi- ments. 1030 LOW ITK-Syk EXPRESSION LEADS TO SEVERE DISEASE

Based on the finding that high, but not low, ITK-Syk expression via STAT3 and IRF-4, which is augmented by downregulation led to a strong activation of thymocytes and the induction of of the Blimp-1 antagonist Bcl-6. Blimp-1 in vitro, we tested whether also in mice, high ITK-Syk These data suggested that high ITK-Syk expression in thymo- expression directly activates the thymocytes, which might induce cytes induces Blimp-1–mediated premature terminal differentia- premature T cell differentiation in the thymus. Thus, we analyzed tion. Thus, Blimp-1 downregulation might be required to escape thymocytes from young IS(hi) mice for expression of Blimp-1, this premature terminal differentiation in the thymus. We tested and consistent with our previous data, we found elevated lev- this hypothesis in isolated splenic T cells from control and dis- els of Blimp-1 in thymocytes from IS(hi) mice compared with eased (29-wk-old) IS(hi) mice that have developed clonal T cell control mice on both the protein and RNA level (Fig. 6B, 6C). lymphomas (Fig. 7C) (17). No Blimp-1 protein expression was This was accompanied by thymic atrophy and a severe reduction detected in the tumors from diseased IS(hi) mice (Fig. 7D). Hence, of DP and SP thymocyte numbers as well as peripheral T cell a possible explanation for the delayed disease development in IS numbers in IS(hi) mice by 4 to 5 wk (Fig. 6D, Supplemental Fig. (hi) mice as compared with IS(lo) mice might be that the majority 4C, 4D) (17). In contrast, Blimp-1 expression was not elevated in of T cells are deleted in the thymus by activation of Blimp-1– thymocytes from IS(lo) mice compared with the control, and mediated premature terminal differentiation and that only those also total thymocyte numbers were similar between control and cells that failed to induce Blimp-1 develop tumors. IS(lo) mice (Fig. 6B–D). Discussion High ITK-Syk expression activates Blimp-1 via STAT3 In this study, we show that mice with T cell–specific ITK-Syk

We next aimed to identify the signaling pathways linking ITK-Syk oncogene expression developed an aggressive polyclonal T cell Downloaded from to the expression of Blimp-1. It is shown that STAT3 induces the lymphoproliferation with concomitant B cell expansion and sys- expression of Blimp-1 and that Syk phosphorylates and activates temic inflammation within 7–9 wk after birth. The phenotype is STAT3 (39, 40). Therefore, we analyzed the phosphorylation strikingly different from our previous model for T cell–specific status of STAT3 upon expression of IS(hi) and IS(lo) constructs in ITK-Syk expression showing a profound loss of thymocytes and wt thymocytes and detected elevated STAT3 phosphorylation only the development of clonal T cell lymphomas by 20–27 wk of age

upon high ITK-Syk expression (Fig. 7A). This suggests that high (17). Comparison of both mouse models revealed that the different http://www.jimmunol.org/ ITK-Syk expression activates STAT3, which then induces ex- phenotypes are caused by different expression levels of ITK-Syk. pression of Blimp-1 in thymocytes. Interestingly, Blimp-1 gene Usually, developing T cells become positively or negatively expression is further augmented by cooperation of STAT3 with selected depending on the strength and duration of their TCR- IFN regulatory factor 4 (IRF-4) (41). We analyzed IRF-4 ex- derived signals (43). Strong TCR-derived signals are linked to the pression and found that only in thymocytes from IS(hi) mice, IRF- elimination of developing T cells from the repertoire by negative 4 transcript levels were significantly elevated (Fig. 7B), suggesting selection (32). Interestingly, young IS(hi) mice showed a severe that STAT3 and IRF-4 cooperatively induce the expression of reduction of total DP and SP thymocytes, whereas the T cell de- Blimp-1. In addition, expression of the Blimp-1 antagonist B cell velopment in the thymus of mice with low ITK-Syk expression was lymphoma-6 (Bcl-6) was selectively decreased only in thymocytes not affected. These data suggested that high ITK-Syk expression by guest on September 28, 2021 from IS(hi) mice (Fig. 7B) (42). Together, this indicates that high might mimic strong TCR signaling and lead to negative selection, ITK-Syk expression leads to the induction of Blimp-1 expression whereas the signals induced by low ITK-Syk expression might be

FIGURE 6. High ITK-Syk expression induces premature T cell differentiation in the thymus. (A) FACS analysis of wt thymocytes retrovirally transduced with an empty vector control (EV; gray filled), IS(lo) (red line), and IS(hi) (blue line), intracellularly stained for Blimp-1. Data are representative of three independent experiments. (B) Expression of Blimp-1 in thymocytes from high ITK-Syk–expressing mice. Immunoblot analysis of thymocytes from control, IS(lo), and IS(hi) mice using an anti–Blimp-1 Ab (n = 4/group). BAP31 served as loading control. Data are representative of three independent experiments. (C) Total RNA from control (n = 7), IS(lo) (n = 6), and IS(hi) (n = 6) thymocytes was isolated to analyze Blimp-1 mRNA levels with specific primers by quantitative RT-PCR using the SYBR Green detection method. Samples were run as duplicates. Statistical significance was analyzed using Mann–Whitney U test (NS, p = 0.3). (D) Absence of thymocytes in 4- to 5-wk-old IS(hi) mice. Total (NS, p = 0.31), DP (NS, p = 0.5), and SP (NS, p = 0.59) thymocyte numbers from 4- to 5-wk-old control, IS(lo), and IS(hi) mice (n $ 6 for each group) are shown. Cell numbers for control and IS(lo) mice are the same as shown in Fig. 1E. Statistical significance was analyzed using Mann–Whitney U test. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 1031

FIGURE 7. High ITK-Syk expression induces Blimp-1 via STAT3. (A) FACS analysis of wt thymocytes retrovirally transduced with an empty vector Downloaded from control (EV; gray filled), IS(lo) (red line), and IS(hi) (blue line), intracellularly stained for p-STAT3. Data are representative of three independent experiments. (B) Total RNA from control (n = 7), IS(lo) (n = 6), and IS(hi) (n = 6) thymocytes was isolated to analyze IRF-4, STAT3, and Bcl-6 mRNA levels with specific primers by quantitative RT-PCR using the SYBR Green detection method. Samples were run as duplicates. Statistical significance was analyzed using Student t test [control versus IS(lo): p = 0.44 (IRF4), p = 0.8 (STAT3), p = 0.79 (Bcl-6); control versus IS(hi): p = 0.19 (STAT3), p = 0.05 (Bcl-6); IS(lo) versus IS(hi): p = 0.28 (STAT3)]. (C) Clonal T cell lymphoma in 29-wk-old IS(hi) mice. FACS analysis of splenocytes (gated on living cells) from diseased IS(hi) mice and control littermates stained for Thy1.2 and B220. (D) Immunoblot analysis of splenic (sp) T cells from control and diseased , IS(hi) mice (n = 2) using an anti–Blimp-1 Ab. BAP31 served as loading control. Data are representative of two independent experiments. *p 0.05, http://www.jimmunol.org/ ***p , 0.001. lower than the threshold required for negative selection. Although that expression of a dominant-negative form of STAT3 inhibits we detected a profound phosphorylation of a variety of important Blimp-1 mRNA induction (49). Besides the activation of STAT3, TCR-downstream signaling molecules upon high ITK-Syk expres- high ITK-Syk expression also led to an upregulation of IRF-4 sion, the transcription of genes that are key mediators of thymocyte expression, which, together with STAT3, controls the expression negative selection was not significantly elevated in thymocytes from of Blimp-1 (41). Hence, strong Syk activity mediated by high IS(hi) mice (33, 34). These data indicated that in the case of strong ITK-Syk expression is required for STAT3–IRF-4–mediated induc- signaling after high ITK-Syk expression, the premature death of tion of Blimp-1 gene expression, leading to premature T cell dif- by guest on September 28, 2021 thymocytes is triggered by other signaling cascades than that ac- ferentiation in the thymus. Although we found Blimp-1 to be induced tivated during thymocyte negative selection. upon high ITK-Syk expression, we cannot exclude that additional Interestingly, thymocytes from IS(hi) mice showed elevated genes are differentially expressed in the two mouse strains and that expression of Blimp-1, which is usually highly expressed in ef- this may contribute to the elimination of high oncogene-expressing fector and memory T cell subsets and is crucial for controlling cells. Additional experiments are required to characterize these genes T cell homeostasis by promoting activation-induced cell death (36, and the involved molecular mechanisms. 44, 45). Blimp-1–deficient effector T cells are resistant to activation- Low ITK-Syk expression in thymocytes did not lead to the induced cell death, which leads to the accumulation of effector and induction of Blimp-1 or loss of thymocytes in IS(lo) mice. memory T cells in Blimp-1–deficient mice that develop autoimmune However, low ITK-Syk–expressing T cells that migrate to the and inflammatory symptoms (45). This suggests that Blimp-1 might periphery showed signs of activation and secreted inflammatory play a role in the selection of early T cells and that the strong signals cytokines. The threshold at which different Ags trigger T cell induced by high ITK-Syk expression trigger premature thymocyte activation depends on various factors including number, affinity, differentiation by the activation of Blimp-1. Interestingly, the tumor- and kinetics of the TCR–peptide–MHC interaction and can be forming T cells in diseased IS(hi) mice lack expression of Blimp-1, modulated by signals from costimulatory molecules such as suggesting that single T cell clones in the thymus of IS(hi) mice that CD28, CTLA-4, or LFA-1 (50–54). The finding that T cells in the fail to induce Blimp-1 escape this elimination and proliferate by periphery of IS(lo) mice are moderately activated suggests that the strong ITK-Syk signaling. Hence, to escape cell elimination, high signals induced by low ITK-Syk expression, although not suffi- ITK-Syk–expressing thymocytes have to bypass the oncogene- cient to induce terminal differentiation in the thymus, might poise induced Blimp-1–mediated premature terminal differentiation. the T cells for stimulation, thereby reducing the requirements for In line with this, most PTCLs were reported to lack Blimp-1 activation. This could explain the polyclonal expansion and acti- expression or additionally express a second isoform of Blimp-1, vation of low ITK-Syk–expressing T cells that secrete inflam- which has a reduced function on multiple target genes (46–48). matory cytokines, which in turn might account for the elevated Interestingly, the expression of this so-called Blimp-1b isoform is B cell numbers and serum Ig titers as well as the systemic in- associated with poor prognosis in T cell lymphomas (47). Fur- flammation, already in very young IS(lo) mice. Interestingly, this thermore, the majority of patients harboring the t(5, 9)(q33;q22) phenotype is similar to mice with T cell–specific elevated PI3K translocation were reported to express the Blimp-1 repressing signaling, which also leads to both T and B cell lymphoprolifer- factor Bcl-6 (13), and, in line with this, we did not detect Blimp-1 ation and elevated cytokine and Ig titers (55). protein in both ITK-Syk–positive PTCL samples. Together, our data suggest that the expression level of an on- High ITK-Syk expression led to the induction of Blimp-1 gene cogene is crucial for the development and the course of disease. expression by activating STAT3. Consistent with this, it was reported Although high ITK-Syk expression in thymocytes led to premature 1032 LOW ITK-Syk EXPRESSION LEADS TO SEVERE DISEASE

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