Proteomics Analysis of Interleukin (IL)-7-induced Signaling Effectors Shows Selective Changes in IL-7Rα449F Knock-in T Cell Progenitors*S

Interleukin (IL)-7 is a cytokine that plays a central role in the development, survival, and proliferation of T and B cell lymphocytes. Overexpression of IL-7 in mice (transgenic (Tg) IL-7) leads to both increased proliferation of early T and B cell progenitors and T and B cell lymphomas. Genetic evidence indicates that known IL-7 receptor (IL-7R)-dependent proteins, including prosurvival protein BCL-2, may not be solely responsible for the effects of IL-7. Other studies indicate that known IL-7-induced signaling proteins dock to a specific tyrosine (Tyr449) residue on the α-subunit of the IL-7R. We have previously shown in an IL-7Rα449F knock-in model that IL-7-induced lymphomas require Tyr449 phosphorylation and that loss of this phosphorylation confers protection from disease. However, the mechanism by which this lymphoma protection occurs remains unclear. Using this genetic model, we aimed to identify novel prosurvival factors important for IL-7-mediated lymphocyte development and lymphomagenesis. An iTRAQ (isobaric tags for relative and absolute quantitation) proteomics analysis was performed comparing CD4−CD8− double negative T cell progenitors from mice overexpressing IL-7 (Tg IL-7) (lymphoma-prone) with Tg IL-7 mice with a mutated IL-7 receptor (Tg IL-7/IL-7Rα449F) (lymphoma-protected). Several proteins involved in survival, proliferation, and apoptosis were found to be differentially expressed between the two samples, and three proteins of particular interest, GIMAP4, BIT1, and FKBP51, were validated by immunoblot analysis.

A properly functioning immune system relies on a delicate system of balance and regulation at both the level of lymphocyte development and lymphocyte survival. Cytokines play a central role in the development, survival, and proliferation of lymphocyte lineages. One of these cytokines, interleukin-7 (IL-7), 1 is of particular interest because defects in IL-7 production or its overexpression can lead to either immunodeficiency or lymphomas, respectively. IL-7 is a growth factor essential for lymphocyte development and survival (1,2). IL-7 deficiency results in T and B cell immunodeficiency in mice and T cell immunodeficiency in humans (2,3). Mice lacking IL-7 are strikingly lymphopenic with thymic cellularity reduced 20-fold (2). The development of both T and B cells is also severely impaired in IL-7 receptor (IL-7R) knock-out mice, which show an early block in T cell development at the CD4 Ϫ CD8 Ϫ double negative (DN) T cell progenitor stage (4). Conversely in vivo addition of IL-7 leads to increased proliferation of early T and B cell progenitors in mice (5,6). Indeed excessive IL-7 signaling has been implicated in a number of human cancers such as T cell acute lymphoblastic leukemia, Hodgkin disease, and Burkitt lymphoma (7)(8)(9)(10)(11)(12)(13) as well as in autoimmune pathology (14), although the exact mechanism by which IL-7 is involved remains unclear.
To determine the role of IL-7 signaling in tumorigenesis, we study a transgenic mouse model that overexpresses IL-7 (Tg IL-7). These mice present with increased T and B cell proliferation, resulting in aggressive tumor formation and almost 100% mortality by 7 months of age due to T and B cell lymphomas (15). This indicates that excessive IL-7 signaling in vivo is sufficient to induce the generation of immune cell tumors. It remains unclear, however, which components of the IL-7 signaling pathway are responsible for this deregulated cellular growth and development in the presence of IL-7 overexpression. Gaining a better understanding of the molecular processes driving lymphoma development in Tg IL-7 mice could provide critical insight into the biology of lymphomas and thus allow for the development of much needed novel therapeutic strategies. IL-7 signaling pathways are induced when IL-7 binds to the IL-7R, a heterodimer composed of the IL-7R␣ chain and the common ␥ chain (␥ c ) (for a review, see Ref. 16). A number of studies clearly demonstrate that Tyr 449 on the IL-7R␣ chain is the main phosphorylation target of the associated tyrosine kinases upon ligand binding (17,18). Once phosphorylated, this 449 YXXM motif may act as a docking site for both signal transducer and activator of transcription 5 (STAT5) and phosphatidylinositol (PI) 3-kinase (17,19). One of the primary targets up-regulated by STAT5 is the well known antiapoptotic protein BCL-2 (20). PI 3-kinase is activated upon binding to phosphorylated Tyr 449 and consequently activates the downstream signaling protein AKT (16). AKT itself triggers a number of prosurvival and growth functions, including inactivation of proapoptotic proteins BAD and BIM and the cell cycle inhibitor p27 kip1 (16). Initial studies to determine which of the known downstream signaling proteins mediate IL-7 function revealed that deficiencies in either STAT5, PI 3-kinase, BCL-2, or AKT do not phenocopy the severe defects in early T and B cell development observed in IL-7 or IL-7R␣ knock-out mice (21)(22)(23)(24)(25). In addition, overexpression of BCL-2 in IL-7R␣ knock-out mice only partially complements thymocyte cellularity (26,27). These data suggest that these known effectors are not necessary for IL-7 function, that BCL-2 is incompletely sufficient, and that novel signaling effectors downstream of IL-7 may be involved. Given these unknowns, alternative global approaches are required to gain a better understanding of IL-7 signaling pathways.
To assess the role of IL-7-induced signaling effectors, we generated a mouse model in which the Tyr 449 residue on IL-7R␣ is substituted with a phenylalanine (IL-7R␣ 449F ). Loss of phosphorylated IL-7R␣ Tyr 449 completely abrogates all downstream signaling pathways dependent on the phosphorylated Tyr 449 residue for activation (28). Interestingly IL-7R␣ 449F lymphocytes bypass a developmental defect at an early stage in thymopoiesis seen in IL-7R knock-out mice such that lymphocyte development is largely unimpaired (28).
To determine the involvement of Tyr 449 -dependent signaling pathways in lymphoma development, we crossed IL-7R␣ 449F mice with Tg IL-7 mice (Tg IL-7/IL-7R␣ 449F ). Strikingly IL-7R␣ 449F mice overexpressing IL-7 are completely diseasefree over their lifespan in marked contrast to Tg IL-7 mice with wild-type IL-7R␣ that succumb to lymphomas at an early age. 2 This indicated that IL-7-induced lymphoma development is entirely dependent on phosphorylation of Tyr 449 in the IL-7R␣ chain and the effectors recruited to this site. Accordingly we formulated the hypothesis that IL-7R␣ Tyr 449 is required for signaling through previously unidentified prosurvival factors that are important for IL-7 to support lymphocyte development and lymphomagenesis.
To test this hypothesis, a proteomics analysis was per-formed using isobaric tags for relative and absolute quantitation (iTRAQ) to identify qualitative and quantitative differences in proteins from Tg IL-7 and Tg IL-7/IL-7R␣ 449F CD4 Ϫ CD8 Ϫ DN T cell progenitors from young, disease-free mice. iTRAQ technology is a major advancement in the study of quantitative gene expression at the proteome level. It is a stable isotope peptide tagging system in which the primary amines of peptides are chemically tagged, with up to four different tags available, and then analyzed by tandem mass spectrometry. This allows simultaneous identification and quantification of proteins from different samples in a single mass spectrometry run. Furthermore as all tryptic peptides are labeled rather than just cysteine-containing peptides as with older ICAT technology, iTRAQ reagents allow for expanded proteome coverage and increased confidence in protein identification. DN thymocytes were examined because they are the precursors of mature T cells and as such are the most likely to develop T cell lymphoma-forming mutations to generate cancer stem cells (29 -32). Due to limited sample and a desire to remain completely unbiased these analyses were performed on whole cell lysate.
Several proteins involved in survival, proliferation, and apoptosis were found to be quantitatively different between the two sample sets, and three proteins of particular interest, GIMAP4, BIT1, and FKBP51, were validated by Western blot analysis. Identifying unknown survival effectors downstream of IL-7 could lead to both a better understanding of the biology of lymphomagenesis and the identification of novel therapeutic targets to treat and/or prevent lymphoma development.

EXPERIMENTAL PROCEDURES
Mice-Animals were housed at the University of British Columbia, Microbiology and Immunology department animal facility in accordance with University of British Columbia Animal Care and Biosafety Committee certificates. Tg IL-7 mice on a FVBN background were obtained from Dr. Benjamin Rich (15) and backcrossed for 20 generations to C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME). IL-7R␣ 449F mice were generated, backcrossed for seven generations to C57BL/6, and then crossed with Tg IL-7 mice to create Tg IL-7/ IL-7R␣ 449F mice. Prelymphoma mice of 7.5 weeks of age were used to avoid frank lymphomas.
DN Thymocyte Enrichment-Thymi were harvested and prepared as single cell suspensions. Individual sample aliquots were stained and assessed by flow cytometry for the absence of frank lymphomas as determined by clonal expansion of thymocyte populations. The remaining cells were labeled with ␣CD8 and ␣CD4 autoMACS magnetic beads (Miltenyi Biotec, Auburn, CA). The labeled thymocytes were run through the "DepleteS" program on an autoMACS automated magnetic cell sorter, and the negative (unlabeled) fraction was collected. The CD4/CD8-depleted thymocytes were then stained for surface markers CD4 and CD8 and lineage (Lin) mixture (B220, Gr-1, TER119, and ␥␦ TCR) and sorted by flow cytometry. The Lin Ϫ CD4 Ϫ CD8 Ϫ (DN) thymocyte fraction was collected and lysed in cold Nonidet P-40 lysis buffer (1ϫ PBS, 1% Nonidet P-40, and 1ϫ proteinase inhibitor mixture (Calbiochem)). Total cell lysates were precleared by centrifugation (13,000 ϫ g), and the supernatant was stored at Ϫ80°C and later pooled.
Protein Quantification-Protein concentration was determined using the Pierce BCA protein assay, and spectrometry readings were taken at A 562 on a Hitachi U-2000 spectrophotometer.
Flow Cytometry-Cell samples were sorted on a FACSAria cell sorter and analyzed on an LSRII flow cytometer (BD Biosciences), and data were analyzed with FlowJo software (Tree Star).
Tandem Mass Spectrometry-iTRAQ labeling and mass spectrometry were performed by the University of Victoria Genome BC Proteomics Centre. Samples were reduced, alkylated, trypsin-digested, and labeled using the iTRAQ Reagents Multiplex kit according to the manufacturer's instructions (Applied Biosystems, Foster City, CA). Each sample was labeled with a different isobaric tag (Tg IL-7 proteins with the 117 iTRAQ tag and Tg IL-7/IL-7R␣ 449F proteins with the 115 iTRAQ tag) and separated first by strong cation exchange HPLC and then further fractionated using reverse phase microcapillary HPLC (33). The reverse phase microcapillary HPLC was coupled on line to an ESI-MS/MS mass spectrometer (API QStar Pulsar, Applied Biosystems) (33). All mass spectrometry runs were performed with the same parameters: detected protein threshold, Ͼ1.30 (95.0%); methyl methanethiosulfate-modified cysteine as fixed modifications; and biological modifications "ID focus" settings. Parameters such as tryptic cleavage specificity, precursor ion mass accuracy, and fragment ion mass accuracy are built-in functions of the protein analysis software.
Data Analysis-QStar spectra were processed by ProteinPilot TM Software 1.0 3 (Applied Biosystems, Software Revision 6684), which searched measured versus theoretical fragment spectra from the International Protein Index (IPI) mouse protein database version 3.15 from the European Bioinformatics Institute (68,248 entries) using the following criteria: trypsin cleavage specificity, methyl methanethiosulfate-modified cysteine as fixed modifications, and biological modifications ID focus settings. The following criteria were required to consider a protein significant: two or more high confidence (Ͼ95%) unique peptides had to be identified, the protein identification had to have a p Ͻ 0.01, and the -fold difference had to be greater than 1.2. This software corrects all protein ratios and individual peptide ratios for bias as part of the software processing. It corrects for pipetting error when mixing different labeled samples, and it identifies the median average protein ratio and corrects it to unity. It also uses all data from a mass spectrometry run to calculate the bias correction factor for each ratio.
Error factor (EF) is a 95% confidence limit of the measurement error term of a given 117/115 ratio. Rather than report a plus/minus range for an average, which is inaccurate for ratios reported in the linear scale, the EF term indicates that the actual average value lies between (reported ratio)/(EF) and (reported ratio) ϫ (EF) 95% of the time. Error factor is calculated as follows: error factor ϭ 10 95% Confidence Error where 95% Confidence Error ϭ S MW ϫ (Student's t factor for n Ϫ 1 degrees of freedom). S MW is the weighted standard deviation of the weighted average of log ratios. The error factor is blank when there is just one value because standard deviation is undefined in this case.
The Unused protein score is ProteinPilot's measurement of protein identification confidence taking into account all peptide evidence for a protein, excluding any evidence that is better explained by a higher ranking protein. The Unused protein score depends on a number of factors, including where the protein is ranked in the list of detected proteins, what spectra the protein represents, and which of those spectra are already represented by higher ranked proteins.
Gene ontology (60) analysis of identified proteins was performed using Blast2GO Version 1.3.1 (34). Protein sequences were acquired using IPI accession numbers, and gene ontology numbers were derived by performing a BLASTp search against the non-redundant database with an expectation value maximum of 1 ϫ 10 Ϫ3 and a high scoring segment pair length cutoff of 33. Protein sequences were then annotated according to the following parameters: a pre-eValue-Hit-Filter of 1 ϫ 10 Ϫ6 , a pro-Similarity-Hit-Filter of 15, an Annotation Cutoff of 55, and a GO Weight of 5. Directed acyclic graphs (DAGs) were then generated using a sequence filter of 5, a score ␣ of 0.6, and a node score filter of 0.
Western Blotting-DN thymocyte proteins were extracted in Nonidet P-40 lysis buffer, incubated on ice for 10 min, and centrifuged to remove cellular debris. Samples were quantified and normalized for total protein level. Proteins were resolved by SDS-PAGE and electrophoretically transferred to a nitrocellulose membrane. Polyclonal antibodies were used for detection and visualized with Alexa Fluor 680 goat anti-rabbit/ mouse IgG antibodies (Molecular Probes) on a LI-COR Odyssey infrared imager. Protein levels were quantified and normalized to total protein loaded by Coomassie Blue staining and LI-COR quantitation.

RESULTS
Purification of ␣␤ DN Thymocytes-Hematopoietic stem cells give rise to T cell progenitors in the thymus by initially differentiating into DN thymocytes. To evaluate changes in signaling effector levels in Tg IL-7/IL-7R␣ 449F thymocyte progenitors that account for differences in functional and transformation outcomes, DN thymocytes were isolated from both Tg IL-7 and Tg IL-7/IL-7R␣ 449F thymi using sequential purification steps. First more mature single positive and double positive thymocytes were removed by magnetic cell sorting (MACS) depletion of CD4 ϩ CD8 ϩ T cells (Fig. 1). This crude DN population was then further purified by flow cytometry cell sorting to remove residual CD4 ϩ or CD8 ϩ cells along with B cells, granulocytes, ␥␦ T cells, and red blood cells (Fig. 1).
Characterization of DN thymocyte populations from the two mouse strains showed that DN cellularity greatly differed between the Tg IL-7 and Tg IL-7/IL-7R␣ 449F mice. Tg IL-7 mice have decreased numbers of DN thymocytes as compared with wild-type counterparts and unusually high proportions of lineage-positive cells (B cells and granulocytes). Tg IL-7/IL-7R␣ 449F mice, on the other hand, have a distribution of thymocyte populations very similar to that of wild-type mice but have a 2-fold increase in overall thymocyte numbers. Fig. 1 shows representative flow cytometry analysis plots of samples at various steps of MACS and flow cytometry purification. The MACS depletion enriched the DN thymocyte target population: percentages of DN thymocytes in Tg IL-7/IL-7R␣ 449F were increased from 3.9 to 61%, and percentages of Tg IL-7 DN thymocytes increased from 2.7 to 9.7%. A number of DN thymocyte enrichments were performed to obtain sufficient amounts of protein from these rare T cell progenitors.
350 g of Tg IL-7/IL-7R␣ 449F protein was obtained through four separate enrichments, 17 mice in total. Conversely 150 g of Tg IL-7 protein (the minimum required to perform iTRAQ analysis) was obtained through eight separate enrichments, 45 mice in total.
Protein Identification and Analysis-iTRAQ mass spectrom-etry data (Fig. 2) were analyzed using Applied Biosystems ProteinPilot Software 1.0, which searched against the IPI mouse protein database for peptide sequence identification.  Proteins of interest were those with two or more high confidence unique peptides identified with a p Ͻ 0.01 and a -fold difference greater than 1.2.
Proteins were then analyzed by gene ontology using Blast2GO software. This software assigns putative roles to identified proteins according to two ontologies: biological process and molecular function. Results were visualized by DAGs, which map proteins according to gene ontology terms by grouping proteins under very broad terms and moving down by levels, grouping proteins under increasingly specific terms. DAGs enable the visualization of the hierarchical structure of terms and provide a more global perspective of general trends within a given protein set. Directed acyclic graphs were generated from the total protein set (537 proteins), the Tg IL-7/IL-7R␣ 449F up-regulated protein set (51 proteins), and the Tg IL-7/IL-7R␣ 449F down-regulated protein set (65 proteins). The numbers of proteins found under each term from level 4 of the DAGs were then plotted for total proteins and Tg IL-7/IL-7R␣ 449F up-regulated and down-regulated proteins (Fig. 3, a and b).
A number of differences in biological process trends were obvious between total proteins and those up-or down-regulated in Tg IL-7/IL-7R␣ 449F DN thymocyte progenitors. There was approximately a 25% relative increase in these cells in the number of proteins up-regulated in cellular metabolism, macromolecule metabolism, and primary metabolism compared with down-regulated proteins and roughly a 15% relative increase compared with total proteins. The relative increase in the number of up-regulated proteins in Tg IL-7/IL-7R␣ 449F DN thymocyte progenitors involved in biosynthesis was more than 50% higher than that of down-regulated proteins. Although 5% of the up-regulated proteins in these progenitors were involved in the cell cycle, there were no down-regulated proteins involved in this process. Conversely 8% of Tg IL-7/IL-7R␣ 449F down-regulated proteins were involved in cell organization and biogenesis with no up-regulated proteins found. All three protein subsets were similar in terms of the percentage of proteins involved in establishment of localization, regulation of cellular physiological process, regulation of metabolism, and transport.
Many differences among the three analyzed protein sets were also apparent upon graphing molecular function DAG data. The percentage of Tg IL-7/IL-7R␣ 449F down-regulated proteins involved in cation binding, metal ion binding, and RNA binding was ϳ30% higher relative to that of both upregulated proteins and total proteins. Conversely the percentage of proteins involved in purine nucleotide binding was 43% higher in up-regulated proteins relative to that in down-regu-lated proteins. 11% of up-regulated proteins were involved in hydrolase activity, acting on acid anhydrides, and 14% were involved in unfolded protein binding with no down-regulated proteins in Tg IL-7/IL-7R␣ 449F progenitors found in these categories. 15% of down-regulated proteins were found to be involved in cytoskeletal protein binding.
Several proteins with putative roles in cell survival, death, or proliferation were among the up-and down-regulated protein sets. GIMAP4 was the first protein identified that fit these criteria. It was determined to be 11.3-fold lower in Tg IL-7/IL-7R␣ 449F DN thymocytes than in Tg IL-7 DN thymocytes based on the 117/115 iTRAQ tag ratio (Table I). GIMAP4 is a recently discovered small GTPase that may play a role in positive selection and apoptosis, although its role in the T cell lineage has yet to be fully characterized (35). The second protein found to fit the above criteria was Annexin A6, whose expression was 2.4-fold lower in Tg IL-7/IL-7R␣ 449F DN thymocytes. Annexin A6 is a family member of the Ca 2ϩ -dependent membrane-binding proteins and may play a role in mediating Ras signaling pathways. There is also evidence that Annexin A6 can act as a recruiting/scaffolding protein in a number of signaling pathways controlling differentiation, proliferation, and apoptosis (36). STAT1 expression was also found to be decreased by 2.3-fold in Tg IL-7/IL-7R␣ 449F DN thymocytes. STAT1 is a transcription factor that is believed to play a role in cytokine response (37). Another protein determined to have decreased expression in the Tg IL-7/IL-7R␣ 449F DN thymocytes was CDC42, whose expression was 1.3-fold lower. CDC42 is involved in mediating cell proliferation and cycling (38,39).
Two proteins of interest were selected from those with increased expression in the Tg IL-7/IL-7R␣ 449F DN thymocytes: BIT1 and FKBP51 with a -fold increase of 1.3 and 2.1, respectively. FKBP51 is an immunophilin that is inhibited by either FK506 or rapamycin, both potent immunosuppressive drugs that inhibit T cell proliferation (40). Inhibition of FKBP51 results in NFB activation and thus increased apoptosis and decreased cell proliferation. BIT1 is a mitochondrial protein that mediates apoptosis through its interaction with Grouch family proteins AES and TLE1. Upon loss of integrin-mediated cell attachment, BIT1 is released from the mitochondria to form a complex with AES; this BIT1-AES complex inhibits TLE1 and results in increased apoptosis.
Protein Validation-Proteins previously implicated in mediating cell survival or apoptosis that were quantitatively different between Tg IL-7 and Tg IL-7/IL-7R␣ 449F DN thymocytes according to iTRAQ ratios were validated by three separate immunoblots using fresh DN lysates. Quantification of the immunoblots was performed using a LI-COR Odyssey infrared imager to generate "Integrated Intensity" values (pixels/ mm 2 ). GIMAP4 had, on average, 56.4-fold (ranging from 30.4 to 123) lower expression in Tg IL-7/IL-7R␣ 449F DN thymocytes by immunoblot analysis quantification (Fig. 4) of the two proteins with increased expression in the Tg IL-7/IL-7R␣ 449F DN thymocytes, BIT1 and FKBP51, show an average -fold increase of 3.1 (ranging from 1.8 to 14.3) and 2.1 (ranging from 1.1 to 3.7), respectively (Fig. 4). Interestingly in all validations, the average differences in protein expression level as determined by immunoblot analysis were found to be equal or greater than those originally reported from iTRAQ analysis. DISCUSSION IL-7 is an essential cytokine for proper development and functioning of the immune system. We are using a Tg IL-7 mouse model as a model of IL-7-related human immune system cancers such as T cell acute lymphoblastic leukemia, Hodgkin disease, and Burkitt lymphoma (7)(8)(9)(10)(11)(12)(13). These mice have increased proliferation in both the T and B cell compartments that leads to lymphoma development and subsequent death by 7 months of age as a result. To investigate the signaling pathways involved in IL-7-induced lymphomas, we generated a mouse model carrying an amino acid substitution in a critical motif in the IL-7R␣ chain (IL-7R␣ 449F ) important for Accession numbers represent Swiss-Prot entries. Tg IL-7 to Tg IL-7/IL-7R␣ 449F protein ratio represents differential expression as determined by 117/115 iTRAQ label ratio. p value represents a measure of significant differential protein expression levels. EF represents a measure of the error in the 117/115 ratio. EF range represents (iTRAQ ratio/EF) to (iTRAQ ratio ϫ EF). Prec M r represents the precursor molecular weight for the peptide sequence. Peptides identified represents the number of unique peptides identified for each protein. Confidence score (a measure of peptide identification certainty) for each unique peptide was Ͼ95%. IMAP, immunity-associated protein ND, not determined.

Signaling Effector Changes in IL-7R␣ Mutant Pro-T Cells
the initiation of a number of IL-7 signaling pathways. Having observed that Tg IL-7/IL-7R␣ 449F mice are completely protected from lymphoma development, we aimed to identify changes in novel signaling effectors downstream of this receptor mutation that may contribute to the observed lymphoma protection. Within the hematopoietic compartment, all mature, terminally differentiated cells arise from a very small population of rare hematopoietic stem cells with the unique ability to selfrenew. Tumor-forming cells also seem to follow a similar hierarchy with a very small population of cancer stem cells initiating and supporting tumor growth (30,32). Thymocyte precursors were studied because they are the population likely responsible for initiating and sustaining T cell lymphoma growth. Although current therapeutic techniques targeting rapidly dividing cells are often quite successful in eliminating tumors, relapse is a frequent problem, indicating that cancer stem cells are not being simultaneously eradicated. It is clear that gaining a better understanding of the molecular and cellular properties of these rare cells would provide tremendous insight into the biology of cancer and allow the development of more effective therapies.
We performed a global proteomics analysis comparing DN thymocyte progenitor whole cell lysate from our genetic models of lymphoma-prone and lymphoma-protected mice. DN thymocyte progenitors were isolated from 7.5-week-old mice to ensure that samples were not clonally transformed cells and hence misrepresentative. Using iTRAQ and tandem mass spectrometry, we were able to identify both qualitative and quantitative differences between the two whole cell lysate protein samples. Through this approach we aimed to characterize and analyze protein differences to better understand the molecular effects of this receptor mutation as well as possibly discover novel IL-7-induced signaling effectors. Because whole cell lysates are typically very complex and have a high dynamic range, our discovery of so many pieces of the IL-7 signaling puzzle is very encouraging but also suggests that many more pieces of the puzzle remain to be discovered. We performed gene ontology analysis comparing all proteins identified with those found to be either up-or downregulated in Tg IL-7/IL-7R␣ 449F DN thymocytes. From biological process analysis, five categories were most clearly affected by the IL-7R␣ 449F knock-in mutation. In the categories of cellular metabolism, macromolecule metabolism, biosynthesis, and primary metabolism the numbers of up-regulated proteins were significantly higher relative to the numbers of down-regulated proteins. In addition, only up-regulated proteins were found to be involved in the cell cycle. This would suggest that during IL-7 stimulation of normal lymphocytes, phosphorylation of the IL-7R␣ Tyr 449 residue activates signaling pathways that in some way negatively regulate these processes. The under-representation of metabolism and cell cycle proteins in Tg IL-7 DN progenitors may hence account for the reduced cellularity evident in these mice. Clearly the abrogation of Tyr 449 -dependent signaling pathways results in an increase in the number of proteins involved in these biological processes. Paradoxically the up-regulation of proteins involved in these processes is indicative of increased cell growth and proliferation. This apparent conundrum may be compensated for by the putative loss of the ability to induce survival signals and induction of apoptosis and highlights the complex homeostatic mechanisms regulating the DN progenitor pool. Compensation by these processes in Tg IL-7/IL-7R␣ 449F DN thymocytes will be further explored.
A number of down-regulated proteins were involved in cell organization and biogenesis, whereas no up-regulated proteins were found. This suggests that the phosphorylation of Tyr 449 is required to mediate normal functioning of cell organization and biogenesis and that loss of these signals results in a decrease in the level of proteins involved in these processes.
Some general trends were also apparent in the analysis of molecular function between total proteins and those either up-or down-regulated in Tg IL-7/IL-7R␣ 449F DN thymocytes. First up-regulated proteins involved in purine nucleotide binding were higher relative to down-regulated proteins. Only up-regulated and total proteins were found to be involved in . DN T cell progenitor lysates were prepared from C57BL/6 (B6), Tg IL-7, and Tg IL-7/IL-7R␣ 449F mice, and 30 g of protein were resolved by polyacrylamide gel electrophoresis and analyzed by Western blot using rabbit polyclonal anti-GIMAP4, anti-BIT1, and anti-FKBP51 antibodies. Quantification was performed using the LI-COR Odyssey Integrated Intensity values in pixels/mm 2 . Numbers below panels represent normalized integrated intensity values averaged from three separate immunoblots of independently derived, pooled lysates. The ratio of expression of each protein in Tg IL-7 to Tg IL-7/IL-7R␣ 449F DN thymocytes is shown below the bracket. Loading normalization was determined by Coomassie staining of total protein loaded. hydrolase activity, acting on acid anhydrides, and unfolded protein binding. There were significantly more down-regulated proteins involved in cation binding, metal ion binding, and RNA binding compared with up-regulated proteins. There were also a number of down-regulated proteins but no upregulated proteins involved in cytoskeletal protein binding.
The trends revealed by analysis of this latter class of proteins yielded some unexpected and novel insights. Closer analysis of the data set (Supplemental Table 1) revealed that a large number of cytoskeletal proteins such as gelsolin; plastin; actin-related protein 2/3 complex subunits 3, 4, and 5; and ␥-actin were down-regulated in Tg IL-7/IL-7R␣ 449F DN thymocytes with iTRAQ ratios ranging from 4.8-to 1.6-fold lower. Because a tumor cell's capacity for migration and invasion is closely associated with changes in the cytoskeleton, a deregulation of cytoskeletal components could be playing a major role in allowing cancer invasion and lymphoma development in Tg IL-7 mice. Down-regulation of proteins normally involved in interacting selectively with components of the cytoskeleton such as actin or tubulin in Tg IL-7/IL-7R␣ 449F DN thymocytes could deregulate processes such as cell adhesion, immune synapse formation, motility, and migration potentially affecting transformation development. Such loss of cellular polarity has clear precedent in transformation of adherent cells, but a role in lymphoid tumorigenesis has not been established.
We were able to identify proteins from the iTRAQ data set that have been previously implicated in regulating survival, proliferation, or death and as such could be potential targets for specifically treating lymphomas. GIMAP4 was one of the first proteins of interest to fit the above criteria and was determined to be 56-fold lower in Tg IL-7/IL-7R␣ 449F thymocyte progenitors compared with Tg IL-7 (Fig. 4). Previous research on this protein has revealed that it is a small GTPase whose expression increases upon positive selection of T cells (41). Within the four DN populations, GIMAP4 was only found to be expressed during the DN IV stage (41). GIMAP4-deficient mice exhibit a delay between the transition from apoptotic to dead cells; as such they have greater amounts of apoptotic cells and decreased amounts of dead cells (42). The processes of T cell development, selection, and activation appear to be unaffected in GIMAP4-deficient mice (42). To our knowledge, this is the first time a connection has been shown between GIMAP4 and IL-7. The results of our quantitative proteomics analysis indicate that GIMAP4 expression levels are regulated through phosphorylation of the IL-7R␣ chain Tyr 449 residue. As such, the abrogation of GIMAP4 induction resulting from loss of Tyr 449 -dependent signals may play a major role in lymphoma protection from IL-7 overexpression. Although known IL-7 signaling effectors activated by Tyr 449 such as STAT5 or PI 3-kinase are involved in numerous different signaling pathways, targeting GIMAP4 may confer a high therapeutic index for treating or preventing lymphomas given that mice deficient in GIMAP4 have normal, unaffected lymphocytes (42). This will be examined further in ongoing studies.
Two other promising candidates that were identified and validated by immunoblot analysis were BIT1 and FKBP51. These proteins were found to be elevated in Tg IL-7/IL-7R␣ 449F thymocyte progenitors compared with controls (Fig.  4). BIT1 is a proapoptotic mitochondrial protein that is involved in regulating apoptosis through its interaction with Groucho family transcriptional regulators AES and TLE1. Upon the loss of integrin-mediated cell adhesion, BIT1 is released from the mitochondria and is then able to form a complex with AES, which is speculated to function in a proapoptotic manner by inhibiting the antiapoptotic TLE1 (43). Both proper cell adhesion and the antiapoptotic proteins BCL-2 and BCL-xL appear to prevent the translocation of BIT1 and thus BIT1-AES complex formation (44). Until now BIT1 has not been shown to be regulated by IL-7. Our results suggest that BIT1 is normally negatively regulated in thymocytes by IL-7R␣ Tyr 449 -dependent signals, and thus upon overexpression of IL-7, inhibition of this proapoptotic protein may permit increased cell survival.
FKBP51 is a T cell-specific immunophilin that can bind to the immunosuppressive drugs FK506 or rapamycin (40). The immunophilin-drug complex mediates its effects by inhibiting calcineurin, a key signaling molecule in T cell activation (40). There is evidence that FKBP51 is required for IB␣ degradation and consequently NFB activation (45). Furthermore FKBP51 overexpression has been shown to lead to NFB activation (46). Such activation of NFB may induce proapoptotic pathways, depending on the cellular context, and render Tg IL7/IL-7R␣ 449F thymocyte progenitors unable to support lymphoma development (47,48). Whether IL-7 stimulation of normal, non-Tg thymocytes can induce GIMAP4 and suppress NFB activation via FKBP51 and BIT1 is currently under investigation.
A global proteomics analysis was used to identify novel IL-7-induced signaling effectors. Although a more focused receptor pulldown approach would address very immediate, early events proximal to the IL-7R, we favored a global approach because we were more interested in evaluating later events, distal to the IL-7R, to determine the range of effector families that were affected. We were able to identify a number of proteins that are specifically dependent on the IL-7R␣ Tyr 449 residue. Three proteins, GIMAP4, FKBP51, and BIT1, have been previously associated with roles in survival and apoptosis and as such were validated by immunoblot analysis. These proteins will be further characterized with regard to their involvement in IL-7-mediated lymphoma development. In addition, our proteomics study of lymphoma-prone and -protected thymocyte progenitors has great relevance for studying changes in protein expression characteristic of progenitor cells destined to generate lymphomas. Identified proteins have the potential to be novel therapeutic targets for treating lymphomas and will be evaluated for their ability to target T cells specifically and leave other biological processes and functions unaffected.