TNFR-Associated Factor Family Expression in Normal Tissues and Lymphoid Malignancies

This information is current as Juan M. Zapata, Maryla Krajewska, Stanislaw Krajewski, of September 27, 2021. Shinichi Kitada, Kate Welsh, Anne Monks, Natalie McCloskey, John Gordon, Thomas J. Kipps, Randy D. Gascoyne, Ahmed Shabaik and John C. Reed J Immunol 2000; 165:5084-5096; ;

doi: 10.4049/jimmunol.165.9.5084 Downloaded from http://www.jimmunol.org/content/165/9/5084

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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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. TNFR-Associated Factor Family Protein Expression in Normal Tissues and Lymphoid Malignancies

Juan M. Zapata,* Maryla Krajewska,* Stanislaw Krajewski,* Shinichi Kitada,* Kate Welsh,* Anne Monks,‡ Natalie McCloskey,§ John Gordon,§ Thomas J. Kipps,¶ Randy D. Gascoyne,† Ahmed Shabaik,ʈ and John C. Reed2*

TNFR-associated factors (TRAFs) constitute a family of adapter that associate with particular TNF family receptors. Humans and mice contain six TRAF , but little is known about their in vivo expression at the single cell level. The in vivo locations of TRAF1, TRAF2, TRAF5, and TRAF6 were determined in human and mouse tissues by immunohistochemistry. Striking diversity was observed in the patterns of immunostaining obtained for each TRAF family protein, suggesting their expression is independently regulated in a cell type-specific manner. Dynamic regulation of TRAFs was observed in cultured PBLs, where anti-CD3 Abs, mitogenic lectins, and ILs induced marked increases in the steady-state levels of TRAF1, TRAF2, TRAF5, Downloaded from and TRAF6. TRAF1 was also highly inducible by CD40 in cultured germinal center B cells, whereas TRAF2, TRAF3, TRAF5, and TRAF6 were relatively unchanged. Analysis of 83 established human tumor cell lines by semiquantitative immu- noblotting methods revealed tendencies of certain cancer types to express particular TRAFs. For example, expression of TRAF1 was highly restricted, with B cell lymphomas consistently expressing this TRAF family member. Consistent with results from tumor cell lines, immunohistochemical analysis of 232 non-Hodgkin lymphomas revealed TRAF1 overexpression in 112 (48%) http://www.jimmunol.org/ compared (49 ؍ cases. TRAF1 protein levels were also elevated in circulating B cell chronic lymphocytic leukemia specimens (n as determined by immunoblotting. These findings contribute to an improved ,(0.01 ؍ with normal peripheral blood B cells (p understanding of the cell-specific roles of TRAFs in normal tissues and provide evidence of altered TRAF1 expression in lymphoid malignancies. The Journal of Immunology, 2000, 165: 5084–5096.

o date, six members of the TNFR-associated factor mits their association with TNF family receptors is the TRAF do- (TRAF)3 family of signal-transducing adapter proteins main, a novel protein fold of ϳ180 aa (1). The x-ray crystallo- T have been identified in humans and mice (1). Several graphic structure of the TRAF domain of TRAF2 reveals a trimeric TRAF family proteins interact directly with the cytosolic domains assembly, with each TRAF domain monomer containing a surface by guest on September 27, 2021 of various TNF family cytokine receptors, including CD27, CD30, crevice responsible for binding peptidyl motifs found in the cyto- CD40, CD120b (TNFR2), lymphotoxin-␤ (LT␤R) and solic domains of the TNF family receptors to which TRAF2 is the herpes virus entry mediator. Through interactions with other known to bind (2–4). Similarities and differences in the peptidyl adapter proteins, TRAFs also indirectly associate with additional specificities of individual TRAFs account for their selective asso- complexes, including TNFR1 (CD120a), DR3, ciations with particular TNFR family members, yielding diversity, and IL-1R (CD121). The structure within these proteins that per- redundancy, and competition among TRAFs with respect to li- gand-inducible recruitment to various TNFR family receptor com- *The Burnham Institute, Program on and Cell Death Regulation, La Jolla, plexes (5–10). California 92037; †British Columbia Cancer Agency, Department of Pathology, Van- While capable of associating with multiple cytokine receptor ‡ couver, British Columbia, Canada; Science Applications International Corp., Na- complexes, several TRAFs can also bind to a variety of protein tional Cancer Institute-Frederick Cancer Research and Deveopment Center, Freder- ick, MD 21702; §Medical Research Council Centre for Immune Regulation, kinases, including the NF-␬B-inducing kinases NK␬B-inducing University of Birmingham, Birmingham, United Kingdom; and ¶Departments of ʈ kinase, receptor interacting protein 1, and -recruiting do- Medicine, Hematology, and Oncology and Pathology, University of California, San ␤ Diego, CA 92093 main (containing IL-1 converting enzyme-associated kinase), as Received for publication March 13, 2000. Accepted for publication August 9, 2000. well as the c-Jun N-terminal kinase (JNK) pathway activators mi- togen-activated protein (MAP) kinase kinase kinase-1, apoptosis The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance signal-regulating kinase-1, and the germinal center kinase-related with 18 U.S.C. Section 1734 solely to indicate this fact. kinase (GCKRK; Refs. 11–17). Thus, TRAFs physically and func- 1 This work was supported by National Institutes of Health/National Cancer Institute tionally connect TNF family cytokine receptors to intracellular Grant CA-69381 and the U.K. Medical Research Council (to J.G.), and NS36821 (to protein kinases, thereby linking these receptors to downstream sig- S. Krajewski). J.M.Z. was a fellow of the Lymphoma Research Foundation of Amer- ica and is currently supported by the Lady Tata Memorial Foundation, and J.G. is a naling pathways. Medical Research Council Non-Clinical Research Professor. The in vivo roles of TRAFs are beginning to be elucidated 2 Address correspondence and reprint requests to Dr. John C. Reed, The Burnham through the generation of transgenic mice that overexpress these Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: [email protected] proteins or dominant-negative mutants of them, as well as through targeted ablation (knockouts) in mice (18–24). However, to 3 Abbreviations used in this paper: TRAF, TNFR-associated factor; DLCL, diffuse large cell lymphoma; NHL, non-Hodgkin lymphoma; B-CLL, B cell chronic lym- date, little is known about the normal patterns of TRAF expression phocytic leukemia; CD40L, CD40 ligand; FSCCL, follicular small cleaved-cell lym- in lymphoid, hemopoietic, and other types of tissues in vivo. More- phoma; LT␤R, lymphotoxin-␤ receptor; JNK, c-Jun N-terminal kinase; ECOG, East- ern Cooperative Oncology Group; ECL, enhanced chemiluminescence; GC, germinal over, little effort has been made thus far to examine how the ex- center; NCI, National Cancer Institute; KLH, keyhole limpet hemocyonin. pression of TRAFs might change in association with malignant

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 5085 transformation, an issue of potential importance for better under- Formulation (29), then converted to the updated Revised European-Amer- standing host immune responses to tumors. ican Lymphoids neoplasm classification where possible (30). Previously, we generated specific Abs to TRAF3 and TRAF4, Immunohistochemistry defining their normal patterns of expression in human tissues in vivo (25, 26). In this report, we identified Abs that selectively Tissue sections were immunostained using a diaminobenzidine-based de- recognize TRAF1, TRAF2, TRAF5, or TRAF6, and used these tection method as described in detail, using either an avidin-biotin complex reagent (Vector Laboratories, Burlingame, CA) or the Envision-Plus-HRP reagents for assessing the in vivo locations of expression of these system (Dako, Carpinteria, CA) using an automated immunostainer (Dako TRAF family proteins in normal human and murine tissues. We Universal Staining System) (25, 26, 31, 32, 33). The dilutions of anti- also characterized the expression of TRAF1, TRAF2, TRAF5, and TRAF antiserum typically used were 1:1500 (v/v) for anti-TRAF1, 1:3000 TRAF6 in 83 human tumor cell lines, and found evidence of ele- for TRAF2, 1:500 for anti-TRAF5, and 1:1000 for anti-TRAF6. Nuclei vations of TRAF1 expression in non-Hodgkin lymphomas (NHLs) were counterstained with either hematoxylin or methyl green. For all nor- mal tissues examined, the immunostaining procedure was performed in and B cell chronic lymphocytic leukemia (B-CLL). The findings parallel using preimmune serum to verify the specificity of the results. lay the foundation for a more complete understanding of the di- Initial confirmations of Ab specificity also included experiments in which versity of cellular responses to the TNF/Toll family receptors, antiserum was preadsorbed with 5–10 ␮g/ml of either synthetic peptide which rely on TRAF family proteins for their immunogen or recombinant protein immunogen. The immunostaining re- sults were arbitrarily scored according to intensity as 0, negative; 1ϩ, weak; mechanisms. 2ϩ, moderate; and 3ϩ, strong. Results presented for each normal tissue were based on immunohistochemical analysis of multiple immunostained Materials and Methods slides (n Ն 3 for each tissue). For lymphomas, samples were additionally

scored for percentage of immunopositive malignant cells, estimating the Downloaded from Ab and immunogen preparation percentage in increments of 10% (0%, 10%, 20%, 30%, etc.) in overview Polyclonal antisera were generated in rabbits using synthetic peptides or and from a minimum of five representative high power fields. Comparisons recombinant protein immunogens. Unless otherwise specified, peptides were made with the germinal centers (GCs) from non-neoplastic lymph ϭ were synthesized with an N-terminal cysteine appended to permit conju- nodes (n 9). gation to maleimide-activated carrier proteins keyhole limpet hemocyonin KLH and OVA (Pierce, Rockford, IL) as described previously (27) and In vitro translations with a C-terminal amide (NH ) rather than free carboxylic acid. Among the 2 TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6 cDNAs subcloned http://www.jimmunol.org/ peptides used as immunogens were: MASSSGSSPRPAPDENEFPFGC, into either pKSII-Bluescript (Stratagene, La Jolla, CA) or pcDNA-3 (In- corresponding to residues 1–22 of human TRAF1; CRADNLHPVSPGSPL vitrogen, San Diego, CA) were transcribed and translated in vitro using 1 TQEK, representing aa 51–69 of murine TRAF1; CVTPPGSLELLQPGF ␮g plasmid DNA, 25 ␮l TNT-reticulocyte lysates and either T3 or T7 RNA SKTLLGTKLEAK, corresponding to aa 6–31 of human TRAF2; CSFLE polymerase (Promega, Madison, WI) according to the manufacturer’s pro- AQASPGTLNQVGPELLQR, representing residues 250–271 of murine 35 tocol in the presence of S-labeled L-methionine (ϳ1 mCi/mmol; Amer- TRAF2; CMAHSEEQAAVPCAFIRQNSG, corresponding to aa 1–20 of sham, Arlington Heights, IL). murine TRAF5; and CDAKPELLAFQRPTIPRNPK, representing residues 451–469 of human and murine TRAF6. Antipeptide antiserum specific for Immunoblotting TRAF3 and TRAF4 have been described (25, 26). An additional anti-TRAF3 serum was produced using recombinant hu- Whole cell lysates were prepared from frozen human tissues obtained at man TRAF3 (residues 341–568)-His6 protein. Recombinant TRAF3 (341– autopsy, as described previously (25, 26, 33). For analysis of cultured cell by guest on September 27, 2021 568) was produced as a fusion protein with a C-terminal his6 tag. This lines, cells were lysed in RIPA solution (25 mM Tris, pH 7.2, 150 mM protein was expressed in BL21 (DE3) cells by induction with 1 mM iso- NaCl, 5 mM EDTA, 1% sodium deoxycholate, 0.1% SDS, and 1% Triton propyl ␤-D-thiogalactoside. Following cell growth and lysis, the clarified X-100) containing protease inhibitors (1 mM PMSF, 0.28 trypsin inhibitor cell lysate was applied to a nickel-nitrilotriacetic acid column (Qiagen, units/ml aprotinin, 50 ␮g/ml leupeptin, 1 mM benzamidine, and 0.7 ␮g/ml Valencia, CA) and eluted with an imidazole gradient. The pooled TRAF3 pepstatin). Cell and tissue lysates were normalized for total protein content fractions were dialyzed against 50 mM Tris at pH 8.8 and applied to a (50 ␮g/lane) and subjected to SDS-PAGE/immunoblot analysis using 10/10 fast protein liquid chromatography mono Q column (Pharmacia, Pis- 1:500–1:2000 (v/v) dilutions of antisera and secondary HRP-conjugated goat cataway, NJ) and eluted with an NaCl gradient. anti-rabbit Ab (1:3000 v/v dilution; Bio-Rad, Richmond, CA), with detection New Zealand White female rabbits were injected s.c. with a mixture of accomplished using an enhanced chemiluminescence (ECL; Amersham- 0.25 ml KLH peptide (1 mg/ml), 0.25 ml OVA peptide (1 mg/ml), or based) multiple Ag detection immunoblotting method that allows for multiple recombinant protein (0.1–0.25 ␮g protein/immunization) and 0.5 ml reprobings of blots without Ab stripping, as described previously (25, 34). Freund’s complete adjuvant (dose divided over 10 injection sites) and then For immunoblot data qualifications, TRAF cDNAs were in vitro trans- boosted three times at weekly intervals followed by another 3–20 boostings lated in the presence of [35S]methionine and analyzed by SDS-PAGE. The at monthly intervals with 0.25 mg each of KLH peptide, OVA peptide, or region of the gel containing each labeled TRAF protein was sliced, and the recombinant protein immunogens in Freund’s incomplete adjuvant, before radioactivity contained in the band was quantified in a scintillation counter. collecting blood and obtaining immune serum. Commercially available The amount of TRAF protein contained in the in vitro translation mixture Abs for TRAF1 (H-132), TRAF2 (H-20), TRAF5 (H-257), and TRAF6 was determined according to the radioactivity incorporated into the protein (H-274) (Santa Cruz Biotechnology, Santa Cruz, CA) were purchased for and the number of methionines contained by each TRAF protein. Known comparison and used for some experiments as indicated below. amounts of each in vitro translated TRAF (5–50 ␮g) were used to create standard curves in immunoblot assays where the ECL-based Ag detection Tissues and patient specimens method was used. ECL data on x-ray films were quantified by scanning densitometry using the IS-1000 image analysis system (Alpha Innotech, Normal tissues for immunohistochemical analysis were derived either from San Leandro, CA), and the results from the in vitro translated protein human biopsy and autopsy material (n Ն 3) or from adult mice of various standard-containing blot were used to normalize all data and thus estimate strains. Only tissues that appeared to be histologically free of disease were the nanograms of TRAF proteins per milligram of total cellular protein. used. These tissues were fixed in either neutral buffered formalin, zinc- Data from two independent standard-containing blots were within 20% buffered formalin (Z-fix; Anatech, Battle Creek, MI), B5, or Bouin’s so- agreement. lution (Sigma, St. Louis, MO), and embedded in paraffin. For part of the analysis, we constructed a tissue microarray containing 130 specimens, Cell isolation and culture representing 0.6- or 1-mm (diameter) cylindrical cores acquired from par- affin blocks of normal human or mouse tissues, which were sectioned at 4- PBLs were isolated from the heparinized blood of normal volunteers and to 5-␮m thickness (28). In addition to normal tissues, archival paraffin cultured in the presence of anti-CD3 (OKT3) Ab, PHA, and IL-2 as de- blocks were obtained for 236 NHL specimens, representing patients’ ma- scribed in detail previously (35). Normal human GC B lymphocytes were terials from several Eastern Cooperative Oncology Group (ECOG)-spon- isolated from tonsils by a method involving magnetic microbead-based sored clinical trials attained through protocol E6491. Histological diagno- immunodepletion with anti-IgD, anti-CD39, anti-CD3, and anti-CD14 sis was confirmed by review by an expert hemopathologist before (36). B cells were cultured in RPMI/10% FBS medium with or without 100 immunostaining, according to the National Cancer Institute (NCI) Working ng/ml of recombinant CD40 ligand (CD40L; Immunex, Seattle, WA). 5086 TRAF EXPRESSION PATTERNS IN NORMAL TISSUES AND LYMPHOID MALIGNANCIES

Peripheral blood B cells were isolated from peripheral blood using anti- (Fig. 1). Additional commercially available antisera for TRAF1 CD19-conjugated magnetic beads (Dynal, Lake Success, New York) ac- (H-132), TRAF2 (SC C20), TRAF5 (SC7220), and TRAF6 (H- cording to the manufacturer’s instructions. B-CLLs were isolated as de- 274) were also determined to bind selectively to their intended scribed (37). All CLL specimens contained Ն95% leukemic cells as determined by FACS analysis using anti-CD19 and anti-CD5 Abs. Of the TRAFs, based on immunoblot analysis of in vitro translated 49 leukemic specimens, 21 represented previously untreated patients and TRAF1 through TRAF6 (Fig. 1). The specificity of these anti- 28 were derived from B-CLL patients with refractory disease. TRAF antisera was also confirmed through experiments where Established tumor cell lines were cultured in either RPMI or DMEM epitope-tagged versions of TRAF1, TRAF2, TRAF5, or TRAF6 with 5 or 10% FBS (38). These cell lines included the NCI panel of 60 human tumor cell lines (39) plus 23 additional human tumor lines main- were transiently expressed in 293T cells and lysates from the trans- tained in our laboratory. fected cells were subjected to SDS-PAGE/immunoblot analysis (data not shown). Statistical analysis Comparisons of protein expression data derived from the NCI panel of 60 Expression of TRAFs in normal tissues human tumor cell lines were made with other biomarker data using Spear- Detergent lysates were prepared from normal tissues, normalized man and Pearson test statistics for nonbinary patterns and Fisher’s exact test for pairs of binary data. In both cases, p values were calculated from for total protein content, and expression of TRAFs was analyzed two-tailed t-distributions and adjusted using a Bonferroni correction for by SDS-PAGE/immunoblotting using antisera with specificity for multiple correlations (39). Correlations of the expression patterns of the TRAF1, TRAF2, TRAF5, or TRAF6. These experiments revealed different TRAF proteins with responses of these cell lines to various cy- tissue-specific differences as well as some similarities in the rela- totoxic drugs and compounds can be found at http://dtp.nci.nih.gov. TRAF1 protein levels in B-CLLs were compared by an unpaired t test, tive steady-state levels of these TRAF family members in human Downloaded from using immunoblot data that had been quantified by previously described tissues. For example, the most abundant amounts of the TRAF1, methods (37, 38, 40). TRAF2, and TRAF6 proteins were detected in thymus, testis, and epidermis (Fig. 2). By comparison, TRAF1 and TRAF6 were far Results less prevalent (as a percentage of total proteins) in most other Characterization of anti-TRAF Abs tissues, whereas TRAF2 was more widely expressed and was Antisera were raised in rabbits using synthetic peptides and re- found at levels that were within a few fold of those seen in the combinant proteins representing fragments of TRAF1, TRAF2, high-expressing tissues. Levels of TRAF5 were highest in thymus http://www.jimmunol.org/ TRAF5, or TRAF6 as immunogens. To explore the specificity of and epidermis. these antisera, immunoblot analysis was performed using in vitro As shown in Fig. 2, the expected endogenous ϳ50- to 53-kDa translated TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF1, ϳ60- to 64-kDa TRAF2, ϳ64-kDa TRAF5, and ϳ66- TRAF6. The antisera raised against human TRAF1 aa 1–22 kDa TRAF6 proteins were the most abundant bands detected in (Bur32), human TRAF2 aa 6–31 (Bur 34), murine TRAF5 aa 1–20 SDS-PAGE/immunoblot assays. However, additional minor bands (1847), as well as human and murine TRAF6 aa 451–469 (Bur30) of lower molecular mass were seen in some cases, presumably were determined to react specifically with the intended TRAF pro- representing partial degradation products of these TRAFs, which may arise during tissue harvesting or processing (Fig. 2). Higher teins, and did not cross-react with other members of the family by guest on September 27, 2021 molecular mass forms of TRAF2 arise from posttranslational mod- ifications, including ubiquitination (data not shown). Rare iso- forms of TRAFs arising from alternative mRNA splicing cannot be excluded. Similar results were obtained in those cases where two or more TRAF-selective Abs were available (data not shown), fur- ther confirming the validity of these results.

FIGURE 1. Characterization of anti-TRAF antisera by immunoblot analysis of in vitro translated TRAF family proteins. Plasmids containing cDNAs encoding human TRAF1, TRAF2, TRAF3, TRAF4, TRAF5, and TRAF6 were used for in vitro translation of these proteins in the presence 35 of [ S]-L-methionine. Equal volumes of the translation lysates were ana- lyzed by SDS-PAGE (10% gels), transferred to nitrocellulose filters, and analyzed by immunoblotting using antisera directed against TRAF1 (H- FIGURE 2. Immunoblot analysis of TRAF1, TRAF2, TRAF5, and 132), TRAF2 (C20), TRAF5 (H-257), or TRAF6 (bur30). Abs were de- TRAF6 expression in normal human tissues. Detergent lysates were pre- tected by an ECL method. Bottom, Autoradiogram resulting from exposure pared from various normal human tissues, normalized for total protein of a blot to x-ray film, confirming production of all in vitro translated content (50 ␮g), and subjected to SDS-PAGE/immunoblot assay using 35S-labeled proteins. Similar results were obtained with additional Abs not antisera specific for TRAF1, TRAF2, TRAF5, or TRAF6. Ab detection shown here. was accomplished by an ECL method. The Journal of Immunology 5087

Table I. TRAFs immunoreactivity in normal human and mouse tissues

Intensity

Organ/Tissue Structure/Cell Type TRAF1 TRAF2 TRAF3 TRAF4 TRAF5 TRAF6

Skin Epidermis Keratinocytes Stratum basale Basal cell layer 1 0–2 0–1 3 1–2 0–1 Stratum spinosum Spinous layer 1 0–1 1–2 1–2 1–2 0–2 Stratum granulosum Granular layer 1 0–1 3 1–2 0 0–3 Dermis Stroma Fibroblasts 0 0 1–2 0 0 0–2 Sweat gland Epithelium 1 0–1 0–2 2–3 0–2 0–2 Sebaceous gland Epithelium 0–1 0 0–1 0 1 0–1 Musculoskeletal Cartilage Chondrocytes 0–1 0–1 2–3 1–3 0–2 0–3 Fibroblasts 0 0 1–2 0 0 0–1 Bone Osteocytes 0 0 0 0 0 0 Osteoclasts 0 0 0 0 0 0–2 Osteoblasts 0 0 0 0 0 0 Striated muscles Muscle fibers 0–1 1 0–2 0 2–3 0–2 Cardiovascular Downloaded from Heart Myocytes 0–1 1 2–3 0–1 1–3 0–3 Capillary endothelium 0–2 0 0–2 0 0 0–1 Respiratory system Trachea (Pseudostratified columnar 0–1 0 3 0–3 2 0–2 epithelium) Lungs Bronchi (Pseudostratified or simple 0–1 0 1 0–3 0–2 0–2 http://www.jimmunol.org/ columnar epithelium) Alveoli Type I/II pneumocytes 0 0 0 0 0 0 Alveolar macrophages 0–2 0–3 0–2 0 0–3 2–3 Alimentary Tract Salivary gland (submandibular gland) Secretory gland acini Serous cells 0–2 0 0 0 0 0 Mucous cells 0 0 0 0 0 0 Salivary duct epithelium 1 0 1–2 1–3 0–1 0–1 Esophagus (stratified squamous epithelium) Basal cell layer 1 0–1 3 3 2 0–1

Spinous layer 1 0–1 0–1 1 1 0–2 by guest on September 27, 2021 Granular layer 1 0–1 0–1 1 1 0–2 Muscularis externa Smooth muscle cells 1 0 1 0–1 0–2 0–2 Stomach (cardiac region) Gastric pits/foveolar cells 0–1 0 2–3 0–2 0 0–3 Cardiac glands 0–1 0–1 1 0 0–1 0–1 Submucosal plexus (Meissner’s plexus) Ganglion cells 1 0 0–2 0 0 0–1 Small intestine Absorptive epithelium 1–2 0–1 1–2 0 0–2 SN 0–3 Colon Absorptive cells 0–2 0–1 1–2 0–2 0–1 0–3 Lamina propria: Lymphocytes 0 0 0–2 0 0 0 Plasma cells 0 0–2 0–2 0–1 0 0–3 Macrophages/DC 0–3 0–2 0 0–2 0–3 0–3 Auerbach’s plexus Ganglion cells 1 0 0–1 0 0 0–2 Liver Hepatocytes 0 1 1–2 0–1 0–2 0–1 Sinusoidal endothelium 0 0 0 0 0 0 Bile duct epithelium 0 0 1 0–2 0 0 Kupfer cells 0 0 0 0 0 0–1 Pancreas Exocrine Acinar cells 0 0 0 0 1–3 0 Ductal epithelium 0–1 0–2 0–1 2–3 0 0–2 Endocrine Islets of Langerhans 0–2 0–3 0–3 0 0 0–3 Urinary Kidney Glomeruli Mesangial cells 0 0 0 0 0 0–1 Endothelial cells 0 0 0 0 0–1 0 Bowman’s capsule Parietal layer (squamous epithelium) 0–2 0 0 0 0 0–3 Visceral layer (podocytes) 0 0 0 0 0 0 Collecting tubules Proximal convoluted tubules 0–1 0 0–1 1–2 1–3 0–1 Loop of Henle—thin limb 0–1 0 0–1 0 0–3 0–2 Distal convoluted tubules 1–2 0–2 1–2 1–2 1–3 1–3 Collecting ducts Epithelial cells 0–2 0 2 2 2–3 0–2 Reproductive male Testis Leydig cells 0 0 0–1 1–2 0–1 0–2 Sertoli cells 0–1 0 0–1 0 1–2 0 Spermatogonia 0–1 0 0–1 0 0 0–2 Spermatocytes 0–3 0–3 0–1 1–3 0–1 0–3 Spermatids 0 0 0–1 0 0 0–3 Spermatozoa 0 0 0 1–2 0 0–2 5088 TRAF EXPRESSION PATTERNS IN NORMAL TISSUES AND LYMPHOID MALIGNANCIES

Table I. Continued

Intensity

Organ/Tissue Structure/Cell Type TRAF1 TRAF2 TRAF3 TRAF4 TRAF5 TRAF6

Prostate Tubuloalveolar glands Basal cells 0 0 1–3 1–3 0 0–3 Luminal secretory cells 0 0 0–1 0 0 0–3 SN Fibromuscular stroma Myoepithelial cells 1 0 1–3 0–2 0–3 0–2 Reproductive female Vagina (epithelium) Basal cell layer 1 0–1 2 3 0–1 0–1 Spinous layer 1 0–1 1 1–2 0–1 0–2 Granular layer 1 0–1 1 1–2 0–1 1–2 Cervical mucous glands 0–1 0 0–1 0–2 0 0–3 SN Uterus Endometrium Simple columnar epithelium 0–2 0–1 0–1 0–1 0–1 0–3 Stromal cells 0 0 0 0–1 0 0–2 Myometrium Smooth muscle cells 1 0–1 1–2 0–1 0–1 0–2 Ovary/oviduct Not tested Mammary gland Tuboloalveolar glands Cuboidal/columnar epithelium 0 0–1 1–2 2–3 0 0

Lactiferous ducts Columnar epithelium 0 0–1 1 0–1 0 0 Downloaded from Myoepithelial cells 0 0 0–1 0 0–1 0 Loose/fibrous stroma Fibroblasts 0 0 0 0 0 0–2 Hematolymphoid tissues Thymus Cortex Cortical thymocytes/lymphoblasts 0 0 0 0 0 2–3 Macrophages 0 0–1 0 0 0 0 Medulla Epithelioreticular cells 0 0–3 2 3 0–3 0–2 Medullary thymocytes 0 0 0–1 0 0 0 http://www.jimmunol.org/ Interdigitating dendritic cells 3 0 0 0 0–2 0 Lymph nodes and tonsils GC Large noncleaved cells 0–2 0–3 0–2 0 0–2 0–2 Small noncleaved cells 0 0–1 0–1 0 0–1 0–1 Small cleaved cells 0 0–1 2–3 0 0–2 0–1 Follicular dendritic cells 0 0 0 0 0–2 0 Macrophages 0–2 2–3 0 0 0–2 0 Mantle zone Lymphocytes 0 0 0–3 0 0–1 0 Interfollicular region Small lymphocytes 0 0 0 0 0 0

Large transformed lymphocytes 0 0 0–2 0 0–2 0–1 by guest on September 27, 2021 Sinus histiocytes 0–3 0–3 0 0–2 1–2 0–3 Plasma cells 0 0–1 3 0, occ2 0 0–3 Dendritic cells 2–3 0 0 0 0, occ 3 0–3 Spleen White pulp Periarteriolar sheets/lymphocytes 0 0 0–1 0 0–1 0 Follicles: GC Lymphocytes 0–2 1–2 0–2 0 0–1 0 Marginal zone Lymphocytes 0 0 0–1 0 0 0 Macrophages 0 0–3 0 0 0–3 0–3 Plasma cells 0 0–2 0–2 0 0 0–2 Interdigitating dendritic cells 0–3 0 0 0 0 0–1 Reticular cells 0 0 0 0 0, occ 0–1 Red pulp/spleen cords Sinus lining cells 0 0–3 0 0 0 0–3 Macrophages 0–3 0–3 0 0–2 0–3 0–3 Megakaryocytes 0 0 0 0 0–1 0–1 Plasma cells 0 2–3 2–3 0 0 0 Bone marrow Erythroid precursors 0 0 0–1 0–1 0 0 Myeloid precursors 0 0–3 0–2 0 0–2 0–2 Megakaryocytes 2–3 0 2–3 0–1 1–3 0–3 Mature neutrophils 0 1–2 0–3 0–3 0–3 0–3 Plasma cells 0 2–3 2–3 0 0 0 Monocytes 0 2–3 1–2 0–1 0 0 Peripheral blood Granulocytes 0 2 1–3 2–3 1–3 0–2 Monocytes 0–2 2–3 2 0–1 0 0 Lymphocytes 0 0–2 0–1 0 0 0 Erythrocytes 0 0 0 0 0 0 Central nervous system Cortex and basal ganglia Gray matter Neurons 0 0–2 0–1 0, occ 2 0 0–3 Neuropil 0 0 0 0 0 0 White matter Axons 0 0 1–3 0 0 0–2 Myelin sheath 0 0 0 0 0 0 Macroglia Astrocytes 0 0 0 0–2 0 0–1 Oligodendroglia 0 0 0 0 0 0 The Journal of Immunology 5089

Table I. Continued

Intensity

Organ/Tissue Structure/Cell Type TRAF1 TRAF2 TRAF3 TRAF4 TRAF5 TRAF6

Microglia Microglial cells 0 0 0 0 0 0 Cerebellum Cortex Purkinje cells 0 0–2 1–3 0, occ 2 0 0–3 Purkinje cell dendrites 0 0 0–1 0 0 0–2 Granular cells 0 0 0 0 0 0 Golgi cells 0 0 0 0 0 Cortex (continued) Stellate and basket cells 0 0–2 0–1 0 0 0 Astrocytes (Bergman glia) 0 0 0 0 0–2 0 Medulla Dentate nucleus 0 0 0 0 0 0 Spinal cord White matter Axons 0 0 1–2 0–1 0 0–3 Myelin sheath 0 0 0 0 0 0–2 Gray matter Ventral horn motoneurons 0 0 0–1 0 0 0–2 Dorsal horn sensory neurons 0 0 0 0–1 0 0–2 Peripheral nervous system Dorsal root and cranial nerve/autonomic 0–1 0 1–2 0–1 0–1 0–2

ganglia; ganglion cells Downloaded from Peripheral nerves Axons 0 0–2 3 1–3 0 0–3 Myelin sheath 0 0 0 0 0 0 Endocrine system Thyroid Follicle cells 0 0 0 0 0 0 Adrenal Cortex 0 0 0–1 0 2 0–2 Medulla Chromaffin cells 0, occ 2 0 2–3 0 0–1 0 Adenohypophysis 0–1 0–2 0–3 0 0 0–2 http://www.jimmunol.org/ Neurohypophysis Nonmyelinated nerve fibers 0 0 0–1 0 0 0–2 Pineal gland Pinealocytes 0–1 0–1 0 0 0 0

Immunohistochemical localization of TRAFs in tissue sections relative amounts of these TRAF family proteins in various types of Using antisera specific for TRAF1, TRAF2, TRAF5, or TRAF6, cells in vivo. the in vivo patterns of expression of these proteins were examined in normal human or murine tissues by immunohistochemistry Inducible expression of TRAFs in PBLs and GC B cells by guest on September 27, 2021 methods. Overall, the cell type-specific patterns of expression of To explore whether changes in TRAF family protein might occur these four TRAF family members were strikingly different, indi- during lymphocyte activation, PBLs (representing mostly T cells) cating that they are all independently regulated to a large extent. were cultured for various times with or without PHA and IL-2 or Table I presents a comprehensive summary of the immunostaining with OKT3 monoclonal anti-CD3 Ab. As shown in Fig. 4A, un- results for TRAF1, TRAF2, TRAF5, and TRAF6, and also in- stimulated PBLs contained little or no detectable TRAF1, TRAF2, cludes for comparison our previously published observations for TRAF5, or TRAF6 when initially placed into culture and during up TRAF3 and TRAF4 (25, 26). In the vast majority of instances, to 6 days of culture in the absence of mitogens or lymphokines immunostaining for TRAFs was confined to the cytosol, though (with the exception of TRAF6, where a slight increase in protein occasionally nuclear immunostaining was also found (Table I). levels was also detected after several days of culture without stim- Fig. 3 presents some examples of TRAF1, TRAF2, TRAF5, and ulus). In contrast, marked increases in all of these TRAF family TRAF6 immunostaining data for the lymphoid and hemopoietic proteins were induced by anti-CD3 Ab and by the combination of organs of lymph node, spleen, thymus, and bone marrow. These PHA and IL-2. However, note that because elevations in TRAFs tissues are emphasized because many of the TNF family cytokine occurred relatively late in these cultures, it is unlikely that PHA, receptors to which TRAFs bind are prominently involved in the anti-CD3, or IL-2 are directly responsible. physiology of these organs. Note that although several TRAFs TRAF expression was also studied in freshly isolated GC B were expressed in the GC lymphocytes of lymph nodes (all except cells. Unstimulated GC B cells contained TRAF2, TRAF3, TRAF4), small resting lymphocytes in the interfollicular regions of TRAF5, and TRAF6 but not TRAF1. In contrast, stimulation of nodes were typically immunonegative for all TRAFs (Fig. 3; Table these B cells with CD40L triggered striking increases in TRAF1 I). Thus, expression of TRAFs in lymphocytes may be dynami- expression, but resulted in no significant changes in the levels of cally regulated in vivo with changes in lymphocyte activation. In- TRAF2, TRAF3, TRAF5, and TRAF6 (Fig. 4B). Probing the same terestingly, TRAF6 was the only TRAF family member expressed blots with Abs specific for other proteins, such as caspase-10 and at immunodetectable levels in cortical thymocytes, whereas poly(ADP-ribose) polymerase, demonstrated the specificity of TRAF3 was the only member detected in medullary thymocytes these CD40L-induced changes in TRAF expression and also con- (Fig. 3; Table I). Thus, TRAF expression may be modulated during firmed loading of approximately equal amounts of total protein in thymocyte differentiation. Differences in the intensity of TRAF all lanes (data not shown). immunoreactivities in myeloid progenitor cells and mature gran- ulocytes in bone marrow similarly suggest fluctuations in the ex- pression of these proteins during hemopoietic differentiation. Ad- Analysis of TRAF protein levels in human tumor cell lines ditional details are provided in Table I. It should be noted that Lysates were prepared from 83 human tumor cell lines, represent- because of the nonquantitative nature of immunohistochemistry, ing a wide range of malignancies, including the entire NCI 60 cell these data should be interpreted as only approximations of the line anticancer drug screening panel (38). After normalization for 5090 TRAF EXPRESSION PATTERNS IN NORMAL TISSUES AND LYMPHOID MALIGNANCIES Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. Immunohistochemical analysis of TRAF family proteins in normal lymphoid and hemopoietic tissues. Representative examples of TRAF1 and TRAF2 (human tissues), and TRAF5 and TRAF6 (murine tissues), immunostaining are shown for lymph node, thymus, spleen, and bone marrow. Ab detection was accomplished by a diaminobenzidine method (brown), and nuclei were counterstained with hematoxylin (blue). Photomicrographs represent ϫ60 to ϫ400 original magnification. Some of the salient features include 1) for TRAF1, overexpression in dendritic cells located in the interfollicular zone of the lymph node, T cell zone in the spleen, and thymic medulla; only multinucleated megakaryocytes are positive in bone marrow; 2) for TRAF2, only a subset of dendritic cells in the lymph node, spleen, and thymic cortex is strongly stained. In the bone marrow, myeloid precursors, granulocytes, monocytes, and plasma cells express TRAF2; 3) for TRAF5, histiocytes in lymph node sinuses and in the red pulp of spleen as well as epithelioreticular cells in the thymic medulla and cortex contain TRAF5 staining. In bone marrow, TRAF5-positive cells include myeloid precursors, mature neutrophils, and megakaryocytes; and 4) for TRAF6, in nodes, GC lymphocytes are commonly positive as are the occasional dendritic cells. Endothelial cells are strongly TRAF6 immunopositive in blood vessels in the spleen. Nearly all immature cortical T cells in thymus but not the differentiated thymocytes in the medulla are strongly positive for TRAF6. In bone marrow, megakaryocytes demonstrate an intense staining, whereas myeloid precursors and neutrophils are moderately positive.

total protein content (50 ␮g/lane), samples were subjected to im- anticipated molecular masses for each of these TRAF family mem- munoblot analysis using various anti-TRAF Abs. Fig. 5 shows a bers, in some cases additional bands were also observed in lesser representative example of some immunoblot data obtained for amounts, possibly representing posttranslationally modified TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6. Though the most versions of TRAFs, low abundance isoforms arising from alterna- abundant bands seen in immunoblot analyses corresponded to the tive mRNA splicing, or partially degraded proteins. The Journal of Immunology 5091

the pre-B cell leukemia 380, which arose from an antecedent non- Hodgkin B cell lymphoma (41). The pre-B cell line 697 and all T cell lines tested failed to express TRAF1, as did all myeloid leu- kemia lines tested except the SR line. However, the AML2 cell line did contain a smaller molecular mass anti-TRAF1 reactive protein, which remains to be characterized. Statistical comparisons of TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 protein levels with other biomarkers previously as- sessed in the 60 cell line drug screening panel revealed no corre- lations. However, interestingly, TRAF6 data were positively cor- related with TRAF2 ( p ϭ 0.0006; r ϭ 0.375) and TRAF3 ( p ϭ 0.0001; r ϭ 0.4112). TRAF2 was also correlated with TRAF3 ( p ϭ 0.0007; r ϭ 0.3665).

TRAF1 expression in NHLs The abundant expression of TRAF1 in B cell lines prompted us to explore the expression of this TRAF family member in NHLs. FIGURE 4. Induction of TRAF expression in PBLs and GC B cells. Patient specimens used for these studies represented archival par- Lysates were prepared, normalized for total protein content (25 ␮g), and affin blocks obtained from a variety of ECOG trials. A total of 232 Downloaded from subjected to SDS-PAGE/immunoblot assay using antisera specific for TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6. A, PBLs were cultured lymphoma specimens were immunostained using anti-TRAF1 Ab, with or without anti-OKT3 Ab (2.5 ␮g/ml) or with PHA (5 ␮g/ml) and and the immunostaining results were scored with respect to the recombinant IL-2 (50 U/ml) for various times as indicated. B, GC B cells percentage of TRAF1-immunopositive neoplastic cells (0–100%). were cultured for 1 day with or without CD40L (100 ng/ml). Based on comparisons with non-neoplastic nodes, the presence of Ն30% immunopositive malignant lymphocytes was chosen as a

cut-off for dichotomizing immunostaining data into positive vs http://www.jimmunol.org/ Using in vitro translated radiolabeled TRAF proteins to create negative groups. Using this method, 112 of 232 (48%) lymphomas standard curves for extrapolation, the relative amounts of each were determined to be TRAF1 positive, having percentages of TRAF were estimated for each tumor cell line. Only the most TRAF1 positive exceeding those found in non-neoplastic reactive prevalent, expected molecular mass band for each TRAF was lymph node biopsies (n ϭ 6). Fig. 7 shows some representative quantified, thus the results do not include the lower abundance examples of TRAF1 immunostaining in a reactive node compared modified or alternative isoforms of TRAF1, TRAF2, TRAF3, with lymphomas, including a TRAF1-negative follicular small TRAF5, and TRAF6. Fig. 6 summarizes the results, expressing the cleaved-cell lymphoma (FSCCL), a TRAF1-positive follicular data as nanograms of TRAF protein per milligram of total protein. mixed small/large cell lymphomas, and a TRAF1-positive diffuse TRAF2, TRAF3, TRAF5, and TRAF6 were widely expressed large cell lymphoma (DLCL). In normal and reactive nodes, note by guest on September 27, 2021 among various types of cancer cell lines, although the levels of that although follicular dendritic cells and extrafollicular histio- TRAF2 and TRAF3 tended to be higher by 2- to 3-fold overall cytes were stained intensely for TRAF1, only occasional large (ac- compared with TRAF5 and TRAF6. In contrast, expression of tivated) GC lymphocytes were TRAF1 immunopositive. Similarly, TRAF1 was far more restricted. The most consistent and highest only occasional malignant B cells were stained for TRAF1 in the levels of expression of TRAF1 were found in B cell lines. Indeed, FSCCL specimen shown in Fig. 7, though TRAF1-positive den- all B cell lines tested contained detectable levels of TRAF1, in- dritic cells and histiocytes were prevalent in the same tissue sec- cluding the B cell lymphoma lines RL, Raji, DND39, MuTu, SU- tion. In contrast, a substantial proportion of the malignant lym- DHL.1, and RS11846, the lymphoblastoid B cell line BJAB, and phocytes was strongly stained for TRAF1 in the follicular mixed

FIGURE 5. Examples of immunoblot analysis of TRAF family protein expression in tumor cell lines. Representative immunoblot data are presented for ex- pression of TRAF1, TRAF2, TRAF3, TRAF5, and TRAF6 in human tumor cell lines. Tumor cell lysates were normalized for total protein content (50 ␮g) before SDS-PAGE/immunoblotting. 5092 TRAF EXPRESSION PATTERNS IN NORMAL TISSUES AND LYMPHOID MALIGNANCIES Downloaded from http://www.jimmunol.org/

FIGURE 6. Summary of TRAF family protein expression in 83 human tumor cell lines. x-ray films resulting from immunoblot analysis were analyzed by scanning densitometry as described (37). Data were compared with a standard curve generated using 35S-labeled, in vitro translated TRAFs, and the nanograms of TRAF per milligram of total protein were calculated and displayed as bar histograms for 83 human tumor cell lines. by guest on September 27, 2021 small/large cell lymphomas and DLCL specimens presented a limited comparison, TRAF1 protein levels appeared to be higher (Fig. 7). in many B-CLLs than in normal B cells (Fig. 8). Moreover, treat- Table II summarizes the TRAF1 immunostaining results, cor- ment-refractory B-CLLs contained significantly higher levels of relating the TRAF1 data with lymphoma histology. Because of the TRAF1 compared with previously untreated B-CLLs ( p ϭ 0.03 by archival nature of these specimens, outdated methods for his- unpaired t test) or normal peripheral blood B lymphocytes ( p ϭ topathological categorization were used (mostly NCI working for- 0.01). Thus, TRAF1 protein levels may be pathologically elevated mulation) at the time these specimens were collected and entered in some B-CLLs, with higher TRAF1 possibly representing a pro- into ECOG databases. Thus, only tentative conclusions can be gression event or adaptation associated with resistance to drawn with respect to histology. However, approximately equiv- chemotherapy. alent proportions of lymphomas with nodular (follicular) (51/107; 48%) vs diffuse (61/125; 49%) cytoarchitecture were TRAF1 im- Discussion munopositive. Comparisons of lymphomas according to cell size In this report, we elucidated for the first time the in vivo patterns (small cells vs large cells) suggested a greater tendency of large of expression of several members of the TRAF family of signal cell lymphomas to be TRAF1 immunopositive than small cell lym- transducing adapter proteins, including TRAF1, TRAF2, TRAF5, phomas (73/133 (55%) large cell vs 19/59 (32%) small cell) ( p ϭ and TRAF6. Because similar data have previously been published 0.004). However, using the histopathological diagnoses to corre- for TRAF3 and TRAF4 (25, 26), the data provided here now com- late TRAF1 immunostaining with lymphoma cell size may have plete the expression analysis for all currently known TRAF family underestimated these differences, because in 31 of 40 (78%) mixed proteins. The analysis of TRAF expression by immunohistochem- small and large cell lymphomas, TRAF1-immunopositive cells istry reveals tissue and cell type-specific expression of all members nevertheless were predominantly larger malignant cells (Fig. 7). of the TRAF family, and suggests great diversity in the repertoire Altogether, TRAF1 expression appears to be up-regulated com- of TRAFs present within various types of cells at particular points pared with normal nodes in roughly half of NHLs. in their differentiation. With six family members, if each were independently regulated, 64 combinations of TRAF family protein TRAF1 protein levels are elevated in B-CLLs expression would be theoretically possible, thus providing enor- TRAF1 expression in B-CLL specimens was also evaluated using mous opportunities for individualizing the intracellular signal immunoblotting methods (37), including 21 previously untreated transduction pathways that specific types of cells use for respond- and 28 treatment-refractory patients. Comparisons were made with ing to the TNF/Toll family cytokine receptors, which rely on purified normal peripheral blood B lymphocytes (n ϭ 4). Though TRAFs for their functions. The Journal of Immunology 5093 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. Comparisons of TRAF1 immunostaining in normal and malignant lymph nodes. Representative examples of TRAF1 immunostaining in normal lymph nodes and lymphomas are presented. Data represent photomicrographs taken at ϫ150 (top) and ϫ400 (bottom) original magnification for (from left to right) a non-neoplastic reactive node biopsy, FSCCL, a follicular lymphoma with mixed small cleaved and large cells (FMCL), and a DLCL. The inset shown for the reactive node represents a higher power view (ϫ1000 original) of follicular dendritic cells, with strong TRAF1 immunostaining. Only occasional lymphocytes in nodes display TRAF1 immunopositivity, whereas follicular and extrafollicular dendritic cells and histiocytes commonly exhibit strong TRAF1 immunoreactivity. In FSCCL, most of the malignant small cleaved cells are immunonegative. In FMCL, the larger transformed lymphocytes are prominently stained for TRAF1. In DLCL, most of the malignant cells contain moderate to strong intensity TRAF1 immunoreactivity.

One of the reasons why differences in the expression of com- mus, and spleen, as well as megakaryocytes, smooth muscle cells, binations of TRAF family proteins can have important implica- neurons in peripheral autonomic ganglia, and selected types of tions relates to hetero-oligomerization among TRAFs. In particu- epithelial cells (Table I). Similarly, TRAF5 directly binds the cy- lar, interactions of some TRAF family members with certain TNF tosolic domain LT␤R (45), but relies on hetero-oligomerization family receptors are dependent on hetero-oligomerization with other TRAFs. For example, although TRAF1 can directly interact with the cytosolic domain of CD30, it does not directly bind the Table II. TRAF 1 expression in lymphomasa cytosolic domains of TNFR1, TNFR2, and CD40, depending in- stead on recruitment to these receptors through association with % Cases TRAF2 (5, 6, 8, 9, 42). Thus, TRAF1 depends on TRAF2 for its Lymphoma Type Tested Positive Positive association with certain TNF family receptors. Moreover, TRAF1/ TRAF2 heterodimers have been shown to recruit the antiapoptotic All combined 232 112 48 FCCb (grade I) 30 11 37 proteins cIAP1 and cIAP2 to TNFR1 and TNFR2 receptors (43). FCC (grade II) 34 16 47 The simultaneous combination of TRAF1, TRAF2, cIAP1, and FCC (grade III) 43 24 56 cIAP2 protein expression has been reported to be required for op- Diffuse mixed (large and small 6467 timal suppression of caspase-8 activation induced by TNFR1, thus cell lymphoma) DLCL 90 49 44 preventing TNFR1-induced apoptosis (44). Thus, it may be of rel- Mantle cell lymphoma 18 3 17 evance to mechanisms of apoptosis suppression in the context of Small lymphocyte lymphoma 11 5 45 responses to TNF-␣ that several types of cells appear to express Normal node 9 0 0 TRAF1 in the absence of TRAF2 (based on immunohistochemical a Immunopositivity defined as Ն30% stained cells. analysis of normal tissues), including dendritic cells in nodes, thy- b FCC, Follicular center cell lymphoma. 5094 TRAF EXPRESSION PATTERNS IN NORMAL TISSUES AND LYMPHOID MALIGNANCIES

In contrast to peripheral blood T cells, GC B cells constitutively expressed TRAF2, TRAF3, TRAF5, and TRAF6. Previous studies have shown that GC B cells do not express TRAF4 (26). Thus, these cells, which respond to CD40, possess a diversity of TRAFs, suggesting that they are poised to rapidly respond to CD40L within the microenvironment of the GC. Among the five TRAF family proteins surveyed, only TRAF1 was induced by CD40L. Recent studies have demonstrated that TNF-␣ and CD40L induce TRAF1 mRNA expression (53), consistent with our results. Many TNF family receptors are expressed on malignant cells, providing opportunities for modulating tumor cell growth, Ag-pre- senting capacity, and sensitivity to apoptotic agents such as anti- cancer drugs. TNF derives its name from early investigations of this cytokine that suggested that it might be selectively cytotoxic to tumor cells, sparing normal cells. Differences in sensitivity to FIGURE 8. TRAF1 protein levels are elevated in B-CLLs. Detergent TNF-␣-induced cell death have been linked to TRAF-dependent lysates were prepared from B cells purified from normal PBLs, previously signal transduction responses, including NF-␬B-dependent and untreated B-CLLs, and refractory B-CLLs. After normalization for total -independent mechanisms (54–56). Thus, alterations in the levels protein content (25 ␮g), samples were subjected to SDS-PAGE/immuno- and ratios of various TRAF family proteins can translate into dif- Downloaded from blot analysis using anti-TRAF1 antiserum. Data were collected by an ECL ferences in tumor cell sensitivity to TNF-mediated cytotoxicity. method with exposure to x-ray film, followed by quantitation by scanning For example, TRAF1 and TRAF2, in combination, reportedly pro- densitometry. Data are expressed relative to positive control cell lysate mote resistance to TNF-induced apoptosis at a proximal step, derived from the RS11846 lymphoma cell line (set at 100%), which served squelching activation of the apical caspase in the death receptor as an internal standard on all blots, as described (37). pathway, namely, pro-caspase-8 (44). Thus, elevated levels of

TRAF1 or TRAF2 might promote tumor cell resistance to TNF-␣ http://www.jimmunol.org/ with TRAF3 for its association with CD40 (8). The interdepen- and related death ligands such as Apo3L (DR3-Ligand), whose dence of TRAF3 and TRAF5 coexpression is underscored by the receptors use very similar signaling mechanisms (57). Conversely, observation that TRAF5 can activate kinases involved in NF-␬B overexpression of TRAF3 can interfere with NF-␬B induction by induction and JNK activation, whereas TRAF3 does not (45, 46). TNFR1, CD40, and TRAF2, in at least some circumstances (58), Thus, CD40-expressing cells that contain TRAF5 in the absence of suggesting that reductions in TRAF3 levels might provide tumor TRAF3 presumably cannot use TRAF5 for CD40 signal transduc- cells with improved resistance to TNF-mediated cytotoxicity. Sim- tion responses. Examples of TRAF5 expression in the absence of ilarly, TRAF3 has been implicated as an important mediator of the immunodetectable TRAF3 were found by immunohistochemical growth-suppressing and apoptosis-sensitizing effects of LT␤R and analysis in interdigitating reticulum cells of nodes (paracortex), CD40 on epithelial cancer cells (59–61). However, because by guest on September 27, 2021 thymus, and spleen; follicular dendritic cells of GCs; as well as in NF-␬B and other TRAF-activated signaling pathways also can up- macrophages and histiocytes, all cell types that are known to ex- regulate expression of molecules involved in Ag presentation, pro- press CD40 and respond to CD40L. Thus, one presumes that these moting NF-␬B induction may have disadvantages in terms of tu- types of cells rely upon other NF-␬B- and JNK-activating TRAF mor avoidance of immune recognition. The immunoblot analysis family members, besides TRAF5, which are known to bind the of TRAF protein levels in 83 tumor cell lines, when compared with cytosolic domain of CD40. the patterns of TRAF expression observed in normal tissues by Our studies with isolated PBLs and GC B cells suggest that immunohistochemical methods, suggests some possible tumor- expression of TRAFs can be dynamically regulated in response to specific alterations in TRAF family protein expression. For in- various stimuli that induce lymphocyte activation, proliferation, or stance, though TRAF1 was not present at immunodetectable levels differentiation. Though little or no TRAF1, TRAF2, TRAF3, in normal mammary epithelium, some breast cancers contained TRAF5, or TRAF6 protein was found in circulating resting PBLs, abundant amounts of TRAF1 protein. Similarly, although TRAF2 expression of all of these TRAF family members was induced by was not detectable by immunostaining in normal prostatic epithe- TCR complex ligation using anti-CD3 Ab or other T cell mitogens. lium, most prostate cancer cell lines contained high levels of Because TRAFs participate in signaling by a variety of TNF fam- TRAF2 protein. Conversely, although normal colonic epithelial ily receptors (CD27, CD30, and OX4B), which provide costimu- cells were uniformly TRAF3 immunopositive, most colon cancer latory functions in T cell responses (47–50), the inducibility of cell lines lacked expression of TRAF3, suggesting cancer-associ- TRAF expression suggests a mechanism for ensuring that only ated down-regulation of TRAF3 expression. Ag-stimulated T cells are competent to respond to costimulatory Examination of TRAF1 expression in lymphomas suggests that signals for TNF family cytokines. In this regard, in vitro studies of striking up-regulation occurs commonly in these neoplasms, par- mature T cells isolated from TRAF knockout mice indicate that ticularly during transformation of the malignant cells to large cell TRAF3 is essential for proliferative responses of T cells, whereas disease. Although normal lymphocytes can be induced to express TRAF6 is not (20, 23). Moreover, CD28-independent costimula- TRAF1, immunohistochemical analysis of normal lymph nodes tion of resting T cells by the TNFR family member 4-1BB has and other peripheral lymphoid organs suggests that relatively small been demonstrated to require TRAF2 (51). Thus, a critical role percentages of normal (presumably activated) lymphocytes ex- exists for TCR-inducible expression of certain TRAF family mem- press this particular member of the TRAF family in vivo. In con- bers in T cell-proliferative responses. Furthermore, poststimula- trast, much higher percentages of malignant lymphoma cells com- tory degradation of TRAFs may help to ensure that T cells are monly expressed TRAF1, implying a deregulation in TRAF1 competent to respond to costimulatory TNF family ligands during expression. B cell lymphoma cell lines were also the most consis- only a brief window, as has been documented recently for the case tent expressers of TRAF1 among the 83 human tumor cell lines of CD30-dependent degradation of TRAF2 (52). examined in vitro, suggesting that TRAF1 overexpression does not The Journal of Immunology 5095 require unique microenvironments found in lymphoid organs in 13. Shi, C. S., A. Leonardi, J. Kyriakis, U. Siebenlist, and J. H. Kehrl. 1999. TNF- vivo and implying autonomous deregulated expression of this mediated activation of the stress-activated protein kinase pathway: TNF receptor- associated factor 2 recruits and activates germinal center kinase related. J. Im- TRAF family member in these malignancies. Furthermore, circu- munol. 163:3279. lating B-CLLs also commonly contained high levels of TRAF1, 14. Baud, V., Z.-G. Liu, B. Bennett, N. Suzuki, Y. Xia, and M. Karin. 1999. Sig- naling by proinflammatory cytokines: oligomerization of TRAF2 and TRAF6 is and recently it has been reported that Reed-Sternberg cells of sufficient for JNK and IKK activation and target gene induction via an amino- Hodgkin’s disease and EBV-transformed lymphoid cells also ex- terminal effector domain. Genes Dev. 13:1297. press high levels of TRAF1 (62). The molecular explanation for 15. Hsu, H., J. Huang, H. Shu, V. Baichwal, and D. V. Goeddel. 1996. TNF-depen- dent recruitment of the protein kinase RIP to the TNF receptor-1 signaling com- the aberrant expression of TRAF1 in lymphomas and B-CLLs re- plex. Immunity 4:387. mains to be revealed. The chromosomal where the TRAF1 16. Hoeflich, K. P., W. C. Yeh, Z. Yao, T. W. Mak, and J. R. Woodgett. 1999. gene resides in humans is not frequently involved in chromosomal Mediation of TNF receptor-associated factor effector functions by apoptosis sig- nal-regulating kinase-1 (ASK1). Oncogene 18:5814. translocations or other cytogenetic abnormalities typically seen in 17. Nishitoh, H., M. Saitoh, Y. Mochida, K. Takeda, H. Nakano, M. Rothe, lymphomas or leukemias (63). The promoter of the TRAF1 gene K. Miyazono, and H. Ichijo. 1998. ASK1 is essential for JNK/SAPK activation contains several NF-␬B binding sites and is highly inducible by by TRAF2. Mol. Cell 2:389. 18. Speiser, D. E., S. Y. Lee, B. Wong, J. Arron, A. Santana, Y. Y. Kong, NF-␬B (53). Thus, deregulation of signal transduction pathways P. S. Ohashi, and W. Choi. 1997. A regulatory role for TRAF1 in antigen-induced that control NF-␬B represents at least one hypothetical mechanism apoptosis of T cells. J. Exp. Med. 185:1777. 19. Lee, S. Y., A. Reichlin, A. Santana, K. A. Sokol, M. C. Nussenzweig, and by which TRAF1 overexpression could occur in lymphomas and Y. Choi. 1997. TRAF2 is essential for JNK but not NF-␬B activation and reg- B-CLLs. Though the functional significance of constitutively ele- ulates lymphocyte proliferation and survival. Immunity 7:703. vated levels of TRAF1 remains to be fully elucidated, T cells of 20. Xu, Y., G. Cheng, and D. Baltimore. 1996. Targeted disruption of TRAF3 leads to postnatal lethality and defective T-dependent immune responses. Immunity transgenic mice engineered to overexpress TRAF1 display resis- 5:407. Downloaded from tance to Ag-induced apoptosis in vitro and in vivo (18). Thus, 21. Yeh, W. C., A. Shahinian, D. Speiser, J. Kraunus, F. Billia, A. Wakeham, elevated TRAF1 levels may endow malignant lymphocytes with J. L. de la Pompa, D. Ferrick, B. Hum, N. Iscove, et al. 1997. Early lethality, functional NF- ␬B activation, and increased sensitivity to TNF-induced cell death enhanced protection from apoptosis induced either intrinsically by in TRAF2-deficient mice. Immunity 7:715. Ag receptor-mediated induction of TNF family death receptors and 22. Nguyen, L. T., G. S. Duncan, C. Mirtsos, M. Ng, D. E. Speiser, A. Shahinian, ligands (18) or extrinsically by other immune effector cells. Future M. W. Marino, T. W. Mak, P. S. Ohashi, and W. C. Yeh. 1999. TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-me- studies of TRAF1 expression in cohorts of lymphoma and B-CLL diated responses. Immunity 11:379. http://www.jimmunol.org/ patients receiving uniform therapy are needed to establish whether 23. Lomaga, M., W.-C. Yeh, I. Sarosi, G. Duncan, C. Furlonger, A. Ho, S. Morony, C. Capparelli, G. Van, S. Kaufman, et al. 1999. 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