IFN-α-Induced Signal Transduction, Expression, and Antitumor Activity of Immune Effector Cells Are Negatively Regulated by Suppressor of This information is current as Signaling of September 24, 2021. Jason M. Zimmerer, Gregory B. Lesinski, Sri Vidya Kondadasula, Volodymyr I. Karpa, Amy Lehman, Abhik RayChaudhury, Brian Becknell and William E. Carson III

J Immunol 2007; 178:4832-4845; ; Downloaded from doi: 10.4049/jimmunol.178.8.4832 http://www.jimmunol.org/content/178/8/4832 http://www.jimmunol.org/ References This article cites 65 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/178/8/4832.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IFN-␣-Induced Signal Transduction, Gene Expression, and Antitumor Activity of Immune Effector Cells Are Negatively Regulated by Suppressor of Cytokine Signaling Proteins1

Jason M. Zimmerer,*† Gregory B. Lesinski,† Sri Vidya Kondadasula,† Volodymyr I. Karpa,† Amy Lehman,‡ Abhik RayChaudhury,§ Brian Becknell,† and William E. Carson III2†¶

Proteins belonging to the suppressors of cytokine signaling (SOCS) family have been shown to regulate cytokine signal trans- duction in various cell types but their role in modulating the response of immune cells to IFN-␣ has not been fully explored. We hypothesized that SOCS proteins would inhibit the antitumor activity of IFN-␣-stimulated immune cells. Transcripts for SOCS1, SOCS2, SOCS3, and cytokine-inducible Src homology 2-containing were identified in total human PBMC (PBMCs, NK 3 5 cells, and T cells) within 1–2 h of stimulation with IFN-␣ (10 –10 U/ml). Immunoblot analysis confirmed the expression of these Downloaded from factors at the protein level. Transcripts for SOCS proteins were rapidly but variably induced in PBMCs from patients with metastatic melanoma following the i.v. administration of IFN-␣-2b (20 million units/m2). Overexpression of SOCS1 and SOCS3, but not SOCS2, in the Jurkat T cell line inhibited IFN-␣-induced phosphorylated STAT1 and the transcription of IFN-stimulated . Conversely, small inhibitory RNA-mediated down-regulation of SOCS1 and SOCS3 in Jurkat cells and normal T cells enhanced the transcriptional response to IFN-␣. Loss of SOCS1 or SOCS3 in murine immune effectors was associated with enhanced IFN-induced phosphorylated STAT1, transcription of IFN-stimulated genes, and antitumor activity. Of note, IFN-␣ http://www.jimmunol.org/ treatment eliminated melanoma tumors in 70% of SOCS1-deficient mice, whereas IFN-treated SOCS-competent mice all died. The .antitumor effects of IFN-␣ in tumor-bearing SOCS1-deficient mice were markedly inhibited following depletion of CD8؉ T cells These results indicate that the antitumor response of immune effector cells to exogenous IFN-␣ is regulated by SOCS proteins. The Journal of Immunology, 2007, 178: 4832–4845.

ecombinant IFN-␣ is used to treat patients with meta- The receptor for IFN-␣ is widely expressed on both tumor cells static malignant melanoma and is associated with an and immune effector cells (8, 12). Binding of IFN-␣ to its receptor overall response rate of 10–15% (1–3). High-dose IFN-␣ activates Jak1 and , which in turn phosphorylate R by guest on September 24, 2021 is also used as an adjuvant in patients who have undergone resec- tyrosine residues within the cytoplasmic region of the receptor. tion of high-risk lesions (nodal disease or primary tumors of These phosphotyrosine residues provide docking sites for STAT1 Breslow thickness Ͼ4 mm) (1, 3–6). However, it has been difficult and STAT2, latent cytoplasmic transcription factors that are phos- to determine the optimal dose of IFN-␣ for melanoma patients or phorylated by the Jaks (13). The prototypical IFN-␣-signaling re- devise strategies to enhance the antitumor effects of IFN-␣ because action results in the formation of IFN-stimulated gene factor 3, a its cellular targets and mechanism of action are largely unknown. DNA-binding complex that consists of STAT1␣ (or STAT1␤), Although exogenous administration of IFN-␣ can act directly on STAT2, and IFN regulatory factor 9 (14). IFN-stimulated gene melanoma cells to inhibit proliferation and up-regulate the expression factor 3 subsequently translocates to the nucleus and binds to IFN- of MHC class I Ags, its stimulatory properties on effector cells of the stimulated response elements located in the promoter regions of immune system are thought to be critical for its antitumor activity IFN-responsive genes (15). These signaling events induce the ex- (7–10). Dunn et al. (11) have also shown that endogenously produced pression of a variety of immunoregulatory genes and largely de- IFN-␣ is required for the prevention of carcinogen-induced tumors termine the pattern of immune cell activation following exposure and that host immune effector cells are critical targets of IFN-␣ during to IFN-␣ (9, 16–19). We have previously demonstrated a high the development of protective antitumor responses. degree of variability in the formation of phosphorylated STAT1 (P-STAT1)3 in patient immune effector cells following IFN-␣-2b immunotherapy and have shown that Jak-STAT signal transduc- *Integrated Biomedical Sciences Graduate Program, †Human Cancer Genetics Pro- tion is down-regulated at higher dose levels of IFN-␣ (20). These gram, Department of Molecular Virology, Immunology, and Medical Genetics, ‡Cen- data suggested that negative regulatory pathways might influence ter for Biostatistics, §Department of Pathology, ¶Department of Surgery, The Ohio State University, Columbus, OH 43210 signal transduction and gene expression in human immune cells following exposure to IFN-␣. Received for publication August 31, 2006. Accepted for publication January 30, 2007. Investigators have identified a family of proteins termed sup- 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 pressors of cytokine signaling (SOCS) that negatively regulate with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by a seed grant from the Immunology Program of The 3 Abbreviations used in this paper: P-STAT1, phosphorylated STAT1; SOCS, sup- Ohio State University-Comprehensive Cancer Center and the Harry J. Lloyd Chari- pressor of cytokine signaling; SH2, Src homology 2; CIS, cytokine-inducible SH2- table Trust (to G.B.L.) and National Institutes of Health Grants P01 CA95426, K24 containing protein; ISG, IFN-stimulated gene; siRNA, small inhibitory RNA; hu, CA93670 (to W.E.C.), and P30 CA16058. human; EGFP, enhanced GFP; DC, dendritic cell; GH, growth hormone; MU, million 2 Address correspondence and reprint requests to Dr. William E. Carson III, Depart- units. ment of Surgery, The Ohio State University, N924 Doan Hall, 410 West 10th Avenue, Columbus, OH 43210. E-mail address: [email protected] Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 4833 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 1. SOCS transcripts are rapidly induced in PBMCs following IFN-␣ stimulation. PBMCs from normal donors were treated with IFN-␣ (103–105 U/ml) or PBS and SOCS mRNA levels were measured by real-time PCR at four time points (0.5, 1, 2, 4 h) using primers specific for (A) SOCS1, (B) SOCS2,(C) SOCS3,or(D) CIS. Data were expressed as the mean fold increase relative to baseline levels (PBS treatment). All real-time PCR data were normalized to the level of ␤-actin mRNA (housekeeping gene). E, Phosphorylation of STAT1 at Tyr701 was measured in parallel by flow cytometry. Mean-specific fluorescence (Fsp) is illustrated on the y-axis. Appropriate isotype control Abs were used to determine background staining. All flow cytometric data were derived from at least 10,000 events gated on the lymphocyte populations determined by light scatter properties (forward scattervs .Statistically significant results vs PBS treatment ,ء .(side scatter). Error bars denote the 95% confidence interval of triplicate experiments (three donors PBMCs were isolated from normal donors, stimulated with IFN-␣ (104 U/ml), and harvested at various times (1, 3, 5, 7, 24 h). Following immunopre- cipitation, SOCS protein levels were measured by immunoprecipitation and subsequent immunoblot analysis using Abs directed against (F) SOCS1, SOCS2, SOCS3, or CIS. Lysates from a SOCS-overexpressing human melanoma cell line (HT144) were used as positive controls. Due to the immuno- precipitation protocol, levels of ␤-actin were measured separately to control for loading. Densitometric data (fold induction) for each condition is shown at the bottom of each lane. Blots shown are representative of separate experiments using PBMCs from three normal donors.

Jak-STAT signal transduction (21). The SOCS family of proteins bind to phosphotyrosine residues in cytokine receptors or Jaks consists of eight members, including SOCS1-SOCS7 and cyto- (22–27) and a C-terminal SOCS box domain that may function to kine-inducible Src homology 2 (SH2)-containing protein (CIS). target SOCS-bound proteins for proteasomal degradation (28, 29). All SOCS proteins have a central SH2 domain that allows them to SOCS1 and SOCS3 also contain a kinase inhibitory region that is 4834 IFN-␣ ANTITUMOR RESPONSE IS NEGATIVELY REGULATED BY SOCS

We now demonstrate that SOCS1–3 and CIS are rapidly in- duced in whole PBMCs, T cells, and NK cells at the transcript and protein level following treatment with IFN-␣. The IFN-␣-induced activation of STAT1 and the subsequent regulation of IFN-stim- ulated genes (ISGs) was significantly reduced in SOCS1- and SOCS3-overexpressing lymphoid cell lines, whereas inhibition of SOCS1 and SOCS3 activity by small inhibitory (siRNA) knock- down led to an enhanced response. Furthermore, IFN-␣-induced signal transduction, gene regulation, and antitumor activity were enhanced in SOCS1- and SOCS3-deficient mice. These results demonstrate that SOCS proteins are critical negative regulators of the immune response to exogenous IFN-␣.

Materials and Methods Reagents and cell lines Recombinant human (hu) IFN-␣-2b (specific activity of 2 ϫ 108 IU/mg) 0 was purchased from Schering-Plough. Human growth hormone was pur- chased from Apollo Cytokine Research. IFN-A/D (sp. act. 1.1 ϫ 108

U/mg; PBL Biomedical) was used in all murine tumor challenge experi- Downloaded from ments and was administered via the i.p. route at a dose of 2 ϫ 104 U/day. IFN-A/D, or universal type I IFN, is a human hybrid recombinant type I IFN constructed from recombinant hu-IFN-␣ A and hu-IFN-␣ D. It is active on a wide variety of mammalian cells (PBL Biomedical). The human melanoma cell lines were gifts from Dr. S. Ferrone (Roswell Park Cancer Institute, Buffalo, NY) and were cultured in RPMI 1640 with 10% FBS and antibiotics (35). The Jurkat T cell lymphoma cell line (clone E6-1) was http://www.jimmunol.org/ obtained from the American Type Culture Collection. The murine mela- noma cell line JB/MS was obtained from V. Hearing (National Cancer Institute, Bethesda, MD) and grown as an adherent monolayer in DMEM supplemented with 10% FBS, sodium bicarbonate, 4 mM L-glutamine, 1% vitamins, 1% sodium pyruvate, 1% nonessential amino acids, and antibiotics (36).

Ab for depletion of CD8ϩ T cells Rat anti-mouse CD8 Ab (clone 2.43) was purchased from the National Cell ϩ

Culture Center (NCCC; Minneapolis, MN). For depletion of CD8 T cells, by guest on September 24, 2021 100 ␮g of Ab was injected i.p. on days Ϫ3, Ϫ1, ϩ1, ϩ3, and every 4 days thereafter in relation to the tumor challenge. Rat IgG was used as a control. CD8 depletion was confirmed by flow cytometric analysis of PBMCs ob- tained from venous blood.

Animals SOCS1-deficient mice die of overwhelming inflammation unless the en- dogenous release of IFN-␥ is eliminated (37). SOCS3Ϫ/Ϫ is an embryonic lethal mutation (38). Therefore, SOCS1ϩ/Ϫ IFN-␥Ϫ/Ϫ and SOCS3ϩ/Ϫ mouse breeding pairs (C57BL/6, Sv129 background) were obtained from Dr. J. Ihle (St. Jude Children’s Research Institute, Memphis, TN) and bred to produce their SOCS competent (SOCS1ϩ/ϩIFN-␥Ϫ/Ϫ and SOCS3ϩ/ϩ) and SOCS-deficient (SOCS1ϩ/ϪIFN-␥Ϫ/Ϫ, SOCS1Ϫ/ϪIFN-␥Ϫ/Ϫ, and SOCS3ϩ/Ϫ) counterparts. Genotyping of the SOCS1 and SOCS3 mice and confirmation of SOCS1 and SOCS3 protein deficiencies was performed as previously described (Refs. 37 and 38 and data not shown). Mice of 5–6 wk of age were used in all experiments. Spleens from male and female mice of each genotype were removed aseptically and mechanically dis- persed through 70 ␮M cell strainers. Splenocytes were washed with PBS and 5% FBS, pelleted by centrifugation and resuspended in RPMI 1640 and 10% FBS. All experiments were performed in compliance with the guidelines of the Institutional Laboratory Animal Care and Use Committee FIGURE 1. (continued) of The Ohio State University (Protocol 2004A0151).

Murine tumor models An i.p. model of murine malignant melanoma was used to test the antitu- able to inhibit Jak activity (26, 27, 30). The expression of SOCS1 mor effects of IFN-A/D in SOCS-deficient mice (36). Mice were injected i.p. with 106 JB/MS melanoma tumor cells and randomly selected to re- and SOCS3 has been shown to mediate potent inhibitory effects on ceive either PBS or IFN-A/D (2 ϫ 104 U/day, i.p.). Mice were examined IFN-␣-stimulated signal transduction and gene regulation in sev- daily, and those exhibiting signs of progressive disease were euthanized via eral experimental systems (31–34), however, the effect of exoge- CO2 inhalation. Survival experiments used at least six mice per group. nous IFN-␣ on SOCS expression in resting immune cell subsets Because IFN-A/D treatment was effective at protecting against lethal tumor challenge in SOCS1ϩ/Ϫ and SOCS1Ϫ/Ϫ mice, this model was modified to has yet to be defined in the context of cancer immunotherapy. We permit the outgrowth of tumors for immunohistochemical analysis. For hypothesized that SOCS proteins exert a negative effect on IFN- these studies, mice were injected i.p. on day 0 with 106 JB/MS melanoma ␣-induced immune activity. cells. Beginning on day 7, mice were treated i.p. for 3 days with IFN-␣ The Journal of Immunology 4835 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. SOCS transcripts are differentially induced in NK cells and T cells following IFN-␣ stimulation. NK cells (CD56ϩ) and T cells (CD3ϩ) were isolated from normal donors (n ϭ 3) and treated with 104 U/ml IFN-␣ or PBS. Cells were harvested at four time points (1, 2, 4, 6 h) for real-time PCR analyses of (A) SOCS1,(B) SOCS2,(C) SOCS3, and (D) CIS transcript levels. Data were expressed as the mean fold increase relative to baseline levels (PBS treatment). 4836 IFN-␣ ANTITUMOR RESPONSE IS NEGATIVELY REGULATED BY SOCS

Quantitation kit (Molecular Probes), and reverse transcribed as previously described (39). The resulting cDNA was used as a template to measure gene expression by real-time PCR using predesigned primer/probe sets (Assays On Demand; Applied Biosystems) and 2ϫ TaqMan Universal PCR Master Mix according to the manufacturer’s recommendations as pre- viously described (40). Predesigned primer/probe sets for human ␤-actin were used as an internal control in each reaction well (Applied Biosys- tems). Real-time PCR data was analyzed using the Sequence Detector soft- ware version 1.6. Flow cytometric analysis of P-STAT1 Phosphorylation of STAT1 at Tyr701 was measured using an intracellular flow cytometric assay as previously described, with modifications (20, 41). A rabbit anti-P-STAT1 (Tyr701) primary Ab (Cell Signaling Technology) was used in combination with a goat anti-rabbit Alexa Fluor 488-conju- gated secondary Ab (Molecular Probes). For assays involving enhanced GFP (EGFP)-positive cells, a goat anti-rabbit allophycocyanin-conjugated secondary Ab was used (Santa Cruz Biotechnology). Immunoprecipitation and immunoblot analysis Following treatment, cells were harvested and lysed in TN1 lysis buffer

⅐ Downloaded from (125 mM NaCl, 50 mM Tris (pH 8), 10 mM EDTA, 10 mM Na4P2O7 10 ␮ H2O, 10 mM NaF, 1% Triton X-100, 3 mM Na3VO4,5 g of aprotinin and leupeptin), and centrifuged at 10,000 rpm. For immunoprecipitation exper- iments, supernatants were collected and treated with 5 ␮g of the appropri- ate Ab, processed per manufacturer’s recommendations (Abcam), and then subjected to immunoblot analysis as previously described (42). Design of SOCS constructs http://www.jimmunol.org/ Overexpression of SOCS1, SOCS2, and SOCS3 proteins was achieved using the PINCO retroviral vector as previously described (43). Briefly, PBMCs from a normal healthy donor were stimulated with IFN-␣ and RNA was isolated and converted to cDNA for use as a template in a PCR to isolate the human SOCS1, SOCS2, and SOCS3 genes. Primers were designed to incorporate 1) AT overhangs; 2) 5Ј BamHI and 3Ј EcoRI re- striction sites; 3) A C-terminal myc tag; and 4) a Kozak sequence before the initiation codon. SOCS transcripts were amplified using PCR according to FIGURE 3. Differential SOCS expression and STAT1 activation in the following schema: 94°C for 5 min; 30 cycles of 94°C for 30 s, 55°C for melanoma patients undergoing IFN-␣ immunotherapy. PBMCs from (A) 30 s, 72°C for 1 min, followed by a final extension at 72°C for 7 min. PCR five melanoma patients were obtained immediately before and 1 h follow- products were gel purified (Qiagen Gel Extraction kit) and ligated into the by guest on September 24, 2021 ing administration of high-dose IFN-␣ (20 MU/m2 i.v.) and analyzed for PCR2.1 vector by TA cloning according to the manufacturer’s recommen- SOCS1–3 and CIS mRNA levels by real-time PCR. In addition, freshly dations (Invitrogen Life Technologies). Following sequence verification, plasmids were digested with BamHI and EcoRI to remove SOCS-encoding isolated PBMCs from (B) normal human donors (n ϭ 5) were treated in dsDNA and these inserts were then ligated into the BamHI EcoRI-digested ␣ 4 vitro with IFN- -2b (10 U/ml) and tested for the induction of SOCS PINCO expression plasmid. Ligation reactions were transformed into top transcripts by real-time PCR. Data were expressed as the mean fold in- 10 Escherichia coli and plasmid DNA was sequenced with the following crease relative to baseline levels. All real-time PCR data were normalized primers (PINCO forward, 5Ј-ACCTTACACAGTCCTGCTGA-3Ј; PINCO to the level of ␤-actin mRNA. reverse, 5Ј-TGAACTAATGACCCCGTAATT-3Ј) to verify each SOCS- expressing construct. Following sequence confirmation, each construct was prepared for virus production by endotoxin-free Maxiprep (Qiagen). (2 ϫ 104 U) or PBS. Tumors were harvested on day 10, formalin fixed, embedded in paraffin, and sectioned. Generation of SOCS-expressing retroviral constructs and transduction of Jurkat cells Isolation of immune subsets SOCS-overexpressing PINCO retroviral constructs were generated by tran- PBMC were isolated from source leukocytes of healthy adult donors sient transfection of the Phoenix-Ampho packaging cell line as previously (American Red Cross) or from the peripheral blood of patients receiv- described (43). The Jurkat cell line was transduced with SOCS-overex- ing high-dose IFN-␣-2b (Ohio State University Institutional Review pressing retroviral constructs as previously described by Becknell et al. Board-approved protocol 99H0348) via density gradient centrifugation (43). Infection efficiency was determined by flow cytometry for EGFP with Ficoll-Paque Plus (Amersham Biosciences). Lymphocyte subsets expression. Cells were sorted based on EGFP positivity and used as ϩ Ϫ were enriched for individual cell populations (CD3 /CD56 T cells and described. CD56ϩ/CD3Ϫ NK cells) by negative selection with the appropriate Ro- sette Sep reagents (Stem Cell Technologies) per the manufacturer’s SOCS down-regulation by RNA interference recommendations. Enriched cell populations were cultured in RPMI SOCS1 Ј 1640 medium supplemented with 10% human AB serum (Pel-Freez High-purity siRNA oligonucleotides that target (5 -CTGGTT Ͼ GTTGTAGCAGCTTAA-3Ј) and SOCS3 (5Ј-TCGGGAGTTCCTGGAC Clinical Systems). Cell purity was routinely 95% as determined by Ј flow cytometry. CAGTA-3 ) sequences were purchased from Qiagen. An oligonucleotide that does not match any sequence was used as a control Real-time PCR siRNA (5Ј-AACACAGTGGAGCGAATTCCT-3Ј; Qiagen). Jurkat cells and normal T cells were transfected with siRNA (2 ␮g) via electroporation Following TRIzol extraction (Invitrogen Life Technologies) and RNeasy using the Nucelofector Amaxa device and cell-specific nucleofector re- purification (Qiagen), total RNA was quantitated via the RiboGreen RNA agent according to the manufacturer’s recommendations.

All real-time PCR data were normalized to the level of ␤-actin mRNA. E, Flow cytometric analysis of P-STAT1 levels in purified NK and T cell subsets Statistically significant results vs PBS ,ء .(was performed in parallel. Error bars denote the 95% confidence interval of triplicate experiments (three donors treatment. ‡, Significant differences between T cells and NK cells. The Journal of Immunology 4837 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 4. Overexpression of SOCS1 and SOCS3 protein in Jurkat cells inhibits the response to IFN-␣. The Jurkat T cell lymphoma cell line was transduced with PINCO retroviral constructs encoding SOCS1, SOCS2, or SOCS3 protein. Cells transduced with the empty PINCO vector served as a negative control. Cell populations were routinely Ͼ95% pure post-FACS for GFP. A, Transcript levels of SOCS1, SOCS2, and SOCS3 in transduced cells as measured by real-time PCR. Data are expressed as the mean fold increase relative to baseline levels. All real-time PCR data were normalized to the level of ␤-actin mRNA in duplicate experiments. B, Protein levels of SOCS1, SOCS2, and SOCS3 as measured by immunoblot analysis of whole cell lysates. These results are representative of duplicate experiments. C, Flow cytometric analysis of P-STAT1 formation in transduced cells following stimulation with 4838 IFN-␣ ANTITUMOR RESPONSE IS NEGATIVELY REGULATED BY SOCS

Statistical analysis titumor effects of IFN-␣ (44–46), NK cells, and T cells were iso- ϭ 4 To estimate the changes in SOCS1–3, CIS, and P-STAT1 over time, linear lated from fresh PBMCs (n 3 donors), treated in vitro with 10 mixed effects models were applied to the data (see Figs. 1 and 2). For each U/ml IFN-␣ for varying periods of time (1, 2, 4, 6 h), and analyzed model, the presence of a significant quadratic trend was assessed, as the for SOCS transcripts by real-time PCR. SOCS1–3 and CIS tran- changes over time appeared to be nonlinear. Based on the models, point scripts were significantly up-regulated in T cells following incu- estimates of fold increases were calculated with 95% confidence intervals. bation with IFN-␣ as compared with PBS-stimulated cells ( p Ͻ Statistical analyses for Figs. 3–7 were assessed by ANOVA; pairwise com- ϭ parisons were performed using a two-sided ␣ ϭ 0.05 level of significance. 0.001; Fig. 2, A–D). In contrast, only SOCS1 ( p 0.0263), SOCS2 Kaplan-Meier estimates of the survival function were calculated for the ( p ϭ 0.0688), and SOCS3 ( p ϭ 0.0031) were up-regulated over different mouse groups, and log-rank tests were used to assess group dif- baseline in NK cells. Significantly greater induction of SOCS1 ferences in survival. For all analyses, an ␣ ϭ 0.05 level of significance was ( p ϭ 0.0106), SOCS2 ( p Ͻ 0.0001), and CIS ( p Ͻ 0.0001) were used. All analyses were performed using SAS version 9.1 (SAS Institute). observed in the T cell compartment as compared with the NK com- Results partment at the 2-h time point. SOCS3 transcripts were up-regulated to a similar degree within T cells and NK cells in response to IFN-␣ SOCS1, 2, 3, and CIS are rapidly induced in human PBMCs ( p ϭ 0.1521). T cells and NK cells were simultaneously evaluated for following in vitro stimulation with IFN-␣ levels of P-STAT1 by flow cytometry (Fig. 2E). This analysis re- Freshly isolated PBMCs from normal human donors (n ϭ 3) were vealed a significantly greater induction of P-STAT1 in T cells as com- 1 5 treated in vitro with varying concentrations of IFN-␣-2b (10 –10 pared with NK cells at each time point and a more rapid return to U/ml) and tested for the induction of SOCS transcripts by real-time baseline in T cells beginning at 2 h ( p Ͻ 0.0001).

PCR (Fig. 1, A–D, represent the combined results of these normal Downloaded from donors). SOCS1–3 and CIS were rapidly induced (1 h or less) in Expression of SOCS transcripts in patient PBMCs post-IFN normal PBMCs following in vitro stimulation with IFN-␣ (Fig. 1, therapy A–D, and data not shown). Significant induction of these genes as To characterize the expression of SOCS transcripts in response to compared with PBS-treated cells was observed following treat- exogenous IFN-␣, peripheral venous blood was obtained from pa- ment with doses of IFN-␣ as low as 103 U/ml ( p Ͻ 0.0001), tients with metastatic melanoma (n ϭ 5) immediately before and 4 5 2 however, maximal expression occurred in response to 10 or 10 1 h following the first dose of IFN-␣-2b (20 million units (MU)/m http://www.jimmunol.org/ U/ml IFN-␣ ( p Ͻ 0.0001). For example, PBMCs treated for 2 h i.v.). PCR analysis revealed that SOCS1, SOCS2, SOCS3, and CIS with 104 U/ml IFN-␣ expressed high levels of SOCS1 (10.32 Ϯ transcripts were all expressed at the one hour time point following 2.19-fold induction), SOCS2 (13.10 Ϯ 3.46), SOCS3 (2.79 Ϯ IFN-␣ therapy (Fig. 3A). Of note, there was considerable interpa- 0.87), and CIS (5.98 Ϯ 2.97) as compared with PBS-treated cells. tient variation with respect to the induction of SOCS1 (range ϭ SOCS transcripts were also reproducibly induced at lower doses of 8.6- to 171.0-fold increase compared with baseline levels), SOCS2 IFN-␣ (101–102 U/ml; data not shown) but to a lesser extent. (range ϭ 3.0- to 9.6-fold increase), SOCS3 (range ϭ 1.1- to 4.6- SOCS4–7 and protein inhibitor of activated STAT1 (PIAS1) tran- fold increase), and CIS (range ϭ 1.2- to 11.5-fold increase). In scripts were not induced even when high doses of IFN-␣ were used addition, freshly isolated PBMCs from normal human donors (n ϭ (data not shown). IFN-␣-induced expression of SOCS transcripts 5) were treated in vitro with IFN-␣-2b (104 U/ml) and tested for by guest on September 24, 2021 by PBMCs was transient in nature as levels of SOCS1–3 and CIS the induction of SOCS transcripts by real-time PCR (Fig. 3B). The reverted to baseline within 2–4 h of stimulation. PBMCs from induction of SOCS1 in normal donors following in vitro IFN-␣ these same donors were simultaneously tested for activation of stimulation was also variable. Interestingly, there appeared to be STAT1 using a flow cytometric assay that employs an Ab specific greater induction of SOCS species in PBMCs following in vivo for P-STAT1. Robust phosphorylation of STAT1 was observed administration of IFN-␣. within 30 min of IFN-␣ treatment ( p Ͻ 0.001 vs PBS-treated PBMCs), however, levels of P-STAT1 had returned to baseline Overexpression of SOCS proteins in the Jurkat cell line inhibits ␣ within2hofstimulation (Fig. 1E), a finding that was consistent the response to IFN- with the expression of multiple SOCS species at this time point. The Jurkat T cell lymphoma cell line was transduced with retro- The enhanced expression of SOCS species was confirmed at the pro- viral constructs expressing SOCS1, 2, or 3 to further evaluate the tein level by immunoblot analysis. These studies revealed that role of SOCS in IFN-␣-mediated signal transduction and gene ex- SOCS1, SOCS2, SOCS3, and CIS were rapidly induced in PBMCs pression. As a control, Jurkat cells were also transduced in parallel following exposure to IFN-␣ (Fig. 1F). In each case, there was in- with the unmanipulated vector (PINCO-EGFP). Cells were then creased expression of SOCS protein at the 1 h time point. Maximal harvested from culture, enriched for EGFP expression by FACS induction of SOCS3 and CIS occurred 3 h posttreatment, whereas sorting (Ͼ95% pure) and tested for their responsiveness to IFN-␣. SOCS1 and SOCS2 expression peaked at 5 and 1 h, respectively. Increased expression of these SOCS species was confirmed by real-time PCR and immunoblot analysis (Fig. 4, A and B). Cells SOCS1, 2, 3, and CIS are differentially induced in human T were treated with various concentrations of IFN-␣ (102–104 U/ml) ␣ cells and NK cells following in vitro stimulation with IFN- or PBS for 30 min and then analyzed for the level of P-STAT1 by To characterize the induction of negative regulators in the immune flow cytometry and downstream ISG transcripts by real-time PCR cell compartments thought to be responsible for mediating the an- (18 h time point). Phosphorylation of STAT1 was not appreciably

IFN-␣ (102–104 U/ml) or PBS for 30 min. D, Analysis of 2Ј-5Ј-oligoadenylate synthetase 1 (OAS1), IFN-stimulated gene 20 (ISG20), and IFN-induced protein with tetratricopeptide repeats 2 (IFIT2) transcript levels in transduced cells following stimulation with IFN-␣ (104 U/ml) or PBS for 18 h. Error Statistically significant results. E, The 1106 MEL melanoma cell line was transduced ,ء .bars denote the 95% confidence interval of duplicate experiments with a PINCO retroviral construct encoding the SOCS2 protein. Cells transduced with the empty PINCO vector served as a negative control. Cells were treated with PBS or human GH (huGH; 100 ng/ml) for 15 min. Levels of P-STAT5 were measured by immunoblot analysis. ␤-actin was used as a loading control. SOCS2 overexpression was also confirmed by immunoprecipitation and subsequent immunoblot analysis. Nonspecific bands (NSB) represent the L chain of the rabbit anti-SOCS2 Ab used in the immunoprecipitation. The Journal of Immunology 4839 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 4840 IFN-␣ ANTITUMOR RESPONSE IS NEGATIVELY REGULATED BY SOCS affected by transduction of Jurkat cells with the empty PINCO- petent mice expressed identical baseline levels of STAT1 and EGFP vector, however, cells that overexpressed SOCS1 or SOCS3 IFN-␣R (data not shown). Similar studies were conducted with showed significantly reduced levels of P-STAT1 in response to SOCS3ϩ/ϩ and SOCS3ϩ/Ϫ splenocytes and revealed increased ac- IFN-␣ treatment ( p Ͻ 0.0001; Fig. 4C). The induction of ISGs was tivation of STAT1 ( p ϭ 0.0002, Fig. 5B) and enhanced transcrip- also significantly inhibited in cells that overexpressed SOCS1 or tion of ISGs in response to IFN-␣ as compared with SOCS3ϩ/ϩ SOCS3 ( p Ͻ 0.045 for all genes; Fig. 4D). For example, the in- splenocytes ( p Ͻ 0.0001 for all genes; Fig. 5D). duction of 2Ј-5Ј-oligoadenylate synthetase 1 (OAS1) was 2-fold less in SOCS1 overexpressing Jurkat cells and 4-fold less in siRNA-mediated down-regulation of SOCS1 and SOCS3 in ␣ SOCS3 overexpressing Jurkat cells as compared with PINCO- Jurkat cells and normal T cells enhances the response to IFN- transduced control cell lines ( p ϭ 0.0002 and p Ͻ 0.0001, respec- The Jurkat cell line was transfected with siRNA oligonucleotides tively). In contrast, overexpression of SOCS2 did not have a sig- targeting SOCS1 or SOCS3 and placed in culture for 16 h before nificant effect on the activation of STAT1 or transcription of analysis. Control conditions included cells transfected with a con- downstream ISGs. To demonstrate that the PINCO-SOCS2 con- trol siRNA sequence as well as mock-transfected cells. The siRNA struct was producing a functional SOCS2 protein, we evaluated the constructs inhibited the expression of SOCS1 and SOCS3 in Jurkat known effects of SOCS2 on growth hormone (GH)-induced cells at the transcript and protein level (Fig. 6, A and B). Cells were STAT5 phosphorylation (47). The 1106 MEL melanoma cell line then stimulated with IFN-␣ (104 U/ml) and analyzed for levels of was used instead of the Jurkat cell line due to the fact that Jurkat the IFN-stimulated genes G1P2, OAS1, and IFIT2 at the 18 h time cells have very low levels of STAT5 protein (Ref. 48 and data not point. As seen in Fig. 6C, knockdown of SOCS1 and SOCS3 led shown). As shown in Fig. 4E, overexpression of SOCS2 resulted to a significant increase in the transcription of the indicated genes Downloaded from in a significant decrease in GH-induced STAT5 phosphorylation. ( p Ͻ 0.05 for all genes) as compared with the control conditions. These data confirmed that the SOCS2 vector produces a functional Similar results were obtained following transfection of primary T protein that is capable of inhibiting relevant signaling pathways. cells from normal human donors with siRNA targeting SOCS1 or Further in vitro studies were also conducted to analyze the rela- SOCS3 (data not shown). tionship between IFN-␣-induced SOCS transcripts and the tran- SOCS deficiency augments the antitumor effects of IFN-␣ in vivo scription of IFN-␣-responsive genes. Because SOCS transcripts http://www.jimmunol.org/ are rapidly induced, human PBMCs were analyzed for the expres- The antitumor effects of IFN-␣ in the setting of SOCS1 deficiency sion of SOCS transcripts by real-time PCR 2 h following IFN-␣ were studied in a murine model of malignant melanoma in which stimulation (104 U/ml). Expression of G1P2 mRNA was evaluated JB/MS cells (1 ϫ 106) were injected i.p. into SOCS1ϩ/ϪIFN-␥Ϫ/Ϫ or in these same normal donors at the 4-h time point, which is when SOCS1Ϫ/ϪIFN-␥Ϫ/Ϫ mice (n ϭ 7 mice/group) (38). One day after this transcript is maximally induced by IFN-␣. As expected, there tumor challenge, mice received daily i.p. injections of IFN-A/D (2 ϫ was a strong inverse correlation between the expression of tran- 104 U) or PBS. SOCS1ϩ/ϩ IFN-␥Ϫ/Ϫ mice and wild-type mice script for SOCS1 and SOCS3 and the induction of G1P2 mRNA (SOCS1ϩ/ϩ IFN-␥ϩ/ϩ) served as controls. As expected, treatment of (parametric Pearson correlation; p ϭ 0.0200 and p ϭ 0.0218, re- both tumor-bearing IFN-␥Ϫ/Ϫ and wild-type mice with IFN-A/D led spectively; data not shown). SOCS2 and CIS mRNA expression to a significant improvement in survival as compared with treatment by guest on September 24, 2021 did not correlate with G1P2 expression. These results indicate that with PBS alone (Fig. 7A; p ϭ 0.0027). The antitumor effects of IFN- the transcriptional response of lymphoid cell lines to IFN-␣ is A/D therapy were significantly enhanced in SOCS1-deficient mice as negatively regulated by specific SOCS proteins. 57–71% of these mice were cured of their tumors, whereas PBS- treated mice all died at 10–14 days (Fig. 7B, p ϭ 0.0002 for Splenocytes from SOCS-deficient mice exhibit increased SOCS1ϩ/Ϫ mice; Fig. 7C, p ϭ 0.0002 for SOCS1Ϫ/Ϫ mice). Of note, ␣ responsiveness to IFN- there was no evidence that mice with SOCS deficiencies experienced The genotype of SOCS1- and SOCS3-deficient mice was con- increased systemic toxicity in response to IFN-␣ treatments: these firmed by PCR and altered expression of SOCS protein was con- mice behaved normally and histochemical analysis of their visceral firmed by immunoblot analysis (data not shown). Splenocytes organs revealed no increase in tissue damage or inflammatory cell from SOCS1ϩ/ϩIFN-␥Ϫ/Ϫ, SOCS1ϩ/ϪIFN-␥Ϫ/Ϫ, and SOCS1Ϫ/Ϫ infiltrate (data not shown). Immunohistochemistry of treated tumors IFN-␥Ϫ/Ϫ mice were examined for the ability to respond to IFN-␣ revealed increased infiltration of CD45ϩ immune cells with IFN-␣ with activation of STAT1 and the transcription of IFN-␣-regulated treatment but there was no difference between SOCS-competent and genes. As seen in Fig. 5A, the generation of P-STAT1 in response SOCS-deficient mice. SOCS1-deficient mice that survived following to IFN-␣ was markedly increased in SOCS1Ϫ/Ϫ splenocytes IFN-A/D treatment were observed for a total of 90 days during which as compared with SOCS1ϩ/Ϫ splenocytes ( p ϭ 0.002) and no recurrent tumors were identified. These mice were then rechal- SOCS1ϩ/ϩ splenocytes ( p ϭ 0.004), while SOCS1ϩ/Ϫ splenocytes lenged with 1 ϫ 106 JB/MS cells and remained tumor free for 100 did not show enhanced activation of STAT1 ( p ϭ 0.154 vs days in the absence of additional IFN-␣ treatments (identically treated SOCS1ϩ/ϩ splenocytes). The transcription of downstream IFN- tumor-naive WT mice rapidly developed lethal disease), suggesting stimulated genes (Gzmb, Ifit2, Mx2) was also significantly en- that an adaptive immune response to the tumor had been induced. hanced in SOCS1Ϫ/Ϫ splenocytes as compared with splenocytes Similarly, SOCS3-deficient mice exhibited enhanced survival from SOCS1ϩ/Ϫ and SOCS1ϩ/ϩ mice ( p Ͻ 0.05 for all genes; Fig. (median survival ϭ 29 days) in response to IFN-A/D therapy 5C). Of note, splenocytes from SOCS1-deficient and SOCS1-com- compared with wild-type littermates (median survival ϭ 21

FIGURE 5. SOCS1- and SOCS3-deficient mice exhibit an augmented response to IFN-␣. A and B, The formation of P-STAT1 in SOCS-competent and SOCS-deficient murine splenocytes was measured by flow cytometry following stimulation of with IFN-A/D (104 U/ml) or PBS for 30 min. C and D, Transcription of granzyme B (Gzmb), Ifit2, and myxovirus resistance 2 (Mx2) genes were measured in splenocytes from SOCS1- and SOCS3-deficient mice following an 18 h stimulation with IFN-A/D (104 U/ml) or PBS via real-time PCR. Data were expressed as the mean fold increase relative to baseline levels (PBS treatment). All real-time PCR data were normalized to the level of ␤-actin mRNA. Error bars denote the 95% confidence interval of duplicate .Statistically significant results ,ء .experiments The Journal of Immunology 4841 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 6. siRNA-mediated inhibition of SOCS1 and SOCS3 augments IFN-␣ responsiveness in vitro. Jurkat cells were transfected via elec- troporation with siRNA specific to SOCS1 and SOCS3. As a negative control, Jurkat cells were transfected in parallel with a control siRNA or no siRNA (mock transfection). A, Specific down-regulation of SOCS by siRNA was confirmed at the transcript level by real-time PCR and (B)atthe protein level by immunoprecipitation and immunoblot analysis of SOCS1 and SOCS3. These results are representative of duplicate experiments. C, IFN-␣ responsiveness at the level of gene transcription was measured in siRNA-transfected cell lines via real-time PCR. Fold increase in ISGs were determined following an 18-h stimulation with IFN-␣ (104 U/ml) or PBS (IFN-␣-inducible protein (clone IFI-15K) (G1P2), 2Ј-5Ј-oligoadenylate synthetase 1 (OAS1), and IFN response factor 7 (IRF7)). Data were expressed as the mean fold increase relative to baseline levels (PBS treatment). All real-time PCR data were normalized to the level of ␤-actin mRNA. Error bars denote the 95% confidence interval of duplicate experiments. .Statistically significant results ,ء days; Fig. 7D; p ϭ 0.0091). However, all SOCS3-deficient mice tent and SOCS1-deficient mice were depleted of CD8ϩ T cells via treated with IFN-A/D eventually succumbed to their tumors. i.p. injection of an anti-CD8 Ab. Control mice were treated with

ϩ the appropriate isotype control Ab. Mice were then challenged i.p. In vivo depletion of CD8 T cells significantly inhibits the with tumor and received daily i.p. injections of IFN-A/D (or PBS) ␣ ϩ antitumor action of IFN- of SOCS1-deficient mice beginning the next day. Depletion of CD8 T cells markedly in- We next examined the role of CD8ϩ T cells in mediating the hibited the antitumor effects of IFN-A/D in SOCS1-deficient mice antitumor effects of IFN-␣ in this murine model. SOCS1-compe- (Fig. 7E; p ϭ 0.0002). In contrast, mice receiving the control Ab 4842 IFN-␣ ANTITUMOR RESPONSE IS NEGATIVELY REGULATED BY SOCS Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 7. SOCS deficiency enhances the antitumor effect of IFN-A/D in a murine model of malignant melanoma. A, SOCS1ϩ/ϩ IFN-␥Ϫ/Ϫ,(B) SOCS1ϩ/Ϫ IFN-␥Ϫ/Ϫ,(C) SOCS1Ϫ/Ϫ IFN-␥Ϫ/Ϫ, and (D) SOCS3ϩ/Ϫ mice were injected i.p. with 1 ϫ 106 JB/MS cells on day 0. Wild-type mice served as controls. Beginning on day 1, mice received daily i.p. injections of PBS or 2 ϫ 104 U of IFN-A/D. As expected, treatment of tumor-bearing SOCS1ϩ/ϩ mice with IFN-␣ led to a significant improvement in survival as compared with treatment with PBS alone (p ϭ 0.0002; A). The antitumor effects of IFN-␣ therapy were significantly enhanced in SOCS1-deficient mice as 57–71% of these mice were cured of their tumors (B and C), whereas PBS-treated mice all died at 10–14 days (p ϭ 0.0002 for SOCS1ϩ/Ϫ mice; p ϭ 0.0002 for SOCS1Ϫ/Ϫ mice). Each group contained seven mice. The antitumor effects of IFN-␣ therapy were significantly enhanced in SOCS3-deficient mice vs SOCS3ϩ/ϩ mice (p ϭ 0.0091; D). Each group contained four mice. Before tumor inoculation, (E) SOCS1Ϫ/ϪIFN-␥Ϫ/Ϫ and (F) SOCS1ϩ/ϩIFN-␥Ϫ/Ϫ mice were injected i.p. with 100 ␮g of a rat anti-mouse CD8 Ab (clone 2.43) or rat IgG on days Ϫ3, Ϫ1, ϩ1, ϩ3, and every fourth day thereafter. CD8ϩ T cell depletion (defined as Ͻ0.5% CD8ϩ cells in the peripheral blood) was confirmed by flow cytometry in all mice before tumor challenge. Mice were injected with 1 ϫ 106 JB/MS cells on day 0. Beginning on day 1, mice received daily injections of PBS or 2 ϫ 104 U of IFN-A/D. The antitumor effects of IFN-␣ therapy were significantly diminished in CD8-depleted mice as compared with CD8-competent mice, regardless of SOCS1 genotype (p Ͼ 0.0002). Each group contained seven mice. exhibited prolonged survival following therapy with IFN-A/D 7F; p ϭ 0.0062). These data suggest that CD8ϩ T cells play a ( p ϭ 0.0002) as observed previously. Depletion of CD8ϩ T cells critical role in mediating the antitumor effects of IFN-␣ and that also inhibited the effectiveness of IFN-A/D in IFN-␥Ϫ/Ϫ mice (Fig. this effect is enhanced in SOCS1-deficient mice. The Journal of Immunology 4843

Discussion exert potent antitumor actions in response to IFN-␣ in the absence We have previously demonstrated that Jak-STAT signal transduc- of SOCS1 activity. tion within host immune cells is critical to the antitumor effects of Importantly, dendritic cell (DC) function is also regulated by Ϫ/Ϫ IFN-␣ in a murine model of malignant melanoma (9). This led us SOCS1. SOCS1 mice have greater numbers of DCs. Also, ␥ to examine the role of SOCS proteins in regulating the antitumor these DCs respond more strongly to IL-4 and IFN- and produce properties of IFN-␣-stimulated immune effector cells. The present higher levels of B cell maturation/differentiation factors such as study demonstrated that SOCS1, SOCS2, SOCS3, and CIS were Baff/BLys (a member of the TNF superfamily) as compared rapidly induced in a dose-dependent manner by IFN-␣ in normal with normal mice (60). Shen et al. (61) subsequently demonstrated lymphocytes at the transcript and protein level. The inhibitory ef- that down-regulation of SOCS1 in DCs using lentiviral-delivered ␣ fects of SOCS proteins were highly specific, as overexpression of siRNA led to enhanced production of TNF- , IL-6, and IL-2 in response to LPS and IFN-␥ treatments. In addition, coculture of SOCS1 and SOCS3, but not SOCS2, in the Jurkat T cell lym- ϩ phoma cell line inhibited IFN-␣-induced activation of STAT1 and SOCS1-deficient DCs with Ag-specific CD8 T cells led to en- the expression of IFN-stimulated genes. Conversely, siRNA-me- hanced T cell proliferation and cytokine secretion as compared with wild-type DCs. Immunization with tyrosinase-related protein- diated down-regulation of SOCS1 and SOCS3 in Jurkat cells and ϩ T cells enhanced the transcriptional response to IFN-␣. The func- 2-pulsed, SOCS1-silenced DCs abrogated the growth of a TRP2 tional importance of SOCS proteins in regulating the actions of B16 melanoma in C57BL/6 mice (61). A similar study confirmed IFN-␣ was suggested by the improved ability of SOCS1- and these results and identified several genes that were expressed to a greater degree in SOCS1-deficient DCs as compared with SOCS3-deficient mice to eliminate melanoma tumor cells follow- ϩ ϩ SOCS1 / DCs (62). Thus, SOCS1-deficiency appears to promote Downloaded from ing treatment with IFN-␣. both the processing of tumor Ags by DCs and their recognition by To date, there have been limited studies investigating the regu- effector T cells. Analysis of IFN-␣-induced DC activity in SOCS- lation of SOCS family protein expression in IFN-␣-stimulated im- deficient mice is therefore warranted. mune effector cells. IFN-␣ has been shown to induce the expres- Reduced levels of SOCS1 in target cells appear to promote the sion of SOCS species in megakaryocytes, LPS-stimulated therapeutic efficacy of IFN-␣ immunotherapy. Evidence in favor macrophages, in an IL-2-primed human T cell line, and a human

of this concept comes from a study by Roman-Gomez et al. (63) http://www.jimmunol.org/ chronic myelogenous leukemia cell line (32, 34, 49–52). Overex- demonstrating that constitutive expression of SOCS1 in PBMCs pression of SOCS proteins has been shown to inhibit cytokine obtained from chronic myeloid leukemia patients correlated with a responses in hepatoma cells and activated T cells (33, 53–57). significantly shorter progression-free survival and poor cytoge- However, the effects of SOCS proteins on unmanipulated human netic response to IFN-␣ therapy (63). Similarly, it has been shown lymphocytes have not been fully explored. We demonstrated that by Imanaka et al. (57) that high-level expression of SOCS1 within SOCS1, SOCS2, SOCS3, and CIS are differentially induced in both hepatocytes from chronic hepatitis C patients correlated with a T cells and NK cells. The finding of variable SOCS induction in diminished antiviral response to IFN-␣. Also, Fenner et al. (64) malignant melanoma patient PBMCs after the administration of recently demonstrated that, compared with wild-type mice,

␣ by guest on September 24, 2021 high-dose IFN- -2b supports the clinical relevance of these find- SOCS1-deficient mice are better able to use endogenous IFN-␣ to ings and further suggests that the immune response to exogenous clear an otherwise lethal viral infection. These data are in agree- may be influenced by individual differences in the ex- ment with our findings and point to a role for SOCS1 in regulating pression of SOCS proteins within lymphocyte populations. the antitumor effects of IFN-␣ therapy and the outcome of viral In the present report, inhibition of SOCS expression in T cells infections. Interestingly, selective down-regulation of SOCS1 ␣ had marked effects on IFN- -induced gene expression. Further- within the T cell compartment does not lead to an overt inflam- ␣ more, the antitumor effects of IFN- were significantly enhanced matory response, as occurs in SOCS1-deficient mice that possess ϳ in SOCS1-deficient mice: 70% of mice achieved long-term res- a functioning IFN-␥ gene (65). Therefore, targeted down-regula- olution of their tumor-burden while no adverse side-effects were tion of SOCS proteins within specific cell types may help to avoid observed. SOCS1-deficient mice that were tumor free following the toxicity associated with global down-regulation of SOCS1. ␣ IFN- treatment survived a second challenge with JB/MS mela- The present study demonstrated that overexpression of SOCS1 noma cells. SOCS3 deficiency also promoted the antitumor effects and SOCS3 had negative effects on IFN-␣-induced Jak-STAT sig- of IFN-␣. These enhanced antitumor effects of IFN-␣ were medi- nal transduction and gene regulation, while experimental reduc- ϩ ated, at least in part, by CD8 T cells. Several lines of evidence tions in SOCS activity enhanced the response to IFN-␣. Reduced point to SOCS1 as an important regulator of T cell development expression of SOCS1 or SOCS3 in tumor-bearing mice enhanced and function. Mice with a targeted deficiency of SOCS1 within the the antitumor activity of exogenous IFN-␣. These findings suggest T cell compartment exhibit an increased ratio of CD8/CD4 mature that modulation of SOCS activity may have beneficial effects in the thymic cells and a significant increase in the prevalence of setting of cytokine-mediated immune phenomena. SOCS proteins CD44highCD8 memory T cells within the periphery (58). Memory may also play a role in mediating endogenous IFN activity which T cells from these mice displayed a 5-fold increased proliferative is known to be important in the immune surveillance for cancer response to IL-2 and IL-15 as compared with wild-type mice. Sim- cells (11). ilarly, the proliferation of T cells from SOCS1Ϫ/ϪIFN-␥Ϫ/Ϫ mice in response to anti-CD3 Ab was markedly enhanced in the pres- ence of IL-12, whereas this cytokine had minimal effects on acti- Acknowledgment vated T cells from normal mice (58, 59). Recently, Davey et al. We thank The Ohio State University Comprehensive Cancer Center Real- ϩ (58) discovered that SOCS1-deficient CD8 T cells proliferate Time Core Facility for assisting in the operation of the ABI PRISM 7900 when transplanted to normal mice and that this response is driven Sequence Detection System. by IL-15 and self ligands that normally drive homeostatic prolif- eration in T cell-deficient mice. The ability of CD8 depletion to abrogate the prosurvival effects of IFN-␣ in tumor-bearing, Disclosures SOCS1-deficient mice suggests that this immune compartment can The authors have no financial conflict of interest. 4844 IFN-␣ ANTITUMOR RESPONSE IS NEGATIVELY REGULATED BY SOCS

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