Major Differences in the Responses of Primary Human Leukocyte Subsets to IFN- β Anette H. H. van Boxel-Dezaire, Joana A. Zula, Yaomin Xu, Richard M. Ransohoff, James W. Jacobberger and George R. This information is current as Stark of October 1, 2021. J Immunol published online 18 October 2010 http://www.jimmunol.org/content/early/2010/10/18/jimmun ol.0902314 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2010/10/19/jimmunol.090231 Material 4.DC1 http://www.jimmunol.org/ Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average by guest on October 1, 2021

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 18, 2010, doi:10.4049/jimmunol.0902314 The Journal of Immunology

Major Differences in the Responses of Primary Human Leukocyte Subsets to IFN-b

Anette H. H. van Boxel-Dezaire,* Joana A. Zula,* Yaomin Xu,† Richard M. Ransohoff,‡ James W. Jacobberger,x and George R. Stark*

Treatment of cell lines with type I IFNs activates the formation of IFN-stimulated factor 3 (STAT1/STAT2/IFN regulatory factor-9), which induces the expression of many . To study this response in primary cells, we treated fresh human blood with IFN-b and used flow cytometry to analyze phosphorylated STAT1, STAT3, and STAT5 in CD4+ and CD8+ T cells, B cells, and monocytes. The activation of STAT1 was remarkably different among these leukocyte subsets. In contrast to monocytes and CD4+ and CD8+ T cells, few B cells activated STAT1 in response to IFN-b, a finding that could not be explained by decreased levels of IFNAR2 or STAT1 or enhanced levels of suppressor of cytokine signaling 1 or relevant tyrosine phosphatases in B cells. Microarray and real-time PCR analyses revealed the induction of STAT1-dependent proapoptotic mRNAs in monocytes but not in Downloaded from B cells. These data show that IFN-stimulated gene factor 3 or STAT1 homodimers are not the main activators of in primary B cells of healthy humans. Notably, in B cells and, especially in CD4+ T cells, IFN-b activated STAT5 in addition to STAT3, with biological effects often opposite from those driven by activated STAT1. These data help to explain why IFN-b increases the survival of primary human B cells and CD4+ T cells but enhances the apoptosis of monocytes, as well as to understand how leukocyte subsets are differentially affected by endogenous type I IFNs during viral or bacterial infections and by type I IFN treatment of patients with multiple sclerosis, hepatitis, or cancer. The Journal of Immunology, 2010, 185: 000–000. http://www.jimmunol.org/

nterferons are pleiotropic cytokines that play important roles factor 3 (ISGF3) is the major transcription factor activated in re- in infection and inflammation. Three classes of IFNs are known: sponse to IFN-a/b (3, 4). ISGF3, a complex of phosphorylated I type I IFNs includes IFN-a,-b,-v,-t,-d,-k, and -ε (1); type STAT1, STAT2, and unphosphorylated IFN regulatory factor II is IFN-g; and type III is IFN-l.IFN-a and -b use the IFNAR1 (IRF)-9, binds to the IFN-stimulated response element (ISRE) and IFNAR2c receptor subunits to signal (2), each of which binds present in the promoters of many ISGs. In response to type I IFNs, constitutively to a single member of the JAK family of kinases: activated STAT1 can also form homodimers that bind to gamma- IFNAR1 to tyrosine kinase 2 and IFNAR2 to JAK1. Ligand bind- activated sequence (GAS) elements in some ISG promoters (3, 5). by guest on October 1, 2021 ing induces the phosphorylation of JAK1, tyrosine kinase 2, intra- It is becoming clear that, in addition to ISGF3 and STAT1 cellular tyrosine residues of each receptor subunit, and STATs. homodimers, other transcription factors play important roles as cy- Activated STATs dimerize, dissociate from the receptor, and trans- toplasmic messengers between the receptor and the nucleus (4), locate to the nucleus to induce the expression of IFN-stimulated helping to explain why type I IFNs, which were discovered on genes [ISGs (3)]. Current data suggest that IFN-stimulated gene the basis of their potent antiviral activities, are now known to act much more broadly, as pleiotropic cytokines that regulate many different cellular functions. For example, STAT3 is activated in *Department of Molecular Genetics, †Statistical Genetics and Bioinformatics, De- partment of Quantitative Health Sciences, and ‡Department of Neurosciences, Neuro- response to type I IFNs in most cell lines, forming STAT3 homo- inflammation Research Center, Lerner Research Institute, Cleveland Clinic Foun- dimers or heterodimers with activated STAT1 (4). In contrast, x dation, Cleveland, OH 44195; and Case Comprehensive Cancer Center, Case West- activation of STAT4 and STAT5 by IFN-a/b is found mostly in ern Reserve University, Cleveland, OH 44106 NK and T cells (6–8). Interestingly, the activation of STAT6 in- Received for publication July 21, 2009. Accepted for publication August 27, 2010. duced by type I IFNs has only been described in B cell lines This work was supported by Pilot Grant PP1086 and Career Transition Fellowship (9). STAT homo- and heterodimers bind to GAS elements in the Award TA3032A1/1 (to A.H.H.vB-D.) from the National Multiple Sclerosis Society and by Grants P01 CA06220 (to G.R.S.) and P30 CA43703 (to Gene Expression and promoters of ISGs, but it is clear that different STAT dimers have Genotyping Facility of the Case Comprehensive Cancer Center) from the National different preferences for specific GAS elements (5). The differ- Institutes of Health. ential activation of STAT4, STAT5, and STAT6 in different cell The microarray data presented in this article have been deposited into National lines suggests the possibility of cell type-specific activation of Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE23307) under accession No. GSE23307. STATs by IFN-a/b in vivo. Of note, evidence for a cell type- Address correspondence and reprint requests to Dr. George R. Stark, Department of specific response to IFN-a was described with respect to differ- Molecular Genetics, Lerner Research Institute, Mail Code NE20, Cleveland Clinic ential ISG induction in human T cells and dendritic cells (10). Foundation, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: [email protected] In this study, we investigated how primary human leukocytes The online version of this article contains supplemental material. signal in response to IFN-b. Undiluted freshly drawn human whole Abbreviations used in this paper: D-PBS, Dulbecco’s PBS; ETS, E–twenty-six; GAS, blood was stimulated with IFN-b in vitro to mimic the situation gamma-activated sequence; HI, healthy individual; IRF, IFN regulatory factor; ISG, IFN-stimulated gene; ISGF3, IFN-stimulated gene factor 3; ISRE, IFN-stimulated in vivo as closely as possible. Because the activation of STATs response element; PY-STAT, phosphotyrosine-STAT; rtPCR, real-time PCR; SHP1, occurs only transiently, the isolation of many different leukocyte Src homology region 2 domain-containing phosphatase 1; SOCS, suppressor of cy- subsets after stimulation of whole blood in combination with tokine signaling; TCP45, T cell protein tyrosine phosphatase of 45 kDa. Western blot analysis is not feasible because, by the time the Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 subsets could be isolated, the optimal time point for activation of

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902314 2 IFN-b–INDUCED RESPONSES IN HUMAN BLOOD CELL SUBSETS

STATs would have passed. Therefore, we used a flow cytometry- Intracellular detection of PY-STATs using flow cytometry based technique that enables the detection of intracellular phos- The published method of Chow et al. (24), which was developed to measure photyrosine-STAT (PY-STAT)1, STAT3, and STAT5 at the single- intracellular phospho-ERK in whole blood cells, was adapted slightly cell level, allowing cells to be fixed at the optimal time for STAT to measure the induction of phospho-STATs in human whole blood. A activation. IFN-b–induced activation of STAT1, STAT3, and STAT5 number of commercially available anti-human CD3, CD4, CD8, CD19, was chosen because these three transcription factors regulate cell and CD14 Abs were screened. The best anti-CD3 and anti-CD14 clones were those that performed optimally after fixation and erythrocyte lysis survival in opposite directions (11–13). Furthermore, this approach but before methanol incubation. In contrast, the best anti-CD8, anti-CD4, allowed us to address whether differential activation of these STATs and anti-CD19 Abs performed best after methanol incubation. As pre- might explain how IFN-b enhances the survival of mature B cells viously published (25), IFN-b–induced phospho-STATs were optimally and T cells (14–19) while increasing apoptosis in monocytes and detected in leukocytes that were permeabilized with 90% methanol (data not shown). many cancer cell lines (20–23). Notably, we found that IFN-b in- After stimulation with IFN-b1a in vitro or leaving cells untreated for the duced significant differences in the activation of STAT1 and STAT5 same time period, whole blood or cell lines were fixed in 4 or 2% form- in different leukocyte subsets and that these differences are related aldehyde, respectively, by adding 10% prewarmed methanol-free formal- to the induction of pro- and antiapoptotic genes, respectively. Our dehyde (Polysciences, Warrington, PA), followed by incubation at 37˚C for results provide important insights into the differential effects that 10 min. After fixation, cell lines were washed twice with 50 ml ice-cold wash buffer (Dulbecco’s PBS [D-PBS], 5% FBS, 0.09% NaN3). Eryth- type I IFNs may have on leukocyte subsets during infection and rocytes were lysed by adding 0.12% Triton X-100 (0.1% Triton X-100 final upon treatment of multiple sclerosis, hepatitis, and some cancers concentration; Pierce, Rockford, IL) dissolved in 13 D-PBS and in- with type I IFNs. cubating for 30 min at room temperature with rocking. Lysed erythrocytes were removed by washing three times with 50 ml ice-cold wash buffer,

followed by spinning at 300 3 g for 10 min at 4˚C. One hundred micro- Downloaded from liters of fixed cells was subsequently added to each 5-ml Falcon FACS Materials and Methods tube (equivalent to 100 ml HT or 130 ml whole blood per tube), contain- Cell culture and IFN-b stimulation ing FITC- or Pacific blue-conjugated anti-CD3 (clone UCHT1; BD Bio- sciences), anti–CD14-FITC (clone RM052; Beckman Coulter, Miami, FL), The HT cell line was acquired from the American Type Culture Collec- or anti–CD14-AF700 (clone TU¨ K4, Invitrogen, Carlsbad, CA) Abs in the tion (Manassas, VA) (CRL-2260, human B lymphoblast derived from a amounts recommended by the manufacturers. Cells were incubated for 30 patient with diffuse mixed lymphoma) and cultured in RPMI 1640 medium min at room temperature in the dark and washed twice: once with 2 ml ice- supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 4500 mg/l cold wash buffer per tube and once with 2 ml ice-cold 13 D-PBS. While http://www.jimmunol.org/ glucose, and 10% FBS. This cell line was maintained in 10- or 15-cm vortexing at high speed, 1 ml 90% methanol in 13 D-PBS was added per 6 culture dishes and was always stimulated at a concentration of 1 3 10 tube, and the mixture was incubated at 220˚C overnight. The next day, 6 cells/ml with IFN-b1a (Avonex, 30 mg/0.5 ml Prefilled Syringe, 12 3 10 the contents of each tube were washed twice with 2 ml wash buffer (with IU/ml; Biogen Idec, Cambridge, MA). Stimulated cells were subsequently spinning at 300 3 g, 4˚C). For the blocking step, the cell pellets were fixed for flow-cytometry analysis or lysed for Western blot analysis. resuspended in 50 ml wash buffer and incubated for 10 min at room Heparinized whole blood was obtained from healthy donors according to temperature in the dark. A combination of Abs directed against human an Institutional Review Board-approved protocol (Cleveland Clinic). CD8 (clone B9.11, PE-Cy5 conjugated; Beckman Coulter), CD19 (clone Within 15 min after venipuncture, undiluted whole blood was stimulated in J4.119, PE-Cy5 or PE-Cy7 conjugated; Beckman Coulter), or CD4 (clone 6- or 10-cm culture dishes or 50-ml canonical tubes (BD Biosciences, San 13B8.20, PE-Cy5 conjugated; Beckman Coulter) and the following Alexa Jose, CA) in vitro with recombinant human IFN-b1a (Biogen Idec), IFN- Fluor 647- or PE-conjugated Abs against PY(701)-STAT1, PY(705)- by guest on October 1, 2021 3 3 a1(33 10 IU/ml), IFN-a2 (Intron A, 10 3 10 IU/ml; Schering-Plough, STAT3, or PY(694)-STAT5 (clones 4A, 4, and 47, respectively, BD Bio- 3 Kenilworth, NJ) or IFN-g (10 3 10 IU/ml; Genentech, South San Fran- sciences) were added in amounts advised by the manufacturer, followed by cisco, CA), as indicated. For each staining, 130 ml of whole blood was incubation at room temperature in the dark for 1 h. Anti-CD8 or anti-CD4 used. All in vitro stimulations were performed in an incubator at 37˚C Abs were used; the alternative T cell subset was identified by selecting (Thermo Fisher Scientific, Asheville, NC); no cell clumping or adhesion to the CD82CD3+ or CD42CD3+ population, respectively. To detect total tissue culture plates was observed. After stimulation, whole blood was STAT1, cells were incubated with anti–STAT1-PE (N terminus of STAT1, fixed and lysed for intracellular detection of PY-STATs. clone 1/Stat1; BD Biosciences) after permeabilization with 90% methanol. To detect the activation of STAT2, rabbit polyclonal anti-PY(689)-STAT2 Western blot analysis of HT cells and isolated leukocyte (Upstate Biotechnology) was added at 6.5 mg/ml. Experiments demon- strating the specificity of this anti–PY-STAT2 Ab are shown in Supple- subsets mental Fig. 1A and 1B. For the fluorochrome-conjugated Abs, the last 3 After stimulation of HT cells with IFN-b1a or after leukocyte subsets were wash step was performed with 3 ml wash buffer per tube (300 g; 10 min; purified from unstimulated whole blood, the cells were washed once with 4˚C). To detect PY-STAT2, cells were incubated with 5 ml of a 1:10 di- PBS, and the cell pellets were lysed for 30 min at 4˚C in 250 ml (per 5 3 lution of goat anti-rabbit IgG-PE (Jackson ImmunoResearch Laboratories, 106 cells) lysis buffer containing 50 mM HEPES (pH 7.9), 250 mM po- West Grove, PA) at room temperature for 30 min. After the final washing tassium chloride, 0.1% Nonidet P-40, 10% glycerol, 0.1 mM EDTA, 10 step, each cell pellet was resuspended in 350 ml wash buffer and measured mM sodium fluoride, 5 mM sodium orthovanadate, 1 mM phenylmethane- on an LSRI or LSRII (both from BD Biosciences) flow cytometer. Samples sulfonyl fluoride, 20 mg/ml aprotinin, 20 mg/ml pepstatin, and 20 mg/ml stained with a single color were used for compensation. Intact cells were leupeptin. Cellular debris was pelleted by centrifugation at 13,000 3 g at gated on forward and side scatter, and 50,000 cells were measured. Flow data were analyzed with WinList (Verity Software House, Topsham, ME). 4˚C for 10 min. Cell extracts were fractionated by electrophoresis in 10 or + 12% SDS-PAGE and transferred to polyvinylidene difluoride membranes The percentage of IFN-b–induced phospho-STAT cells was determined (Millipore, Bedford, MA). The following Abs were used: rabbit polyclonal by subtracting the percentage of positive cells in unstimulated cells, which , anti-SOCS1 and rabbit polyclonal anti–SH-PTP1 (clones H-93 and C-19, was set at 2%. An example of an analysis is shown in Fig. 2. respectively; Santa Cruz Biotechnology, Santa Cruz, CA), mouse mono- clonal anti-T cell protein tyrosine phosphatase of 45 kDa (TCP45) (clone Detection of IFNAR2 and caspase 3 activation by flow CF4-1D; EMD Chemicals, Gibbstown, NJ), mouse monoclonal anti–N- cytometry terminal STAT1 (clone 42, BD Biosciences), rabbit polyclonal anti–PY701- STAT1, rabbit polyclonal anti–PY705-STAT3, rabbit polyclonal anti-STAT3, Unstimulated and undiluted whole blood (150 ml) was incubated with rabbit polyclonal anti–PY694-STAT5, rabbit polyclonal anti-STAT5 (Cell mouse anti–IFNAR2-PE (clone MMHAR-2; R&D Systems, Minneapolis, Signaling Technology, Beverly, MA), rabbit polyclonal anti–PY689-STAT2 MN) or control mIgG2a-PE (clone G155-178; BD Biosciences), as rec- and rabbit polyclonal anti-STAT2 (Upstate Biotechnology, Lake Placid, ommended by the manufacturer, in combination with the same anti-CD NY), and mouse monoclonal anti–b-actin (clone AC-74; Sigma-Aldrich, Abs as mentioned above, for 30 min at 4˚C. Whole blood cells were St. Louis, MO). HRP-coupled goat anti-rabbit or goat anti-mouse IgG subsequently fixed, and erythrocytes were lysed as mentioned above, and (Rockland Immunochemicals, Gilbertsville, PA) was used for visualiza- after the washing steps, the cell pellet was resuspended in 350 ml wash tion, using the ECL (ECL Plus) Western blot analysis detection system buffer and measured on a LSRII (BD Biosciences) flow cytometer the (PerkinElmer, Waltham, MA). same day. The Journal of Immunology 3

To detect induction of apoptosis, whole blood that was diluted 1:3 with condition. Genes in this filtered list were further grouped into those that plain RPMI 1640 was not stimulated or was stimulated with 2000 IU/ml were changed in all B cells and monocytes, those that were changed in IFN-b for different time periods up to 48 h at 37˚C or was kept at 50˚C for monocytes only, and those that were changed in B cells only (healthy 1 h (positive control). Cells were then washed one time with 25 ml 13 individual [HI] #1 and HI #2). The Enrichment Analysis D-PBS, and whole blood was divided (150 ml/tube) and incubated with Software Toolkit (available at http://omicslab.genetics.ac.cn/GOEAST/) the same anti-CD Abs as mentioned above for 30 min at 4˚C. Whole was used to sort these groups of genes according to gene ontology, par- blood cells were subsequently fixed, and erythrocytes were lysed as ticularly apoptosis, proliferation, and cell-cycle regulation. mentioned above; after the washing steps, each pellet was eventually Real-time PCR (rtPCR) was used to confirm changes in gene expression resuspended in 100 ml Permeabilization Medium B (Invitrogen) and in- obtained by microarray analysis. rtPCR was done with RNA isolated from cubated with 20 ml anti-activated caspase 3-PE Ab (0.25 mg) for 30 min at B cells and monocytes (present in whole blood of six healthy individuals, room temperature. Finally, after each tube was washed with 3 ml stain and purification occurred after stimulation of whole blood, as mentioned buffer, the cell pellet was resuspended in 350 ml stain buffer and measured above), which were left untreated or were stimulated with 2000 IU/ml IFN-b on a BD Biosciences LSR II flow cytometer the same day. Induction of for 3 h. Thus, rtPCR was performed with 24 samples to detect changes activated caspase 3 in leukocyte subsets by IFN-b was determined by in seven mRNAs. The following seven TaqMan Gene Expression Assays subtracting the percentage of caspase 3+ cells in unstimulated cells from from Applied Biosystems (Foster City, CA) were used (gene, assay ID those in IFN-b–stimulated cells. number): BAK1, Hs00832876_g1; CASP3, Hs00263337_m1; CDKN1A, Hs00355782_m1; BCL2L13, Hs00209789_m1; STK3, Hs00169491_m1; Statistical analysis IL2RA, Hs00907779_m1; and NAMPT, Hs00237184_m1. Two candidate genes were chosen for endogenous control determination based on studies GraphPad InStat 3 was used (GraphPad Software, La Jolla, CA). The about rtPCR performed with RNA from B cells and monocytes: eukaryotic Friedman test, which is a nonparametric repeated-measures ANOVA for 18S rRNA and HPRT1. An Applied Biosystems ABI 7900HT unit with paired samples test, was used to test whether the four blood cell subsets automation attachment was used for rtPCR. This unit is capable of col- (monocytes, B cells, and CD8+ and CD4+ T cells) differed with respect lecting spectral data at multiple points during a PCR run. To execute the to activation of STAT1, STAT3, and STAT5. When the Friedman test first step and make archive cDNA, 150 ng total RNA was reverse tran- Downloaded from showed a significant difference (p , 0.05), post hoc analysis was sub- scribed in a 25-ml reaction using Applied Biosystems enzymes and re- sequently performed using the Dunn test to detect which blood subsets agents, in accordance with the manufacturer’s protocols. RNA samples differedsignificantlyfromeachother.Whencalculatingthep values, the were accurately quantitated using an ND-1000 spectrophotometer (Nano- Dunn test takes into account the number of comparisons one is making drop Technologies, Wilmington, DE). The cDNA reaction from above was (Bonferroni adjustment). The Pearson correlation test was used to de- diluted by a factor of 10. For the PCR step, 9 ml this diluted cDNA was termine whether the percentages of PY-STAT+ leukocyte subsets that used for each of three replicate 15-ml reactions carried out in a 384-well

were induced after stimulation with IFN-b for 45 min correlated with the plate. Standard PCR conditions were used for the Applied Biosystems http://www.jimmunol.org/ percentage of activated caspase 3+ subsets after longer periods of stim- assays: 50˚C for 2 min, 95˚C for 10 min, followed by 40 cycles of 95˚C ulationwithIFN-b. The coefficient of determination (R2) and the two- for 15 s alternating with 60˚C for 1 min each. The comparative cycle tailed p values are shown (if significantly correlated). threshold method was used for relative quantitation. 18S rRNA had very little variation in expression across the sample sets; therefore, it was Blood cell subset isolation, gene-array analysis, and real-time chosen as the endogenous control. For rtPCR data analysis, RNA abun- PCR dance was normalized for each gene with respect to the endogenous control in that sample (18S), and mean values for fold changes were cal- Twenty-two milliliters of undiluted whole blood from each of two healthy culated for each gene (IFN-b stimulated over control treated). PCR con- donors was stimulated with 2000 IU/ml IFN-b1a (Avonex, Biogen Idec) for firmation of gene-expression array data required that the direction of the 3 h, and 22 ml of blood was left unstimulated for 3 h. An aliquot of blood change in expression had to be the same with rtPCR as with gene- was taken out after 45 min of stimulation with IFN-b to determine the expression arrays and be increased $2-fold. by guest on October 1, 2021 activation of STAT1, STAT3, and STAT5 by flow cytometry. Immediately following stimulation for 3 h, 10 ml whole blood was incubated with 500 ml whole blood anti-CD14 or anti-CD19 microbeads (Miltenyi Biotec, Results Auburn, CA) for 15 min at 4˚C to isolate monocytes and B cells, re- Exposure to low doses of IFN-b causes differential activation of spectively. After washing with cold running buffer (PBS, 2 mM EDTA, STAT1, STAT3, and STAT5 in primary human blood cell subsets 0.5% BSA, 0.09% sodium azide; Miltenyi Biotec) to remove unbound microbeads and after bringing the volume of the whole blood back to the To verify Ab specificity, we compared our flow cytometry method starting volume by adding cold running buffer, the cells of interest were to Western blot analysis, using the human leukemic cell line HT, positively selected using the AutoMACS Pro Separator (Miltenyi Biotec) which was stimulated with 1000 IU/ml of IFN-b (Fig. 1). The two and program posselWB. During the entire isolation procedure, the methods yielded the same overall pattern. Of note, the highest blood cells were kept cold. The procedure is very fast (maximally 20 min), + which helps to preserve the quality of the RNA. The purity (90–99%) of percentage of PY-STAT HT cells was found 30 min after stim- the positively selected fraction and the yield were excellent, because the ulation with IFN-b, as usually found in human cell lines. The negative sorted fraction was totally depleted of each subset of interest. advantage of flow cytometry is that it reveals the percentage of Total RNA was isolated from the isolated unstimulated (control) or IFN-b– each cell subset in which a certain STAT is activated (Figs. 1, 2). stimulated blood cell subset by dissolving the cells in TRIzol (Invitrogen; 3 6 To begin to investigate how primary human monocytes, B cells, 1–10 10 cells in 1 ml), following the protocol of the manufacturer. One + + microgram of total RNA (100 ng is minimally needed) was sent to the and CD4 and CD8 T cells respond to IFN-b, we stimulated Cleveland Clinic Genomics Core. A single round of in vitro transcription undiluted whole blood samples from nine healthy individuals with amplification was carried out using the Illumina RNA Amplification Kit 500 IU/ml of IFN-b for 25 min (Fig. 3A). We observed significant (Ambion, Austin, TX) to amplify mRNA and, thus, to obtain ample differences in the fractions of leukocyte subsets in which STAT1 amounts of cRNA to perform the whole gene-expression (p # 0.0001), STAT3 (p = 0.003), and STAT5 (p = 0.006) were assay using the humanRef-8 v2 expression bead chips microarray (Illu- + mina, San Diego, CA), which has 22,184 transcript probes, representing activated. Unexpectedly, remarkably few B cells and CD4 T cells 18,189 genes in total. The microarray data discussed in this publication showed activation of STAT1 in comparison with monocytes. The have been deposited in the National Center for Biotechnology Informa- differences in activation of STAT3 were very similar to those seen tion’s Gene Expression Omnibus and are accessible through GEO Series for the activation of STAT1; much fewer B cells and CD4+ T cells accession number GSE23307. Expression data normalization and differ- ential expression analysis were handled through the Illumina BeadStudio displayed activation of STAT3 compared with monocytes. Thus, Gene Expression module V3.2. The data were first normalized by using the of the blood cell subsets investigated, the highest percentage of Illumina background normalization algorithm and then the differential- PY-STAT1+ and PY-STAT3+ cells were found among monocytes, expression analyses were performed using Illumina’s custom model. whereas the percentages of CD8+ T cells positive for PY-STAT1 Downstream data processing and reporting were handled in R packages and PY-STAT3 were intermediate (Fig. 3A). In contrast, many (http://www.r-project.org). Genes for downstream analyses were filtered to + include only those with both differential-expression analysis p values , more CD4 T cells than B cells showed activation of STAT5 when 0.001 and fold changes .2 compared with the unstimulated (control) whole blood was stimulated with 500 IU/ml IFN-b for 25 min. 4 IFN-b–INDUCED RESPONSES IN HUMAN BLOOD CELL SUBSETS

ples from three healthy individuals were stimulated with 500 IU/ ml of IFN-b for various times, up to 75 min (Fig. 4). The greatest activation of STAT3 and STAT5 was detected in all blood cell subsets after 45 min of exposure to IFN-b; it declined gradually thereafter. Optimal activation of STAT1 in CD8+ T cells and monocytes was also found after 45 min. Interestingly, although the IFN-b–induced activation of STAT3 in CD4+ T cells and B cells was very low after 25 min (#15%), it increased by $ 1.8-fold (between 22 and 32% on average) after 45 min. In contrast, the activation of STAT1 in CD4+ T cells, as well as B cells, remained very low for the entire period tested (,10% positive cells). Because the optimal time point for activation of STATs in whole blood cells was 45 min, we tested whether the same significant differences in PY-STAT1, PY-STAT3, and PY-STAT5 induction could be found as after 25 min of stimulation with IFN-b. To this end, whole blood from seven healthy subjects was stimulated with 500 IU/ml IFN-b for 45 min (Fig. 3B). There were significant differ- ences in the fractions of blood cell subsets that showed activation of STAT1 (p = 0.0005) and STAT5 (p = 0.025). Thus, even after longer stimulation with IFN-b, much fewer B cells and CD4+ T cells Downloaded from showed induction of PY-STAT1 than did monocytes, whereas more CD4+ T cells than CD8+ T cells showed activation of STAT5 (Fig. 3B). Although monocytes still showed the highest percentage of PY- STAT3+ cells, the difference, compared with B cells and CD4+ FIGURE 1. Flow cytometric and Western blot analyses of PY-STATs T cells, was no longer significant. Therefore, the activation of show the same pattern. The leukemic human B cell line HT was stimulated STAT3 by IFN-b in CD4+ T cells and B cells is delayed compared http://www.jimmunol.org/ with 1000 IU/ml of IFN-b1a. Cells were lysed and subjected to Western with activation of STAT3 in CD8+ T cells and monocytes, in par- blot analysis (A) or fixed and stained to determine the percentage of cells ticular after 25 min (Fig. 3A), but it eventually catches up after 45 with PY-STATs by flow cytometry (B). min (Figs. 3A,3B, 4).

Exposure to low doses of IFN-b causes differential activation Exposure to high-dose IFN-b also causes differential of STAT1 and STAT5 in primary human blood cell subsets at activation of STAT1, STAT3, and STAT5 in primary human the optimal time for PY-STAT induction blood cell subsets

Although 25 min of stimulation with IFN-b is optimal for STAT Because the observed induction of PY-STATs at the optimal times by guest on October 1, 2021 activation in many cell lines, it is possible that this is not optimal occurred in #50% of the cells after stimulation with 500 IU/ml in CD4+ T cells and B cells in whole blood. Therefore, blood sam- IFN-b, we explored whether stimulation with higher concentra-

FIGURE 2. Analysis of activation of STAT1 and STAT5 in human leukocytes. Undiluted whole blood from a healthy donor was stimulated with 500 IU/ ml of IFN-b1a or was untreated for 45 min. After processing as described in Materials and Methods, the cells were analyzed by flow cytometry. CD14+ (monocytes) and CD4+/CD3+ (CD4+ T cells) were determined within the live gate. The percentages of IFN-b–induced PY-STAT+ cells within each leukocyte subset was determined by subtracting the percentages of positive unstimulated cells, which were set at ,2%. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 3. In vitro stimulation of whole blood with IFN-b reveals distinctive STAT-activation patterns in different leukocyte subsets. Un- diluted whole blood of healthy donors was left untreated or was stimulated by guest on October 1, 2021 with 500 IU/ml of IFN-b1a for 25 min (9 persons) (A) or 45 min (7 persons) (B) or with 2000 IU/ml IFN-b1a for 45 min (20 persons) (C), and induction of PY-STAT1, PY-STAT3, and PY-STAT5 was determined. The geometric means 6 SEM of the various donors are shown for the per- FIGURE 4. Activation of STATs in primary human blood cells after centages of leukocyte subsets positive for each PY-STAT. The Friedman stimulation with IFN-b is optimal after 45 min. Undiluted whole blood test revealed significant differences in the activation of STATs among the from three healthy donors was stimulated or not with 500 IU/ml IFN-b1a four leukocyte subsets. The Friedman test was followed by post hoc for different time periods. The geometric means 6 SEM of the three do- analysis, revealing significant differences in STAT1, STAT3, or STAT5 nors are shown for the percentages of leukocyte subsets positive for each activation between the subsets (indicated by arrows). pp , 0.05; ppp , PY-STAT. 0.01; pppp , 0.001.

(Fig. 5). At 2000 IU/ml of IFN-b, there were significant differ- tions of IFN-b would yield a higher percentage of cells positive ences in the fractions of blood cell subsets that showed activation for PY-STATs. We stimulated whole blood of three healthy indi- of STAT1 (p , 0.0001), STAT3 (p = 0.0026), and STAT5 (p , viduals with increasing doses of IFN-b (up to 10,000 IU/ml) for 0.0001). Remarkably, in contrast to the lower dose, using this high 45 min (Fig. 5). At a concentration of 200 IU/ml, virtually no acti- dose of IFN-b to stimulate whole blood of many donors also vation of STATs was detected in any of the blood cell subsets tested. revealed high numbers of PY-STAT1+ CD4+ T cells (Fig. 3C). All four subsets responded with activation of STAT3 and STAT5 at However, even at such a high concentration of IFN-b, much fewer 500 IU/ml, and more cells became positive at the higher concen- B cells than monocytes and CD4+ and CD8+ T cells activated trations. Furthermore, the activation of STAT1 by IFN-b was also STAT1. Also, fewer B cells than monocytes activated STAT3 and dose responsive in CD8+ T cells and monocytes. Remarkably, it STAT5 (Fig. 3C). Of interest, the highest fraction of PY-STAT5+ was still observed that very few B cells and CD4+ T cells (#10%) cells was still found among the CD4+ T cell subset (Fig. 3C). showed activation of STAT1, even at the higher concentrations of In summary, irrespective of IFN-b concentration or stimulation IFN-b (2,000–10,000 IU/ml). time, peripheral blood B cells do not show appreciable activation To test whether the same differences in STAT1 and STAT5 of STAT1 in response to IFN-b, indicating that ISGF3 or STAT1 activation could be observed with 2,000 IU/ml as with 500 IU/ml homodimers are not the main transcription factors driving ISG IFN-b, whole blood from 20 healthy subjects was stimulated for induction in the majority of primary human B cells or in CD4+ 45 min with the higher concentration (Fig. 3C); 2,000 IU/ml was T cells at lower IFN-b concentrations. In contrast, these subsets used instead of 10,000 IU/ml because the activation of STAT1 in show activation of STAT3 and STAT5, and the highest activation monocytes and B cells was lower with the latter concentration of STAT5 occurs in CD4+ T cells. 6 IFN-b–INDUCED RESPONSES IN HUMAN BLOOD CELL SUBSETS

leukocyte subsets did we observe a correlation between the total percentages of PY-STAT1+ cells after 45 min and the percentages of activated caspase 3+ cells at any of the later time points (data not shown). This result might be explained by the fact that many of the PY-STAT1+ cells are actually doubly positive for PY-STAT3 and PY-STAT5, resulting in opposing effects on apoptosis induction at an individual cell level. Therefore, a double staining was also performed with anti–PY-STAT1/PY-STAT3 or anti–PY-STAT1/PY- STAT5 Abs to detect doubly positive cells after IFN-b stimula- tion. Fig. 6B shows that the induction of PY-STAT1+/PY-STAT32, PY-STAT1+/PY-STAT3+, PY-STAT1 2/PY-STAT3+, PY-STAT1 +/PY- STAT52,PY-STAT1+/PY-STAT5+,orPY-STAT12/PY-STAT5+ pos- itive cells among the four leukocyte subsets is strikingly different. For instance, the percentage of PY-STAT1+/PY-STAT3 2 cells is highest in CD4+ T cells, followed by CD8+ T cells, and it is lowest in B cells and monocytes (Fig. 6B). In contrast, the percentage of PY-STAT1+/PY-STAT52 cellsishighestinCD8+ T cells, followed by CD4+ T cells and monocytes, but again is lowest in B cells. Remarkably, the generation of PY-STAT12/PY-STAT3 + and PY- STAT12/PY-STAT5 + positive cells could only be observed in the Downloaded from Bcellssubset(Fig.6B), because in this subset IFN-b induced the lowest percentage of PY-STAT1+ cells. The monocyte subset showed the highest amount of apoptosis induction by IFN-b;Fig.6A illus- trates that after 8 h of stimulation, significant apoptosis induction could be observed for the first time in all four donors tested. Strik- ingly, the generation of PY-STAT1+/PY-STAT32 monocytes after 45 http://www.jimmunol.org/ min correlated very significantly with the fraction of activated cas- pase 3+ monocytesafter8hofIFN-b stimulation (p = 0.0008; Fig. 6C). Of note, after 10 or 12 h of IFN-b stimulation, the number of activated caspase 3+ monocytes doubled or tripled, compared with 8 h of stimulation (Fig. 6A), suggesting that apoptosis was induced eventually, even in PY-STAT1+/PY-STAT3+ or PY-STAT1+/ PY-STAT5+ monocytes. None of the other subsets showed a corre- lation between induction of PY-STAT1+/PY-STAT3 2 or PY-STAT1+/ by guest on October 1, 2021 PY-STAT52 after 45 min with apoptosis induction after 8 h (data not shown), but this could be because individual donors showed varia- tion in the B cell or CD4+ or CD8+ T cell subsets with respect to significant activation of caspase 3. Therefore, the highest observed FIGURE 5. Dose responses for stimulation of PY-STATs with IFN-b in activation of caspase 3 within the T and B cell subsets of four healthy whole blood cell subsets. Undiluted whole blood of three healthy donors subjects (based on Fig. 6A,CD4+ T cells: at 6 h, 6 h, 10 h, and 12 h; was stimulated or not with increasing concentrations of IFN-b1a for 45 CD8+ Tcells:at4h,6h,10h,and10h;andBcells:at10h,10h, min. PY-STAT1, PY-STAT3, and PY-STAT5 were determined in leukocyte 12 h, and 12 h) was plotted against the percentage of PY-STAT1+/ 6 2 subsets as indicated. Geometric means SEM of three healthy donors are PY-STAT3 cells after 45 min, revealing a significant correlation shown for the percentages of PY-STAT+ blood cells. only within the CD8+ T cell subset (Fig. 6C; p = 0.0357). Because this subset has the highest percentage of PY-STAT1+/PY-STAT52 cells, many of the PY-STAT1+/PY-STAT32 CD8+ T cells are likely Differential activation of STAT1, STAT3, and STAT5 in to be PY-STAT1+/PY-STAT3 2/PY-STAT5 2 and prone to apoptosis b leukocyte subsets by IFN- is associated with differences in induction. In contrast, CD4+ T cells and B cells have the lowest per- apoptosis induction centages of PY-STAT1+/PY-STAT52 cells and, consequently, pos- Activation of STAT1 usually leads to induction of cell-cycle arrest sibly the lowest amount of apoptosis induction due to the antiapo- and apoptosis, whereas enhanced survival and proliferation result ptotic effects in PY-STAT1 +/PY-STAT5+ cells. from PY-STAT3 and PY-STAT5 induction. Therefore, we explored whether the differential activation of these STATs could be re- Differential activation of STAT1 in primary human monocytes sponsible for previously unexplained differences in induction of and B cells is connected to differential induction of b apoptosis in the various primary human leukocyte subsets by IFN- apoptosis-promoting mRNAs by IFN- b. To this end, whole blood from four healthy subjects was We investigated whether the observed differential activation of stimulated with IFN-b for 4, 6, 8, 10, 12, 24, or 48 h; apoptosis STAT1 in B cells and monocytes would result in differences in induction in the various subsets was determined by the activation mRNA induction and, in particular, in the induction of proapoptotic of caspase 3 (Fig. 6A). Stimulation with IFN-b induced the greatest mRNAs. To this end, undiluted whole blood from two healthy apoptosis in monocytes, much less in CD8+ T cells, and the least in individuals was stimulated with 2000 IU/ml of IFN-b for 3 h, and B cells and CD4+ T cells (Fig. 6A), in agreement with previous pure B cells and monocytes were isolated by using magnetically reports. Using the blood of the same donors, the induction of PY- labeled Abs. An aliquot of blood was taken out after 45 min of STAT1, PY-STAT3, and PY-STAT5 was also determined after stimu- stimulation with IFN-b to detect the activation of STAT1, STAT3, lation with IFN-b for 45 min in vitro. Interestingly, in none of the and STAT5 by flow cytometry. Supplemental Fig. 2A shows the The Journal of Immunology 7

A B

C Downloaded from http://www.jimmunol.org/

FIGURE 6. Differences in activation of STAT1 in leukocyte subsets are associated with variation in induction of apoptosis. A, Undiluted whole blood from four healthy donors was stimulated or not with 2000 IU/ml IFN-b for varying times. Induction of activated caspase 3 in leukocyte subsets by IFN-b was determined by subtracting the percentage of caspase 3+ cells in unstimulated cells from those in IFN-b–stimulated cells; results are shown for in- dividual donors. B, Undiluted whole blood from five healthy donors was stimulated or not with 2000 IU/ml IFN-b for 45 min. Geometric means 6 SD of five donors are shown for PY-STAT1+/PY-STAT32, PY-STAT1+/PY-STAT3+, PY-STAT12/PY-STAT3+, PY-STAT1+/PY-STAT52, PY-STAT1+/PY-STAT5+, 2 and PY-STAT1 /PY-STAT5+–positive subsets. C, Data from four healthy individuals (data of Fig. 6A and 6B combined) are depicted: induction of PY- by guest on October 1, 2021 STAT32/PY-STAT1+-positive monocytes and CD8+ T cells after stimulation with IFN-b for 45 min correlated with caspase 3 activation after 8 and 4–10 h, respectively. expected differential activation of STATs in B cells and monocytes Likewise, increased expression of CASP3 is dependent on STAT1 from two healthy individuals. Remarkably, mRNA induction by activation (28). Although it has not been conclusively shown IFN-b was very different in primary human B cells and mono- that expression of BAK1, BCL2L13 (BCL-RAMBO), and STK3 are cytes. Three hours of stimulation with IFN-b caused 1462 mRNAs dependent on the activation of STAT1 homodimers, this conclusion to be up- or downregulated by $2-fold in monocytes or B cells is very likely to be correct because all of these mRNAs are induced (836 upregulated, 626 downregulated). Fig. 7A shows the Venn after IFN-g stimulation (29) (http://www.interferome.org). diagram of the 836 mRNAs that were increased by $2-fold in After IFN-b stimulation, some mRNAs were induced $2-fold monocytes and B cells in response to IFN-b. Remarkably, of the in B cells and monocytes (TNFSF13B, IRF1, TNFSF10, FAS; mRNAs that changed in the monocytes of HI #1, 337 of 596 Table I). TNFS10 (TRAIL) and FAS cause apoptosis in cancer (57%) were increased in monocytes only, whereas 233 of 596 cells but not necessarily in normal immune cells (30, 31). Remark- (39%) were shared between monocytes and B cells of HI #1, and ably, after sorting the mRNAs that were increased by $2-fold in 229 of 596 (38%) were shared with B cells of HI #2. In contrast, B cells only according to their ontology, we did not find any mRNA the responses in B cells of HI #1 and HI #2 were very similar, to be related to apoptosis induction or cell-cycle arrest. In contrast, because 316 mRNAs of 405 (HI #1) or 410 (HI #2) were increased IL2RA and PBEF1 are two mRNAs that were increased in primary by 2-fold (78 and 77%, respectively). human B cells only (Table I), and TNFSF13B (BAFF) was in- The mRNAs that increased by $2-fold in monocytes only creased 3.5-fold more in B cells compared with monocytes (Table were sorted according to their ontology, using the Gene Ontology I). These mRNAs are all related to increased survival and pro- Enrichment Analysis Software Toolkit program. The following liferation (32–36). Of note, IL2RA and PBEF1 are induced by type I mRNAs, increased in monocytes only, are classified as inducers of IFNs only and not by IFN-g (http://www.interferome.org). In re- programmed cell death: CDKN1A, BAK1, BCL2L13, CASP3,and sponse to IL-2 or IL-6, the mRNAs of IL2RA and PBEF1 are known STK3 (Table I); they are all known to be very potent apoptosis- to be increased after binding of activated STAT5 or STAT3 (32, 35), inducing (26–29). The mRNA of CDKN1A (p21 or Cip1) respectively, to the promoters. Because IL-10 increases the ex- was increased 3-fold in monocytes, whereas the mRNA expression pression of TNFSF13B mRNA, the enhanced transcription is likely in B cells of both healthy individuals remained unchanged. The dependent on PY-STAT3 binding to the TNFSF13B promoter (36). induction of p21 is dependent on binding of activated STAT1 to the Because only B cells show the formation of PY-STAT12/PY- GAS element in the p21 promoter after IFN-g stimulation (27). STAT3+ and PY-STAT1 2/PY-STAT5 + cells after stimulation with 8 IFN-b–INDUCED RESPONSES IN HUMAN BLOOD CELL SUBSETS

individuals. After 45 min of stimulation, the activation of STAT1, STAT3, and STAT5 showed the typical pattern (Supplemental Fig. 2B). Induction of BCL2L13 and IL2RA mRNA was not replicated by rtPCR, but we confirmed increased BAK1, CASP3, CDKN1A, and STK3 mRNAs (in monocytes only) and an increase in PBEF1 mRNA (in B cells only) after IFN-b stimulation (Fig. 7B). In summary, the differential activation of STAT1 by IFN-b in pri- mary human monocytes and B cells is associated with very sig- nificant differences in mRNA induction. High activation of STAT1 in monocytes is related to an increase in the expression of potent inducers of apoptosis, whereas poor STAT1 activation in B cells could explain why the activation of STAT3 and STAT5 leads to increased induction of certain proliferation-stimulating genes in B cells only. IFNAR2 and STAT1 levels are similarly expressed in primary human leukocytes, and STAT2 is activated normally in B cells To begin to understand the mechanism that causes few B cells to activate STAT1 in response to IFN-b, we tested surface expression of IFNAR2. IFNAR1 is crucial for ligand binding, but IFNAR2, Downloaded from with its long cytoplasmic tail, possesses two conserved tyrosine FIGURE 7. Striking differences in gene induction in purified B cells and residues that are crucial for activation of STAT1, STAT2, and monocytes after in vitro stimulation with IFN-b. Undiluted whole blood STAT3 (1–4). IFNAR2 expression on leukocyte subsets present from two healthy donors was stimulated or not with 2000 IU/ml IFN-b1a in whole blood of six healthy individuals was studied by flow for 3 h. A, After 3 h, pure B cells and monocytes were isolated using cytometry. Fig. 8A shows that 100% of monocytes, B cells, and magnetically labeled Abs. RNA isolated from the purified subsets was used CD4+ and CD8+ T cells expressed IFNAR2; therefore, lack of its http://www.jimmunol.org/ for analysis of gene expression using microarray. The fold increase in expression cannot be the reason why few B cells activated STAT1. mRNA was calculated comparing expression in unstimulated subsets with Although IFNAR2 expression was equal, the functionality of this stimulated subsets. The Venn diagram shows increases in mRNA by $2- receptor chain might be different in B cells. We previously found fold and illustrates different mRNA-induction patterns in B cells (CD19) that the activation of STAT2 preceded the activation of STAT1 in and monocytes (CD14). B, Differences in expression of apoptosis-inducing mRNAs (BAK1, CASP3, CDKN1A, STK3) and the survival-promoting fibrosarcoma cells and that STAT2-null cells are severely ham- mRNA PBEF1 between B cells and monocytes after stimulation with IFN- pered in their ability to activate STAT1 (37). Fig. 8B shows the b, revealed by microarray analysis (Table I), was confirmed by rtPCR, percentage of cells positive for PY-STAT2 after stimulating whole using mRNA isolated from purified monocytes and B cells, normalized blood of six healthy individuals with 2000 IU/ml IFN-b for 45 by guest on October 1, 2021 relative to 18S rRNA. The median fold changes in mRNA expression in min. Monocytes, B cells, and CD4+ and CD8+ T cells demon- monocytes and B cells derived from six healthy subjects are shown for strated activation of STAT2 in response to IFN-b (Fig. 8B); there- stimulated subsets compared with control subsets. fore, failure to activate STAT2 is not the cause of low STAT1 activation in B cells. Another possible explanation for the very IFN-b (Fig. 6B), it is likely that activated STAT5 and STAT3 cause low activation of STAT1 in B cells is that many fewer B cells the increases in IL-2RA, PBEF1, and BAFF in B cells only. express STAT1 protein. We used flow cytometry to compare the To confirm these microarray data from two healthy individuals, four different leukocyte subsets for the percentages of cells that express total STAT1 (Fig. 8C), finding that all subsets were pos- the induction of specific mRNAs in monocytes and B cells after + 3 h of stimulation with IFN-b was replicated in six healthy itive for STAT1. Although the percentages of total STAT1 B cells were slightly lower compared with the other subsets, this differ- ence cannot explain why usually ,15% of the B cells showed activation of STAT1 in response to IFN-b. Finally, Western blot Table I. Fold increases of mRNAs in primary human monocytes and analysis of total STAT1 expression in isolated monocytes, B cells, b B cells from two HIs after 3 h of stimulation with IFN- and CD4+ or CD8+ T cells from two healthy individuals revealed similar levels of STAT1 expression in all subsets (data not shown). Monocytes B Cells B Cells mRNA (HI #1) (HI #1) (HI #2) Types I and II IFNs induce similar amounts of PY-STAT1+ CDKN1A (p21)a 3.1 1.0 0.8 B cells BAK1b 2.5 0.9 1.1 BCL2L13b 2.4 1.1 1.4 To test whether other type I IFNs and type II IFN also stimulate CASP3a 2.3 1.5 1.5 few B cells to activate STAT1, undiluted whole blood of six STK3b 2.7 1.3 1.7 healthy subjects was stimulated with 2000 IU/ml IFN-g for 30 min IL2RAc 1.7 4.7 5.8 d (the optimal time for activation of STATs by IFN-g; data not PBEF1 1.6 3.2 3.2 shown), and 2000 IU/ml IFN-a1, IFN-a2b, or IFN-b for 45 min. TNFSF13B (BAFF)e 2.8 10.4 10.0 TNFSF10 (TRAIL) 6.8 18.2 23.7 Fig. 9 shows that within all four leukocyte subsets tested (B cells, FAS 4.4 4.7 3.4 monocytes, CD4+ or CD8+ T cells), all type I IFNs induced simi- IRF1 2.9 2.6 2.8 lar percentages of cells to activate STAT1. In B cells and mono- + aKnown STAT1-dependent gene. cytes, type II IFN stimulated similar percentages of PY-STAT1 bProbable STAT1-dependent gene. cells as did type I IFNs (Fig. 9). Because T cells that produce IFN- cKnown STAT5-dependent gene. dKnown STAT3-dependent gene. g, such as Th1 and Tc1 cells, are unresponsive to IFN-g due to + + eProbable STAT3-dependent gene. changed IFN-gR expression levels (38), CD4 and CD8 T cells The Journal of Immunology 9

FIGURE 9. Similar activation of STAT1 in B cells after stimulation with IFN-g, IFN-a1, IFN-a2b, and IFN-b. Undiluted whole blood from six healthy subjects was stimulated for 30 min with 2000 IU/ml IFN-g or for 45 min with 2000 IU/ml IFN-a1, IFN-a2b, or IFN-b (optimal time points for activation of STATs). Activation of STAT1 was determined in the various leukocyte subsets for all six healthy subjects individually as the + percentages of PY-STAT1 subsets. Downloaded from

tion in B cells by types I and II IFNs. We tested whether the ex- pression of SOCS1, SHP1, and TCP45 was enhanced in B cells, compared with other leukocyte subsets, in isolated monocytes, B cells, and CD4+ and CD8+ T cells of one healthy individual, as

well as isolated monocytes and B cells from two other healthy http://www.jimmunol.org/ subjects. However, Western blot analysis showed no SOCS1 ex- pression in any of the unstimulated subsets, whereas it did in an IFN-g–stimulated monocytic cell line, and SHP1 and TCP45 were expressed at similar levels in all subsets (data not shown).

Discussion Mechanistic aspects of differential STAT induction in response FIGURE 8. Differences in STAT1 activation cannot be explained by to IFN-b differences in IFNAR2 expression levels, STAT2 activation, or STAT1 by guest on October 1, 2021 levels. A, Surface IFNAR2 expression was determined on monocytes, B The aims of this study were to investigate how certain primary cells, CD4+ T cells, and CD8+ T cells present in unstimulated whole blood human blood cells signal in response to IFN-b and to explore of six healthy individuals. B, Activation of STAT2 in leukocyte subsets was how such signals might be related to apoptosis or cell survival. determined by stimulating undiluted whole blood of six healthy donors Nonimmune cells and cell lines have primarily been used to study with 2000 IU/ml of IFN-b1a or not for 45 min. C, STAT1 expression was type I IFN signaling and to elucidate the mechanisms by which determined in monocytes, B cells, CD4+ T cells, and CD8+ T cells present these IFNs regulate transcription. We developed a flow cytometry- in unstimulated blood of four healthy subjects. Geometric means 6 SEM based assay to detect, at the single-cell level, the activation of for the percentages of leukocyte subsets that are positive for surface specific STATs in primary human leukocytes. IFN-a/b–induced IFNAR2 (A), PY-STAT2 (B), or intracellular STAT1 (C) are shown. activation of STAT1, which results mainly in the formation of the ISGF3 complex, but also leads to the formation of STAT1 ho- generated lower amounts of PY-STAT1+ cells in response to IFN-g modimers in adherent and nonadherent cell lines, is a hallmark of than in response to type I IFNs (Fig. 9). Remarkably, also on an type I IFN signaling. The human leukemic B cell line HT and the individual donor level, IFN-a1, -a2b, -b, and -g activated equal leukemic CD4+ T cell line Jurkat form abundant amounts of percentages of PY-STAT1+ B cells (Fig. 9). We found that only 7% PY-STAT1 in response to IFN-b (Fig. 1; A.H.H. van Boxel-Dezaire of healthy subjects (3 of 41) showed activation of STAT1 in .60% and G.R. Stark, unpublished data). Unexpectedly, we found that of their B cells in response to IFN-b. As shown in Fig. 9, two only a small fraction of primary human B cells activated STAT1 in individuals had .60% of their B cells positive for PY-STAT1 in response to IFN-b and that this activation was independent of the response to IFN-a/b and IFN-g. The four individuals who showed concentration of IFN used (500–10,000 IU/ml) or the time of lower activation of STAT1 in B cells in response to IFN-b (i.e., stimulation (10–75 min; Fig. 4). We tested IFN-b–induced sig- ∼10–20% of the cells positive for PY-STAT1) showed a similar naling in 41 individuals. Although most of the time we saw that response after stimulation with IFN-a1, -a2b, and -g. Therefore, it a maximum of 25% of the B cells responded by activating STAT1, can be concluded that the same mechanism that influences the in 7% of these individuals, we detected .65% of the B cells activation of STAT1 in B cells after ligation of the type I IFNAR positive for PY-STAT1 induction (3 of 41). It may be that these also influences its activation by the IFN-gR. persons have an underlying disease that has not been diagnosed or Suppressor of cytokine signaling 1 (SOCS1), which diminishes that this response is normal during a subclinical virus infection. activation of STAT1 by the types I and II IFNRs by influencing the Nevertheless, despite individual variations, we found that many activation of the JAKs (39), and Src homology region 2 domain- fewer B cells showed activation of STAT1 compared with mono- containing phosphatase 1 (SHP1) and TCP45, two protein tyrosine cytes and CD4+ and CD8+ T cells after stimulation with 2000 IU/ml phosphatases known to decrease tyrosine phosphorylation of STAT1 IFN-b (Fig. 3C). To begin to understand the mechanism, we studied (40, 41), are all candidates to explain the lack of PY-STAT1 induc- IFNAR expression and found that 100% of primary human B cells, 10 IFN-b–INDUCED RESPONSES IN HUMAN BLOOD CELL SUBSETS monocytes, and CD4+ and CD8+ T cells expressed the IFNAR2 PY-STAT3 or anti–PY-STAT1/PY-STAT5 Abs, we were able to chain (Fig. 8A), which has a long cytoplasmic tail containing two detect the activation of STAT1/STAT3 and STAT1/STAT5 to- conserved tyrosine residues that are crucial for activation of STAT1, gether, in individual cells. Notably, the percentage of PY-STAT1+/ STAT2, and STAT3 (1–4). Although IFNAR2 expression was found PY-STAT3– monocytes found in response to IFN-b after 45 min to be equal, the functionality of this receptor chain could still be correlated significantly with the percentage of activated caspase 3+ different in B cells. An explanation for the low activation of STAT1 monocytes after 8 h (Fig. 6C). Enhanced STAT3 protein levels in could be decreased activation of STAT2, because activation of monocytes have been demonstrated to suppress DNA binding of STAT1 depends on the activation of STAT2 in fibrosarcoma cells STAT1 homodimers by sequestering STAT1 into STAT1/STAT3 and primary fibroblasts (37, 42). However, STAT2-deficient peri- heterodimers (48). Therefore, it is to be expected that the PY- toneal macrophages retained the ability to activate STAT1, high- STAT1+/PY-STAT32 monocytes would become apoptotic first and lighting intriguing differences in the ability of the IFNAR to activate that PY-STAT1+/PY-STAT3+ monocytes are protected from apo- STAT1 in fibroblasts and monocytic cells (42). Nothing is known ptosis induction only if PY-STAT3 levels are high enough to se- about this function of the IFNAR in other leukocyte subsets, but we quester activated STAT1. However, after longer stimulation with found low activation of STAT1 in primary human B cells at every IFN-b (.10 h), the percentage of apoptotic cells is doubled or IFN-b concentration and in CD4+ T cells at lower IFN-b concen- tripled compared with the percentage at 8 h, indicating that even trations, despite normal activation of STAT2. At the optimal time PY-STAT1+/PY-STAT3+ monocytes eventually die. Very few B cells point for IFN-b–induced STAT activation, we found the highest are PY-STAT1 +/PY-STAT32 and, because STAT1 activation is so activation of STAT5 in primary human CD4+ T cells and we also low in these cells, it is more likely that enhanced PY-STAT3 levels found activation of STAT5 and STAT3 in primary human B cells, could suppress DNA binding of STAT1 homodimers by sequester- suggesting that the type I IFNR is functional in B cells. ing STAT1 into STAT1/STAT3 heterodimers in PY-STAT1+/PY- Downloaded from It will be important to investigate whether low STAT1 activation STAT3+ B cells than in monocytes (48). It is interesting that CD4+ is an intrinsic property of mature B cells and CD4+ T cells or T cells that show the highest percentage of PY-STAT1+/PY-STAT3 2 whether it is a result of other factors present in whole blood. cells in response to IFN-b after 45 min display the lowest apoptosis Notably, our results comparing IFN-a1, IFN-a2b, and IFN-g with induction, along with B cells (Fig. 6A,6B). Both leukocyte subsets IFN-b showed very similar low activation of STAT1 in B cells have very low numbers of PY-STAT1+/PY-STAT5 2 cells and sig- (Fig. 9), suggesting that the same mechanism is involved. By nificant numbers of PY-STAT1+/PY-STAT5 + cells, suggesting that http://www.jimmunol.org/ influencing activation of the JAKs, SOCS1 can diminish the ac- the activation of STAT5 in the majority of CD4+ T cells and B cells tivation of STAT1 by the types I and II IFNRs (39). SHP1 and protects against apoptosis induction. Indeed, the antiapoptotic and TCP45 are protein tyrosine phosphatases that decrease tyrosine mitogenic properties of type I IFNs in mouse T cells are dependent phosphorylation of STAT1 (40, 41). Although SOCS1, SHP1, and on the activation of STAT3 and STAT5 (47). Strangely, despite the TCP45 are excellent candidates to explain the lack of PY-STAT1 fact that monocytes also generate very high numbers of PY-STAT1+/ induction in B cells by types I and II IFNs, we could not find PY-STAT5+ cells in response to IFN-b, they are the most sensitive to evidence of their enhanced protein expression in B cells only. apoptosis induction. This disparity between monocytes, B cells, and Based on our experiments, we propose that physical properties of CD4+ T cells might be explained by the fact that our anti–PY-STAT5 by guest on October 1, 2021 STAT1 protein itself are altered or that a selective negative reg- Ab recognizes activated STAT5A and STAT5B, and human mono- ulator of STAT1 tyrosine phosphorylation is present in the ma- cytic cells activate only STAT5A (49), in contrast to human T cells, jority of B cells. B cells are a heterogeneous population, and it which activate STAT5A and STAT5B in response to type I IFNs (8). will be important to characterize the minor fractions that show In accordance with the above-mentioned data, when monocytes activation of STAT1 by studying the expression of CD markers, and B cells were compared for proapoptotic mRNA induction by chemokine receptors, and adhesion molecules. Perhaps the STAT1- IFN-b, we only found enhancement of CDKN1A, BAK1, CASP3, activating cells are immature, because type I IFNs inhibit B and and STK3 mRNA in monocytes (Fig. 7B, Table I). Evidence that T cell lymphopoiesis (43). In contrast, IFN-a induces STAT1- the induction of these mRNAs depends upon phosphorylated dependent proliferation in dormant hematopoietic stem cells (44), STAT1 homodimers is as follows. First, IFN-g does not induce suggesting that the response to type I IFNs may change during the CDKN1A (or p21) in STAT1-deficient U3A fibrosarcoma cells, but maturation of leukocyte subsets. Future experiments could address it does enhance p21 expression in U3A cells in which STAT1 has this issue by analyzing subsets separated on the basis of lineage been reintroduced (27), and enhancement of CASP3 expression is and differentiation markers expressed on their surfaces. dependent on PY-STAT1 formation (28). Second, because BAK1 expression is induced directly in HT-29 cells by IFN-g (29), its Biological consequences of differential STAT activation induction probably depends on the formation of STAT1 homo- Type I IFNs cause apoptosis in many cancer cell lines and are dimers. Finally, because types I and II IFNs enhance STK3 ex- used to treat several types of tumors (22, 23). Similarly, type I pression (http://www.interferome.org), it is likely that the induc- IFNs induce apoptosis in primary monocytes (20, 21) but, in con- tion of this mRNA occurs through activation of STAT1. p21, trast, increase the survival and proliferation of primary B cells and BAK1, CASP3, and STK3 are known to be involved at different T cells (14–19). In agreement, we found the highest activation of stages of the intrinsic apoptotic pathway. For instance, increased caspase 3, a hallmark of apoptosis induction, in monocytes, fol- p21 leads to cell-cycle arrest in the G1 phase of fibrosarcoma and lowed by CD8+ T cells, and the least amount in CD4+ T cells and Burkitt’s lymphoma cells, and the induction of G1 arrest in Bur- B cells after stimulation with IFN-b. Interestingly, during virus kitt’s B cell lymphoma by type I IFNs is followed by induction of infection of mice, only CD8+ T cells with low STAT1 activation apoptosis (26). BAK1 is a member of the BCL-2 family of proa- proliferate in response to type I IFNs, as a result of lower STAT1 poptotic proteins that, upon activation by IFN-a, forms oligomers or protein expression (45). The apoptosis-inducing capacity of type I heterodimers that interact with the mitochondria, leading to the IFNs is largely attributed to the activation of STAT1 (11, 46), release of cytochrome c and apoptosis induction (50, 51). Notably, it whereas the activation of STAT3 and STAT5 by type I IFNs is was shown that apoptosis induction through activation of STAT1 is related to survival and proliferation (12, 13, 47). By performing mediated by activation of the effector caspase 3, among others (46). double staining of IFN-b–stimulated cells with anti–PY-STAT1/ Interestingly, STK3 is a direct substrate of caspase 3 and, following The Journal of Immunology 11 cleavage, translocates to the nucleus and induces chromatin con- propose that the low activation of STAT1 and much higher activa- densation, followed by internucleosomal DNA fragmentation (52, tion of STAT3 and STAT5 and, consequently, the absence of in- 53). However, increased levels of STK3 can also accelerate apo- duction of genes participating sequentially in the intrinsic apoptosis- ptosis induction through the activation of caspase 3 (52). induction pathway (as observed in monocytes) are important me- Induction by IFN-b of CDKN1A, BAK1, CASP3, and STK3 all chanisms to enable primary human B cells to survive in response to involved in the intrinsic apoptotic pathway, seems to occur only in IFN-b. More in-depth studies using Chip assays to determine the monocytes, whereas the induction of certain proapoptotic genes residence of specific STATs on specific promoters are necessary to was not found in B cells exclusively. Nevertheless, in monocytes understand in detail how STAT1, STAT3, and STAT5 activated by and B cells, TRAIL mRNA was increased by 7–24-fold after IFN-a/b exert their proapoptotic and mitogenic effects in specific IFN-b stimulation (Table I). Although TRAIL expression induces immune subsets. In addition, it will be vital to unravel the mecha- apoptosis in tumor- and virus-infected cells, it exhibits no ap- nism of low STAT1 activation in the great majority of primary hu- parent adverse affect on normal cells (30, 54). Moreover, TRAIL man B cells. engagement on T cells can even lead to increased proliferation, Although to enable survival, few B cells activate STAT1 in re- but it is not known whether this is also true for B cells (55). sponse to types I and II IFNs, it is nevertheless important that the However, the expression of TRAIL on monocytic cells might still antiviral effects of IFNs are preserved. mRNAs derived from virus- have inhibitory effects, because the rapid maturation of monocytes responsive genes, such as MX1, OAS, EIF2AK2, ISG15, IFI44, and into short-lived dendritic cells by IFN-b is associated with TRAIL IFITM3, increased $2-fold in B cells and monocytes in response to expression (21). TRAIL induction by IFN-b in fibrosarcoma cells IFN-b (data not shown). It seems that only the promoter of MX1 is dependent on ISGF3 binding to the ISRE element in the pro- harbors a classical ISRE element (57), and increased MX1 tran- moter (56). The fact that our data suggest that TRAIL mRNA was scription in B cells, despite low STAT1 activation by IFN-b, could Downloaded from increased in B cells by IFN-b, despite low activation of STAT1, be the result of activation of similar transcription factor com- could be explained by the binding of STAT2dimer/IRF-9 or plexes as mentioned above for TRAIL. All of the other typical STAT2/STAT6/IRF-9 to the ISRE (4, 5), because primary human virus-responsive genes are suggested to belong to a new subtype of B cells can activate STAT2 and STAT6 (Fig. 8B). Alternatively, ISRE termed “E–twenty-six (ETS)/IRF response elements,” which formation of STAT3, STAT5, or STAT6 homodimers (5) in re- can bind IRF dimers (similar to classic ISRE) or an ETS/IRF dimer sponse to IFN-b could be responsible for the observed increase in (57). Because B cells express IRF4, IRF8, PU.1, and other ETS http://www.jimmunol.org/ IRF-1 in primary human B cells (Table I) and IRF1 dimers could family members (57), an IRF/ETS dimer might bind to these subsequently bind to the ISRE in the TRAIL promoter (57). In promoters in primary B cells after IFN-b stimulation. Therefore, addition to TRAIL induction, we found that, in monocytes and the inability of most B cells to activate STAT1 does not lead to B cells, the mRNA for the death receptor FAS was increased by 3– a deficient antiviral response in B cells, but the mechanism has still 4-fold (Table I). Upon FASL binding, the extrinsic apoptosis path- to be elucidated. These results have important implications for way could be triggered by recruitment of FAS-associated death understanding more fully the influence of IFN-a/b on leukocyte domain protein, activation of caspase 8, and cleavage of the proa- subsets during virus infection in humans, as well as the effects of poptotic BCL-2 family member Bid (31, 50). However, we did not treatment with IFN-a/b on these subsets in patients with multiple by guest on October 1, 2021 observe any simultaneous increase in FASL mRNA expression in sclerosis, hepatitis, or cancer. B cells or monocytes. Of note, FAS also has nonapoptotic functions (31), because individuals with homozygous caspase-8 reduction- Acknowledgments of-function mutations display defects in FAS signaling and im- We thank Dr. Ian M. Kerr for many helpful suggestions and Dr. Ganes paired proliferation of B, T, and NK cells (58). C. Sen for critically reviewing this manuscript. In addition, we thank Mike + 2 + Very low percentages of PY-STAT1 /PY-STAT3 and PY-STAT1 / Sramkoski (Case Comprehensive Cancer Center Flow Cytometry Core, 2 PY-STAT5 were observed in B cells, the only leukocyte subset in Case Western Reserve University) and Cathy Shemo and Sage O’Bryant which the PY-STAT12/PY-STAT3 + and PY-STAT1 2/PY-STAT5+ (Flow Cytometry Core, Lerner Research Institute of the Cleveland Clinic) combinations were induced upon IFN-b stimulation (Fig. 6B). These for advice and technical assistance. We thank Dr. Pieter Faber (Genomics findings together are likely to be responsible for decreased induction Core Facility, Cleveland Clinic) for microarray analyses. Moreover, we of apoptosis and increased induction of the PY-STAT3– or PY- thank Vai Pathak and Dr. Patrick Leahy (Gene Expression and Genotyping Core Facility, Case Comprehensive Cancer Center, Case Western Reserve STAT5–dependent mRNAs that are responsible for increased survival University) for assistance with the rtPCR experiments. Finally, we thank and proliferation. Notably, the probable PY-STAT3–dependent BAFF Drs. Thomas Hamilton and Ernest Borden (Department of Immunology (36), which our preliminary data suggest to be increased 3.5-fold and Taussig Cancer Institute and Cleveland Clinic, respectively) for their more in B cells compared with monocytes (Table I), was shown to kind gifts of human rIFN-g, rIFN-a1, and rIFN-a2b. overcome any negative effects from FAS signaling and increase the survival of B cells (59). Another activated STAT3-dependent mRNA Disclosures (35), PBEF1, increased in B cells only (Fig. 7B, Table I) and not in The authors have no financial conflicts of interest. monocytes in response to IFN-b.Notably,PBEF1 inhibits the in- duction of apoptosis in neutrophils and epithelial cells by reducing References the activity of caspases 3 and 8 (34). In addition, PBEF1 synergizes 1. Pestka, S., C. D. Krause, and M. R. Walter. 2004. , -like with IL-7 in pre-B cell colony formation (33). We did not observe cytokines, and their receptors. Immunol. Rev. 202: 8–32. increased BCL2 or BCL2L1 expression, which was previously sug- 2. Novick, D., B. Cohen, and M. Rubinstein. 1994. The human interferon alpha/ gested to be the antiapoptotic mechanism of IFN-b in B cells (15). beta receptor: characterization and molecular cloning. Cell 77: 391–400. 3. Stark, G. R., I. M. Kerr, B. R. Williams, R. H. Silverman, and R. D. Schreiber. The promoters of BCL2 and BCL2L1 are typical targets of acti- 1998. How cells respond to interferons. Annu. Rev. Biochem. 67: 227–264. vated STAT5 in response to several growth factors, and the STAT5- 4. van Boxel-Dezaire, A. H., M. R. Rani, and G. R. Stark. 2006. Complex mod- dependent induction of resistance to apoptosis functions through ulation of cell type-specific signaling in response to type I interferons. Immunity these proteins (13). Because we studied only early mRNA tran- 25: 361–372. 5. Decker, T., P. Kovarik, and A. Meinke. 1997. GAS elements: a few nucleotides scription in response to IFN-b, the induction of BCL2 and BCL2L1 with a major impact on cytokine-induced gene expression. J. Interferon Cytokine mRNA in B cells might need .3 h of stimulation with IFN-b.We Res. 17: 121–134. 12 IFN-b–INDUCED RESPONSES IN HUMAN BLOOD CELL SUBSETS

6. Cho, S. S., C. M. Bacon, C. Sudarshan, R. C. Rees, D. Finbloom, R. Pine, and 33. Samal, B., Y. Sun, G. Stearns, C. Xie, S. Suggs, and I. McNiece. 1994. Cloning J. J. O’Shea. 1996. Activation of STAT4 by IL-12 and IFN-alpha: evidence for and characterization of the cDNA encoding a novel human pre-B-cell colony- the involvement of ligand-induced tyrosine and serine phosphorylation. J. enhancing factor. Mol. Cell. Biol. 14: 1431–1437. Immunol. 157: 4781–4789. 34. Jia, S. H., Y. Li, J. Parodo, A. Kapus, L. Fan, O. D. Rotstein, and J. C. Marshall. 7. Yu, C. R., J. X. Lin, D. W. Fink, S. Akira, E. T. Bloom, and A. Yamauchi. 1996. 2004. Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experi- Differential utilization of -signal transducer activator of transcrip- mental inflammation and clinical sepsis. J. Clin. Invest. 113: 1318–1327. tion signaling pathways in the stimulation of human natural killer cells by IL-2, 35. Nowell, M. A., P. J. Richards, C. A. Fielding, S. Ognjanovic, N. Topley, IL-12, and IFN-alpha. J. Immunol. 157: 126–137. A. S. Williams, G. Bryant-Greenwood, and S. A. Jones. 2006. Regulation of 8. Matikainen, S., T. Sareneva, T. Ronni, A. Lehtonen, P. J. Koskinen, and pre-B cell colony-enhancing factor by STAT-3-dependent interleukin-6 trans- I. Julkunen. 1999. Interferon-alpha activates multiple STAT proteins and upre- signaling: implications in the pathogenesis of rheumatoid arthritis. Arthritis gulates proliferation-associated IL-2Ralpha, c-myc, and pim-1 genes in Rheum. 54: 2084–2095. human T cells. Blood 93: 1980–1991. 36. Zhou, L., R. Zhong, W. Hao, H. Wang, X. Fan, L. Zhang, and Q. Mi. 2009. 9. Gupta, S., M. Jiang, and A. B. Pernis. 1999. IFN-alpha activates Stat6 and leads Interleukin-10 and interferon-gamma up-regulate the expression of B-cell activating to the formation of Stat2:Stat6 complexes in B cells. J. Immunol. 163: 3834– factor in cultured human promyelocytic leukemia cells. Exp. Mol. Pathol. 87: 54–58. 3841. 37. Li, X., S. Leung, I. M. Kerr, and G. R. Stark. 1997. Functional subdomains of 10. Schlaak, J. F., C. M. Hilkens, A. P. Costa-Pereira, B. Strobl, F. Aberger, STAT2 required for preassociation with the alpha interferon receptor and for A. M. Frischauf, and I. M. Kerr. 2002. Cell-type and donor-specific transcrip- signaling. Mol. Cell. Biol. 17: 2048–2056. tional responses to interferon-alpha. Use of customized gene arrays. J. Biol. 38. Bach, E. A., S. J. Szabo, A. S. Dighe, A. Ashkenazi, M. Aguet, K. M. Murphy, Chem. 277: 49428–49437. and R. D. Schreiber. 1995. Ligand-induced autoregulation of IFN-gamma re- 11. Kim, H. S., and M. S. Lee. 2007. STAT1 as a key modulator of cell death. Cell. ceptor beta chain expression in T helper cell subsets. Science 270: 1215–1218. Signal. 19: 454–465. 39. Alexander, W. S. 2002. Suppressors of cytokine signalling (SOCS) in the im- 12. Bowman, T., R. Garcia, J. Turkson, and R. Jove. 2000. STATs in oncogenesis. Nat. Rev. Immunol. Oncogene 19: 2474–2488. mune system. 2: 410–416. 13. Debierre-Grockiego, F. 2004. Anti-apoptotic role of STAT5 in haematopoietic cells 40. David, M., H. E. Chen, S. Goelz, A. C. Larner, and B. G. Neel. 1995. Differential and in the pathogenesis of malignancies. Apoptosis 9: 717–728. regulation of the alpha/beta interferon-stimulated Jak/Stat pathway by the SH2 14. Morikawa, K., H. Kubagawa, T. Suzuki, and M. D. Cooper. 1987. Recombinant domain-containing tyrosine phosphatase SHPTP1. Mol. Cell. Biol. 15: 7050–7058. interferon-alpha, -beta, and -gamma enhance the proliferative response of human 41. Kra¨mer, O. H., S. K. Knauer, G. Greiner, E. Jandt, S. Reichardt, K. H. Gu¨hrs, Downloaded from B cells. J. Immunol. 139: 761–766. R. H. Stauber, F. D. Bo¨hmer, and T. Heinzel. 2009. A phosphorylation- 15. Su, L., and M. David. 1999. Inhibition of B cell receptor-mediated apoptosis by acetylation switch regulates STAT1 signaling. Genes Dev. 23: 223–235. IFN. J. Immunol. 162: 6317–6321. 42. Park, C., S. Li, E. Cha, and C. Schindler. 2000. Immune response in Stat2 16. Hibbert, L., and G. R. Foster. 1999. Human type I interferons differ greatly in knockout mice. Immunity 13: 795–804. their effects on the proliferation of primary B cells. J. Interferon Cytokine Res. 43. Lin, Q., C. Dong, and M. D. Cooper. 1998. Impairment of T and B cell de- 19: 309–318. velopment by treatment with a type I interferon. J. Exp. Med. 187: 79–87. 17. Tough, D. F., P. Borrow, and J. Sprent. 1996. Induction of bystander T cell 44. Essers, M. A., S. Offner, W. E. Blanco-Bose, Z. Waibler, U. Kalinke,

proliferation by viruses and type I interferon in vivo. Science 272: 1947–1950. M. A. Duchosal, and A. Trumpp. 2009. IFNalpha activates dormant haemato- http://www.jimmunol.org/ 18. Marrack, P., J. Kappler, and T. Mitchell. 1999. Type I interferons keep activated poietic stem cells in vivo. Nature 458: 904–908. T cells alive. J. Exp. Med. 189: 521–530. 45. Gil, M. P., R. Salomon, J. Louten, and C. A. Biron. 2006. Modulation of STAT1 19. Scheel-Toellner, D., D. Pilling, A. N. Akbar, D. Hardie, G. Lombardi, protein levels: a mechanism shaping CD8 T-cell responses in vivo. Blood 107: M. Salmon, and J. M. Lord. 1999. Inhibition of T cell apoptosis by IFN-beta 987–993. rapidly reverses nuclear translocation of protein kinase C-delta. Eur. J. Immunol. 46. Sironi, J. J., and T. Ouchi. 2004. STAT1-induced apoptosis is mediated by 29: 2603–2612. caspases 2, 3, and 7. J. Biol. Chem. 279: 4066–4074. 20. Moore, R. N., H. S. Larsen, D. W. Horohov, and B. T. Rouse. 1984. Endogenous 47. Tanabe, Y., T. Nishibori, L. Su, R. M. Arduini, D. P. Baker, and M. J. David. regulation of macrophage proliferative expansion by colony-stimulating factor- 2005. Cutting edge: role of STAT1, STAT3, and STAT5 in IFN-alpha beta induced interferon. Science 223: 178–181. responses in T lymphocytes. J. Immunol. 174: 609–613. 21. Santini, S. M., C. Lapenta, M. Logozzi, S. Parlato, M. Spada, T. Di Pucchio, and 48. Ho, H. H., and L. B. Ivashkiv. 2006. Role of STAT3 in type I interferon F. Belardelli. 2000. Type I interferon as a powerful adjuvant for monocyte- responses. Negative regulation of STAT1-dependent inflammatory gene activa-

derived dendritic cell development and activity in vitro and in Hu-PBL- tion. J. Biol. Chem. 281: 14111–14118. by guest on October 1, 2021 SCID mice. J. Exp. Med. 191: 1777–1788. 49. Meinke, A., F. Barahmand-Pour, S. Wo¨hrl, D. Stoiber, and T. Decker. 1996. 22. Pfeffer, L. M., C. A. Dinarello, R. B. Herberman, B. R. Williams, E. C. Borden, Activation of different Stat5 isoforms contributes to cell-type-restricted signaling R. Bordens, M. R. Walter, T. L. Nagabhushan, P. P. Trotta, and S. Pestka. 1998. in response to interferons. Mol. Cell. Biol. 16: 6937–6944. Biological properties of recombinant alpha-interferons: 40th anniversary of the 50. Li, J., and J. Yuan. 2008. Caspases in apoptosis and beyond. Oncogene 27: 6194– discovery of interferons. Cancer Res. 58: 2489–2499. 6206. 23. Clemens, M. J. 2003. Interferons and apoptosis. J. Interferon Cytokine Res. 23: 51. Panaretakis, T., K. Pokrovskaja, M. C. Shoshan, and D. Grande´r. 2003. 277–292. Interferon-alpha-induced apoptosis in U266 cells is associated with activation of 24. Chow, S., D. W. Hedley, P. Grom, R. Magari, J. W. Jacobberger, and the proapoptotic Bcl-2 family members Bak and Bax. Oncogene 22: 4543–4556. T. V. Shankey. 2005. Whole blood fixation and permeabilization protocol with 52. Lee, K. K., T. Ohyama, N. Yajima, S. Tsubuki, and S. Yonehara. 2001. MST, red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in a physiological caspase substrate, highly sensitizes apoptosis both upstream and leukocyte subpopulations. Cytometry A 67: 4–17. downstream of caspase activation. J. Biol. Chem. 276: 19276–19285. 25. Krutzik, P. O., J. M. Irish, G. P. Nolan, and O. D. Perez. 2004. Analysis of 53. Kakeya, H., R. Onose, and H. Osada. 1998. Caspase-mediated activation of a 36- protein phosphorylation and cellular signaling events by flow cytometry: tech- kDa myelin basic protein kinase during anticancer drug-induced apoptosis. niques and clinical applications. Clin. Immunol. 110: 206–221. Cancer Res. 58: 4888–4894. 26. Subramaniam, P. S., P. E. Cruz, A. C. Hobeika, and H. M. Johnson. 1998. Type I in- 54. Griffith, T. S., S. R. Wiley, M. Z. Kubin, L. M. Sedger, C. R. Maliszewski, and terferon induction of the Cdk-inhibitor p21WAF1 is accompanied by ordered G1 ar- N. A. Fanger. 1999. Monocyte-mediated tumoricidal activity via the tumor ne- rest, differentiation and apoptosis of the Daudi B-cell line. Oncogene 16: 1885–1890. crosis factor-related cytokine, TRAIL. J. Exp. Med. 189: 1343–1354. 27. Chin, Y. E., M. Kitagawa, W. C. Su, Z.-H. You, Y. Iwamoto, and X.-Y. Fu. 1996. 55. Chou, A. H., H. F. Tsai, L. L. Lin, S. L. Hsieh, P. I. Hsu, and P. N. Hsu. 2001. Cell growth arrest and induction of cyclin-dependent kinase inhibitor p21 WAF1/ CIP1 mediated by STAT1. Science 272: 719–722. Enhanced proliferation and increased IFN-gamma production in T cells by signal 28. Huang, Y. Q., J. J. Li, and S. Karpatkin. 2000. Thrombin inhibits tumor cell transduced through TNF-related apoptosis-inducing ligand. J. Immunol. 167: growth in association with up-regulation of p21(waf/cip1) and caspases via 1347–1352. a p53-independent, STAT-1-dependent pathway. J. Biol. Chem. 275: 6462–6468. 56. Rani, M. R., S. Pandalai, J. Shrock, A. Almasan, and R. M. Ransohoff. 2007. 29. Ossina, N. K., A. Cannas, V. C. Powers, P. A. Fitzpatrick, J. D. Knight, Requirement of catalytically active Tyk2 and accessory signals for the induction J. R. Gilbert, E. M. Shekhtman, L. D. Tomei, S. R. Umansky, and M. C. Kiefer. of TRAIL mRNA by IFN-beta. J. Interferon Cytokine Res. 27: 767–779. 1997. Interferon-gamma modulates a p53-independent apoptotic pathway and 57. Kanno, Y., B. Z. Levi, T. Tamura, and K. Ozato. 2005. Immune cell-specific apoptosis-related gene expression. J. Biol. Chem. 272: 16351–16357. amplification of interferon signaling by the IRF-4/8-PU.1 complex. J. Interferon 30. Yu, J. W., and Y. Shi. 2008. FLIP and the death effector domain family. On- Cytokine Res. 25: 770–779. cogene 27: 6216–6227. 58. Chun, H. J., L. Zheng, M. Ahmad, J. Wang, C. K. Speirs, R. M. Siegel, 31. Peter, M. E., R. C. Budd, J. Desbarats, S. M. Hedrick, A.-O. Hueber, J. K. Dale, J. Puck, J. Davis, C. G. Hall, et al. 2002. Pleiotropic defects M. K. Newell, L. B. Owen, R. M. Pope, J. Tschopp, H. Wajant, et al. 2007. The in lymphocyte activation caused by caspase-8 mutations lead to human immu- CD95 receptor: apoptosis revisited. Cell 129: 447–450. nodeficiency. Nature 419: 395–399. 32. Rusterholz, C., P. C. Henrioud, and M. Nabholz. 1999. Interleukin-2 (IL-2) 59. Hancz, A., Z. He´rincs, Z. Neer, G. Sa´rmay, and G. Koncz. 2008. Integration of regulates the accessibility of the IL-2-responsive enhancer in the IL-2 receptor signals mediated by B-cell receptor, B-cell activating factor of the tumor necrosis alpha gene to transcription factors. Mol. Cell. Biol. 19: 2681–2689. factor family (BAFF) and Fas (CD95). Immunol. Lett. 116: 211–217.