Interferon (IFN) 13 Acts Downstream of IFN-~/-induced Class II Transactivator Messenger RNA Accumulation to Block Major Histocompatibility Complex Class II Gene Expression and Requires the 48-kD DNA-binding Protein, ISGF3-~ By Hong-Tao Lu,*:~James L. Riley,wGerald T. Babcock,* Michael Huston,II George R. Stark,* Jeremy M. Boss,w and Richard M. Ransohoff*~: From the *Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195; ~.Department of Bioflogy, Cleveland State University, Cleveland, Ohio 44115; w of Microbiology and Immunology, Emory University,Atlanta, Georgia 30322; and IIDepartment of Immunology, Berlex Downloaded from http://rupress.org/jem/article-pdf/182/5/1517/1107516/1517.pdf by guest on 26 September 2021 Biosciences, Richmond, California 94804 Summary Interferon (IFN) ",/, a cardinal proinflammatory cytokine, induces expression of the gene prod- ucts of the class II locus of the major histocompatibility complex (MHC), whereas IFN-0t or -[3 suppresses MHC class II expression. The mechanism of IFN-13-mediated MHC class II in- hibition has been unclear. Recently, a novel factor termed class II transactivator (CIITA) has been identified as essential for IFN-~/-induced MHC class II transcription. We studied the sta- tus of IFN-~/-induced CIITA messenger P, NA (mI(NA) accumulation and CIITA-driven transactivation in IFN-13-treated cells and used cell lines that had defined defects in the type I IFN response pathway to address the roles of IFN signaling components in the inhibition of MHC class II induction. IFN-~ treatment did not suppress IFN-~-induced accumulation of CIITA mR~A. After cells were stably transfected with CIITA, endogenous MHC class II genes were constitutively expressed, and MHC class II promoters, delivered by transfection, were ac- tively transcribed in CIITA-expressing cells. Expression of these promoters was significantly impaired by pretreatment with IFN-[3. These results suggest that IFN-[3 acts downstream of CIITA mRNA accumulation, and acts in part by reducing the functional competence of CIITA for transactivating MHC class II promoters. IFN stimulated gene factor 3 (ISGF3) was essential for IFN-[3 to mediate inhibition of MHC class II induction, regardless of whether MHC class II transcription was stimulated by IFN-~ or directly by CIITA expression. Results of these experiments suggest that inhibition of MHC class II in IFN-[3-treated cells requires expression of gene(s) directed by the ISGF3-IFN-stimulated response element pathway, and that these gene product(s) may act by blocking CIITA-driven transcription of MHC class II promoters. iochemical and genetic studies of signaling by the IFNs as a paradigm for a "direct effector" model of transcrip- B recently culminated in a convincing and lucid descrip- tional regulation by cytokines, involving protein-protein tion of the process by which type I IFNs induce transcrip- interactions to form a transcriptional activator, IFN-stimu- tion of a set of immediate response genes, termed the IFN- lated gene factor 3 (ISGF3), which can activate transcrip- responsive genes (ISGs) 1 (1). This pathway has been proposed tion via an inducible enhancer, the IFN-stimulated re- sponse element (ISRE). Upon receptor binding by IFN-ot or IFN-~, latent cytoplasmic transcription factors, collec- 1Abbreviations used in this paper: CAT, chloramphenicol acetyltransferase; tively termed ISGF3-ot, were shown to be phosphorylated CIITA, class II transactivator; EMSA, electrophoresismobility shift assay; on tyrosine residues and to accumulate in the nucleus, GAS, T-activated sequence; ISG, IFN-responsive gene; ISGF3, IFN- stimulated gene factor 3; ISRE, IFN-stimulatedresponse element; mRNA, where they formed a complex with a 48-kD DNA-binding messenger RNA; MS, multiple sclerosis; PTK, protein tyrosine kinase; protein, designated ISGF3-~/. These components were des- STAT, signal transducer and activator of transcription. ignated ISGF3, which was shown to be essential to induce 1517 j. Exp. Med. The Rockefeller University Press 0022-1007/95/11/1517/09 $2.00 Volume 182 November 1995 1517-1525 transcription from ISG promoter/enhancer elements con- lines with selective defects in the IFN-y pathway for MHC taining ISILEs. class II expression (26, 27). MHC class II gene induction by Unexpectedly, this research revealed that type I (IFN-a IFN-y required intermediary protein synthesis in most cells or IFN-[3) and type II (IFN-3') IFNs, despite signaling (28, 29). Additionally, the regulatory cis element for MHC through unique receptors, share certain transcriptional reg- class II transcription was quite different from the y-acti- ulatory components (2). In particular, a 91-kD ISGF3-0~ vated sequence (GAS) element that governed the direct component, designated signal transducer and activator of (protein synthesis-independent) response to IFN-~/ (1). transcription (STAT) 10~, and a nonreceptor protein ty- Taken together, studies of IFN-~/induction of MHC class rosine kinase (PTK) termed JAK1, proved to be absolutely II transcription indicated an indirect mechanism requiring required for response to both types I and II IFNs (2-4). synthesis of a protein factor, in addition to the direct JAK- Other components, such as the transcription factors ISGF3-',/ STAT pathway. Class II transactivator (CIITA), a novel and p113/STAT-2, as well as the PTK tyk2, were impli- transcription factor deficient in one bare lymphocyte syn- cated particularly in signaling by the type I IFNs. The PTK drome complementation group, has recently been identi- JAK2 was used specifically by IFN-~/(1, 5, 6). These results fied by a variety of genetic and biochemical strategies as the explained previously described signaling interactions be- IFN-y-inducible factor that was necessary for MHC class tween the partially overlapping pathways used by the two II transcription (30-33). IFN types (7). Based on recent reports, ClITA is proposed to activate Downloaded from http://rupress.org/jem/article-pdf/182/5/1517/1107516/1517.pdf by guest on 26 September 2021 The ISGs encode products that mediate the biological MHC class II transcription by interacting (directly or indi- consequences of IFN treatment, including antiviral, growth- rectly) with factors bound to regulatory DNA elements up- regulatory, and immune-modulatory effects (8). Prominent stream of the structural genes (34, 35). Furthermore, CIITA among these effects, IFN treatment regulates expression of contains a potent NH2-terminal transactivation domain that MHC antigens (8). Either of the two IFN subtypes, type I functions in heterologous context (34, 35). Taken in aggre- or type II IFN, up-regulate expression of MHC class I anti- gate, these observations prompted the attractive hypothesis gens (9). It has been shown that MHC class II antigens that physical interaction between CIITA and promoter- were responsive to IFN-% whereas IFN-ot and -13 were bound components positions the transactivator domain to typically unable to induce MHC class II expression (9). In provide a stimulatory interface between the MHC class II fact, for many cell types, type I IFN was shown to block gene-specific factors and the basal transcription apparatus the induction of MHC class II expression by IFN-~/(10-12). (34, 35). These contrasting consequences oflFN-~/and IFN-[3 treat- Experiments described in this report addressed the role ment for MHC class II expression might be ofpathogenetic of CIITA in the pathway by which IFN-[3 inhibited MHC significance, as IFN-~/and IFN-[~ recently exhibited highly class II expression. Additionally, cell lines with defined de- divergent effects in regulating the human inflammatory de- fects in type I IFN signaling were used to address the func- myehnating disorder multiple sclerosis (MS). MS patients tions of individual ISR.E-ISGF3 signaling components. treated with IFN-~/experienced elevated disease activity, ac- Initial experiments were performed in the parental fibrosar- companied by immune stimulation (13, 14). Patients treated coma cell line 2fTGH, which responds to IFN-~ with ex- with IFN-~ experienced fewer and milder attacks, with di- pression of MHC class II antigens, and this induction can minished disease progression as monitored by magnetic-res- be readily suppressed by IFN-[3. As anticipated, CIITA onance brain scanning (15, 16). IFN-[3 may ameliorate MS messenger RNA (mRNA) accumulated in 2fTGH cells af- by down-regulating pathogenic MHC class II expression in ter induction with IFN-~/. We found that IFN-13 treatment the affected central nervous system tissues (17). did not suppress IFN-~-induced CIITA mRNA accumu- In this regard, we and others have shown that IFN-[3- lation. 2fTGH cells that were stably transfected with a mediated blockade of IFN-~/-induced MHC class II ex- CIITA expression construct displayed MHC class II anti- pression occurred at the transcriptional level (18, 19). Tran- gens in the absence oflFN-% and MHC class II promoter/ scriptional suppression of MHC class II by IFN-[3 was reporters were strongly activated in CIITA-transfected relatively gene specific and did not require sequence con- ceils. IFN-[3 pretreatment impaired the expression of MHC tent, beyond the conserved MHC class II cis-regulatory el- class II promoter/reporter constructs in CIITA-transfected ements that were also required for response to IFN-',/(20). cells, and this inhibition was observed at modest concentra- MHC class II expression has been intensely investigated tions of IFN-13. Mutant cell lines were used to show that in recent years (9, 21-24). Expression of MHC class II was functional ISGF3-~/was required for IFN-[3 to mediate in- shown to be regulated developmentally and environmen- hibition of MHC class II expression, either induced by tally, in a remarkably specific and stringent fashion. Tran- IFN-~/treatment or directly by CIITA expression. scriptional activity of the MHC class II genes has been Taken together, these results indicate that IFN-[3 treat- shown to determine their expression, and several lines of ment blocks MHC class II expression at a point subsequent evidence have indicated selective transcriptional control of to the accumulation of CIITA mRNA. Our observations the MHC class II genes.