Genomic Effects of IFN- in Multiple Sclerosis Patients 1 Bianca Weinstock-Guttman,* Darlene Badgett, † Kara Patrick,* Laura Hartrich, †‡ Roseane Santos, † Dennis Hall, † Monika Baier, § Joan Feichter,* and Murali Ramanathan 2 * † The purpose of this report was to characterize the dynamics of the gene expression cascades induced by an IFN--1a treatment regimen in multiple sclerosis patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to therapy. In this open-label pharmacodynamic study design, peripheral blood was obtained from eight relapsing- remitting multiple sclerosis patients just before and at 1, 2, 4, 8, 24, 48, 120, and 168 h after i.m. injection of 30 g of IFN--1a. The total RNA was isolated from monocyte-depleted PBL and analyzed using cDNA microarrays containing probes for >4000 known genes. IFN--1a treatment resulted in selective, time-dependent effects on multiple genes. The mRNAs for genes implicated in the anti-viral response, e.g., double-stranded RNA-dependent protein kinase, myxovirus resistance proteins 1 and 2, and guanylate binding proteins 1 and 2 were rapidly induced within 1– 4 h of IFN- treatment. The mRNAs for several genes involved in IFN- signaling, such as IFN-/ receptor-2 and Stat1, were also increased. The mRNAs for lymphocyte activation markers, such as IFN-induced transmembrane protein 1 (9 –27), IFN-induced transmembrane protein 2 (1– 8D),  2 -microglobulin, and CD69, were also increased in a time-dependent manner. The findings demonstrate that IFN- treatment induces specific and time-dependent changes in multiple mRNAs in lymphocytes of multiple sclerosis patients that could provide a framework for rapid monitoring of the response to therapy. The Journal of Immunology, 2003, 171: 2694 –2702. R ecombinant human IFN- has emerged as one of the most commonly prescribed forms of therapy for relaps- ing multiple sclerosis (MS) 3 patients on the basis of sev- eral double-blind, placebo-controlled, multicenter trials (1–3). Treatment with recombinant IFN--1a has been shown to slow the accumulation of physical disability in MS, reduce the fre- quency of relapses, and decrease the accumulation of disease bur- den and atrophy as evaluated by magnetic resonance imaging. IFN- was also shown to delay the conversion to clinically definite MS in patients with a first demyelinating event (4, 5). The effects of IFN- treatment are complex, and its pharmaco- dynamics at the genomic level in humans are poorly understood. In MS patients in particular, the benefit associated with IFN- ther- apy is difficult to monitor (6, 7), and despite extensive examination of the effects of IFN- in vitro with techniques such as flow cy- tometry, ELISA, RT-PCR, and proliferation and adhesion assays, the cellular, molecular, and immune mechanisms mediating the clinical effects of IFN- in MS are poorly delineated. Patients with relapsing MS respond better to IFN- treatment than patients with progressive disease. However, relapsing MS patients also exhibit considerable interindividual heterogeneity in their clinical re- sponses to IFN- therapy. Large phase III studies indicate a 30 – 40% general clinical benefit, although the response varied among patients. Magnetic resonance imaging data also underscore the het- erogeneity of responses to IFN- therapy in MS patients; 40% of patients show complete suppression of new gadolinium-enhancing lesions, whereas 20% of patients have 70% suppression (8). The goal of this study was to characterize the dynamics of the gene expression cascades induced by an IFN--1a treatment reg- imen in MS patients and to examine the molecular mechanisms potentially capable of causing heterogeneity in response to ther- apy. Our results demonstrate that the IFN--1a treatment causes large-scale, time-dependent changes in immunomodulatory gene expression in circulating lymphocytes that can be effectively as- sessed using the array technique. The gene expression changes are orchestrated with immunological changes at the protein and cel- lular levels. Materials and Methods Study population With informed consent, peripheral blood anticoagulated with heparin was obtained by venipuncture from eight patients (six women and two men; mean age  SD, 49.0  11.0 years) with active relapsing remitting MS (Expanded Disability Status Scale range, 1.5– 6.5; mean Expanded Dis- ability Status Scale  SD, 2.9  1.6) who were to receive IFN- therapy. Patients had not previously received IFN- and were clinically stable for the preceding 4 wk. Peripheral blood samples were obtained just before treatment and at 1, 2, 4, 8, 24, 48, 120, and 168 h after a 30-g dose of i.m. IFN--1a (Avonex; Biogen, Cambridge, MA). Patients were administered 30 g of IFN--1a i.m. weekly, and additional samples were obtained before the 3 and 6 mo doses. Cell analysis A complete blood count and five-point differential were obtained from a clinical laboratory on Coulter STKS and Max M instruments (Beckman Coulter Clinical Diagnostics, Brea, CA). RNA isolation PBMC were rapidly isolated using gradient separation on cell preparation tubes (BD Biosciences, Mountain View, CA). Monocytes were depleted *Jacobs Neurological Institute, Buffalo General Hospital, Buffalo, NY 14203; De- partments of † Pharmaceutical Sciences and ‡ Pathology, State University of New York, Buffalo, NY 14260; and § Department of Statistics, The Cooper Institute, Golden, CO 80401 Received for publication March 25, 2003. Accepted for publication June 26, 2003. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by Grant RG3258A2 from the National Multiple Sclerosis Society. R.S. received scholarship support from CAPES, Ministry of Education, Brazil. 2 Address correspondence and reprint requests to Dr. Murali Ramanathan, Depart- ment of Pharmaceutical Sciences, 543 Cooke, Buffalo, NY 14260. E-mail address: murali@acsu.buffalo.edu 3 Abbreviations used in this paper: MS, multiple sclerosis; AUEC, area under the effect curve; GBP, guanylate binding protein; IFNAR, IFN-/ receptor; Jak, Janus kinase; Mx-2, myxovirus (influenza) resistance protein 2; Tyk2, tyrosine kinase 2. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00