Contrasting Roles for CD4 vs. CD8 T-Cells in a Murine Model of Virally Induced ‘‘T1 Black Hole’’ Formation Istvan Pirko1*, Yi Chen 2 , Anne K. Lohrey2, Jeremiah McDole3, Jeffrey D. Gamez1, Kathleen S. Allen 4 , Kevin D. Pavelko4, Diana M. Lindquist5, R. Scott Dunn5, Slobodan I. Macura6, Aaron J. Johnson4 1 Department of Neurology, Mayo Clinic, Rochester, Minnesota, United States of America, 2 Department of Neurology, University of Cincinnati, Cincinnati, Ohio, United States of America, 3 Department of Pathology and Immunology, Washington University in St. Louis, St. Louis, Missouri, United States of America, 4 Department of Immunology, Mayo Clinic, Rochester, Minnesota, United States of America, 5 Imaging Research Center, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio, United States of America, 6 Department of Biochemistry, NMR Core Facility, Mayo Clinic, Rochester, Minnesota, United States of America Abstract MRI is sensitive to tissue pathology in multiple sclerosis (MS); however, most lesional MRI findings have limited correlation with disability. Chronic T1 hypointense lesions or ‘‘T1 black holes’’ (T1BH), observed in a subset of MS patients and thought to represent axonal damage, show moderate to strong correlation with disability. The pathogenesis of T1BH remains unclear. We previously reported the first and as of yet only model of T1BH formation in the Theiler’s murine encephalitis virus induced model of acute CNS neuroinflammation induced injury, where CD8 T-cells are critical mediators of axonal damage and related T1BH formation. The purpose of this study was to further analyze the role of CD8 and CD4 T-cells through adoptive transfer experiments and to determine if the relevant CD8 T-cells are classic epitope specific lymphocytes or different subsets. C57BL/6 mice were used as donors and RAG-1 deficient mice as hosts in our adoptive transfer experiments. In vivo 3-dimensional MRI images were acquired using a 7 Tesla small animal MRI system. For image analysis, we used semi-automated methods in Analyze 9.1; transfer efficiency was monitored using FACS of brain infiltrating lymphocytes. Using a peptide depletion method, we demonstrated that the majority of CD8 T-cells are classic epitope specific cytotoxic cells. CD8 T-cell transfer successfully restored the immune system’s capability to mediate T1BH formation in animals that lack adaptive immune system, whereas CD4 T-cell transfer results in an attenuated phenotype with significantly less T1BH formation. These findings demonstrate contrasting roles for these cell types, with additional evidence for a direct pathogenic role of CD8 T-cells in our model of T1 black hole formation. Citation: Pirko I, Chen Y, Lohrey AK, McDole J, Gamez JD, et al. (2012) Contrasting Roles for CD4 vs. CD8 T-Cells in a Murine Model of Virally Induced ‘‘T1 Black Hole’’ Formation. PLoS ONE 7(2): e31459. doi:10.1371/journal.pone.0031459 Editor: Robyn Klein, Washington University, United States of America Received September 2, 2011; Accepted January 8, 2012; Published February 13, 2012 Copyright: 2012 Pirko et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: National Institutes of Health, National Institute of Neurological Disorders and Stroke R01 NS058698. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pirko@mayo.edu Introduction Multiple sclerosis is the leading cause of disability among young adults in the western world [1]. MRI findings in MS represent important components of the diagnostic criteria and are extensively used in clinical disease monitoring [2]. In general, T2 hyperintense lesion-based MRI features in MS ,including lesion load or number of lesions, show only a limited correlation with neurological outcome and future disability risk; however, the presence of chronic lesional T1 hypointensity or T1 black holes (T1BH) is associated with a more disabling course [3,4]. T1BH is thought to represent axonal damage [5,6]. The exact pathomech- anism leading to the formation of T1BH remains poorly understood due to the lack of appropriate experimental model systems and inability to mechanistically address this issue in clinical research. TMEV infection in mice is an accepted model of multiple sclerosis [7,8]. In this model, mice of susceptible genetic background develop a biphasic disease; the late or chronic phase is characterized by chronic-progressive demyelination. In contrast to the most commonly used experimental allergic encephalomy- elitis (EAE) models, the initial target of the immune response in the TMEV models is the viral infection itself; however, in later stages of the disease, the immune response also includes myelin self- epitopes via epitope spread [9]. C57BL/6 mice, infection with Theiler’s Murine Encephalomyelitis Virus (TMEV) results in the MRI phenomenon of T1BH formation [10]. It is important to note that C57BL/6 mice represent a resistant strain, meaning that chronic-progressive demyelination doesn’t develop in this model. The observed T1 black holes appear in the context of acute neuroinflammation in the early stage of viral infection; however, axonal damage, the most specific and pathognomonic process that characterizes T1 black holes in MS, is also observed in these areas in our model [10,11]. Similarly to the original observations in MS, tissue studies in our model demonstrate extensive axonal and neuronal damage in these areas [12]. As demonstrated by our team earlier, CD8 T- cells are the main contributors in the pathogenesis of T1 hypointense lesions in this model [10]. CD8 T- cells have also been associated with axonal injury in the TMEV model [13,14,15,16,17], contribute to the development of autoimmune responses in this model [18] and also play a critical role in inducing severe blood-brain barrier permeability under specific conditions [19,20,21,22]. PLoS ONE | www.plosone.org 1 February 2012 | Volume 7 | Issue 2 | e31459