Traditionally, CD4 T cells, including Th1 and Th17 cells are considered the most significant mediators in the pathogenesis of MS and its key animal model, EAE (experimental allergic encephalomyelitis) [23]. While subsets of CD 4 T cells are common targets in therapeutic efforts directed at MS, recent pathology reports also indicate an important role for CD8 T cells. CD8 T cells are the most numerous lymphocyte subpopulations in MS lesions and in normal appearing MS tissue, regardless of MS subtype or stage of lesion formation [15,24]. CD8 T-cells were also observed in close proximity to damaged neurons in axons both in MS and our model [15,25]. Despite these observations, the role of CD8 T-cells remains controversial, as some studies suggest that CD8 T-cells are mainly regulators and suppressors of the inflammatory activity. [15,24] Therefore, the contribution of CD8 T cells in MS pathogenesis in the form of axonal disruption and new lesion formation needs to be further defined. Our current study had two main goals: (1) to establish whether the pathogenic CD8 T-cells in our model are classic viral epitope specific cytotoxic lymphocytes or other CD8+ immune cell types, and (2) to investigate the potential role of CD4 T cells in the process of T1BH formation. In our experiments, we utilized transgenic mice with no adaptive immune system (RAG-12/2 mice). We adoptively transferred CD8+ or CD4+ cells from donor C57BL/6 mice into RAG-12/2 mice [25]. These experiments were designed to demonstrate if the immune system’s capability to generate T1BH can be restored by the above cell transfers. In a separate experiment, we utilized a peptide depletion technology to selectively eliminate CD8 T-cells recognizing the immunodomi- nant peptide in our model [26]. We know from previous experiments in this model that over 70% of brain infiltrating CD8 T-cells recognize a specific viral capsid peptide, VP2121–130 [26,27,28]. By intravenously injecting this peptide one day prior to disease induction with TMEV, we can effectively eliminate CD8 T-cells that recognize this peptide [26,27]. Our experiments demonstrate a surprising contrast in the contribution by CD8 vs. CD4 T-cells in T1BH formation, and confirmed that T1BH formation is driven mainly by epitope specific CD8 T-cells in our model. Materials and Methods Mice C57BL/6J and RAG-1 deficient mice on C57BL/6 background were purchased from the Jackson Laboratory in Bar Harbor, ME. The number of mice used is as detailed below. The experiments were approved by the local Institutional Animal Care and Use Committee at the University of Cincinnati (06-10-09-01) and at Mayo Clinic, Rochester, Minnesota (A29509). TMEV infection using the Daniels strain was induced via intracerebral inoculation of 200,000 PFU using standard methods described earlier [29,30,31]. Peptide depletion experiment In the experiments determining whether VP2 epitope specific CD8 T-cells contribute to the process of T1BH formation, we used epitope depletion by intravenous administration of VP2121–130 peptide one day prior to TMEV infection, as described in details earlier [27]. In this study, 8 TMEV infected mice underwent VP2 depletion, and 8 TMEV infected mice received irrelevant E7 peptide injection as negative controls. The E7 peptide is derived from the human papilloma virus, which has no relevance to TMEV infection [27]. CD4 vs CD8 T-Cells in T1 Black Hole Formation Isolation of Brain Lymphocytes As published by our team earlier [12], whole brains were strained through a nylon mesh 100-mm filter into RPMI (MediaTech Inc., Herndon, VA), and 700 mg of collagenase type 4 (Worthington, Lakewood, NJ) was added to each 5-ml tissue suspension. Suspensions were incubated in a water bath at 42uC for 45 minutes. Each 5-ml suspension was then added to 50 ml high speed centrifuge tubes (Nalge Nunc International, Rochester, NY) containing a solution of 1 ml of 106 PBS, 9 ml of Percoll (Sigma- Aldrich GmbH, Steinheim, Germany), and 35 ml of RPMI. Cell suspensions were then spun at 10,000 rpm (Sorvall SS-34 rotor) for 30 minutes (6 g max = 11962.6). The lymphocyte layer present in the bottom 5 ml of solution was collected. This lymphocyte layer was then suspended into 50 ml conical tubes (Becton Dickinson, Franklin Lakes, NJ) and diluted with RPMI to a total volume of 50 ml. Cell suspensions were then spun at 3006g for 10 minutes on a Sorvall Legend RT tabletop centrifuge. Media was aspirated off, and cell pellets were resuspended in RPMI media. FACS analysis Isolated brain lymphocytes were resuspended in FACS buffer (16 PBS, 1% FCS and 0.025% sodium azide) and incubated with Db:VP2121–130 tetramer or Db:E7 tetramer for 40 minutes followed by a 20 minute incubation with anti-CD4 and anti- CD8. Cells were then rinsed twice with FACS buffer and fixed in 1% PFA in 16 PBS. Samples were run on a BD LSR II flow cytometer (BD Biosciences, San Jose CA) and analyzed using FACS Diva software (BD Biosciences, San Jose CA). Studies of viral load We performed plaque assays to determine viral load using established methods [32,33]. Briefly, 7-day TMEV infected brains were collected in Dulbecco’s medium and frozen at 280uC. We studied 3 brains per group. The brains were then thawed, homogenized, and sonicated using two 90 second pulses. This solution was then centrifuged at 2000 rpm for 10 minutes and the supernatant was removed. The plaque assays were performed using 10-fold dilutions of brain homogenate. The serial dilutions aliquots were inoculated onto susceptible L2 cell monolayers. Each dilution is plated in duplicate to enhance accuracy. L2 cells were plated at a density of 16105/well in 12-well plates the day before. The cells were incubated at 37uC for 1 h before the addition of 1 ml of DMEM containing 2% FBS and 0.5% low-melting-point agarose. When the plates are incubated, the original infected cells release viral progeny. Viral spread is restricted to neighboring cells by the gel. Consequently, each infectious particle produces a circular zone of infected cells called a plaque. Eventually the plaque becomes large enough to be visible to the naked eye. After 72 h at 37uC, the cells were fixed and stained with cresyl violet to enhance the contrast between the living cells and the plaques. Viral titers were calculated in plaque-forming units (PFU) per milliliter. To determine the virus titer, the plaques are counted at the lowest dilution that forms plaques in the plate wells. To calculate the titer of virus the plagues from the lowest dilution to form plaques were counted. The duplicates for each dilution were counted and averaged. The average was then multiplied by 50 and the dilution factor that was counted. The resulting number is the PFU per well. Adoptive transfer experiments To determine the role of CD8 and CD4 T cells in general, we utilized mice that lack an adaptive immune system, and as such, PLoS ONE | www.plosone.org 2 February 2012 | Volume 7 | Issue 2 | e31459