they lack CD 4 and CD 8 T cells. These RAG-12/2 mice otherwise have the same genetic background as C57BL/6 mice. We harvested CD8 and CD 4 T cells from spleens of C57BL/6 mice, and transferred these into irradiated RAG-1 2/2 recipient mice as described earlier [25]. Briefly, spleens of GFP+ mice (C57BL/6-Tg(UBC-GFP)30Scha/J mice, # 004353, The Jackson Laboratory, Bar Harbor, ME) were removed and strained through a nylon mesh 100-mm filter. CD8+ and CD4+ cells were purified from the resulting lymphocyte population using MACS LS cell purification columns (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s protocol, resulting in 95% purity as determined by flow cytometric analysis (data not shown). C57BL/6 mice were irradiated with 400 rads of c-radiation then received 106 CD8+ or CD4+ positively sorted spleen cells via tail-vein injection. Positive sorting does not affect functions of CD4 or CD8 T cells. MRI acquisition and analysis In each case, the formation of T1BH was monitored by volumetric MRI: a volume acquisition T1BH-s were defined as focal cerebral hypointensity as demonstrated by our team earlier [10]. T1 weighted spin echo sequence was used, with 200 mm isometric resolution using a Bruker Biospec 7 Tesla horizontal bore small animal MRI system, on day 7 following disease induction with TMEV, as described earlier [10,34]. We utilize standard spin echo sequences to conform with the original study demonstrating T1BH in MS [3]. For volumetric analysis, the 3D ROI tool was used in Analyze 9.1 (Mayo Clinic, Biomedical Imaging Resource) [35,36]. Since the analysis is semi-automated, we needed to standardize the analysis methods and use 2 investigators to analyze each dataset at least twice. Their intra-and inter-rater reliability was superior, as reported earlier [37]. The outcome analyzed in our studies was the total volume of T1BH per animal. Results Adoptive cell transfer experiments T1BH-s were observed in all TMEV infected experimental groups (Figure 1). The cell transfer experiments revealed that CD8 CD4 vs CD8 T-Cells in T1 Black Hole Formation T cell recipient mice developed an average T1BH volume over 3 fold higher compared to sham transfer controls (p = 0.0002) (Figure 2). The detected total T1BH volumes per animal were comparable to our original findings in C57B6/J mice [10]. In sharp contrast, our CD4 transfer experiments demonstrated a surprising finding: CD4 T cells actively inhibited T1BH formation in our model. An almost 4-fold reduction was detected (p = 0.0006) in T1BH volume compared to sham treated irradiated infected controls. The observed low-level T1BH formation among recipients of sham transfer is thought to be mediated either by innate immune cells, most likely neutrophils, microglia or macrophages; or via direct viral cytopathic effects. These alternative mechanisms are also the subject of ongoing investiga- tions in our laboratory. We did not perform CD4 or CD8 transfers to uninfected hosts, as homing of immune cells in that condition is not expected. FACS analysis of brain infiltrating lymphocytes Following the MRI acquisition experiments, the animals were sacrificed on the same day and their brains were extracted for flow cytometric analysis of brain infiltrating lymphocyte subsets. (Figure 3.) We were specifically interested in transfer efficacy, and in the proportion of CD8 T-cells recognizing VP2 epitopes. In brains with sham transfer, 1.6% of cells were CD8+ positive vs. in brains with CD8 splenocyte transfer, were 12.6% positivity was demonstrated (Figure 2A). Using tetramer technology, we demonstrated that a total of 2.6% of CD8+ cells were also VP2 121–130 specific. CD4 transfer was also monitored by FACS (Figure 3B). 14.4% of brain infiltrating immune cells were CD4+ and CD82 following CD4+ splenocyte transfer; whereas only 0.2% were CD4+ CD82 after sham transfer. Viral load analysis To determine whether differences in viral load may explain the observed differences in T1BH volume between our CD8 and CD4 adoptive transfer groups, we performed plaque assays. These assays revealed that there was no significant difference (p = 0.62) in Figure 1. Example for T1 black hole formation in 7-day TMEV infected C57BL/6J mice. Top row: axial images, bottom row: coronal images extracted from a representative mouse. To provide guidance, some (but not all) areas containing T1 black holes are indicated with red frames. These are located in diverse areas of the brain, but mostly in periventricular areas, in the thalamus, hippocampus, white matter, corpus callosum and even in the cortex. doi:10.1371/journal.pone.0031459.g001 PLoS ONE | www.plosone.org 3 February 2012 | Volume 7 | Issue 2 | e31459