CD4 vs CD8 T-Cells in T1 Black Hole Formation Figure 2. MRI T1BH volumetry results in adoptive transfer experiments using RAG 1 2/2 recipient mice. The bar chart shows the mean T1BH volumes in CD8 and CD4 adoptive transfer experiments. Controls represent sham transfer (PBS injection) following irradiation and infection. CD8 T cell transfer resulted in 3-fold increase of T1BH formation, while CD4 transfer resulted in a 2-fold decrease. Error bars represent SD. doi:10.1371/journal.pone.0031459.g002 viral load between CD8 and CD4 adoptive transferred RAG-1 deficient mice on day 7 of TMEV infection (Figure 4). Epitope specific CD8 T-cell depletion experiments Depleting the CD8 T cells that recognize the VP2 immunodo- minant peptide (Figure 5) resulted in a significant 2-fold reduction of T1BH formation compared to sham treatment with E7 peptide (p = 0.017). This suggests that the vast majority of CD8 T-cells contributing to T1BH formation are classic epitope specific cytotoxic lymphocytes. Discussion The above results reinforce that CD8 T cells play the predominant role in the pathogenesis of T1BH formation, and clarifies that the leading contribution in this process is by epitope specific cytotoxic CD8 T cells. Since a small degree of T1BH formation is observed even in control animals (infected RAG-1 2/2 mice receiving sham transfer) it is likely that either direct cytopathic effects or cells of the innate immune system, likely neutrophils, also contribute to this process. Meanwhile, when CD4 cells are transferred to animals with deficient adaptive immunity, they appear to play a preventive role in T1BH formation and significantly reduce the observed baseline T1BH formation. T1BH-s are thought to represent axonal and/or neuronal damage in MS [6,38,39]. In the TMEV model, viral replication mostly takes place in neurons in the stage when our experiments were conducted [40,41], and at this stage and in this strain, demyelination is not observed, unless stimulated by the intrave- nous injection of the immunodominant VP2 peptide, as demonstrated by our team earlier [19,42]. We therefore hypothesize that cytotoxic CD8 T cells targeting the immunodo- minant VP2 epitope are targeting neurons and axons, consistent with our recent published studies [12]. By transferring CD8 T-cells into CD8 deficient infected hosts, we were able to restore T1BH formation. In addition epitope specific elimination of VP2 specific CD8 T-cells resulted in reduced T1BH formation. Altogether, these observations very strongly suggest that T1BH formation in this model is related to neuronal and axonal injury caused directly or indirectly by CD8 T-cells. Given that CD8 T-cells are the most numerous lymphocytes in MS lesions regardless of stage of lesion formation, and given that CD8 T-cells have been observed strongly apposed to neurons and axons in MS tissue, we propose that a similar CD8 T cell driven mechanism as observed in our model may be responsible for T1BH formation in MS [43,44]. Another key analogy between our model and MS-related T1BH PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e31459