1C,D). To confirm the expression of

the antigen, mice that were given the AAV2-ova vector were infused with OT-1 T cells, a CD8+ T cell population specific for the SIINFEKL peptide (ova257-264). These T cells divided and down-regulated CD62L (Fig. 1B, right panels), verifying that the antigen was expressed. We conclude that AAV-2-ova vector stimulated CD8+ but not CD4+ T cell responses. Two different T cell receptor transgenic CD4+ T cells failed to respond to AAV-OVA (Fig. 1). To test whether endogenous R428 research buy CD4+ T cells were helping the CD8+ cells, MHC class II–deficient mice, which lack CD4+ T cells, were given AAV2-ova vector and then OT-1 T cells. Figure 2A shows the response of OT-1 T cells, SP600125 ic50 measured using CFSE, at day 3 (D3), day 5 (D5), and week 8 (W8) after adoptive transfer (shaded profiles). In the liver, OT-1 cells divided as early as day 3, and almost all of the cells were CFSE-low by day 5; these cells were also present at week 8. In control mice given the AAV2-gfp vector (nonshaded profiles), there was very little OT-1 T cell division at days 3 and 5, although the cells were subject to some loss of CFSE staining by week 8. Strikingly, there was no

difference in the division of OT-1 T cells between normal B6 mice and MHC class II–deficient mice. In the spleen and the PLN, there was essentially no cell division in any of the mice at day 3, but divided cells appeared in these tissues on day 5, as previously reported14;

again, there was no difference between normal and MHC class II–deficient mice. Figure 2B shows the outcome of these experiments in terms of the numbers of Flavopiridol (Alvocidib) OT-1 T cells in the liver (left panel of Fig. 2B), spleen (center), and PLN of normal B6 versus MHC class II–deficient mice at day 3, day 5, and week 8 after adoptive transfer. In liver, there was a statistically significant expansion of OT-1 T cells at all three time points, but no significant difference between B6 and MHC class II–deficient mice. In spleen, we observed significant clonal expansion only on day 5, but not later. Again, there was no difference between normal B6 and MHC class II–deficient mice. In PLN, we observed no clear effects on overall OT-1 T cell numbers on days 3 and 5, although there was a significant increase in the numbers of OT-1 T cells in MHC class II–deficient mice on day 5, followed by a significant loss of OT-1 T cells in the AAV2-ova–transduced mice at week 8. The overall conclusion is that MHC class II–restricted helper T cells did not influence the response of OT-1 CD8+ T cells to the AAV2-ova vector.