30 The data reported here support a different ICG-001 supplier model in which the CD8+ T cells respond to an AAV2 vector-encoded transgene expressed in hepatocytes and are primed without CD4+ T cell help by direct presentation of antigen. The absence of cross-priming is consistent with several other features of the AAV vectors. First, cell death and the subsequent phagocytosis of apoptotic bodies is a route by which cellular antigen enters the cross-presentation

pathway,34 but these vectors are noncytopathic so this pathway of antigen presentation would not be favored. Second, whereas transduction of liver cells with adenovirus resulted in robust synthesis of type 1 IFN, this type of response was not elicited by AAV vectors.35 The type 1 IFNs play diverse roles in induction of specific immunity, and one of these roles is the promotion of cross-presentation.36 The evidence in favor of a direct-primed response raises the question of how the T cells could actually be engaged. Although hepatocytes are separated from circulating T cells by an endothelial barrier, this endothelium is fenestrated and direct contact between T cells and underlying hepatocytes can occur.12 In our model, the hepatocytes

are the only cells transduced with the AAV vector. Thus, both the ultrastructural evidence and the expression pattern of AAV favor the model that hepatocytes are directly priming the CD8+ T cells. Our Alpelisib in vivo data also support the interpretation that AAV2-ova did not induce a CD4+ T cell response, and that, furthermore, the CD8+ T cell MCE response was not modified by the absence of CD4+ T cell help. In similar studies using D0.11.10

T cells, the lack of a readily detectable immune response was attributed either to the induction of T cell anergy16 or to the differentiation of the T cells into classic Tr3 cells expressing CD25 and the transcription factor forkhead box P3 (FoxP3).37 However, even if such cells were generated, a local immune response occurred in their presence. The CD8+ T cell response to AAV was associated with elevated expression of the coinhibitory molecule PD-1. The PD-1 molecule is associated with a senescent phenotype, characteristic of inactive T cells found in the liver during chronic infections, such as with lymphocytic choriomeningitis virus and HCV.38, 39 The regulation of PD-1 expression on CD8+ T cells is not fully understood, but our data contribute to this question by showing that the antigen-specific induction of PD-1 on CD8+ T cells in the liver was not different in mice lacking MHC class II; therefore, we do not see an essential role either for CD4+ T helper cells, or for CD4+ T regulatory cells, in the induction of PD-1.