14 The relative contributions of direct vs indirect donor alloantigen presentation to recipients' T cells on the development of chronic rejection (CR) is as yet ambiguous. It has been argued that while direct allorecognition may play an important role in initiating acute cellular rejection (ACR); CR on the contrary, is predominantly an outcome of donor allopeptides being presented in the context of recipient MHC. Given that CR is one of the two most common causes for late organ allograft failure, it is somewhat imperative that the prevailing conundrum concerning its pathogenesis be resolved. To address unequivocally this issue, we transplanted aortas (AO) from C57B1/6 Aβb (H2b, MHC class II-1-)→naive C3H (H2k) recipients (Group V; n=6). It was reasoned that due to lack of MHC class II expression in the donor tissue, the antigen (Ag) presentation would be primarily indirect allowing us to study its role in the development of CR. Additionally, the role of direct Ag presentation in the pathogenesis of this lesion was also studied by transplanting AO from naive C3H→C57Bl/6 Aβb recipients (Group VI; n=6). Controls (n=6/group) included Tx of AO from C3H → C3H (Group 1), C57Bl/6 Aβb→C57Bl/6 Aβb (Group II), C3H→C57Bl/6 WT (Group III) and C57Bl/6 WT→C3H (Group IV). As published previously, these aortic allografts undergo self-resolving ACR with spontaneous acceptance, eventually developing changes pathognomonic of CR by d30 post-Tx. At the time of sacrifice (d30-40 post-Tx), the grafts were harvested for immunohistochemical analysis and the sera and splenocytes were also recovered for the determination of α-donor antibody and proliferative responses by complement-dependent microcytotoxicity and MLR assays respectively. Unlike that from Groups I and II, AO harvested from animals in Groups III and IV had marked circumferential intimal thickening due largely to proliferation of α-smooth muscle actin✦ (α-smA✦) cells. Additionally, both the α-donor and α-third party proliferative responses and the titers of cytotoxic antibodies remained comparable in animals in Groups III and IV. On the contrary, AO obtained from the majority of Group V animals exhibited marked reduction in intimal thickening which was largely eccentric and patchy with preservation of elastic membranes; this finding was associated with unmitigated α-donor proliferative and humoral responses. Interestingly, despite the lack of α-donor proliferative and cytotoxic antibody responses, circumferential intimal thickening of moderate severity was witnessed in the majority of aortic allografts harvested from Group VI recipients. Taken together, these data suggest that despite preservation of α-donor immune responses, presentation of Ag by indirect pathway alone does not precipitate the fulminant development of CR indicating that perhaps both pathways of Ag presentation are required for optimal immune activation. More importantly, the presence of moderate CR in recipients which lack α-donor cellular and humoral responses underscore the importance of numerous immune and non-immune pathways which operate in concert towards the development of this lesion.