BACKGROUND: In the direct pathway, T cells recognize intact donor major 
histocompatability complexes and allogeneic peptide on the surface of donor 
antigen presenting cells (APCs). Indirect allorecognition results from the 
recognition of processed alloantigen by self MHC complexes on self APCs. In this 
study, we wished to evaluate the relative contribution of different intragraft 
cells to the alloactivation of nave and memory T cells though the direct and the 
indirect pathway of allorecognition.
METHODS: The processing of membrane fragments from IFN-treated single donor 
endothelial cells (EC), fibroblasts or renal epithelial cells (RPTEC) was 
evaluated by DiOC labeling of each cell type and flow cytometry following 
interaction with PBMC. Direct pathway activation of nave CD45RA+ or memory 
CD45RO+ CD4+ T cells was evaluated following coculture with IFN-treated and MHC 
class II-expressing EC, fibroblasts or RPTEC. Indirect pathway activation was 
assessed using CD45RA+ or CD45RO+ CD4+ T cells cocultured with autologous 
irradiated APCs in the absence or presence of sonicates derived from IFN-treated 
allogeneic EC, fibroblasts or RPTEC. Activation of T cells was assessed by 
[3H]thymidine incorporation and by ELISpot assays.
RESULTS: We find that CD14+ APCs readily acquire membrane fragments from 
fibroblasts and RPTEC, but fail to acquire membrane fragments from intact EC. 
However, APCs process membranes from EC undergoing apoptosis.There was a notable 
direct pathway alloproliferative response of CD45RO+ CD4+ T cells to IFN-treated 
EC, but not to fibroblasts or RPTEC. Also, there was a minimal direct pathway 
response of CD45RA+ CD4+ T cells to all cell types. In contrast, we found that 
both CD45RA+ and CD45RO+ CD4+ T cells proliferated following coculture with 
autologous APCs in the presence of sonicates derived from IFN-treated EC, 
fibroblasts or RPTEC. By ELISpot, we found that these T cells stimulated via the 
indirect pathway also produced the cytokines IFN, IL-2, IL-4 and IL-5.
CONCLUSIONS: Recipient APCs may readily process membrane fragments from 
allogeneic intragraft cells, but not from EC unless they are undergoing 
apoptosis. This processing is sufficient for indirect pathway alloactivation of 
both CD45RA+ and CD45RO+ CD4+ T cells. Only graft vascular EC mediate direct 
pathway reactivation of CD4+ T cells.