In-Vitro monitoring for indirect presentation of donor-derived antigens by dendritic cells

Indirect presentation is considered to be the major contributor for allorecognition post solid organ transplantation. Yet, currently there are no reliable means to predict or monitor the level of alloresponsiveness following stimulation via the indirect pathway. While about 10% of peripheral blood T cells can recognize allo-MHC molecules via the direct pathway, the frequency of circulating T cells that can recognize allopeptides via indirect presentation is estimated to be about 1.00-fold lower. Previous attempts to evaluate indirect allorecognition in-vitro used peripheral blood B cells or monocytes as APCs, and synthetic peptides corresponding to donor incompatible antigens. This approach neglected the relative inability of B cells/monocytes to stimulate primary T cell responses. Furthermore, it relied upon the investigator's bias as to what may be considered an allo-peptide. In an attempt to construct a more physiologic method, that will not require the synthesis of donor-specific peptides and thus minimize costs, we chose to use dendritic cells (DCs) as APCs. Two advantages are provided by this approach: 1] DCs are able to stimulate naive T cells (that constitute the majority of circulating T cells), and 2] DCs use their own processing mechanisms to digest antigens, therefore, generating more physiologic allopeptides. Thus, recipient-derived DCs were generated from peripheral blood CD14+ cells cultured in GM-CSF and IL-4 containing media. Donor specific peptides were generated via three separate routes. Donor lymphnode cells, obtained at the time of organ harvesting, were induced to undergo apoptosis or necrosis; alternatively cellular extracts were generated. Each of these preparations were "fed" to recipient DCs. Kinetic studies were performed to determine the best combinations of DCs, alloantigens, and autologous T cells. Hence, we have analyzed the maturation status of DCs at the time of pulsing with alloantigens; the length of incubation with alloantigens; and the optimal parameters for co-culture with autologous T cells. Optimal ratio of DCs to alloantigens that yield maximal antigen presentation was determined in serial dilutions. The ability of DCs pulsed with alloantigens to stimulate autologous T cell proliferation was recorded and compared with the stimulation against autologous and superantigens. Our results indicate that the source of alloantigens (apoptotic; extracts; necrotic) has a major impact on the ability of DCs to initiate donor-specific T cell responses. The time frame in which the alloantigen is introduced to the DC prior to induction of cell maturation is also crucial. We propose that our in-vitro DC system provides a more physiologic strategy to predict donor-specific alloresponsiveness and thus may be useful in post transplant monitoring.