Monitoring indirect presentation of alloantigens by utilizing the autologous processing machinery of dendritic cells in-vitro

Background: Judicious individualization of immunosuppression protocols requires the ability to monitor the recipients' specific immune response towards the allograft. While several indirect presentation studies have been reported, currently there are no consistently reliable means to assess the state of transplant acceptance. A potential explanation might be the lack of established kinetics for in-vitro indirect presentation assays. Methods: Dendritic cell (DCs) were used as self-specific "processing machinery" for the generation of allogeneic peptide repertoire tailored by the individual's capacity to process antigens via the indirect pathway. The kinetics of antigen processing and presentation of newly acquired antigens was then assessed to define the following: (1) Optimal time to introduce apoptotic-allogeneic cells to DCs; (2) optimal time for processing before induction of DC maturation; and (3) optimal time for stimulation of autologous T cells by the pulsed DCs. Results: Altering kinetic parameters associated with time allotted for antigen processing; antigen presentation and DCs maturation, showed substantial variations in the level of T cell activation, as was documented by mixed lymphocyte reaction (MLR)-like proliferation assays. Further experiments using allogeneic cellular extracts showed similar variations in T cell stimulation by monitoring T cell precursor frequencies and interferon (IFN)-γ secretion ELISPOT assays. Using the appropriate kinetic parameters, we have also shown difference in T-cell-subsets (CD4, CD8) precursor frequencies and cytokine secretion in response to different antigenic sources. Conclusions: The ability to construct an in-vitro, recipient-tailored, donor-specific, indirect presentation assay is dependent on essential kinetic parameters associated with antigen processing and presentation, as described in this report. Moreover, the use of this approach may circumvent the need for identifying specific immunogenic peptides for cancer-related or other vaccine-development studies.