Molecular Characterization of the Human Alloimmune Response

When T lymphocytes from two individuals are placed in a mixed lymphocytic reaction (MLR) and allowed to stimulate each other a powerful proliferative response is elicited, reflecting the degree of incompatibility at the major histocompatibiltiy loci. Such in vitro proliferations translate in vivo into graft rejection of stem cell and solid organ transplants and to graft versus host disease (GvHD) in recipients of allogeneic stem cell transplants. Defining the clonal T cell responses elicited in an MLR between two individuals is complicated by the multiplicity of T cell HLA- antigen interactions. We used flow cytometry to investigate the MLR between mismatched individuals to better characterize responder T cell proliferation and the subsets involved in the alloresponse. After observing proliferation in the setting of a completely mismatched one-way MLR and ensuring that the design for the MLR was viable, we sought to characterize clonal T cell responses in a simple system. We attempted to elicit T cell responses to autologous antigen-presenting cells (APC) transfected with a single mismatched HLA-A2 molecule. We failed to detect any alloresponding T cells using a flow cytometric system, except when dendritic cells were used. We then used an HLA-A2 negative responder pre-stimulated in an MLR with an HLA-A2 positive individual for re-challenge by the responder?s own APC transfected with an HLA-A2 plasmid. In this way, we sought to identify an expanding HLA-A2 specific T cell population which could be further characterized by T cell receptor cloning. Nevertheless, we failed to detect an alloimmune response in this T cell line stimulated with HLA-A2 transfected APCs three times over a 21- day culture period. These results suggest that either the frequency of alloreactive T cells was below the limit of detection by these methods or that the particular stimulator-responder pairs used in these experiments were non-reactive. Future work will extend the study to more HLA mismatched stimulator-responder pairings to better define the characteristics of alloreactive T cells.