Use of Dendritic Cells Versus Peripheral Blood Lymphocytes To Segregate Alloreactive and Regulatory T Cells during Allogeneic Transplantation.

A fundamental research objective in allogeneic transplantation is the selective depletion of alloreactive T cell populations that are responsible for GVHD while preserving mediators of anti-tumor immunity and immune reconstitution. Alloreactive T cells may be identified by their expression of activation markers, such as CD25, following stimulation by recipient antigen presenting cells. However, CD25 expression is also present on regulatory T cells, which suppress alloreactivity through the expression of inhibitory ligands such as CTLA-4 and GITR. In an effort to develop an optimal strategy to segregate these populations, we have examined the effect of conditions of allogeneic stimulation on the balance of alloreactive and regulatory T cell populations. Stimulation of lymphocytes with irradiated peripheral blood mononuclear cells results in a gradual increase in the percentage of CD4/CD25+ cells over 7 days of culture. Mean percentage of CD4/CD25 at day 3 and 6 was 3% and 11%, respectively. On day 3 of stimulation, these cells demonstrate a characteristic activated phenotype with low intensity of CD25 expression and absence of GITR and CTLA-4 expression. Following isolation by magnetic bead separation, CD25+ cells demonstrated high levels of proliferation in response to restimulation with allogeneic PBMC. In contrast, minimal proliferation was observed following stimulation of the CD25- fraction. On day 6 of culture increased intensity of CD25 expression was observed in a fraction of the stimulated population. A majority of this population now expressed GITR and CTLA-4, consistent with a regulatory phenotype. Lymphocytes stimulated with mature DC demonstrated a markedly different pattern. A marked rise in CD4+/CD25+ cells with high levels of CD25 expression was noted early in the culture period; Mean CD4+/CD25+ cells on day 3 of culture were 9%. These cells were uniformly positive for CTLA-4 and GITR. A further increase in the percentage of cells demonstrating a regulatory phenotype was noted on day 6 of culture. We have demonstrated that the balance of activated and regulatory cells is determined by the nature of the antigen presenting cell and the duration of T cell stimulation. Based on these findings, we plan to conduct a clinical trial in which patients will undergo allogeneic transplantation following the selective removal of alloreactive T cell populations.