Injury to the Thymic Niche for DN2/DN3 Developing T Cells Impairs Reconstitution.

Despite recovery of most hematopoietic functions, prolonged defects in generating functional T lymphocytes is a common occurrence after T cell depleted bone marrow transplant. While the mechanisms of these defects have not all been elucidated, contributing factors include age, T cell depletion of the graft, therapy with radiation and cytotoxic agents, and graft versus host disease (GvHD). We have designed an in vivo functional assessment of the ability of thymic stroma to support de novo T lymphocyte development. Mice deficient for the alpha chain of the IL-7 receptor (IL7Rα−/−) support robust thymic reconstitution after transplant of limited numbers of congenic precursors. We demonstrated that this capacity for reconstitution of immunodeficient strains depends on the paucity of specific endogenous precursors (DN3) cells in the IL7Rα−/− thymus and reflects the presence of functionally normal but empty stromal niches in this immunodeficient strain. We have proceeded to demonstrate that a variety of chemotherapeutic agents as well as aging, impair the ability of IL7Rα−/− thymic stroma to support de novo T cell development. Some agents allow donor chimerism in the thymus, but not rescue of the hypocellularity (eg cyclophosphamide) while others do not affect reconstitution (eg fludarabine). Multi-agent regimens have been administered and in some instances demonstrate an additive detrimental impact on thymic reconstitution. For several agents, damage has been localized to specific stromal niches by isolating changes in lymphoid subsets and stromal keratin expression. Decreased availability of the stromal niche for DN3 progenitors is associated with a decreased frequency of DN3s and an absolute block in recipient IL7Rα−/− T cell development. In addition, RNA from treated and untreated thymic stromal cells has been used to evaluate the gene expression pattern in thymic stroma of IL7Rα−/− mice treated with cytotoxic agents. In one example to be presented we find decreased thymic reconstitution in mice treated with Busulfan with sustained changes in stromal elements. These findings are consistent with sustained damage to thymic stromal cells. Among other changes, busulfan leads to decreased expression of laminin 5 by cortical thymic epithelial cells. The integrin heterodimerα6β4 is a binding partner for laminin 5 and is expressed uniformly by DN2 thymocytes. The significance of laminin 5/α6β4 signaling during T cell development was assessed using mice with a targeted mutation in the integrin β4 signaling domain. In these experiments mutant fetal liver was used to create hematopoietic chimeras and evaluate T cell development. Our studies provide insight into the nature of damage to thymic stroma by cytotoxic regimens and an understanding of the effect of this damage on subsequent immune reconstitution.