Mechanisms Involved in the Establishment of Tolerance Through Costimulatory Blockade and BMT: Lack of Requirement for CD40L-Mediated Signaling for Tolerance or Deletion of Donor-reactive CD4+ Cells

We have previously shown that high levels of multiline-age mixed hematopoietic chimerism and systemic T-cell tolerance can be achieved in mice without myeloablation through the use of anti-CD40L and costimulatory blockade alone (plus CTLA4Ig) or with recipient CD8 depletion and allogeneic bone marrow transplantation. Chimeric mice permanently accept donor skin grafts (> 100 days), and rapidly reject third-party grafts. The mechanisms by which costimulatory blockade facilitates the engraftment of allogeneic hematopoietic cells have not been defined. To further understand the in vivo mechanisms by which the administration of anti-CD40L mAb facilitates the engraftment of donor bone marrow and rapidly tolerizes CD4+ T cells, we analyzed the establishment of chimerism and tolerance in CD40L -/- mice. We demonstrate here that anti-CD40L mAb treatment is required only to prevent CD40L/CD40 interactions, and that no signal to the T cell through CD40L is necessary for the induction of CD4+ tolerance. Peripheral deletion of donor-reactive CD4+ T cells occurs rapidly in CD40L -/- mice receiving bone marrow transplantation (BMT), indicating that this deletion in the presence of anti-CD40L is not due to targeting of activated CD4+ cells by the antibody. Complete CD4+ cell tolerance is observed by both skin graft acceptance and in vitro assays before deletion is complete, indicating that additional mechanisms play a role in inducing CD4+ T-cell tolerance as the result of BMT in the presence of CD40/CD40L blockade.