M. Sykes

Allograft tolerance, meaning a state in which the immune system regards a donor graft as "self", is the "holy grail" of research in transplantation. Tolerance would avoid the need for chronic immunosuppressive therapy, with all of its toxicities, that is currently required to prevent graft rejection. Tolerance would also prevent chronic rejection, the major cause of late graft loss, which remains a major problem despite recent improvements in immunosuppressive drugs. Mixed hematopoietic chimerism, meaning co-existence of allogeneic and recipient hematopoietic stem cells and their progeny, provides a powerful means of achieving transplantation tolerance. Mixed allogeneic chimerism can be induced in mice receiving allogeneic bone marrow transplantation (BMT) after relatively mild, non-myeloablative host conditioning with depleting anti-T cell monoclonal antibodies (mAbs), low dose (3 Gy) total body irradiation (TBI), and local thymic irradiation (TI) (7 Gy). These mice are specifically tolerant to donor and host antigens. The amount of conditioning used to achieve mixed chimerism can be reduced by using high stem cell doses in place of TBI, and by replacing both recipient T cell depletion and thymic irradiation with a short course of costimulatory blockade i.e. blockers of "second signals" to T cells. Lasting chimerism and donor-specific tolerance were achieved with a regimen that requires no host pre-conditioning, by giving a high dose of fully MHC-mismatched donor marrow followed by a single injection of each of two costimulatory blockers. Engrafted allogeneic stem cells provide a life-long source of progenitor cells that seed the thymus, giving rise to antigen-presenting cells (APC) which mediate clonal deletion of donor-reactive T cells. Host antigen-presenting cells also populate the thymus, so intrathymic deletion of cells recognizing both the donor and the host occurs. In the regimens involving costimulatory blockade, peripheral mechanisms, including anergy and deletion, are involved in the tolerization of the pre-existing donor-reactive T cell repertoire.

Mixed chimerism also has a role in the treatment of hematologic malignancies. Graft-versus-host disease (GVHD) is currently the major toxic limitation to the use of this treatment. However, GVHD is also associated with beneficial graft-vs-leukemia/lymphoma (GVL) activity, because GVH-reactive donor T cells eliminate residual malignant cells in the host. In mice, non-myeloablative conditioning including in vivo T-cell depletion of the recipient followed by bone marrow transplantation that is T-depleted in vivo produces a state of mixed chimerism without inducing GVHD. Delayed donor lymphocyte infusions (DLI) given at day +35 mediate GVH responses that result in conversion to complete chimerism, and are associated with marked GVL effects, but without GVHD, which is a disease of epithelial tissues (skin, intestines, liver). DLI given to mixed chimeras induce stronger GVL effects than the same DLI given to fully allogeneic chimeras. This concept was translated into clinical protocols using anti-T-cell antibody as well as thymic irradiation for recipient and donor marrow T-cell depletion, and, most recently, using ex vivo donor CD34+ cell selection from G-CSF mobilized peripheral blood. Preliminary clinical data have confirmed the feasibility of this approach in patients with advanced, chemorefractory lymphoid malignancies. In further studies, we studied the activation, expansion and trafficking patterns of donor T cells in the mouse model. GVH-reactive CD4 and CD8 cells underwent expansion, developed effector function and converted to the memory phenotype in mixed chimeric DLI recipients without causing GVHD, whereas similar expansion was associated with GVHD in freshly conditioned mice. Inflammation in the GVHD target tissues plays a critical role in detemining whether or not activated GVH-reactive T cells remain in the lymphohematopoietic system (where they mediate GVL) or traffic to the GVHD target tissues (R. Chakraverty, C. Lin and M. Sykes, unpublished data)

In view of our clinical and laboratory results, we have begun studies of combined kidney and bone marrow transplantation in patients with renal failure due to multiple myeloma. Paradoxically, although some of the patients had only transient chimerism, renal allograft tolerance has been achieved. We have recently extended combined non-myeloablative BMT and kidney transplantation to the haploidentical setting in patients who have renal failure but do not have a malignancy, using a protocol that we showed leads to transient mixed chimerism without GVHD in the early studies in patients with hematologic malignancies. Encouraging preliminary data provide proof of principle that this approach can be used to induce donor-specific tolerance in humans.

Supported by NHLBI, NCI and the Immune Tolerance Network.

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