Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model

Abstract

Bone marrow transplantation (BMT) has considerable potential for the treatment of malignancies, hemoglobinopathies, and autoimmune diseases, as well as the induction of transplantation allograft tolerance. Toxicities associated with standard preparative regimens for bone marrow transplantation, however, make this approach unacceptable for all but the most severe of these clinical situations. Here, we demonstrate that stable mixed hematopoietic cell chimerism and donor-specific tolerance can be established in miniature swine, using a relatively mild, non-myeloablative preparative regimen. We conditioned recipient swine with whole-body and thymic irradiation, and we depleted their T-cells by CD3 immunotoxin-treatment. Infusion of either bone marrow cells or cytokine-mobilized peripheral blood stem cells from leukocyte antigen-matched animals resulted in stable mixed chimerism, as detected by flow cytometry in the peripheral blood, thymus, and bone marrow, without any clinical evidence of graft-versus-host disease (GvHD). Long-term acceptance of donor skin and consistent rejection of third-party skin indicated that the recipients had developed donor-specific tolerance.

Figure 1

Schematic representation of nonmyeloablative preparative regimen used to establish hematopoietic mixed chimerism in miniature swine.

Figure 2

PB T-cell depletion. (a) Graph of the absolute number of T cells remaining in PB each day during the nonmyeloablative preparative regimen. *Animal 13475 received the same preparative regimen, except without T-cell depletion. (b) Chart showing the actual number of T cells detected in the PB immediately before donor cell infusion on day 0.

Figure 3

WBC and PLT counts during engraftment after nonmyeloablative preparative regimen. (a) WBC and (b) PLT.

Figure 4

Percent of donor PAA⁺ cells detected in the PB after (a) BMT and (b) PBSCT in miniature swine treated with the nonmyeloablative preparative regimen.

Figure 5

Percent of donor PAA⁺ thymocytes detected in thymic biopsy samples taken from miniature swine after nonmyeloablative preparative regimen and hematopoietic cell transplantation.

Figure 6

Thymic regeneration and mixed chimerism establishment after nonmyeloablative preparative regimen. (a) FACS dot plot representation of different size (FSC) fractions of (1) immature (CD1⁺); (2) mature (CD3⁺); and (3) donor-type (PAA⁺) thymocytes before and 72 days after PBSCT in animal 13235. (b) Two-color FACS analysis of thymocytes from animal 13235 on day 72 showing donor PAA+ thymocytes present in both the immature (CD1⁺) and mature (CD3⁺) fractions.

Figure 7

Day 41 after grafting of self skin (a), PBSC donor skin (b), and 2 third-party SLA-matched skin grafts (c and d) placed on animal 13379 82 days after PBSCT. Black rectangular areas are the sites where skin biopsies were taken.