Stable trichimerism after marrow grafting from 2 DLA-identical canine donors and nonmyeloablative conditioning

Abstract

Although hematopoietic cell transplantation (HCT) is generally accomplished using a single donor, multiple donors have been used to enhance the speed of engraftment, particularly in the case of umbilical cord blood grafts. Here we posed the question in the canine HCT model whether stable dual-donor chimerism could be established using 2 DLA-identical donors. We identified 8 DLA-identical littermate triplets in which the marrow recipients received 2 Gy total body irradiation followed by marrow infusions from 2 donors and postgrafting immunosuppression. All 8 dogs showed initial "trichimerism," which was sustained in 5 dogs, while 2 dogs rejected one of the allografts and remained mixed chimeras, and 1 dog rejected both allografts. Immune function in one trichimeric dog, as tested by mixed leukocyte culture response and antibody response to sheep red blood cells, was found to be normal. Five dogs received kidney grafts from one of their respective marrow donors at least 6 months after HCT without immunosuppressive drugs, and grafts in 4 dogs are surviving without rejection. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow grafts from 2 DLA-identical littermates could result in sustained trichimerism, and immunologic tolerance could include a kidney graft from one of the marrow donors.

Figure 1

Percent donor chimerism for granulocytes and mononuclear cells for dog G643 showing stable long-term trichimeric engraftment and dog G664 showing initial trichimerism with subsequent rejection of one donor 4 weeks after TBI. Data points were determined by VNTR-PCR analysis.

Figure 2

Trichimerism in cells derived from hematopoietic cells from peripheral blood, bone marrow, and lymph node. (A) Examples of marrow trichimerism in 3 of the dogs given marrow transplants from 2 DLA-identical donors determined at the weeks after HCT shown. (B) Chimerism analysis for dog G643, 34 weeks after HCT, of granulocytes and mononuclear cells from peripheral blood cells; CD45⁺ (leukocytes), CD34⁺ (progenitor stem cells), and DM5⁺ (myeloid cells) from bone marrow; CD3⁺ (T-cell antigen) and CD21 + (B-cell antigen) from lymph node cells. Cells were labeled with antibodies indicated above and sorted by FACS before determining chimerism.

Figure 3

Mixed leukocyte culture reactivity of lymphocytes from a trichimeric recipient (dog# G643) and one of its DLA-identical donors (G642) and an unrelated DLA-nonidentical (G653) in response to stimulation with irradiated autologous lymphocytes and lymphocytes from the DLA-identical littermates and an unrelated dog. Blood samples for the MLR were collected from donor and recipient 7 months after HCT. Values shown are counts per minute (CPM) ± standard deviation of triplicate samples. Concanavalin A (ConA) served as the positive control.

Figure 4

Log 2 hemaglutinin titers in serum from dog G643 (diamonds) in response to sheep red blood cell (SRBC) injection. Control values (boxes) were obtained from historical data of 5 dogs.