Establishment of Trichimerism in the Dog Leukocyte Antigen (DLA)-Identical Canine Hematopoietic Transplant Model.
Authors: Graves Scott S. , Hogan William , Georges George E. , Kuhr Christian , Diaconescu Razvan , Harkey Michael , Zellmer Eustacia , Storb Rainer 
Published: October 2019

Although hematopoietic cell transplantation is generally accomplished utilizing a single donor and recipient pair, multiple donors have been used to mediate engraftment, particularly in the case of low donor cell counts as in umbilical cord blood transplantation. The general engraftment outcome of HLA nonidentical cord blood transplantation is the predominance of one of the units over the other. Here we pose the question whether in the canine hematopoietic cell transplantation model, can we establish multiple donor chimerism using two DLA-identical donors and a single recipient. We identified 8 triplets of DLA-identical littermates by matching highly polymorphic microsatellite markers within DLA class I and class II regions and confirmed by DLA-DRB1 gene sequencing. The marrow recipients received 2 Gy total body irradiation followed by intravenous infusions of marrow cells from both donors 1 and 2. The median number of donor cells injected was 4.1 (range = 2.0–7.0) X 108 cells/kg. Post grafting immunosuppression consisted of cyclosporine (CSP) and mycophenolate mofetil (MMF) given for 35 and 28 days, respectively. The median time to hematological recovery (blood counts equivalent to pre-HCT levels) was 38 (range = 30–42) days. The degrees of chimerism were determined using variable number tandem repeat-polymerase chain reaction (VNTR-PCR) methods. As shown in the Table, sustained trichimerism occurred in 4 out of 8 dogs with engraftment for a period grater than 26 weeks. For G631, both donor grafts were rejected shortly following discontinuation of CSP and MMF. Dog G513 developed graft versus host disease (GvHD) which was successfully treated with a short course of CSP. Five dogs received kidney allografts from one of the respective HCT donors 6 months after HCT to assess donor specific immune tolerance.

Chimerism Analysis of Dogs Receiving Marrow from Two Donors Duration of Engraftment in Weeks (% Chimerism) Recipient Donor 1 Donor 2 Recipient GVHD Accept Donor 1 Kidney Graft (wks) ND = not determined; [R] = rejection G158 >43 (5%) 37 (0%) [R] >43 (95%) No Yes (20) G193 >44 (72%) 16 (0%) [R] >44 (28%) No Yes (10) G362 >47 (28%) >47 (55%) >47 (17%) No Yes (16) G513 >47 (53%) >47 (22%) >47 (25%) Yes Yes (16) G551 >32 (35%) >32 (20%) >32 (45%) No ND G631 8 (10%) [R] 8 (12%) [R] 8 (>88%) No ND G643 >30 (28%) >30 (40%) >30 (23%) No Yes (4) G664 >26 (50%) 6 (2%) [R?] >26 (48%) No ND

Kidney allografts were found to be essentially free of inflammation when assessed histologically on biopsy. Dog G643, was tested for immune function by a mixed leukocyte reaction and found to be competent against DLA nonidentical stimulator cells but nonresponsive against either of the donor stimulator cells. In summary, following nonmyeloablative conditioning, simultaneous administration of marrow stem cells from two DLA-identical littermates can result in trichimerism. Furthermore, immunological tolerance to multiple hematopoietic cell donors can include a solid organ graft from one of the marrow donors.