A highly effective method to establish long-term, stable mixed hematopoietic chimerism was developed in the dog model. This involves nonmyeloablative allogeneic hematopoietic cell transplantation (HCT), consisting of 2 Gray (Gy) total body irradiation (TBI), dog leukocyte antigen (DLA)-identical marrow, and short-term post-grafting immunosuppression. We hypothesized that CD4+CD25+ Treg cells may be important regulators for the maintenance of cellular immune tolerance after allogeneic HCT. Previously, we showed in 8 mixed chimeras that naive donor lymphocyte infusion (DLI) did not change the level of donor chimerism. However, reconditioning mixed chimeras with 2 Gy TBI followed by DLI “breaks” tolerance and increases the level of donor chimerism. Seven mixed chimeras were reconditioned with 2 Gy TBI followed by DLI. Within 4 weeks after DLI, conversion to 100% donor chimerism was seen in 5 of 7 dogs and 2 dogs had a > 50% sustained increase in donor chimerism. Four recipients developed graft-versus host disease (GVHD). A control group of 3 mixed chimeras reconditioned with 2 Gy TBI without DLI had no change in donor chimerism. These results suggest that reconditioning with 2 Gy TBI followed by DLI can break the tolerance mechanism established in mixed chimeras. Next we asked if CD4+CD25+ Treg cells obtained from mixed chimeras before reconditioning could block the increase in donor chimerism following 2 Gy TBI and DLI. Peripheral blood mononuclear cells (PBMC) from 8 mixed chimeras were obtained by leukapheresis and cultured in bulk mixed leukocyte culture (MLC) with 3rd party DLA-mismatched, unrelated and irradiated CD34+ derived dendritic cells (10:1 responder: stimulator ratio) or PBMC (1:1). On day 4 of MLC, CD25+ cells were isolated by positive immunomagnetic selection. Next, artificial antigen presenting cells (aAPC, KT32) were added to expand the CD4+CD25+ Treg cells. The aAPC expressed Fcγ receptor CD32, canine CD86, and human IL-15, were loaded with the canine-specific mitogenic anti-CD3ε antibody 17.6F9 and irradiated prior to stimulation of CD4+CD25+ Treg. After 7 days, Treg were expanded a median of 23 (range, 8–36)-fold. Expanded CD4+CD25+ Treg were assessed for phenotype and in vitro function. The Treg cells were generated from 8 mixed chimeras and were infused back into the respective dogs (median dose 1× 107/kg) after reconditioning with 2 Gy TBI and immediately prior to DLI. In 6 of 8 dogs there was no change in the level of donor chimerism at 16–20 weeks follow-up; 2 dogs converted to complete donor chimerism within 6 weeks. Treatment with expanded Treg cells blocked conversion to complete donor chimerism after 2 Gy TBI +DLI in 6 of 8 dogs, compared with significant increases in donor chimerism for all 7 dogs after 2 Gy TBI +DLI not given Treg (p=.007). None of the 8 Treg recipient dogs developed GVHD, compared with 4 of 7 not given Treg, (p=0.02). A control group of 4 mixed chimeras were infused with expanded, non-Treg CD25 T cells. To this end, CD25+ T cells were immunomagneticaly depleted on day 4 of MLC. CD25 T cells were expanded with aAPC. Three of 4 dogs converted to the complete donor chimerism within 7 weeks after 2 Gy TBI, non-Treg and DLI. These results suggest that ex vivo expanded CD4+CD25+ Treg cells have in vivo function in a large animal model and can restore the tolerance mechanism in mixed chimeras that is broken by 2 Gy TBI and DLI.

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