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Additional Supporting Information may be found in the online version of this article.

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Figure S1: Detection of chimerism in bone marrow and thymus of mixed chimeras. (A) Representative flow cytometry data demonstrating presence of donor-type (PAA positive) cells in recipient bone marrow greater than 100 days posttransplant. (B) Chimerism was also detected in thymic tissue, where donor-type cells were found to contribute to both single-positive and double-positive thymocyte populations. Representative data, animal 20680.

Figure S2: VCA sparing in cutaneous GvHD. (A) Animal 20313, VCA and surrounding skin 50 days post-VCA transplant. The GvHD skin rash was observed to demarcate clearly at the VCA border. (B) Histological assessment of host skin confirmed this diagnosis, while (C) no signs consistent with rejection or GvHD were identified on VCA biopsy.

Figure S3: Recipients tolerant of VCA accept donor split thickness skin grafts indefinitely and reject third-party skin grafts. (A) 20311 donor graft POD 102. (B) 20311 donor-matched graft POD 24. (C) 20311 single haplotype disparate graft POD 24. (D) 20680 donor graft POD 135. (E) 20680 donor-matched graft POD 42. (F) 20680 single haplotype SLAaa graft POD 56. In both animals, donor skin grafts were placed over 150 days after VCA, third-party grafts were placed after interval of at least 80 days.

Figure S4: Mixed chimeras demonstrate specific unresponsiveness to donor in CML. (A) CML data from animal 20311 demonstrating normal anti-donor and anti-third-party responses prior to conditioning and transplant. (B) Comparable lytic activity was observed prior to conditioning in control animal 20312 which underwent conditioning without HCT and subsequently received a VCA greater than 100 days later. (C) Following HCT chimeric animals demonstrated specific unresponsiveness to donor antigens in CML. Representative data from 20311 over 100 days posttransplant are shown. (D) In contrast, in the absence of HCT, anti-donor responses are maintained. Data shown for conditioned control animal 20312 greater than 100 days postconditioning. Percentage-specific lysis against self targets <5% in all assays.

Figure S5: Lack of evidence for chronic pathological changes in VCAs accepted long term. All VCAs were monitored regularly for histological evidence of acute or chronic rejection throughout experimental follow up. No changes consistent with chronic rejection were identified in any specimen. Representative images from terminal specimens are presented. (A) Dilated apocrine unit without rejection, 20311, 504 days posttransplant (×20). (B) 20313, 115 days posttransplant. (C) Artery with no evidence of allograft vasculopathy, 20680, 486 days posttransplant (×20). (D) 20681, 139 days posttransplant (×15).

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