Mixed chimerism and tolerance without whole body irradiation in a large animal model

Abstract

Mixed hematopoietic chimerism may provide a treatment for patients with nonmalignant hematologic diseases, and may tolerize patients to organ allografts without requiring chronic immunosuppression. However, the toxicity of the usual conditioning regimens has limited the clinical applicability of this approach. These regimens generally include some level of whole body irradiation (WBI), which is thought to facilitate engraftment either by making room for donor hematopoietic stem cells or by providing sufficient host immunosuppression to enable donor cells to engraft. Here, we have established mixed chimerism across both minor and major histocompatibility barriers in swine, by using high doses of peripheral blood stem cells in the absence of WBI. After mixed chimerism was established, swine leukocyte antigen-matched (SLA-matched) donor skin grafts were tolerated and maintained for a prolonged period, whereas third-party SLA-matched skin was rejected promptly. Donor-matched kidney allografts were also accepted without additional immunosuppression. Because of its low toxicity, this approach has potential for a wide range of clinical applications. Our data may indicate that niches for engrafting stem cells are filled by mass action and that WBI, which serves to empty some of these niches, can be omitted if the donor inoculum is sufficiently large and if adequate host T-cell depletion is achieved before transplant.

Figure 1

Diagram of preparative regimen used for PBSCT. ^AAn additional 3 days of leukapheresis were necessary to yield 200 × 10⁸/kg for animal no. 13101.

Figure 2

Peripheral blood T-cell depletion. (a) Absolute number of T cells present in the peripheral blood pretreatment (day –2), and post TI (700 or 1,000 cGy) and pCD3-CRM9 administration (days –1 and 0). (b) Absolute number of T cells remaining in PB on day 0 for each animal.

Figure 3

Clinical course of animals receiving (700 or 1,000 cGy TI + DTCD3) on WBC (a) and platelets (PLT) (b).

Figure 4

Chimerism among animals receiving SLA-matched PBSCT. (a) Peripheral blood; (b) thymus.

Figure 5

Peripheral chimerism among animals receiving one-haplotype–mismatched PBSCT with (a) 700 cGy TI; (b) 1,000 cGy TI.

Figure 6

Chimerism detected by flow cytometry and immunohistochemistry in thymic biopsy tissue from animal no. 13101 on day 158. (a) Double staining of thymus by FACS. (b–e) Histology (immunohistochemistry).

Figure 7

(a) Plasma creatinine levels of single haplotype–mismatched chimeric re cipients that received donor MHC-matched kidney allografts without immunosuppression: Animals no. 13101 and no. 13476 were chimeric animals that received donor MHC-matched kidney allografts; animal no. 10653 was a naive animal that received a single haplotype–mismatched kidney allograft. (b–e) Donor-specific tolerance and immunocompetence of single haplotype–mismatched chimeric animal no. 13101: CTL responses of a naive SLA^ad animal (b) and of animal no. 13101 before PBSCT (c); after PBSCT, but before kidney transplantation (KTx) (d); and after KTx (e). Each graph shows anti-self SLA^ad (open squares), anti-donor SLA^ac (closed squares), and anti–third-party Yucatan miniature swine (open triangles) response.