Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation

Abstract

The toxicity of chronic immunosuppressive agents required for organ transplant maintenance has prompted investigators to pursue approaches to induce immune tolerance. We developed an approach using a bioengineered mobilized cellular product enriched for hematopoietic stem cells (HSCs) and tolerogenic graft facilitating cells (FCs) combined with nonmyeloablative conditioning; this approach resulted in engraftment, durable chimerism, and tolerance induction in recipients with highly mismatched related and unrelated donors. Eight recipients of human leukocyte antigen (HLA)-mismatched kidney and FC/HSC transplants underwent conditioning with fludarabine, 200-centigray total body irradiation, and cyclophosphamide followed by posttransplant immunosuppression with tacrolimus and mycophenolate mofetil. Subjects ranged in age from 29 to 56 years. HLA match ranged from five of six loci with related donors to one of six loci with unrelated donors. The absolute neutrophil counts reached a nadir about 1 week after transplant, with recovery by 2 weeks. Multilineage chimerism at 1 month ranged from 6 to 100%. The conditioning was well tolerated, with outpatient management after postoperative day 2. Two subjects exhibited transient chimerism and were maintained on low-dose tacrolimus monotherapy. One subject developed viral sepsis 2 months after transplant and experienced renal artery thrombosis. Five subjects experienced durable chimerism, demonstrated immunocompetence and donor-specific tolerance by in vitro proliferative assays, and were successfully weaned off all immunosuppression 1 year after transplant. None of the recipients produced anti-donor antibody or exhibited engraftment syndrome or graft-versus-host disease. These results suggest that manipulation of a mobilized stem cell graft and nonmyeloablative conditioning represents a safe, practical, and reproducible means of inducing durable chimerism and donor-specific tolerance in solid organ transplant recipients.

Fig. 1

Algorithm for conditioning, kidney and FCRx transplant, and maintenance immunosuppression. A) Fludarabine was administered day −4, −3, −2 (30 mg/kg/dose) relative to the living donor kidney transplant (day 0). Dialysis was performed at least 6 hours after each dose pre-transplant. Two doses of cyclophosphamide (50 mg/kg) were given day −3 and day +3. FCRx was administered day +1. If the recipient was chimeric and/or tolerant to their donor at 6 months, and if the standard of care protocol biopsy showed no evidence for rejection, the MMF was discontinued. At 9 months, if renal function remained stable, the tacrolimus was reduced (trough levels 0–3ng/ml). At 12 months, the tacrolimus was discontinued if donor chimerism was present, the repeat protocol biopsy was normal, and renal function stable. B) Serum creatinine levels (mg/deciliter) for all active subjects vs. time.

Fig. 2

Summary of nadir, donor chimerism, and clinical outcomes in Subject #3. A) Kinetics of nadir (absolute neutrophil count [ANC]) ≤ 500. The horizontal axis represents days after kidney transplant and vertical axis ANC. B) Whole blood mononuclear cell chimerism using the STR molecular assay (LabCorp). The sensitivity and specificity for these assays was 1–2%. C) Whole blood mononuclear cells were isolated and sorted for B cells (CD19⁺), T cells (CD3⁺), or myeloid-derived cells (CD66B⁺). D) Fresh recipient peripheral blood lymphocytes were exposed to PHA, Candida, recipient stimulators, donor simulators, and third-party allo-stimulators in one-way MLR proliferative assays. Third-party allo-responder controls were performed. A stimulation index ≥ 3 is positive. The stimulation index is calculated as the ratio of antigen-specific proliferation to unstimulated recipient lymphocytes. Error bars are mean ± SD for triplicate assays.

Fig. 2

Summary of nadir, donor chimerism, and clinical outcomes in Subject #3. A) Kinetics of nadir (absolute neutrophil count [ANC]) ≤ 500. The horizontal axis represents days after kidney transplant and vertical axis ANC. B) Whole blood mononuclear cell chimerism using the STR molecular assay (LabCorp). The sensitivity and specificity for these assays was 1–2%. C) Whole blood mononuclear cells were isolated and sorted for B cells (CD19⁺), T cells (CD3⁺), or myeloid-derived cells (CD66B⁺). D) Fresh recipient peripheral blood lymphocytes were exposed to PHA, Candida, recipient stimulators, donor simulators, and third-party allo-stimulators in one-way MLR proliferative assays. Third-party allo-responder controls were performed. A stimulation index ≥ 3 is positive. The stimulation index is calculated as the ratio of antigen-specific proliferation to unstimulated recipient lymphocytes. Error bars are mean ± SD for triplicate assays.

Fig. 3

Representative sections of kidney transplant biopsy at one year after all immunosuppression was withdrawn. Staining with A) hematoxylin-eosin (20×), B) Masson trichrome (10×), and C) PAS (20×) shows no acute or chronic rejection, minimal tubular atrophy, and minimal interstitial fibrosis.

Fig. 4

Non-responsiveness of chimeric recipients to archived pre-transplant recipient stimulators. Subject 5 is shown as a representative MLR. Y-axis denotes the mean stimulation index; x-axis denotes the antigenic challenge used. White bar demonstrates response of the transplant recipient; gray and black bars show response of third-party MHC-disparate individuals allo#1 and allo#2. Please note that allo #1 and #2 were also used as stimulators in these experiments.

Fig. 5

Representative 10 color flow cytometric analysis of FC subpopulations in the transplanted stem cell product. CD8⁺/TCR⁻ FC Total were sorted and re-stained for CD56, CD19, CD3, CD11c, CD11b, HLA-DR, fox p3 and CD123. A) CD56^dim/− FC were analyzed for co-expression of CD19, CD3ε, CD11c, CD11b, HLA-DR, and CD123. B) Flow cytometric analysis of CD56^bright FC.