Mixed chimerism and acceptance of kidney transplants after immunosuppressive drug withdrawal

Abstract

Preclinical studies have shown that persistent mixed chimerism is linked to acceptance of organ allografts without immunosuppressive (IS) drugs. Mixed chimerism refers to continued mixing of donor and recipient hematopoietic cells in recipient tissues after transplantation of donor cells. To determine whether persistent mixed chimerism and tolerance can be established in patients undergoing living donor kidney transplantation, we infused allograft recipients with donor T cells and hematopoietic progenitors after posttransplant lymphoid irradiation. In 24 of 29 fully human leukocyte antigen (HLA)-matched patients who had persistent mixed chimerism for at least 6 months, complete IS drug withdrawal was achieved without subsequent evidence of rejection for at least 2 years. In 10 of 22 HLA haplotype-matched patients with persistent mixed chimerism for at least 12 months, reduction of IS drugs to tacrolimus monotherapy was achieved. Withdrawal of tacrolimus during the second year resulted in loss of detectable chimerism and subsequent rejection episodes, unless tacrolimus therapy was reinstituted. Posttransplant immune reconstitution of naïve B cells and B cell precursors was more rapid than the reconstitution of naïve T cells and thymic T cell precursors. Robust chimerism was observed only among naïve T and B cells but not among memory T cells. No evidence of rejection was observed in all surveillance graft biopsies obtained from mixed chimeric patients withdrawn from IS drugs, and none developed graft-versus-host disease. In conclusion, persistent mixed chimerism established in fully HLA- or haplotype-matched patients allowed for complete or partial IS drug withdrawal without rejection.

Fig. 1.. Patient survival, graft survival, duration of complete withdrawal of IS drugs, and duration of chimerism in HLA-matched patients.

(A) Patient survival. (B) Graft survival. (C) Duration off IS drugs. Patients without bars did not meet criteria to achieve IS drug withdrawal. (D) Duration of chimerism up to the last time point tested.

Fig. 2.. Persistent mixed chimerism and kidney graft acceptance in HLA-matched recipients.

The percentages of donor cells in whole blood, daily doses of CsA or tacrolimus, and serum creatinine concentrations (mg/dL) at serial time points after kidney transplantation are shown in 10 recipients who had at least 2% donor cells in all chimerism tests. Orange lines show whole blood chimerism, and blue lines show the daily dose of calcineurin inhibitor. MMF was discontinued in all patients at 1 month after transplant. Calcineurin inhibitor used in patient nos. 1 to 18 was CsA and was tacrolimus (Tac) in patient nos. 21 to 29.

Fig. 3.. Differences in magnitude T and B cell chimerism are due to differences in kinetics of reconstitution of naïve cells in HLA-matched transplant recipients.

(A) Mean percentages (±SEM) of donor-type cells among purified T cells, B cells, NK cells, CD34⁺ cells, granulocytes, and whole blood (WB) cells before and at serial time points after kidney transplantation (KTx) in all 29 patients. (B) Percentages in 10 patients with chimerism that persisted for at least 24 months. (C) Percentages in patients with chimerism that was lost within 24 months. The Wilcoxon signed-rank test was used to make statistical comparisons. (D) Mean percentages of naïve CD3⁺ T cells among PBMCs. (E) Percentages of naïve CD4+ T cells among PBMCs. (F) Percentages of naïve CD8+ T cells among PBMCs. (G) Percentages of thymic-derived CD3⁺ T cell precursors (cells containing TRECs) among PBMCs. (H) Percentages of thymic-derived CD4⁺ T precursors among PBMCs. (I) Percentages of thymic-derived CD8+ T precursors among PBMCs. (J) Percentages of B cell precursors (pro-B cells) among PBMCs. (K) Percentages of naïve B cells among PBMCs. The Mann-Whitney U test was used to make statistical comparisons. (L) compares the mean percentage of donor-type cells among sorted naïve B cells to mean percentages among sorted pro-B cells, naïve CD4⁺ T cells, memory RO⁺CD4⁺ T cells, naïve CD8⁺ T cells, and memory CD8⁺ T cells. There were 6 to 12 available patient samples analyzed at each time point for the naïve and precursor cell studies. The two-sided Dunnett’s test was used for multiple comparisons. ns, not significant (P > 0.05); *P < 0.05 and **P < 0.01.

Fig. 4.. Differences in magnitude of T and B cell chimerism are due to differences kinetics of reconstitution of naïve cells in HLA haplotype–matched transplant recipients.

(A) Mean percentages (±SE) of donor-type cells among T cells, B cells, NK cells, CD34⁺ cells, granulocytes, and whole blood cells before and at serial time points after kidney transplantation in all 22 patients. (B) Percentages in 10 patients with detectable chimerism for at least 12 months. (C) Percentages in patients in whom chimerism persisted for less than 12 months. The Wilcoxon signed-rank test was used to make statistical comparisons. (D) Mean percentages of naïve CD3⁺ T cells among PBMCs. (E) Percentages of naïve CD4⁺ T cells among PBMCs. (F) Percentages of naïve CD8⁺ T cells among PBMCs. (G) Mean Shannon index diversity scores of the TCRα chain genes. (H) Scores of the TCRβ chain genes. (I) Percentages of B cell precursors among PBMCs. (J) Percentages of naïve B cells among PBMCs. The Mann-Whitney U test was used to make statistical comparisons. (K) compares the mean percentage of donor-type cells among sorted naïve CD4⁺ T cells to memory RO⁺CD4⁺ T cells, naïve CD8⁺ T cells, and RO⁺ memory CD8⁺ T cells. There were 6 to 12 available patient samples analyzed at each time point for the naïve and precursor cell studies. The two-sided Dunnett’s test was used to make statistical comparisons of sorted cells. P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5.. Loss of mixed chimerism during or after the withdrawal of IS drugs in 10 HLA-mismatched patients.

The percentage of donor cells in whole blood (red), daily doses of MMF (blue), trough blood concentrations of tacrolimus (green), and serum creatinine concentrations are shown at serial time points for each patient up to 762 days (24 months). Red and blue symbols are no longer shown after loss of chimerism or discontinuation of MMF, respectively. Arrows show acute rejection episodes (AR) or microvascular injury (MVI). Patient no. 9 died during tapering of tacrolimus. Due to pulmonary embolism (PE).