Long-term results in recipients of combined HLA-mismatched kidney and bone marrow transplantation without maintenance immunosuppression

Abstract

We report here the long-term results of HLA-mismatched kidney transplantation without maintenance immunosuppression (IS) in 10 subjects following combined kidney and bone marrow transplantation. All subjects were treated with nonmyeloablative conditioning and an 8- to 14-month course of calcineurin inhibitor with or without rituximab. All 10 subjects developed transient chimerism, and in seven of these, IS was successfully discontinued for 4 or more years. Currently, four subjects remain IS free for periods of 4.5-11.4 years, while three required reinstitution of IS after 5-8 years due to recurrence of original disease or chronic antibody-mediated rejection. Of the 10 renal allografts, three failed due to thrombotic microangiopathy or rejection. When compared with 21 immunologically similar living donor kidney recipients treated with conventional IS, the long-term IS-free survivors developed significantly fewer posttransplant complications. Although most recipients treated with none or two doses of rituximab developed donor-specific antibody (DSA), no DSA was detected in recipients treated with four doses of rituximab. Although further revisions of the current conditioning regimen are planned in order to improve consistency of the results, this study shows that long-term stable kidney allograft survival without maintenance IS can be achieved following transient mixed chimerism induction.

Keywords: Chimerism; immunobiology; kidney transplantation; living donors; tolerance.

Conflict of interest statement

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

© Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

Figures

Figure 1. Nonmyeloablative conditioning regimens

The initial conditioning regimen (Figure 1, NKD03) consisted of cyclophosphamide (60 mg/kg) administered i.v. on Days −5 and −4 with respect to transplantation; humanized anti-CD2 mAb (MEDI 507) (0.6 mg/kg/dose)on Days −2, −1, 0 and +1; cyclosporine A (CyA) (5mg/kg) i.v. on Day −1 and thymic irradiation (700cGy) on Day −1. Hemodialysis was performed 14 h after each dose of cyclophosphamide. On Day 0, kidney transplantation was followed by i.v. infusion of unprocessed donor bone marrow (DBM; 2–3 × 108 mononuclear cells/kg). Oral CyA (Neoral) was administered postoperatively at 8–12 mg/kg/day with target trough blood levels of 250–350 ng/mL, then tapered and discontinued over several months. The protocol was modified after treatment of the third subject (see the Results section), with the addition of rituximab, 375 mg/m2/dose on Days −7 and −2 (red arrows); and prednisone, 2 mg/kg/dose starting on the day of transplantation and tapering to withdrawal over the next 10 posttransplant days (mod NKD03). Since subjects treated with this mod NKD03 still developed donor-specific antibodies (DSAs) after discontinuation of immunosuppression, the regimen was further modified (Figure 1) to add two more doses of rituximab (375 mg/m2/dose) on Days 5 and 12 (red arrows), plus a more prolonged course of prednisone until Day 20, and tacrolimus in place of CyA (ITN036). Tacrolimus was slowly tapered over several months and completely discontinued at 8 months after confirming no rejection by a 6-month protocol biopsy.

Figure 2. Clinical course after combined kidney and bone marrow transplantation

Blue lines indicate serum creatinine levels (mg/dL). Green bars indicate induction immunosuppression (CyA: cyclosporine, Tac: tacrolimus). Orange/blue bars indicate reinstitution of maintenance immunosuppression (MMF: mycophenolate mofetil, actino: actinomycin, Pred: prednisone), CTLA4lg (belatacept).

Figure 3. Late renal allograft biopsies (all protocol biopsies unless noted)

(A, B, C [Subject 1 at 7.5 years]) Protocol biopsy shows no evidence of rejection by light microscopy (LM) (A). The glomeruli are normal by electron microscopy (EM) (B) and no C4d staining is detectable by immunofluorescence (IF) (C). (D, E, F [Subject 2 at 8 years]) An indication biopsy for proteinuria shows prominent lobular mesangial expansion with widespread glomerular basement membrane (GBM) duplication (D). Granular dense deposits in the duplicated GBM are seen by EM (E, arrow). Granular and segmental staining for C3 along the GBM and in the mesangium is evident by IF, but no immunoglobulin was detected (F), indicative of recurrent C3 glomerulopathy (originally classified as membranoproliferative glomerulonephritis [MPGN], Type I). (G, H, I [Subject 4 at 5 years]) Widespread GBM duplication and glomerulitis are seen by LM (G). EM shows prominent duplication of the GBM without deposits and reactive endothelial cells (H, arrow). C4d deposition is detected in peritubular capillaries (I). These findings are indicative of chronic, active antibody-mediated rejection, which at this time was subclinical. (J, K, L [Subject 5 at 6 years]) The last protocol biopsy shows glomerulitis and widespread duplication of the GBM by LM (J) and EM (K) and no C4d deposition by IF (L). DSA for donor Class II was detected intermittently and a previous biopsy showed C4d deposition. These findings meet the criteria of subclinical, C4d negative, early-stage chronic active antibody-mediated rejection. (M, N, O [Subject 6 at 2 years]) LM shows normal glomeruli with rare foci of interstitial mononuclear inflammation affecting

Figure 4. Detection of donor-specific antibody (DSA)

DSAs as detected by ELISA. NKD03: Subject 1: DSA never detected. Subject 2: DSA transiently detected once at 8 years. Subject 3: *DSA was negative by ELISA but retrospective analysis with single antigen beads showed positive DSA against donor HLA Class I (mean fluorescence intensity > 10 000). The subject developed acute humoral rejection on Day 10 with ELISA detected DSA (B44 and DR4). Pretransplant DSA had been negative by ELISA, but more recent Luminex suggested that positive anti-Class I DSA (B44) before transplantation may have been present before transplantation. Mod NKD03: Subject 4: anti-donor DR17 antibody has been persistently positive since shortly after stopping immunosuppression. Subject 5: weak anti-donor DR53 antibody has been intermittently detectable. ITN036: in contrast to NKD03 subjects, no DSA has been detectable in any ITN036 subject including Subject 10 who developed severe acute cellular rejection.

Figure 5

(A) CD3−CD19+ cell depletion after combined kidney and bone marrow transplantation. In two NKD03 subjects, recovery of CD3−CD19+ cells (B cells) was found by Day 50 (Subject 1, gray) and Day 120 (Subject 2, black). With addition of two doses of pretransplant rituximab (modified NKD03), B cells were partially depleted and recovered by Days 150 (Subject 4, dark blue) and 180 (Subject 5, light blue). With four doses of peritransplant rituximab (ITN036), B cells were nearly completely depleted (less than 1–2/mm2) for 180 days. (B) Serum B cell activating factor (BAFF). Serial serum BAFF levels were measured by ELISA. Serum BAFF levels were <2 ng/mL at all time points assessed in Subject 1. Subject 2 had high BAFF levels initially until Day 100 and waned thereafter to <2 ng/mL after Day 100 and he successfully discontinued his immunosuppression. High BAFF levels were detected for over 300 days in the two subjects treated with the mod NKD03 regimen and in the ITN036 subjects.