Ex Vivo Major Histocompatibility Complex I Knockdown Prolongs Rejection-free Allograft Survival

Abstract

Background: Widespread application of vascularized composite allotransplantation (VCA) is currently limited by the required lifelong systemic immunosuppression and its associated morbidity and mortality. This study evaluated the efficacy of ex vivo (after procurement but before transplantation) engineering of allografts using small interfering RNA to knockdown major histocompatibility complex I (MHC-I) and prolong rejection-free survival.

Methods: Endothelial cells (ECs) were transfected with small interfering RNA targeted against MHC-I (siMHC-I) for all in vitro experiments. MHC-I surface expression and knockdown duration were evaluated using quantitative polymerase chain reaction (qPCR) and flow cytometry. After stimulating Lewis recipient cytotoxic lymphocytes (CTL) with allogeneic controls or siMHC-I-silenced ECs, lymphocyte proliferation, CTL-mediated and natural killer-mediated EC lysis were measured. Using an established VCA rat model, allografts were perfused ex vivo with siMHC-I before transplantation. Allografts were analyzed for MHC-I expression and clinical/histologic evidence of rejection.

Results: Treatment with siMHC-I resulted in 80% knockdown of mRNA and 87% reduction in cell surface expression for up to 7 days in vitro (P < 0.05). Treatment of ECs with siMHC-I reduced lymphocyte proliferation and CTL-mediated cytotoxicity (77% and 50%, respectively, P < 0.01), without increasing natural killer-mediated cytotoxicity (P = 0.66). In a rat VCA model, ex vivo perfusion with siMHC-I reduced expression in all tissue compartments by at least 50% (P < 0.05). Knockdown prolonged rejection-free survival by 60% compared with nonsense-treated controls (P < 0.05).

Conclusions: Ex vivo siMHC-I engineering can effectively modify allografts and significantly prolong rejection-free allograft survival. This novel approach may help reduce future systemic immunosuppression requirements in VCA recipients.

Fig. 1.

MHC-I knockdown in vitro. A, ECs silenced with a 1-time dose of 20 nanomolar siRNA against MHC-I at day 0 were collected at days 1, 3, and 7 posttreatment and compared with nonsense-treated controls using qPCR. Statistically significant reduction in relative MHC-I mRNA expression was observed at all time points as compared with control cells (P < 0.05; n = 3 for each). B, Knockdown of MHC-I on a protein level was analyzed on day 7 by flow cytometry and shown to be dose-dependent, as compared with nonsense controls (n = 3 for each).

Fig. 2.

MHC-I silencing of endothelial cells reduces lymphocyte proliferation and CD8⁺ T cell cytotoxicity. A, MHC-I knockdown significantly reduced lymphocyte counts as compared with untreated and nonsense siRNA-treated RAECs 24 hours after transfection (P < 0.05; n = 3 for each). B, In target:effector ratios of 20:1, 1:1, and 1:20, MHC-I siRNA-treated RAECs demonstrated a marked reduction in cytotoxicity 24 hours after transfection (P < 0.05 for all; n = 3 for each). C, Target:effector ratio of 1:20 resulted in a 58% decrease in endothelial cell cytolysis compared with nonsense-treated controls 24 hours after transfection (P < 0.05; n = 3 for each).

Fig. 3.

Natural killer cell-mediated cytotoxicity remains unchanged with MHC-I knockdown. NK:EC ratios of 1,000:1, 10:1, and 0.1:1 demonstrated no significant differences in NK-mediated cytotoxicity 24 hours after transfection (P > 0.1 for all; n = 3 for each).

Fig. 4.

Ex vivo siMHC-I demonstrates effective knockdown of treated allografts. A, MHC-I mRNA expression is effectively reduced both in vitro and in vivo compared with nonsense-treated controls. Reduced expression was observed in vivo in multiple tissue compartments, including the vascular pedicle, fat, and skin, on postoperative day 3 (P < 0.05 for all; n = 3 for each). B, siMHC-I perfusion resulted in marked reduction of MHC-I expression in vascular tissue specifically on postoperative day 3 as compared with nonsense-treated controls (P < 0.05; n = 3 for each).

Fig. 5.

Immunohistochemistry of allograft vasculature on postoperative day 3. A, Flaps were co-perfused with FITC-lectin (green) to stain the intact vasculature and siGLO-labeled siRNA (red). Ex vivo perfused siRNA localizes to the perivascular space, where it exerts its effect on the endothelial barrier. B, Immunohistologic staining of MHC-I demonstrated marked reduction after ex vivo siMHC-I perfusion (right) as compared with siNonsense controls (left). MHC-I antigen is stained in green. Expression was reduced in multiple layers of the arterial and venous walls, but most prominently in the tunica intima, the innermost layer that is comprised of endothelial cells. C, MHC-I expression was completely masked at the microvascular level of the postcapillary venules.

Fig. 6.

Kaplan-Meier in vivo survival curve. A, MHC-I silencing prolonged rejection-free survival of the allograft by 60% as compared with nonsense treated controls (Log-rank test: P = 0.029; n = 5 for each group). B, Untreated allografts without any systemic immunosuppression (top left) demonstrated clinical signs of acute rejection within the first week after transplant. However, addition of a brief 5-day course of CSA prevented early rejection (top right). Thus, ex vivo perfusion of nonsense and MHC-I-targeted siRNA was followed by a brief 5-day course of CSA. Control nonsense-treated flaps began exhibiting signs of rejection by postoperative day (POD) 14 (representative flap shown bottom left) in contrast to siMHC-I flaps, which took significantly longer to reject (representative flap shown bottom right). CSA, cyclosporine.