Cultured thymus tissue implantation promotes donor-specific tolerance to allogeneic heart transplants

Abstract

Eighty-six infants born without a thymus have been treated with allogeneic cultured thymus tissue implantation (CTTI). These infants, who lack T cells and are profoundly immunodeficient at birth, after CTTI from an unmatched donor develop T cells similar to those of recipient that are tolerant to both their own major histocompatibility antigens and those of the donor. We tested use of CTTI with the goal of inducing tolerance to unmatched heart transplants in immunocompetent rats. We thymectomized and T cell-depleted Lewis rats. The rats were then given cultured thymus tissue from F1 (Lewis × Dark Agouti ) under the kidney capsule and vascularized Dark Agouti (DA) heart transplants in the abdomen. Cyclosporine was administered for 4 months. The control group did not receive CTTI. Recipients with CTTI showed repopulation of naive and recent thymic emigrant CD4 T cells; controls had none. Recipients of CTTI did not reject DA cardiac allografts. Control animals did not reject DA grafts, due to lack of functional T cells. To confirm donor-specific unresponsiveness, MHC-mismatched Brown Norway (BN) hearts were transplanted 6 months after the initial DA heart transplant. LW rats with LWxDA CTTI rejected the third-party BN hearts (mean survival time 10 days); controls did not. CTTI recipients produced antibody against third-party BN donor but not against the DA thymus donor, demonstrating humoral donor-specific tolerance. Taken together, F1(LWxDA) CTTI given to Lewis rats resulted in specific tolerance to the allogeneic DA MHC expressed in the donor thymus, with resulting long-term survival of DA heart transplants after withdrawal of all immunosuppression.

Keywords: Immunology; Tolerance; Transplantation.

Figure 1. T cell reconstruction and functional transplanted thymus tissue in cardiac allograft recipients with CTTI.

(A) Schematic outline of experimental design to show naive T cell reconstitution, thymopoiesis, and donor-specific tolerance induced by CTTI. All LW rats were thymectomized and T cell depleted via anti-CD5 mAb before heart transplantation and CTTI. CTTI from F1(LWxDA) rats and hearts from DA rats were transplanted into thymectomized LW recipients. CsA was given for 4 months after transplantation via osmotic pump. The third-party BN heart was transplanted into the neck 2 to 3 months after CsA discontinuation. Control rats experienced identical procedures except they did not receive CTTI. (B) Circulating T cell repopulation after T cell depletion and thymus and heart transplantation. All animals showed dramatic reduction of circulating T cells after T cell depletion. Cardiac allograft recipients with CTTI (blue line) showed gradual repopulation of circulating T cells. Animals without CTTI also showed some degree of circulating T cells (red line). However, naive and recent thymic emigrants CD4 and CD8 T cells were significantly increased (P < 0.01) in animals with CTTI, whereas control animals showed neither circulating naive CD4 and CD8 T cells nor RTE CD4 and CD8 T cells. (C) Engrafted cultured thymus tissues under the renal capsule on day 180 in a recipient of cardiac allograft. Histology showed a distinct structure separate from renal tissue (original magnification, ×20). Engrafted cultured thymus tissue (right panel) showed a normal thymus structure (H&E), viable T cells (CD3), T cell proliferation (Ki67), and Hassall body formation (black arrow) with a lacy pattern (cytokeratin) on epithelial cells, confirming the viability of thymus with thymopoiesis (original magnification, ×200). Data are presented as means ± SD; n = 8–9 animals per group; student’s t test, *P < 0.05; **P< 0.01; ***P < 0.001, ****P < 0.0001; NS, not significant (P > 0.05). CTTI, cultured thymus tissue implantation; LW, Lewis; LWxDA, Lewis × Dark Agouti; DA, Dark Agouti; CsA, cyclosporine A; BN, Brown Norway; RTE, recent thymic emigrant.

Figure 2. Long-term cardiac allograft survival regardless of thymus cotransplantation.

(A) Graft survival of DA heart in recipients with or without CTTI. Kaplan-Meier survival curve showed significantly prolonged graft survival from animals with or without CTTI and syngeneic controls (LW heart into LW rat) compared with LW rats with DA heart transplants without immunosuppression/thymectomy (DA control). (B) Representative scanned image of explanted DA heart graft on day 180 from animals with and without CTTI. Images were adapted from whole slide scan. (C) ISHLT grading showed a significant reduction of rejection in both recipients with CTTI and without CTTI compared with DA control with no immunosuppression (n = 3–4 per group). Turkey test, ***P < 0.001; NS, not significant (P > 0.05). DA, Dark Agouti; CTTI, cultured thymus tissue implantation; LW, Lewis; ISHLT, International Society for Heart and Lung Transplantation.

Figure 3. Donor-specific tolerance in animals with CTTI versus general hyporesponsiveness in animals without CTTI.

(A) Graft survival of BN (cervical) heart in recipients with or without CTTI. Recipients with CTTI rejected the BN heart rapidly (median survival time = 10 ± 1.0 days), whereas recipients without CTTI did not reject the third-party BN hearts. The BN control (no immunosuppression/thymectomy) shows rejection of BN hearts by LW rats. The syngeneic control shows lack of rejection of LW hearts by LW rats. Kaplan-Meier survival curve showed significant differences in the graft survival. (B) ISHLT grading showed a severe rejection of BN heart in animals with CTTI, whereas there was a significant reduction of rejection in recipients without CTTI compared with BN controls with no immunosuppression (n = 3–5 per group). Turkey test, ***P < 0.001; NS, not significant (P > 0.05). (C) Representative scanned image of explanted BN heart graft at the time of rejection or designated time points posttransplantation. Both BN heart grafts from animals with CTTI (third panel) and allogeneic (second panel) showed severe mononuclear cell infiltration, whereas BN heart grafts from animals without CTTI (fourth panel) or animals with syngeneic CTTI (first panel) showed no sign of rejection. Images were adapted from whole slide scan. CTTI, cultured thymus tissue implantation; BN, Brown Norway; LW, Lewis; ISHLT, International Society for Heart and Lung Transplantation.

Figure 4. T cell infiltration (CD3 IHC) in LW, DA, and BN hearts.

(A) Quantification of inflammatory cells in the primary abdominal DA cardiac allograft. DA grafts from animals both with and without CTTI showed no significantly reduced inflammatory cell infiltration (at POD180) compared with DA control grafts (in LW rats with no immunosuppression), showing a high level of graft infiltration of immune cells. (B) Quantification of inflammatory cells in secondary cervical BN cardiac allografts. Both the BN control grafts (in LW rats with no immunosuppression) and the BN grafts from recipients with CTTI showed significantly elevated inflammatory cell infiltration. The BN grafts from animals without CTTI showed significantly reduced inflammatory cell infiltration compared with BN control. (C) Heart allografts from DA and BN rats were harvested with native heart at the time of BN heart rejection. Grossly, native heart and DA heart (POD 196) did not show a dramatic increase of T cells, whereas BN heart (POD14) showed a massive amount of T cells in recipients with CTTI. Images were adapted from whole slide scan. For panels A and B, 3–5 animals per group were analyzed; Turkey test, *P < 0.05; **P< 0.01; ***P < 0.001; NS, not significant (P > 0.05). LW, Lewis; DA, Dark Agouti; BN, Brown Norway; CTTI, cultured thymus tissue implantation.

Figure 5. Donor-specific humoral tolerance with CTTI.

(A) Representative histogram plots for posttransplant donor-specific alloantibody (anti-DA and anti-BN antibodies) measured by T cell flow crossmatch. LW recipients with or without CTTI did not generate any antibodies against DA antigen (A, second and third plots, respectively, in top row), whereas animals with CTTI were able to generate antibody against BN antigen (A, second panel, bottom row). Serum samples from LW recipients of DA heart transplantation without immunosuppression (A, first panel in top row) and from LW recipients of BN heart transplantation without immunosuppression (A, first panel in bottom row) were used as positive controls (DA control and BN control) for anti-DA or anti-BN antibody, respectively. (B) Level of anti-DA antibody after primary DA heart transplantation. Serum samples from animals with CTTI and without CTTI after DA heart transplantation (POD 180) did not develop antibody against DA cells. (C) Levels of anti-BN antibody after secondary BN heart transplantation. Animals with CTTI showed significantly elevated anti-BN Abs, whereas animals without CTTI showed no antibodies against BN cells. A total 4 to 5 animals per group were analyzed; Student’s t test. CTTI, cultured thymus tissue implantation; DA, Dark Agouti; BN, Brown Norway; LW, Lewis.