Rejection of cardiac xenografts transplanted from alpha1,3-galactosyltransferase gene-knockout (GalT-KO) pigs to baboons

Abstract

The use of alpha1,3-galactosyltransferase gene-knockout (GalT-KO) swine donors in discordant xenotransplantation has extended the survival of cardiac xenografts in baboons following transplantation. Eight baboons received heterotopic cardiac xenografts from GalT-KO swine and were treated with a chronic immunosuppressive regimen. The pathologic features of acute humoral xenograft rejection (AHXR), acute cellular xenograft rejection (ACXR) and chronic rejection were assessed in the grafts. No hyperacute rejection developed and one graft survived up to 6 months after transplantation. However, all GalT-KO heart grafts underwent graft failure with AHXR, ACXR and/or chronic rejection. AHXR was characterized by interstitial hemorrhage and multiple thrombi in vessels of various sizes. ACXR was characterized by TUNEL(+) graft cell injury with the infiltration of T cells (including CD3 and TIA-1(+) cytotoxic T cells), CD4(+) cells, CD8(+) cells, macrophages and a small number of B and NK cells. Chronic xenograft vasculopathy, a manifestation of chronic rejection, was characterized by arterial intimal thickening with TUNEL(+) dead cells, antibody and complement deposition, and/or cytotoxic T-cell infiltration. In conclusion, despite the absence of the Gal epitope, acute and chronic antibody and cell-mediated rejection developed in grafts, maintained by chronic immunosupression, presumably due to de novo responses to non-Gal antigens.

Figure 1. Serum troponin T levels in recipients and palpation scores based on the strength of contraction of cardiac xenografts

Normal troponin T levels are less than 0.10 ng/mL. Contraction of grafts was monitored by palpation and scored 0–3. The arrowheads indicate the day of biopsies and the arrow indicates the day of graftectomy. The eight heterotopic cardiac grafts were divided into the three groups based on their rejection profile: (1) healthy-graft group (B226, B225 and B216), (2) rapidly weakened group (B214 and B218) and (3) gradually weakened group (B229, B223 and B228). B226 and B225 were euthanized and B216 died with beating heart grafts, as a result of anemia (during a period when no donor blood was available), ischemia in a leg (associated with an indwelling femoral artery catheter) or spontaneous intra-abdominal hemorrhage, on day 23, 16 or 56, respectively.

Figure 2. Histological features of grafts showing the development of acute humoral xenograft rejection (AHXR), acute cellular xenograft rejection (ACXR) and chronic xenograft rejection

In graftectomy samples from B226 (A, B:day 16, HE stain), thrombi (arrows in A) were seen in capillaries. Polymorphonuclear leukocytes (arrow in B) and mononuclear cells (arrowhead in B) infiltrated the grafts. In graftectomy samples from B214 (C, D: day 59, HE stain), interstitial hemorrhage and multiple microthrombi in the microvasculature developed. In addition to the infiltration of polymorphonuclear leukocytes (arrow in D), focal mononuclear cells (arrowhead in D) infiltrated the graft. In graftectomy samples from B229 (E, H, K: day 78), B223 (F, I, L, N–P: day 110) and B228 (G, J, M: day 179) (E–J: HE stain; inset in G, K–M: EMG stain), multiple microthrombi, an irregular distribution of interstitial hemorrhage and chronic xenograft vasculopathy (arrowheads in E–G) developed along with a focal mononuclear cell infiltrate (H–J). AHXR was characterized by thrombotic microangiopathy with multiple microthrombi in capillaries (arrows in K–M) and arteries (double arrow in K) in the grafts, and by immunoglobulin and complement deposition (N: IgM; 0: C5b-9). Two-color immunostaining for CD41 (red color) and C4d (green color) showed that almost all of the CD41⁺ thrombi (arrows in P) were detected within vessels that contained deposits of complement. Electron microscopy (Q, R) revealed damaged capillaries in the graft of B228 on day 179 (× 1900). The damage was characterized by the loss of endothelial cells with fibrin thrombi (asterisks in Q) and capillary destruction (arrowheads in R) with interstitial hemorrhage.

Figure 3. Histological features of cardiac grafts showing fibrinoid necrosis and endothelialitis in the arteries

The early fibrinoid necrosis in arteries (A: B214, day 59, EMG stain) showed enlarged endothelial cells (arrowheads) and the exudation of fibrinoid materials (arrows). In advanced AHXR (B–D: B218, day 67; B: HE stain, C: TUNEL stain, D: EMG stain), arteries showed fibrinoid necrosis with thrombus formation (asterisks in B and C), neutrophil infiltration (arrows in B), TUNEL⁺ cells (arrows in C) and deposits of fibrin (D, E), immunoglobulin (F: IgM) and complement (G: C4d), along with neointimal thickening (D). Two-color immunostaining (H) for CD41 (red color) and C4d (green color) showed that CD41⁺ thrombi were detected within small arteries that contained deposits of complement. Endothelialitis (I) was noted in arteries with infiltration of mononuclear cells underneath the endothelium (J) and media (J), infiltration of both CD3 and TIA-1⁺ cytotoxic T cells (arrows in K), and neointimal thickening (L) (I–L: B228, day 179; I, J, L: EMG stain, K: two-color stain with CD3 (brown) and TIA-1 (blue)).

Figure 4. Histological features of grafts showing graft infiltrating cells and the development of acute cellular xenograft rejection (ACXR)

Polymorphonuclear leukocytes (arrow in A), mononuclear cells (arrowheads in B), CD68⁺ macrophages (arrow in C) and a small number of CD3⁺ cells (arrow in D) infiltrated the graft 7 days after transplantation. In the healthy-graft group (E, F: B216, day 56; E: HE stain, F: CD3 stain), polymorphonuclear leukocytes (arrow in E) and mononuclear cells (arrowhead in E), including a small number of CD3⁺ cells (arrow in F), infiltrated the grafts. In rapidly weakened group (G, H: B218, day 67; G: HE stain, H: CD3 stain) and gradually weakened group (I: B223, day 110, HE stain), focal mononuclear cell infiltration was evident in the graft, showing ACXR. Some TUNEL⁺ dead cells (arrows in J, K) were seen in contact with infiltrating mononuclear cells (J, K: B223, day 110, TUNEL stain). Some thrombi (asterisk in L) were present with CD3⁺ cell infiltration (arrow) (L: B223, day 110, CD3 stain). The phenotypes of the infiltrating cells included many CD3⁺ T cells (H, M, T), both TIA-1 and CD3⁺ cytotoxic T cells (N), CD68⁺ macrophages (O), CD4⁺ cells (Q), CD8⁺ cells (R) and a small number of CD20⁺ B cells (P) and NKB-1⁺ NK cells (S) (M–P: B223, day 110, Q–T: B229, day 179; M, T: CD3 stain, N: two-color stain with CD3 (brown) and TIA-1 (blue), O: CD68 stain, P: CD20 stain, Q: CD4 stain, R: CD8 stain, S: NKB-1 stain).

Figure 5. The number of infiltrating cells in the grafts (cells/single field of 0.0625 mm²) and the correlation between CD3⁺ T cells and TUNEL⁺ dead cells

The number of infiltrating cells of all phenotypes except NKB-1⁺ cells increased after transplantation and during the development of graft failure (healthy-graft group: B226, B225 and B216; rapidly weakened group: B214 and B218 and gradually weakened group: B229, B223 and B228). The results are depicted as the mean ± standard error, as calculated from 40 randomly selected fields. There was a statistically significant correlation between the number of CD3⁺ T cells and the number of TUNEL⁺ dead cells in capillaries and the interstitium in the grafts (p < 0.05).

Figure 6. Histological features of grafts showing chronic xenograft vasculopathy (A–I: B223, day 110; A, D, G: HE stain, B, E, F: EMG stain, C, F, I: α-actin stain)

Chronic xenograft vasculopathy in arteries was characterized by the narrowing of the arterial lumen with intimal fibrous thickening consisting of α-actin⁺ cells without elastosis. Neither fibrinoid material nor cellular infiltration was seen in arteries, suggesting fully developed vasculopathy. The distribution of chronic vasculopathy in grafts, including the percent of arteries affected, as well as the severity and types of chronic vasculopathy is also shown. Although a similar percentage of small and large arteries were affected by chronic xenograft vasculopathy, the vasculopathy was, on average, more severe in the smaller arteries. Large number of arteries that showed evidence of vasculopathy also had fully developed or humoral-associated rejection types.

Figure 7. Histological features of chronic xenograft vasculopathy

In chronic humoral rejection-associated vasculopathy (A–H: B229, day 78), fibrinoid materials were present within a thickened intima (A: HE stain, B: EMG stain). TUNEL⁺ dead cells (arrows in C), that may be endothelial cells, were detected on the luminal surface of arteries along with exudative lesions (asterisk in C) (C: TUNEL method). The deposition of fibrin (D), IgM (E), IgG (F), C4d (G) and C5b-9 (H) was detected in arteries. Chronic cellular rejection-associated vasculopathy (I–P: B223, day 110) was characterized by active endothelialitis (arrow in J), presence of TUNEL⁺ dead cells (arrow in K) and cellular infiltration in the arterial intima (I: EMG stain, J: HE stain, K: TUNEL method). Cellular infiltrate in arterial walls was composed of T cells (L: CD3 stain), cytotoxic cells (M: TIA-1 stain), both CD3 and TIA-1⁺ cytotoxic T cells (N: two-color stain with CD3 (brown) and TIA-1 (blue)), macrophages (O: CD68 stain) and a small number of B cells (P: CD20 stain). Both chronic humoral and cellular rejection-associated vasculopathy (Q–T: B223, day 110) was characterized by fibrinoid material deposition and cellular infiltration in arterial intima (Q: EMG stain). Cellular infiltrate in the intima was composed of polymorphonuclear leukocytes (arrow in R), T cells (arrows in S) and macrophages (arrow in T) (R: HE stain, S: CD3 stain, T: CD68 stain).