Antibody-mediated rejection of arterialised venous allografts is inhibited by immunosuppression in rats

Abstract

Objectives and design: We determined in a rat model (1) the presence and dynamics of alloantibodies recognizing MHC complexes on quiescent Brown-Norway (BN) splenic cells in the sera of Lewis (LEW) recipients of Brown-Norway iliolumbar vein grafts under tacrolimus immunosuppression; and (2) the presence of immunoglobulins in the wall of acute rejected vein allografts.

Materials and methods: Flow cytometry was used for the analysis of day 0, 14 and 30 sera obtained from Lewis recipients of isogeneic iliolumbar vein grafts (group A) or Brown-Norway grafts (group B, C) for the presence of donor specific anti-MHC class I and II antibodies. Tacrolimus 0.2 mg/kg daily was administered from day 1 to day 30 (group C). Histology was performed on day 30.

Results: Sera obtained preoperatively and on day 30 were compared in all groups. The statistically significant decrease of anti MHC class I and II antibody binding was observed only in allogenic non-immunosuppressed group B (splenocytes: MHC class I - day 0 (93% ± 7% ) vs day 30 (66% ± 7%), p = 0.02, MHC class II - day 0 (105% ± 3% ) vs day 30 (83% ± 5%), p = 0.003; B-cells: MHC class I - day 0 (83% ± 5%) vs day 30 (55% ± 6%), p = 0.003, MHC class II - day 0 (101% ± 1%) vs day 30 (79% ± 6%), p = 0.006; T-cells: MHC class I - day 0 (71% ± 7%) vs day 30 (49% ± 5%), p = 0.04). No free clusters of immunoglobulin G deposition were detected in any experimental group.

Conclusion: Arterialized venous allografts induce strong donor-specific anti-MHC class I and anti-MHC class II antibody production with subsequent immune-mediated destruction of these allografts with no evidence of immunoglobulin G deposition. Low-dose tacrolimus suppress the donor-specific antibody production.

Figure 1. Dynamic of anti splenic cells MHC class I and II antibodies concentrations.

The percentage binding of the fluorescence-labelled MHC class I (A) and MHC class II (B) antibody to BN splenic cells in the presence of sera from group A, B, or C obtained on day 0, 14, or 30 after transplantation. Sera from the allogeneic non-immunosuppressed group B, taken on day 30, significantly inhibited fluorescence-labelled MHC class I and MHC class II antibody binding to splenic cells, compared to day 0 sera. Error bars represent SEM, *p<0.05, **p<0.01.

Figure 2. Dynamic of anti splenic B-cells MHC class I and II antibodies concentrations.

The percentage binding of the fluorescence-labelled MHC class I (A) and MHC class II (B) antibody to BN splenic B-cells, identified as CD45RA-positive cells, in the presence of sera from group A, B, or C obtained on day 0, 14, or 30 after transplantation. Only sera from the allogeneic non-immunosuppressed group B, taken on day 14 or day 30, significantly inhibited fluorescence-labelled MHC class I and MHC class II antibody binding to splenic B-cells. Error bars represent SEM, *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Dynamic of anti splenic T-cells MHC class I and II antibodies concentrations.

The percentage binding of the fluorescence-labelled MHC class I (A) and MHC class II (B) antibody to BN splenic T-cells, identified as CD3-positive cells, in the presence of sera from group A, B, or C obtained on day 0, 14, or 30 after transplantation. Only sera from the allogeneic non-immunosuppressed group B obtained on day 14 or day 30 showed significant inhibition of fluorescence-labelled MHC class I antibody binding to splenic T-cells. Error bars represent SEM, *p<0.05, **p<0.01, ***p<0.001.

Figure 4. Histological features of rejected non-immunosuppressed alloveins.

Representative light microscopic photograph showing the histological features of alloveins in non-immunosuppressed rats (group B) 30 days following transplantation into the infrarenal abdominal aorta: A - vein stained for immunoglobulins with a fluorescein isothiocyanate-conjugated antibody (Chemicon International Inc, Temecula, California, USA). No free clusters of immunoglobulins were detected in the allovenous wall. B - vein stained for CD4+ cells with anti-CD4 antibody (OX-8, Cymbus Biotechnology LTD, Southampton, UK) as described previously. Massive infiltration of CD4+ immunocompetent cells (stained brown) led to the destruction of allovenous wall with no histological signs of arterialisation. Original magnification ×100.

Figure 5. Histological features of arterialised immunosuppressed alloveins.

Representative light microscopic photograph showing the histological features of alloveins in immunosuppressed rats with tacrolimus (group B) 30 days following transplantation into the infrarenal abdominal aorta. Vein stained for immunoglobulins with a fluorescein isothiocyanate-conjugated antibody (Chemicon International Inc, Temecula, California, USA). No free clusters of immunoglobulins were detected in the thick arterialised wall of venous allograft. Original magnification ×400. L – lumen, I – tunica intima, M – tunica media, A – tunica adventitia.