Murine cytomegalovirus dissemination but not reactivation in donor-positive/recipient-negative allogeneic kidney transplantation can be effectively prevented by transplant immune tolerance

Abstract

Cytomegalovirus (CMV) reactivation from latently infected donor organs post-transplantation and its dissemination cause significant comorbidities in transplant recipients. Transplant-induced inflammation combined with chronic immunosuppression has been thought to provoke CMV reactivation and dissemination, although sequential events in this process have not been studied. Here, we investigated this process in a high-risk donor CMV-positive to recipient CMV-negative allogeneic murine kidney transplantation model. Recipients were either treated with indefinite immunosuppression or tolerized in a donor-specific manner. Untreated recipients served as controls. Kidney allografts from both immunosuppressed and tolerized recipients showed minimal alloimmunity-mediated graft inflammation and normal function for up to day 60 post-transplantation. However, despite the absence of such inflammation in the immunosuppressed and tolerized groups, CMV reactivation in the donor positive kidney allograft was readily observed. Interestingly, subsequent CMV replication and dissemination to distant organs only occurred in immunosuppressed recipients in which CMV-specific CD8 T cells were functionally impaired; whereas in tolerized recipients, host anti-viral immunity was well-preserved and CMV dissemination was effectively prevented. Thus, our studies uncoupled CMV reactivation from its dissemination, and underscore the potential role of robust transplantation tolerance in preventing CMV diseases following allogeneic kidney transplantation.

Keywords: apoptotic cell therapy; cytomegalovirus reactivation; immunosuppression; kidney transplant; transplant immune tolerance.

Figure 1.. Gross appearance and histopathological assessments of kidney allografts on day 60 post-transplantation.

(A) Representative images demonstrating gross appearance of kidney allografts from untreated (Unt), chronic immunosuppression-treated (IS), and donor ECDI-SP-treated (Tol) transplant recipients. The scatter graph represents the weight of the kidney allografts in indicated groups. (B) Representative PAS-stained sections from the indicated groups showing allograft inflammation (original magnification: 200×). Scatter graphs show quantitative assessment of glomerulitis, tubulitis and interstitial inflammation in the kidney allografts. (C) Representative images of kidney allografts demonstrating fibrosis by Sirius Red staining (original magnification: 200×). Scatter graph shows quantitative assessment of glomerular and interstitial fibrosis in the kidney allografts. Pathology score scale, 0=none, 1=minimal, 2=mild, 3=moderate, and 4=severe. N=4–5 kidney allografts per group. *P <0.05 (ANOVA). For (B) and (C), scale Bar = 100 μm.

Figure 2.. Whole blood chemistry measured from samples collected on day 60 post-transplant.

(A) Scatter graph demonstrating serum creatinine (Cr) levels in indicated groups. (B) Scatter graph demonstrating blood urea nitrogen (BUN) levels in indicated groups. Unt: N=12; IS: N=6; Tol: N=7. *P <0.05 (ANOVA).

Figure 3.. Anti-donor antibody levels in recipient sera on day 60 post-transplantation.

(A) Representative overlaid FACS histograms showing the levels of specific anti-donor antibody sub-classes (IgG1, IgG2b, IgG2c, and IgG3) in indicated groups. (B) Scatter graphs showing mean fluorescence intensity (MFI) of specific anti-donor antibody subclasses in indicated groups. Data were normalized using naïve mice serum. N=4–5 recipients per group. *P <0.05 (ANOVA).

Figure 4.. Intragraft infiltration of T cells and their expression of the inflammatory cytokine IFNγ on day 60 post-transplantation.

(A) and (B) Scatter graphs showing total number of infiltrating CD8 and CD4 T cells per allograft in indicated groups. Cell numbers were calculated based on FACS analysis. (C) and (D) Representative FACS plots demonstrating IFNγ expression by CD8 T cells ((C), gated on live CD45⁺CD3⁺CD8⁺ cells) and CD4 T cells ((D), gated on live CD45⁺CD3⁺CD4⁺ cells) in indicated groups. Right scatter graphs showing the total number of intragraft IFN⁺ CD8 and CD4 T cells enumerated by FACS analysis. Data were compiled from 3 independent experiments with a total of 4–5 mice per group. *P <0.05 (ANOVA).

Figure 5.. Intragraft MCMV-specific CD8 T cells in Unt, IS and Tol groups.

(A) Representative FACS plots demonstrating intragraft MCMV epitope m38-specific CD8 T cells (gated on total live CD45⁺CD3⁺CD8⁺ T cells) in indicated groups. Cells were stained with the B6 MHC I tetramer H-2K^b:m38. (B) Scatter graph showing the percentages of m38-specific CD8 T cells in indicated groups. Data were compiled from 4 independent experiments with a total number of 5–6 mice per group. Statistical analysis was performed by ANOVA, and no significance (NS) was observed.

Figure 6.. MCMV DNA (IE1 gene) in the kidney allografts and other organs detected by quantitative polymerase chain reaction.

(A) Viral DNA in the kidney allograft, the liver and the lungs collected on day 28 post-transplantation. (B) Viral DNA in the kidney allograft and the salivary gland on day 60 post-transplantation. All data of kidney allografts were first normalized to the expression levels of β-actin, then compared to the levels of the contralateral kidney removed on the day of transplantation (day 0) from the same BALB/c donor latently infected with MCMV. Data of the liver, lungs and salivary gland were similarly first normalized to β-actin and then compared to those of respective organs harvested from uninfected B6 host. Day 28 data were from 2 recipients per group. Day 60 data were from 4 to 5 recipients per group, and *P <0.05 (ANOVA).

Figure 7.. Expression of IFN-γ by intragraft MCMV-specific CD8⁺ T cells on day 60 post-transplantation.

(A) Representative FACS plots demonstrating the expression of IFNγ by intracellular staining. Plots were gated on H-2K^b:m38⁺CD8⁺CD44⁺ T cells in the kidney allografts. (B) Scatter plot demonstrating total number of IFNγ expressing tetramer⁺CD8⁺CD44⁺ T cells in kidney allografts as shown in (A). (C) Scatter graph showing mean fluorescent intensity (MFI) of IFNγ expression by tetramer⁺CD8⁺CD44⁺ T cells as shown in (A). Data were compiled from 3 independent experiments with a total of 4 mice per group. *P <0.05 (ANOVA).