Disruption of murine cardiac allograft acceptance by latent cytomegalovirus

Abstract

Cytomegalovirus (CMV) reactivation is a well-described complication of solid organ transplantation. These studies were performed to (1) determine if cardiac allograft transplantation of latently infected recipients results in reactivation of CMV and (2) determine what impact CMV might have on development of graft acceptance/tolerance. BALB/c cardiac allografts were transplanted into C57BL/6 mice with/without latent murine CMV (MCMV). Recipients were treated with gallium nitrate induction and monitored for graft survival, viral immunity and donor reactive DTH responses. Latently infected allograft recipients had approximately 80% graft loss by 100 days after transplant, compared with approximately 8% graft loss in naïve recipients. PCR evaluation demonstrated that MCMV was transmitted to cardiac grafts in all latently infected recipients, and 4/8 allografts had active viral transcription compared to 0/6 isografts. Latently infected allograft recipients showed intragraft IFN-alpha expression consistent with MCMV reactivation, but MCMV did not appear to negatively influence regulatory gene expression. Infected allograft recipients had disruption of splenocyte DTH regulation, but recipient splenocytes remained unresponsive to donor antigen even after allograft losses. These data suggest that transplantation in an environment of latent CMV infection may reactivate virus, and that intragraft responses disrupt development of allograft acceptance.

Figure 1. Cytomegalovirus and cardiac graft survival

C57BL6 (H2b) mice received BALB/c (H2d) cardiac allografts or C57BL6 isografts. A. Recipients latently infected with murine cytomegalovirus (MCMV) received allografts (◆D-R+ Allograft) or isografts (▲D-R+ Isograft) and were compared with non-infected allograft recipient (■D-R- Allograft) for graft survival. Kaplan—Meier analysis shows that latently infected D-R+ allograft recipients have significantly worse graft survival than non-infected D-R-allograft recipients (21% versus 92%, p<0.0001). All D-R+ cardiac isografts remain functional beyond 100 days post-transplant. B. Latent infection of donor allografts (oD+R-) showed 100 day graft survival comparable to non-infected D-R-allografts (p=0.59). Sensitization (without infection) using heat killed MCMV (□D-R- HK-MCMV) also had no significant influence upon graft survival. Non-infected C57BL/6 mice not treated with gallium nitrate (historical controls) promptly reject their BALB/c grafts (ΔD-R- GN-). Cardiac graft function was assessed by trans-abdominal palpation.

Figure 2. Pathologic changes in cardiac allografts of recipients latently infected with murine cytomegalovirus (MCMV)

Histology in cardiac allografts (BALB H2^d) from MCMV-naïve C57BL/6 recipients compared to allografts and isografts from MCMV-positive C57BL/6 recipients 21 and 45 days post-transplant. A,D&C. Representative H&E (A&D) 21 and 45 days after transplant show typical cellular infiltration associated with this model, and trichrome sections (G) show mild patchy fibrosis in CMV-naïve recipient allografts. B&E. In contrast, CMV-positive recipient allografts show prominent cellular infiltration 21 days after transplant (B), which is more pronounced by day 45 (E) and is associated with vasculitis (black arrow) with fibrin (red arrow) deposition in lumen. H. Day 45 trichrome staining shows advanced fibrosis (blue staining). C,F,G. Isografts from CMV-positive recipients show bland myocardium, with very lile cellular infiltrate at either time point, and nominal fibrosis at day 45. Specimens were formalin fixed and embedded. Magnification = 40X, except for D & E 100X.

Figure 3. Molecular reactivation of latent murine cytomegalovirus (MCMV) after cardiac transplantation

Allograft and isograft recipients latently infected with MCMV were evaluated 21 days post-transplant by focused expansion assay for MCMV glycoprotein-B (GB) mRNA transcription using nested RT-PCR. A. Allografts from latently infected recipients show definitive molecular MCMV reactivation in 4/8. B. Isografts from latently infected mice showed no evidence of molecular reactivation (0/6). Both allografts and isografts contain MCMV DNA confirming prior infection and transmission in all recipients. Each lane represents DNA or mRNA from one graft. Presence of β-actin confirms recovery of RNA and controls are technique controls. No-RT controls performed concurrently with all RT-PCR confirm absence of DNA contamination (not shown).

Figure 4. Changes in immune regulation in cardiac allograft recipients latently infected with murine cytomegalovirus (MCMV)

Splenocytes isolated from MCMV-naive (D-R-) and MCMV-positive (D-R+) cardiac allograft recipients between 33 and 110 days post-transplant were evaluated using trans-vivo DTH. Briefly, splenocytes (Spl) obtained from individual mice were combined with sub-cellular donor alloantigen (DonAg) alone or with polyclonal antibodies to TGF-β or IL-10, tetanus toxoid (TT) with/without DonAg, or heat inactivated MCMV with/without DonAg, and injected into pinnae of naïve C57BL/6 mice. All mice were sensitized to TT prior to evaluation. A. As previously published, MCMV-negative allograft acceptors demonstrate regulation of donor-reactive DTH responses that are restored by antibodies to TGF-β or IL-10. Further, immune regulation extends to suppress TT-reactive DTH responses when donor alloantigen is present at the DTH site, so-called “linked non-responsiveness”. As expected MCMV-naïve splenocytes did not show a full response when challenged with MCMV antigen. B. Splenocytes from recipients latently infected with MCMV no longer demonstrate TGF-β or IL-10 regulation, but interestingly remain unresponsive to donor antigen. In addition, there is disruption of linked non-responsiveness, as prominent CMV- and TT-reactive DTH responses are not suppressed when co localized with donor antigen at DTH sites. DTH responses are measured after 24 hours as the change in ear thickness (mean ± SD × 10⁻⁴ inches). Each bar represents results from 3-6 mice (D-R-) or 5 mice (D-R+).

Figure 5. Influence of murine cytomegalovirus (MCMV) on regulatory and inflammatory mediator mRNA in cardiac grafts

Real-time RT-PCR was performed on heterotopic cardiac grafts 21 days after transplantation for regulatory genes Foxp3, IDO, IL-10, and TGF-β, or inflammatory genes IFN-α and IL-12. Groups were allograft or isograft recipients latently infected with MCMV (CMV+ Allo or CMV+ Iso respectively) and non-infected allograft recipients (CMV- Allo). Allografts show significantly higher expression of regulatory genes Foxp3, IDO, and TGF-β compared to isografts independent of MCMV. Recipient MCMV does not appear to negatively influence graft expression of regulatory genes Foxp3, IDO, IL-10, or TGF-β, as CMV+ allografts have significantly higher transcript levels than CMV- allografts. In contrast, MCMV does appear to influence inflammatory mediator expression, as CMV+ allografts show significantly increased IFN-α mRNA compared to CMV- allografts. Each point represents mean ± standard error mRNA frequency (mRNA relative to GAPDH) from n=1 mouse (performed in duplicate).

Figure 6. Foxp3 staining of cardiac allografts

Immunohistochemical staining was performed on representative sections from CMV-positive and negative allografts 21 and 45 days after transplantation. A&D. Sections from CMV-negative allografts show numerous CD3+ cells (brown) with scattered Foxp3+ cells (blue). B&E. CMV-positive allografts show a similar staining pattern at day 21, but have more numerous Foxp3+ cells at day 45 after transplant. C&F. 40X views of CMV-positive grafts, clearly showing persistence of Foxp3+ T-cells (blue).