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. 2021 Feb;21(2):515-524.
doi: 10.1111/ajt.16197. Epub 2020 Aug 17.

Acute murine cytomegalovirus disrupts established transplantation tolerance and causes recipient allo-sensitization

Shuangjin Yu  1   2 , Anil Dangi  1 , Melanie Burnette  1 , Michael M Abecassis  3 , Edward B Thorp  4 , Xunrong Luo  1   5
Affiliations

Acute murine cytomegalovirus disrupts established transplantation tolerance and causes recipient allo-sensitization

Shuangjin Yu et al. Am J Transplant. 2021 Feb.

Abstract

We have previously shown that acute cytomegalovirus (CMV) infection disrupts the induction of transplantation tolerance. However, what impact acute CMV infection would have on the maintenance of established tolerance and on subsequent recipient allo-sensitization is a clinically important unanswered question. Here we used an allogeneic murine islet transplantation tolerance model to examine the impact of acute CMV infection on: (a) disruption of established transplantation tolerance during tolerance maintenance; and (b) the possibility of recipient allo-sensitization by CMV-mediated disruption of stable tolerance. We demonstrated that acute CMV infection abrogated transplantation tolerance during the maintenance stage in 50%-60% recipients. We further demonstrated that acute CMV infection-mediated tolerance disruption led to recipient allo-sensitization by reverting the tolerant state of allo-specific T cells and promoting their differentiation to allo-specific memory cells. Consequently, a second same-donor islet allograft was rejected in an accelerated fashion by these recipients. Our study therefore supports close monitoring for allo-sensitization in previously tolerant transplant recipients in whom tolerance maintenance is disrupted by an episode of acute CMV infection.

Keywords: alloantibody; basic (laboratory) research/science; immunobiology; immunosuppression/immune modulation; infection and infectious agents - viral: Cytomegalovirus (CMV); islet transplantation; re-transplantation; rejection: T cell-mediated (TCMR); tolerance.

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Figures

Figure 1.
Figure 1.. Islet allograft survival in donor ECDI-SP tolerized recipients acutely infected with MCMV on day 0, day 14 and day 95.
(A) Schematic treatment timeline of different groups. Islet transplantation was performed on day 0 from BALB/c donors to diabetic B6 recipients. Transplantation tolerance was induced by BALB/c ECDI-SP infusions on day -1 and day 7. Recipients were infected with 1× 108 pfu of the MCMV strain Δm157 intraperitoneally on day 0, day 14 or day 95. (B) Islet allograft survival (y axis) plotted as a function of “days post infection” (x axis). pfu: plaque forming unit. N = 8–20 in each group. *P＜0.05; ns = no significance.
Figure 2.
Figure 2.. Survival of the second BALB/c islet allograft in previously tolerized recipients rejecting their first BALB/c islet allograft in setting of MCMV infection.
(A) Schematic timeline of the second islet transplantation. Tolerized recipients were infected with MCMV and subsequently rejected their BALB/c islet allograft (indicated as “Day -10”). Ten days later (indicated as “Day 0”), a second BALB/c islet allograft was transplanted underneath the kidney capsule of the contralateral kidney (the “Tol-MCMV-Rej” group). As a tolerant control, we contemporaneously re-transplanted a second BALB/c islet allograft into tolerized recipients without an episode of acute MCMV infection (the “Tol” group). Naïve B6 recipients were also transplanted with BALB/c islet allografts without any treatment (the “Naïve” group) for comparison of rejection kinetics. (B) Islet allograft survival (y axis) plotted as a function of “days post transplantation” of the second BALB/c islet allograft (x axis). N = 2–4 in each group. *P＜0.05.
Figure 3.
Figure 3.. Cellular immunity during accelerated graft rejection indicates recipient sensitization.
(A, B) Tolerant recipients infected with MCMV on day 14 post the first BALB/c islet transplant were sacrificed as soon as graft rejection was confirmed (the “Tol+MCMV+Rej” group). Tolerant recipients without MCMV infection (the “Tol” group) were sacrificed contemporaneously as the control. Islet allografts and sera were collected at the time of sacrifice, and examined for graft-infiltrating T cells (A) and circulating DSAs (B). (C, D) Recipients re-transplanted with a second BALB/c islet allograft as outlined in Figure 2A were sacrificed on day 3 post the second transplant. The second islet allografts and sera were collected at the time of sacrifice, and examined for graft-infiltrating T cells (C) and circulating DSAs (D). An additional “naïve” group was also transplanted for comparison in which a BALB/c islet allograft was transplanted to naïve B6 recipients and sacrificed on day 3 post-transplant. Data are presented as mean ± SD in all scatter plots and bar graphs. N = 3 in all groups. MFI, mean fluorescent intensity. *P＜0.05; ns = no significance.
Figure 4.
Figure 4.. MCMV infection reverts the inhibitory effects of donor ECDI-SP on allo-specific T cells.
(A) Schematic experimental timeline. B6 mice were tolerized by intravenous injection of 108 BALB/c ECDI-SP on day -7 and day 1. Allogeneic challenge was provided on day 0 by an intraperitoneal injection of 106 fresh BALB/c splenocytes. These mice were then either infected with 108 pfu MCMV (Δm157) intraperitoneally on day 14 (the “(+)MCMV” group) or not (the “(-)MCMV” group). All mice were sacrificed on day 21, and splenic T cells were purified for donor-stimulated MLR in vitro. (B) Purified T cells were labelled with the proliferation dye V450 and co-cultured with BALB/c enrich dendritic cells (eDCs) at a ratio of 1:1 for 5 days. T cell proliferation was determined by V450 dilution and compared between groups. N = 4 in both groups. (C) Schematic experimental timeline. The experimental timeline is the same as in (A) with the exception of additional adoptive transfer of 5×105 V450-labele 4C CD4 T cells on day -9. (D) Number of 4C CD4 T cells in the spleen. (E) In vivo proliferation of splenic 4C CD4 T cells. (F) In vivo IFN-γ production by splenic 4C CD4 T cells. (D) - (F): N = 3 in all groups. Data are presented as mean ± SD in all scatter plots. *P＜0.05.
Figure 5.
Figure 5.. Tolerized recipient splenic T cells following MCMV infection exhibit memory phenotype and function.
Tolerant recipients infected with MCMV on day 14 post the first BALB/c islet transplant were sacrificed 10 days after graft rejection was confirmed (the “Tol+MCMV+Rej” group). Tolerant recipients without MCMV infection (the “Tol” group) were sacrificed contemporaneously as the control. Naïve B6 mice (“Naïve” group) were sacrificed and similarly examined for comparison. Spleens from mice of various groups were harvested, and splenic T cells were isolated and analyzed. (A) Percentage of CD44high T cells among total splenic T cells. (B) Representative histograms (left panel) and scatter plot (right panel) of CD127 expression among CD44high T cells from various groups. (C) T cells from various groups were labeled with V450 and subjected to in vitro BALB/c eDC re-stimulation (T:eDC ratio 1:1) for 3 days. T cell proliferation (by V450 dilution) is shown in representative histograms (left panel), and quantified and compared in scatter plot (right panel). (A) – (C): N = 3–4 in each group. Data are presented as mean ± SD in all scatter plots. *P＜0.05; ns = no significance.
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