Virus-induced abrogation of transplantation tolerance induced by donor-specific transfusion and anti-CD154 antibody

Abstract

Treatment with a 2-week course of anti-CD154 antibody and a single transfusion of donor leukocytes (a donor-specific transfusion or DST) permits skin allografts to survive for >100 days in thymectomized mice. As clinical trials of this methodology in humans are contemplated, concern has been expressed that viral infection of graft recipients may disrupt tolerance to the allograft. We report that acute infection with lymphocytic choriomeningitis virus (LCMV) induced allograft rejection in mice treated with DST and anti-CD154 antibody if inoculated shortly after transplantation. Isografts resisted LCMV-induced rejection, and the interferon-inducing agent polyinosinic:polycytidylic acid did not induce allograft rejection, suggesting that the effect of LCMV is not simply a consequence of nonspecific inflammation. Administration of anti-CD8 antibody to engrafted mice delayed LCMV-induced allograft rejection. Pichinde virus also induced acute allograft rejection, but murine cytomegalovirus and vaccinia virus (VV) did not. Injection of LCMV approximately 50 days after tolerance induction and transplantation had minimal effect on subsequent allograft survival. Treatment with DST and anti-CD154 antibody did not interfere with clearance of LCMV, but a normally nonlethal high dose of VV during tolerance induction and transplantation killed graft recipients. We conclude that DST and anti-CD154 antibody induce a tolerant state that can be broken shortly after transplantation by certain viral infections. Clinical application of transplantation tolerance protocols may require patient isolation to facilitate the procedure and to protect recipients.

FIG. 1

Skin allograft survival in uninfected and LCMV-infected mice. C57BL/6 mice were treated with a single donor-specific BALB/c spleen cell transfusion and a brief course of anti-CD154 MAb and given a BALB/c skin graft as described in Materials and Methods. Transplantation was performed on small groups of animals over the course of several weeks, as animals became available. A cumulative total of 96 tolerized, transplanted animals was used in this experiment. (A) On the day after transplantation, a cohort of 20 mice was injected with LCMV; the remaining animals were untreated. Skin allograft survival in this group was statistically significantly shorter than that in the pool of all other uninfected transplanted mice (P < 0.001). (B) On day 15 after transplantation, 15 recipients from among the uninfected controls with successful grafts (A) were injected with LCMV. There was no statistically significant difference in overall graft survival between this group and the pool of all remaining uninfected animals (P = 0.06). (C) On day 29 after transplantation, seven of the recipients from among the uninfected controls that had not spontaneously rejected their grafts (B) were selected and injected with LCMV. Graft survival in this group was statistically similar to that observed in the pool of all remaining uninfected control animals (P = 0.54). (D) On days 51 to 57 after transplantation, eight recipients from among the uninfected controls that had not spontaneously rejected their grafts (C) were selected and injected with LCMV. Three of the tolerized recipients infected with LCMV received no other intervention; one recipient rejected its graft 60 days after LCMV infection. Five of the tolerized recipients infected with LCMV, whose grafts then survived 47 days after infection, were further challenged with 10⁶ PFU of VV strain WR injected directly into their grafts. One of these animals rejected its graft 30 days later; the remaining grafts survived through the end of the experiment. Graft survival in the two virus-treated groups was statistically similar to that in the pool of all remaining uninfected controls. Each panel shows graft survival relative to the day of LCMV injection. Cumulative graft survival was calculated by the method of Kaplan and Meier, which takes into account the successive removal of mice from the pool of control animals. Vertical bars indicate censored data (i.e., animals with intact grafts at the conclusion of the study and animals removed at successive intervals for use in the timed infection experiments shown in panels B and C).

FIG. 2

LCMV-induced infiltration of leukocytes into the rejecting allograft. C57BL/6 mice were pretreated with a single donor-specific BALB/c spleen cell transfusion and a brief course of anti-CD154 MAb and then given a BALB/c skin graft as described in Materials and Methods. Mice were either left untreated (A) or infected with LCMV 1 day after transplantation (B and C). (B) The early phase of graft rejection characterized by mononuclear cell infiltration of the epidermal basal layer and hair follicles. This is followed by denudation of the graft (C). (Hematoxylin and eosin stain; magnification, ×160).

FIG. 3

Skin allograft survival in LCMV-infected mice treated with anti-CD8 MAb. C57BL/6 mice were pretreated with a single donor-specific BALB/c spleen cell transfusion and a brief course of anti-CD154 MAb and then given a BALB/c skin graft as described in Materials and Methods. All mice were infected with LCMV 1 day after transplantation (arrow) and randomized into three groups. Group 1 received no further treatment. Group 2 was treated with anti-CD8 MAb on days 0, 1, and 2 relative to infection (days 1 to 3 relative to transplantation). Group 3 was treated with anti-CD8 MAb on days 8, 9, and 10 relative to infection (days 9, 10, and 11 relative to transplantation), by which time the induced T-cell response has usually cleared LCMV infection. Graft survival in groups 2 and 3 differs from that in group 1, where P values are ≤0.025.

FIG. 4

Effect of DST and anti-CD154 MAb on LCMV-induced CTL activity. C57BL/6 mice were pretreated with a single donor-specific BALB/c spleen cell transfusion and a brief course of anti-CD154 MAb and then given a BALB/c skin graft as described in Materials and Methods. Groups of mice bearing intact grafts were infected with LCMV on day 1, 15, 50, or 70 after skin transplantation. T cells from these mice were tested in 7- to 8-h chromium-51-release assays for cytotoxicity against LCMV-infected syngeneic MC57G cells (A, D, G, and J), uninfected MC57G cells (B, E, H, and K), or allogeneic P-815 cells (C, F, I, and L). Open symbols represent the percent lysis mediated by splenocytes from mice treated with DST and anti-CD154 MAb and skin grafted. Solid symbols represent cytotoxicity mediated by control thymectomized mice infected with LCMV. In the day 70 experiment, the ×-× symbols refer to lysis by cells from LCMV-infected normal nonthymectomized mice that were used as the controls.