Apoptosis within spontaneously accepted mouse liver allografts: evidence for deletion of cytotoxic T cells and implications for tolerance induction

Abstract

MHC-mismatched liver grafts are accepted spontaneously between many mouse strains. The underlying mechanism(s) is unclear. In the B10 (H2(b)) to C3H (H2(k)) strain combination used in this study, donor T cells within the liver were rapidly replaced within 2 to 4 days of transplantation with those of the recipient. Freshly isolated liver graft-infiltrating cells harvested on days 4 and 7 exhibited strong CTL responses against donor alloantigens. CTL activity was reduced substantially, however, by day 14, although levels of CTL precursors in the spleen and liver remained high. Examination of the liver allografts by in situ terminal deoxynucleotidyltransferase-catalyzed dUTP-digoxigenin nick end labeling on days 4, 7, and 14 after transplantation revealed prominent apoptotic cells dispersed throughout the nonparenchymal cell population. When acute liver allograft rejection was induced by administration of IL-2 from days 0 to 4 post-transplant (median survival time, 5 days), apoptotic activity (day 4) was reduced substantially, whereas CTL activity was enhanced. Nonparenchymal cells isolated from allografts of unmodified recipients 4, 7, and 14 days after transplantation exhibited significantly higher DNA fragmentation after 18-h culture than cells from liver isografts. Moreover, the level was 4 to 5 times higher than that of cells from IL-2-treated mice (on day 4). These observations suggest that T cell deletion, not regulation, may be responsible for spontaneous liver allograft acceptance. The molecular recognition events that cause apoptosis of infiltrating T cells and why this occurs within liver grafts, but not heart or skin grafts, remain to be elucidated.

FIGURE 1

Flow cytometric analysis (MHC class I staining) of donor (H2K^b+) and recipient (H2K^k+) T cells and subsets in freshly isolated NPC populations from liver allografts (B10→C3H) at various times after transplantation. The results are representative of three separate experiments.

FIGURE 2

Cytotoxic activity of freshly isolated liver NPC (A) and spleen cells (B) from C3H recipients of B10 hepatic allografts. A 4-h ⁵¹Cr release assay was used to determine cytotoxicity against target cells expressing donor (H2^b) alloantigen 4, 7, 14, and 150 days after transplantation. Third-party (H2^d) and syngeneic (H2^k) target cells were also used. Cells were pooled from groups of three transplanted animals, and the experiment was performed three times.

FIGURE 3

Generation of CTL from the spleens of C3H (H2^k) mice that received an orthotopic B10 (H2^b) liver transplant 4, 7, 14, or 150 days previously. Splenic T cells were incubated for 96 h with γ-irradiated (20 Gy) B10 spleen cell stimulators at a low stimulator:responder ratio (1:4) then used as effectors. The effector cells were cultured with target cells expressing the haplotype of donor (EL4; H2^b; □) or with third-party (P815; H2^d; △) or syngeneic (R1.1; H2^k; ●) targets. Cells were pooled from groups of three transplanted animals, and the experiment was performed four times. Results are the mean ± 1 SD.

FIGURE 4

Apoptotic cells identified in portal areas of liver grafts by TUNEL staining, as described in Materials and Methods. A, Isograft (C3H→C3H), day 7; B, allograft (B10→C3H), day 4; C, allograft, day 7; D, allograft plus IL-2 administration, day 4. The cells were counterstained with hematoxylin. Magnification: A, B, and D, ×400; C, ×200.

FIGURE 5

Incidence of apoptotic cells determined, as described in Materials and Methods, by in situ nick end labeling (TUNEL) within the NPC (infiltrate) and parenchymal cell populations of hepatic transplants (isografts, allografts, and allografts plus IL-2) at various times after transplantation.

FIGURE 6

DNA fragmentation in NPC isolated from liver allografts 4, 7, and 14 days after transplantation. The cells were cultured overnight (18 h) without stimulation. DNA fragmentation was then measured as described in Materials and Methods. Cells isolated from liver isografts and from liver allografts of mice treated with systemic mouse rIL-2 (MST 5 days) were also examined. Cells were pooled from groups of three transplanted animals, and the experiment was performed twice. Results are the mean ± 1 SD.