Murine liver allograft transplantation: tolerance and donor cell chimerism

Abstract

Nonarterialized orthotopic liver transplantation with no immunosuppression was performed in 13 mouse-strain combinations. Two strain combinations with major histocompatibility complex class I and class II and minor histocompatibility complex disparity had 20% and 33% survival of more than 100 days, but the other 11 combinations, including four that were fully allogeneic and all with only class I, class II or minor disparities, yielded 45% to 100% survival of more than 100 days. Long-living recipients permanently accepted donor-strain heterotopic hearts transplanted on the same day or donor-strain skin 3 mo after liver transplantation, in spite of detectable antidonor in vitro activity with mixed lymphocyte reaction and cell-mediated lymphocytotoxicity testing (split tolerance). In further donor-specific experiments, liver grafts were not rejected by presensitized major histocompatibility complex class I-disparate recipients and they protected donor-strain skin grafts from second set (or any) rejection. Less frequently, liver transplantation rescued rejecting skin grafts placed 1 wk earlier in major histocompatibility complex class I, class II and minor histocompatibility complex, class II or minor histocompatibility complex-disparate strain combinations. Donor-derived leukocyte migration to the central lymphoid organs occurred within 1 to 2 hr after liver transplantation in all animals examined, persisted in the surviving animals until they were killed (> 375 days), and was demonstrated with double-immunolabeling to be multilineage. The relation of these findings to so-called hepatic tolerogenicity and to tolerance in general is discussed.

Fig. 1

Recipient spleen 7 mo after liver transplantation in the B10→C3H combination stained for donor-specific MHC class II [I_A^b] antigens. The majority of donor cells were found in the periarterial lymphatic sheath and the marginal zone (immunoperoxidase staining for I-A^b, counterstained with hematoxylin; original magnification × 40). Inset: (original magnification × 400 ) shows the individual cell staining (arrow) in greater detail.

Fig. 2

Double immunofluorescent staining (DIF) controls for Lineage identity of donor cells in recipient tissues. (a) No staining was seen in negative controls (see “Materials and Methods”; original magnification × 400). (b) DIF staining of normal B10 lymph node (positive control) with anti-I-A^b (red, non–B cells) and anti-B-220 (green, B cells). Note red cells in the paracortex, which are only positive for I-A^b; the green cells in the cortex, which are only positive for B-220; and the yellow cells (class II-positive B cells), which are double positive for both I-A^b and B-220 (original magnification × 200). (c) DIF staining of the allografted liver with anti-I-A^b (red) and anti-B-220 (green) served as an additional control (original magnification × 400) Note red-only staining of biliary epithelium (donor I-A^b) and green-only staining of infiltrating recipients B-220–positive B cells. Lack of double-staining in bile ducts excludes possibility of nonspecific cross-reactivity of immunohistochemical reagents used. Arrow highlights lipofuscin autofluorescence.

Fig. 3

Lineage identification of donor cells by double immunofluorescent staining (DIF) in recipients’ spleen 7 mo after OLT in B10→C3H combination. (a) DIF staining for donor I-A^b (red) and B-220 (B cells, green; original magnification × 100). A splenic arterial branch is located in the center. Single-positive donor non–B cells (large arrowhead, red-only) and double-positive donor B cells (small arrowhead, yellow) are located in the PALS. Inset: Yellow double-positive donor–B cell in greater detail (original magnification × 400). (b) Donor leukocytes of dendritic cell lineage could also be detected by DIF labeling (original magnification × 400). Red-only donor cell in the PALS is positive for donor I-A^b (large arrowhead), whereas green-only cells stain positive with mAb 2A1 (dendritic cell marker). The double-positive (bright yellow) cell is a donor dendritic cell (small arrowhead), which is stained for both donor class II and dendritic cell–associated antigen 2A1. (c) Donor T cells were also detected with DIF staining (original magnification × 400). Note red cells (large arrowhead), which are positive for donor MHC class I antigens (H-2K^b) alone; green cells, which are positive with mAb Thy 1.2 (T cells) alone; and donor T cell (bright yellow), which is positive for both donor class I and Thy 1.2 (small arrowhead). The Thy 1.2–stained cell could be a fibroblast, but confirmatory evidence of a donor subpopulation of T cells was obtained by double labeling with donor class I and CD4 or CD8.