Rejection responses to allogeneic hepatocytes by reconstituted SCID mice, CD4, KO, and CD8 KO mice.

INTRODUCTION: The purpose of the current study was to investigate the capacity of CD4+, CD8+, or non-T cells to independently initiate acute rejection of allogeneic hepatocytes using reconstituted SCID, CD4 or CD8 knockout (KO) recipient mice. METHODS: Allogeneic hepatocytes (FVB/N, H-2q) were transplanted into C57BL/6.SCID (H-2b), CD4 KO (H-2b), CD8 KO (H-2b), or beige/beige (H-2b) mice. SCID mice with functioning hepatocellular allografts subsequently received purified non-T cells (NTC), CD4+, or CD8+ splenocytes. Some mice were treated with anti-CD4, anti-CD8, and/or anti-nkl.1 mAb. Recipient mice were also assessed for donor-reactive delayed-type hypersensitivity (DTH) responses and donor-reactive alloantibody production. RESULTS: Median hepatocellular allograft survival time (MST) was 28 days in CD4+ reconstituted SCID mice and 14 days in CD8+ reconstituted SCID mice. SCID hosts reconstituted with NTC demonstrated indefinite hepatocellular allograft survival (>120 days). MST was 10 days in untreated beige/beige (NK cell deficient) mice. MST was 14 days in untreated, 35 days in anti-CD4 mAb treated, and 10 days in anti-nkl.1 mAb treated CD8 KO mice. MST was 10 days in untreated, 35 days in anti-CD8 mAb treated, and 7 days in anti-nk1.1 mAb treated CD4 KO mice. Donor-reactive DTH responses were not detected in reconstituted SCID mice, were minimal in CD4 KO mice, and were prominent in CD8 KO mice after rejection of allogeneic hepatocytes. Similarly, donor-reactive alloantibody, was not detected in CD4 KO hosts, but was readily detected in CD8 KO hosts. CONCLUSIONS: These studies show that both CD4+ and CD8+ T cells (but not host NTC) can independently initiate the rejection of allogeneic hepatocytes. While hepatocyte rejection by isolated CD4+ T cells is not surprising, rejection by CD8+ T cells (in the absence of CD4+ T cells) was unusual, and may explain the failure of "standard" immunosuppressive regimens to suppress acute rejection of allogeneic hepatocytes, as noted in prior studies. Furthermore, NK cells do not appear to be required for either CD4+ T cell or CD8+ T cell initiated hepatocyte rejection.

Patterns of immune responses evoked by allogeneic hepatocytes: evidence for independent co-dominant roles for CD4+ and CD8+ T-cell responses in acute rejection.

INTRODUCTION: This is the first in a series of reports that characterizes immune responses evoked by allogeneic hepatocytes using a functional model of hepatocyte transplantation in mice. METHODS: "Donor" hepatocytes expressing the transgene human alpha-1-antitrypsin (hA1AT-FVB/N, H2q) were transplanted into C57BL/6 (H2b) or MHC II knockout (H2b) hosts treated with anti-CD4, anti-CD8, or a combination of anti-CD4 and anti-CD8 monoclonal antibodies (mAbs). Hepatocyte rejection was determined as a loss of circulating ELISA-detectable transgene product (hA1AT). In addition, some C57BL/6 mice underwent transplantation with FVB/N heterotopic cardiac allografts and were treated with anti-CD4 mAb. Cardiac allograft rejection was determined by palpation. Graft recipients were tested for donor-reactive alloantibodies and donor-reactive delayed-type hypersensitivity (DTH) responses. RESULTS: The median survival time (MST) of allogeneic hepatocytes in normal C57BL/6 mice was 10 days (no treatment), 10 days (anti-CD4 mAb), 14 days (anti-CD8 mAb), and 35 days (anti-CD4 and anti-CD8 mAbs). The MST of hepatocytes in B6 MHC class II knockout mice was 10 days (no treatment) and 21 days (anti-CD8 mAb). The MST of cardiac allografts was 11 days (no treatment) and >100 days (anti-CD4 mAb). Donor-reactive DTH responses were readily detected in both untreated and mAb-treated recipients. Donor-reactive alloantibody was barely detectable in untreated hosts. CONCLUSIONS: These studies demonstrate that allogeneic hepatocytes are highly immunogenic and stimulate strong cell-mediated immune responses by both CD4+ and CD8+ T cells, even when treated with agents that can cause acceptance of cardiac allografts. Indeed, CD4+ or CD8+ T cells seem to independently cause hepatocellular allograft rejection. Allogeneic hepatocytes evoked strong donor-reactive DTH responses but were poor stimuli for donor-reactive antibody production. This is an unusual pattern of immune reactivity in allograft recipients.

Efficacy of anti-intercellular adhesion molecule-1 immunotherapy on immune responses to allogeneic hepatocytes in mice.

Adhesion molecules appear to play important roles in vascularized organ allograft rejection, because antibodies directed against them are effective in prolonging survival of vascularized organ allografts in rodents. However, the efficacy of these agents for cellular allografts is unknown. The current studies were undertaken to determine the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on host immune responses to purified hepatocytes. Host mice (C3H, H-2(k)) grafted with hepatocytes in sponge matrix allografts (HC-SMA) received IgG isotype control, anti-ICAM-1, or anti-VCAM-1 monoclonal antibody (mAb) on days 0 through 9 after grafting. Twelve to 14 days later, host cells infiltrating the HC-SMA were assessed for the development of allospecific cytolytic T cells (allo-CTLs). Treatment with anti-ICAM-1 or anti-VCAM-1 mAb resulted in significantly decreased recruitment of host cells into HC-SMA (P < .035). However, only anti-ICAM-1 mAb resulted in abrogation of development of allo-CTLs in HC-SMA (P = .001). C3H (H-2(k)) hosts grafted with allogeneic hepatocytes from control C57BL/6 (H-2(b)) or ICAM-1 knockout [H-2(b)] mice elicited the development of allo-CTLs in HC-SMA (P = not significant). Furthermore, there was no difference in the development of allo-CTLs in HC-SMA of control hosts [C57BL/6, H-2(b)] compared with ICAM-1 knockout hosts (H-2(b)) (P = not significant). Treatment with anti-ICAM-1 mAb had no effect on the development of allo-CTLs in ICAM-1 knockout (H-2(b)) hosts bearing HC-SMA. The immunosuppressive effect of host treatment with anti-ICAM-1 mAb does not appear to be a consequence of simple blockage of donor hepatocyte or host immune cell expression of ICAM-1, but suggests a potential inhibitory effect on host immune cell activation or function, as well as an effect on recruitment of host cells to the allograft.

Effect of tumor necrosis factor alpha and intercellular adhesion molecule-1 expression on immunogenicity of murine liver cells in mice.

Adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) have been implicated in the pathogenesis of various inflammatory liver disease states, including viral and autoimmune hepatitis as well as liver allograft rejection. Tumor necrosis factor alpha (TNF-alpha) is an inflammatory cytokine known to up-regulate adhesion molecules as well as major histocompatibility complex (MHC) class I expression, and has been demonstrated to be important in the rejection of vascularized organ allografts. The current studies address the effect of TNF-alpha and the role of ICAM-1 expression on liver cell immunogenicity in vitro in mixed lymphocyte hepatocyte culture (MLHC), in vitro in mixed lymphocyte liver nonparenchymal cell culture (MLNPC), in vivo in hepatocyte sponge matrix allografts (HC-SMA), and in vivo in liver nonparenchymal cell sponge matrix allografts (NPC-SMA). Purified allogeneic hepatocytes (HC) and liver nonparenchymal cells (NPC) under naive, unstimulated conditions demonstrated different profiles of MHC antigen and adhesion molecule expression, but both liver cell populations stimulated the proliferation and development of allospecific cytotoxic effectors in vitro and in vivo. Despite significant up-regulation of MHC class I and ICAM-1 on both HC and liver NPCs by in vivo treatment with TNF-alpha, the immunogenicity of TNF-alpha-stimulated liver cells was not appreciably different from naive, unstimulated liver cells. In contrast, ICAM-1-negative HC and NPCs were significantly less immunogenic both in terms of lymphocyte proliferative responses and the generation of allospecific cytolytic effectors. These results suggest that constitutive expression of ICAM-1 enhances the immunogenicity of "donor" liver cells but is not absolutely required to elicit immune responses to allogeneic liver cells. Further studies to determine the role of adhesion molecule expression on trafficking of host immune cells to the liver and the role of adhesion molecule expression by host cells are required to clarify their role in immune responses to liver cells.

In vivo immunogenicity of purified allogeneic hepatocytes in a murine hepatocyte transplant model.

BACKGROUND: It has been reported previously that liver grafts and liver cells seem to be tolerogenic, based on the high frequency of spontaneous tolerance after orthotopic liver transplantation in rodents and on the phenomenon of portal venous tolerance in other models. The purpose of the current study was to characterize in vivo immune responses to allogeneic hepatocytes transplanted into the portal circulation. METHODS: In this functional model of hepatocyte transplantation, "donor" hepatocytes from mice transgenic for human alpha1-antitrypsin (hA1AT) were transplanted by intrasplenic injection into host mice and the secreted hA1AT protein measured in host serum to determine hepatocellular graft survival. Host immune responses were assessed by measurement of donor-specific alloantibodies and delayed-type hypersensitivity responses. In some experiments, liver nonparenchymal cells (NPCs) were co-transplanted with the allogeneic hepatocyte transplant. RESULTS: Allogeneic hepatocyte transplant into immunocompetent hosts resulted in loss of host serum hA1AT by days 7-10 after transplant, whereas syngeneic hosts maintained long-term hepatocellular graft survival as reflected by persistence of serum hA1AT for > 20 weeks. Allogeneic hepatocyte transplantation resulted in the development of donor-specific alloantibody and delayed-type hypersensitivity responses, as well as a "second set" response of accelerated hepatocellular graft rejection after a second transplant. Pretransplantation or co-transplantation of donor-matched liver NPCs at the time of allogeneic hepatocyte transplantation did not prolong hepatocellular allograft survival. CONCLUSIONS: Allogeneic hepatocytes introduced into the portal circulation via intrasplenic injection are immunogenic not tolerogenic and stimulate a weak humoral and strong cell mediated host immune response in vivo. Co-transplantation or pretransplantation of allogeneic liver NPCs did not protect allogeneic hepatocytes from immunologic rejection.

A functional model of hepatocyte transplantation for in vivo immunologic studies.

BACKGROUND: Previous studies to determine the significant donor and host factors which contribute to immune responses to allogeneic hepatocytes have been pursued with in vitro and in vivo studies. However, in order to further characterize in vivo host immune responses to transplanted hepatocytes in conjunction with their consequences upon allograft survival, a functional model of hepatocyte transplantation in which the function and survival of transplanted hepatocytes could be serially assessed was pursued. METHODS: A transgenic mouse line expressing the human alpha1-antitrypsin (hA1AT) gene was developed in an FVB/N (H2q) mouse (hA1AT-FVB/N). Hepatocytes from these "donor" transgenic mice were transplanted into host mice by intrasplenic injection, and subsequent survival of the transgenic hepatocytes was determined by assay for the secreted hA1AT protein in host serum by enzyme-linked immunosorbent assay. RESULTS: Transplantation of transgenic "donor" hepatocytes (hA1AT-FVB/N) into (a) syngeneic FVB/N (H2q), (b) allogeneic immunoincompetent C57BL/6.SCID (H2b), or (c) allogeneic T cell-deficient (outbred) hosts resulted in long-term survival (> 16 weeks) of hepatocytes. Transplantation of allogeneic hepatocytes (hA1AT-FVB/N, H2q) into C57BL/6 (H2b), C3H (H2k), or BALB/c (H2d) hosts resulted in loss of hA1AT in host serum by day 10 after transplant. Likewise, transplantation of allogeneic hepatocytes into hosts deficient in B cells, CD8+ T cells, or CD4+ T cells resulted in loss of hA1AT by day 10 after transplant. CONCLUSIONS: This functional model of hepatocyte transplantation is validated for the study of host immune responses to hepatocellular grafts and to assess efficacy of strategies designed to alter these in vivo immune responses. The immunologic rejection of allogeneic hepatocytes appears to be T cell-mediated.

Costimulation pathways in host immune responses to allogeneic hepatocytes.

BACKGROUND: This study investigated the role of the CD28/B7 (blocked by CTLA4Ig) and CD40/CD40L (blocked by MR1) costimulation pathways on in vivo host T-cell- mediated immune responses to allogeneic hepatocytes. METHODS: Survival of allogeneic hepatocytes (H-2q) in C57BL/6 (H-2b) mice untreated or treated with MR1, CTLA4Ig, L6 (control fusion protein), or a combination of MR1 and CTLA4Ig fusion protein was determined. RESULTS: Median survival time for hepatocellular allografts was 10, 84, 10, 10, and 84 days in untreated (n= 10), MR1-treated (n=7) (P<.0001), CTLA4Ig-treated (n=7) (P=0.02), L6-treated (n=3) (P, not significant), and the combination of MR1- and CTLA4Ig-treated (n=6) (P=0.0003) groups, respectively. CONCLUSIONS: Host treatment with MR1, but not CTLA4Ig, prolonged hepatocellular allograft survival. These data suggest that CD28/B7 interactions appear relatively unimportant, whereas CD40/CD40L interactions provide critical costimulator signals for T-cell-dependent immune responses to allogeneic hepatocytes.