[Show abstract][Hide abstract] ABSTRACT: Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection. Host serum alloantibody levels correlated with in vivo allospecific cytotoxic activity in CD8 knockout hepatocyte rejector mice. Host macrophage depletion, but not CD4(+) T cell, NK cell, neutrophil, or complement depletion, inhibited in vivo allocytotoxicity. Recipient macrophage deficiency delayed CD4-dependent hepatocyte rejection and inhibited in vivo allocytotoxicity without influencing alloantibody production. Furthermore, hepatocyte coincubation with alloantibody and macrophages resulted in Ab-dependent hepatocellular cytotoxicity in vitro. These studies are consistent with a paradigm of acute humoral rejection in which CD4(+) T cell-dependent alloantibody production results in the targeting of transplanted allogeneic parenchymal cells for macrophage-mediated cytotoxic immune damage. Consequently, strategies to eliminate recipient macrophages during CD4-dependent rejection pathway may prolong allograft survival.
The Journal of Immunology 08/2008; 181(2):1224-31. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Despite success of early islet allograft engraftment and survival in humans, late islet allograft loss has emerged as an important clinical problem. CD8+ T cells that are independent of CD4+ T cell help can damage allograft tissues and are resistant to conventional immunosuppressive therapies. Previous work demonstrates that islet allografts do not primarily initiate rejection by the (CD4-independent) CD8-dependent pathway. This study was performed to determine if activation of alloreactive CD4-independent, CD8+ T cells, by exogenous stimuli, can precipitate late loss of islet allografts. Recipients were induced to accept intrahepatic islet allografts (islet 'acceptors') by short-term immunotherapy with donor-specific transfusion (DST) and anti-CD154 mAb. Following the establishment of stable long-term islet allograft function for 60-90 days, recipients were challenged with donor-matched hepatocellular allografts, which are known to activate (CD4-independent) CD8+ T cells. Allogeneic islets engrafted long-term were vulnerable to damage when challenged locally with donor-matched hepatocytes. Islet allograft loss was due to allospecific immune damage, which was CD8- but not CD4-dependent. Selection of specific immunotherapy to suppress both CD4- and CD8-dependent immune pathways at the time of transplant protects islet allografts from both early and late immune damage.
American Journal of Transplantation 06/2008; 8(6):1113-28. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As novel acute allograft rejection mechanisms are being discovered, determining the conditions that promote or subvert these distinct rejection pathways is important to interpret the clinical relevance of these pathways for specific recipient groups as well as specific tissue and organ transplants. We have employed a versatile hepatocellular allograft model to analyze how the host immune repertoire and immune locale influences the phenotype of the rejection pathway. In addition, we investigated how peripheral monitoring of cellular and humoral immune parameters correlates with the activity of a specific rejection pathway. Complete MHC mismatched hepatocellular allografts were transplanted into immune competent CD4-deficient, CD8-deficient, or C57BL/6 hosts to focus on CD8-dependent, CD4-dependent, or combined CD4 and CD8-dependent alloimmunity, respectively. Hepatocellular allografts were transplanted to the liver or kidney subcapsular space to investigate the influence of the immune locale on each rejection pathway. The generation of donor-reactive DTH, alloantibody, and allospecific cytotoxicity was measured to assess both cellular and humoral immunity. Graft-infiltrating lymphocytes were phenotyped and enumerated in each recipient group. In the presence of CD8+ T cells, cytolytic cellular activity is the dominant mechanism of graft destruction and is amplified in the presence of CD4+ T cells. The absence of CD8+ T cells (CD8 KO) results in potent humoral immunity as reflected by high levels of cytotoxic alloantibody and graft rejection with similar kinetics. Transplant to the liver compared to the kidney site is distinguished by more rapid kinetics of rejection and alloimmunity, which is predominately cell mediated rather than a mix of both humoral and cell-mediated immunity. These studies define several rejection mechanisms occurring in distinct immune conditions, highlighting the plasticity of acute allograft rejection responses and the need to design specific monitoring strategies for these pathways to allow dynamic immune assessment of clinical transplant recipients and targeted immunotherapies.
[Show abstract][Hide abstract] ABSTRACT: The role of CD4+ T cells in promoting CD8+ T cell effector activity in response to transplant Ags in vivo has not been reported. We used a hepatocellular allograft model known to initiate both CD4-dependent and CD4-independent rejection responses to investigate the contribution of CD4+ T cells to the development, function, and persistence of allospecific CD8+ T cell effectors in vivo. Complete MHC-mismatched hepatocellular allografts were transplanted into C57BL/6 (CD4-sufficient) or CD4 knockout (CD4-deficient) hosts. The development of in vivo allospecific cytotoxicity was determined by clearance of CFSE-labeled target cells. CD8+ T cell cytotoxic effector activity was enhanced in response to allogeneic hepatocellular grafts with a greater magnitude of allocytotoxicity and a prolonged persistence of CTL effector activity in CD4-sufficient hosts compared with CD4-deficient hosts. Cytolytic activity was mediated by CD8+ T cells in both recipient groups. In response to a second hepatocyte transplant, rejection kinetics were enhanced in both CD4-sufficient and CD4-deficient hepatocyte recipients. However, only CD4-sufficient hosts developed recall CTL responses with an augmented magnitude and persistence of allocytotoxicity in comparison with primary CTL responses. These studies show important functional differences between alloreactive CD8+ T cell cytolytic effectors that mature in vivo in the presence or absence of CD4+ T cells.
The Journal of Immunology 08/2007; 179(1):80-8. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The goal of this study was to determine the in vivo conditions that promote activation of the (CD4-independent) CD8+ T cell-mediated rejection pathway. We have previously noted that hepatocellular but not islet allografts readily activate this rejection pathway. In the current study, we utilized these two cell transplant models to investigate whether differences in host cell recruitment to the graft site, expression of T-cell activation markers by CD8+ graft infiltrating cells (GICs), and/or development of delayed-type hypersensitivity (DTH) and cytotoxic T lymphocyte cell-mediated effector functions could account for the differential transplant outcomes. The collective results demonstrate that recruitment of CD8+ T cells to the site of transplant, CD103 or CD69 expression on CD8+ GICs, and activation of alloreactive DTH responses are insufficient to initiate CD4-independent, CD8-dependent transplant rejection. Instead, rejection by alloreactive (CD4-independent) CD8+ T cells correlated with expression of CD25, CD154 and CD43 by CD8+ GICs, in vitro alloproliferation by recipient CD8+ T cells, and the development of in vivo allospecific cytolytic effector function. These results suggest that tissue-derived factors influence the activation and maturation of (CD4-independent) CD8+ T cells into cytolytic effectors, which correlates with transplant rejection.
American Journal of Transplantation 11/2006; 6(10):2268-81. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Short-term immunotherapy targeting both LFA-1 and CD40/CD154 costimulation produces synergistic effects such that long-term allograft survival is achieved in the majority of recipients. This immunotherapeutic strategy has been reported to induce the development of CD4+ regulatory T cells. In the current study, the mechanisms by which this immunotherapeutic strategy prevents CD8+ T cell-dependent hepatocyte rejection in CD4 knockout mice were examined. Combined blockade of LFA-1 and CD40/CD154 costimulation did not influence the overall number or composition of inflammatory cells infiltrating the liver where transplanted hepatocytes engraft. Expression of T cell activation markers CD43, CD69, and adhesion molecule CD103 by liver-infiltrating cells was suppressed in treated mice with long-term hepatocellular allograft survival compared to liver-infiltrating cells of untreated rejector mice. Short-term immunotherapy with anti-LFA-1 and anti-CD154 mAb also abrogated the in vivo development of alloreactive CD8+ cytotoxic T cell effectors. Treated mice with long-term hepatocyte allograft survival did not reject hepatocellular allografts despite adoptive transfer of naive CD8+ T cells. Unexpectedly, treated mice with long-term hepatocellular allograft survival demonstrated prominent donor-reactive delayed-type hypersensitivity responses, which were increased in comparison to untreated hepatocyte rejectors. Collectively, these findings support the conclusion that short-term immunotherapy with anti-LFA-1 and anti-CD154 mAbs induces long-term survival of hepatocellular allografts by interfering with CD8+ T cell activation and development of CTL effector function. In addition, these recipients with long-term hepatocellular allograft acceptance show evidence of immunoregulation which is not due to immune deletion or ignorance and is associated with early development of a novel CD8+CD25high cell population in the liver.
The Journal of Immunology 01/2006; 175(12):7855-66. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Allogeneic hepatocytes initiate both CD4- and CD8-dependent rejection responses. The current studies address the hypothesis that acute damage of allogeneic liver parenchymal cells by the CD4-dependent pathway is alloantibody-mediated and examines immune conditions which promote activation of this pathway.
The role of alloantibody in CD4-dependent hepatocyte rejection was evaluated by assessing hepatocyte (FVB/N, H-2q) survival in CD8-depleted B-cell knockout (KO) (H-2b) recipients and by monitoring hepatocyte survival in C57BL/6.SCID (H-2b) recipients transfused with donor-reactive alloantibody. The development of donor-reactive alloantibody in C57BL/6 (H-2b), CD8-depleted C57BL/6, CD8 KO (H-2b), IFN-gamma KO (H-2b), perforin KO (H-2b), and FasL mutant gld/gld (H-2b) hepatocyte recipients was assessed.
Hepatocyte rejection in B-cell KO mice was significantly delayed by CD8+ T-cell depletion (median survival time [MST], 35 days) when compared to untreated (MST, 8 days) and CD4-depleted (MST, 10 days) recipient mice. Transfusion of donor-reactive alloantibody into SCID recipients with functional hepatocellular allografts was sufficient to precipitate rejection in a dose-dependent fashion. Donor-reactive alloantibody was minimal in the serum of C57BL/6 hepatocyte recipients, but was produced in significant quantities in hepatocyte recipients genetically deficient in or depleted of CD8+ T cells and in recipients with impaired cytotoxic effector mechanisms. In addition, recipients with defects in Th1 immunity, such as IFN-gamma KO recipients, also produced readily detectable alloantibody.
Collectively, these data support the hypothesis that acute immune damage of allogeneic hepatocytes by the CD4-dependent pathway is mediated by alloantibody and that this pathway is favored when Th1- or cell-mediated cytotoxic effector immune mechanisms are impaired.
[Show abstract][Hide abstract] ABSTRACT: Transplant rejection has generally been considered a CD4 T-cell-dependent immune process. CD4-independent, CD8 T-cell rejection pathways have recently gained attention because of their relative resistance to immunosuppression. In the current study, the role of the allograft tissue in activation of these distinct pathways was examined by comparing host-immune responses with allogeneic pancreatic islets or hepatocytes transplanted across the same genetic disparity.
To compare activation of CD4-dependent versus CD8-dependent alloimmunity, islets or hepatocytes retrieved from FVB/N (H-2) mice were transplanted into CD8 or CD4 T-cell-reconstituted severe combined immunodeficiency mice, CD4 or CD8 knockout (KO) mice, and anti-CD4 monoclonal antibody (mAb) or anti-CD8 mAb treated C57BL/6 mice (all H-2). The ability to immunomodulate CD4-dependent allograft rejection (in CD8 KO mice) was examined in the context of several mechanistically distinct immunotherapeutic strategies, including anti-CD4 mAb, donor-specific transfusion and anti-CD154 mAb, and anti-lymphocyte function-associated antigen-1 mAb.
The studies demonstrate that, whereas hepatocytes evoke alloreactive CD4-dependent and (CD4-independent) CD8 T-cell immune responses, allogeneic islets only activate CD4-dependent immune pathways. CD4-dependent host-immune responses initiated by pancreatic islet allografts were readily suppressed by a variety of short-term immunotherapies, whereas hepatocyte-initiated CD4-dependent alloimmune responses were not.
These results demonstrate that immune characteristics of the specific allograft tissue uniquely influence the pattern of host immune responses such that the propensity to activate CD4- or CD8-dependent alloimmune responses can be distinguished. Furthermore, CD4-dependent immune responses activated by different tissues from the same donor strain are distinguished by their susceptibility to specific immunotherapy.
[Show abstract][Hide abstract] ABSTRACT: Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8(+) T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4(+) and CD8(+) T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.
American Journal of Transplantation 08/2004; 4(7):1061-70. · 6.19 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Allogeneic hepatocytes elicit CD4-dependent and (CD4-independent) CD8+ T-cell-initiated graft rejection. The (CD4-independent) CD8+ T-cell pathway is resistant to immunosuppressive strategies that readily and indefinitely suppress CD4+ T-cell-dependent rejection responses. Consequently, successful immunoregulation of hepatocyte-initiated immune responses requires a strategy which regulates both CD4-dependent and CD8-dependent rejection responses. Interference with CD40/CD40 ligand (CD40L) costimulation only transiently suppresses CD4- and CD8-dependent hepatocyte rejection. Interference with CD28/B7 costimulation transiently suppresses CD4-dependent hepatocyte rejection, but is ineffective for suppression of CD8-dependent hepatocyte rejection. To date, hepatocyte survival > 60 days post-transplant has not been achieved by any immunotherapeutic strategy. In the current study, we evaluated a novel immunosuppressive strategy which targets both LFA-1 and CD40L-mediated signals. Targeting LFA-1 suppressed (CD4-independent) CD8+ T-cell-initiated hepatocyte rejection such that allogeneic hepatocyte survival > 60 days was achieved in 70% of CD4 KO mice. Targeting both LFA-1-mediated signals and CD40/CD40L costimulation resulted in synergistic effects, such that hepatocellular survival > 60 days was achieved in 100% of C57BL/6 mice (which have both CD4- and CD8-dependent T-cell pathways available).
American Journal of Transplantation 10/2003; 3(10):1251-8. · 6.19 Impact Factor