CD4 T-Cell Regulation of Cytotoxic T Cells During the Induction Phase of Liver-Induced Spontaneous Tolerance in Rats

Purpose To check for in vivo CD4 T-cell-mediated inhibition of the immune response in rats with spontaneously accepted liver transplants.Methods Using the Lewis to Wistar Furth rat strain combination, we performed transient in vivo depletion of CD4 T-cells by anti-CD4 monoclonal antibodies (mAb) after liver transplantation. We used the CTL assay to detect primed T cells. We also retransplanted a grafted donor liver, parked for 3 days, into a secondary naive recipient rat.Results When Lewis rat livers were transplanted into the recipient Wistar Furth rats, the grafts suffered an early immune attack, followed by spontaneous acceptance without immunosuppression. However, giving anti-CD4 mAb to the recipients at the time of grafting prolonged the acute rejection reaction. Furthermore, giving anti-CD4 therapy on postoperative days (PODs) 21 and 35, but not on PODs 56 and 100, induced transient liver damage in recipients overcoming acute rejection. No primed T cells were detected by the CTL assay in the recipient rats within 2 months after transplantation. Meanwhile, the retransplanted liver had no ability to elicit an immune attack.Conclusions CD4 T cells seemed to downregulate the effector function of T cells, but not T-cell proliferation in this model.     

Treatment with monoclonal antibodies to ICAM-1 and LFA-1 in rat heart allograft rejection

During allograft rejection, leukocyte infiltration in the graft is regulated by various adhesion molecules. Treatment with monoclonal antibodies to ICAM-1 and LFA-1 (CD11 a) induces specific tolerance after murine heart transplantation. In this study, we investigated the possibility of tolerance induction using these antibodies in a fully incompatible rat heart transplant model. Heterotopic, intra-abdominal heart transplantation was performed using Dark Agouti (DA) rats as donors and Lewis (LEW) rats as recipients. Group A (n = 6) received no immunosuppression and served as controls. In group B (n = 6) 500 μg/kg per day 1A29 (anti-ICAM-1) was administered intravenously (i. v.) for 5 days; group C rats received the same dosage of WT.1 (anti-CD11 a) i. v. for 5 days. In group D (n = 6), rats received combined i. v. administration of anti-CD11 a and anti-ICAM-1 (500 μg/kg per day of each, for 5 days. The antibodies used were monoclonal mouse anti-rat antibodies produced from hybridomas. Allograft survival was monitored by daily palpation of the graft. There was no statistically significant difference in allograft survival between the groups (A: 5.7 ± 0.5 days; B: 5.7 ± 0.5 days; C: 5.7 ± 0.5 days; D: 6.2 ± 0.4 days). Treatment with monoclonal antibodies to ICAM-1 and LFA-1 alone or in combination had no effect on allograft survival after heart transplantation between fully incompatible rat strains. We conclude that induction of tolerance using these antibodies seems inconceivable in human heart transplantation. Received: 1 July 1996 Received after revision: 25 September 1996 Accepted: 28 October 1996     

Liver allograft rejection in rats depleted of CD8+ cells

The mechanism(s) of rejection or tolerance induction is a competitive, complex process that presumably involves interactions between multiple subpopulations of T lymphocytes. We investigated the roles of CD8⁺ cytolytic and CD4+ helper T cells in rat strains that tolerate liver allografts and that differ at both the major histocompatibility complex (MHC) (RT1) and minor histocompatibility genes. Orthotopic liver transplantation (OLT) with arterial reconstruction was performed with Brown Norway (BN) (RT1ⁿ) donors and Lewis (RT1¹) recipients, some of which were untreated, others treated with anti-CD8 antibody, and still others treated with anti-CD4 antibody. Liver graft rejection was monitored for 28 days on the basis of two criteria: (1) serum levels of AST enzyme at 3-day intervals and (2) liver biopsies at weekly intervals and at the time of sacrifice at the end of the study period. In the untreated control group, an elevation of AST was found to peak at day 6 after grafting, and it remained elevated until day 28 (AST 542±72 U/l). Histologically, signs of severe rejection were first observed on day 9; these changed to moderate rejection about day 21 and to mild rejection by day 28, when the animals were sacrificed. Recipients pre-treated with anti-CD8 demonstrated a significant elevation of AST within 6 days that, unlike in the control recipients, continued to rise sharply through the observation period (AST 1127±181 U/1, P=0.009 vs control group). Liver biopsies showed mild rejection at day 9 and moderate rejection at days 21 through 28. Recipients pretreated with anti-CD4 showed a time course of enzyme elevation and severity of rejection that was not significantly different from that observed in the control group. However, anti-CD4 treatment resulted in only 75% depletion of CD4⁺ cells in peripheral blood as compared to complete elimination of CD8⁺ cells following anti-CD8 treatment. Functional studies of spleen and liver-infiltrating lymphocytes obtained after 28 days showed low proliferative response in mixed lymphocyte culture with both BN and PVG stimulator spleen and lymph node cells. These results suggest that in this donor/recipient combination, removal of CD8⁺ cells increases the severity of rejection as demonstrated by a progressive rise in AST and histology. Moreover, OLT in this combination results in a profound, nonspecific inhibition of proliferative T-cell responses to MHC alloantigens.     

Mobilization of host stem cells enables long-term liver transplant acceptance in a strongly rejecting rat strain combination

Careful examination of liver, kidney and heart transplants in human recipients has revealed small numbers of host bone marrow derived stem cells in the graft. If the limited recipient repopulation of a donor graft that is currently observed could be facilitated, it is possible that conversion to a predominantly host phenotype would permit long-term graft function without immunosuppression. We proposed to "engineer" repopulation after transplant in a strain combination (dark agouti [DA] to Lewis green fluorescent protein+[LEW GFP+]) which rejects liver grafts strongly, a model that more closely resembles the situation in humans. Treatment on days 0, 1, 2, 3 and 7 after transplantation with low-dose (0.1 mg/kg) tacrolimus (T) designed to blunt rejection combined with plerixafor (P) to mobilize host stem cells resulted in greater than 180 days graft survival with extensive albeit spotty conversion of a small (50%) DA graft to the recipient LEW GFP+ genotype. Subsequent skin grafting revealed donor-specific graft prolongation. The T plus P treatment resulted in higher levels of Lin-Thy1+CD34+CD133+ stem cells and Foxp3+ regulatory T cells in the blood and liver at day 7. Thus, pharmacological mobilization of host stem cells sustains liver allografts by two mechanisms: repopulation of injured donor cells and regulation of the immune response.     

"Infectious tolerance" develops after the spontaneous acceptance of Lewis-to-Dark Agouti rat liver transplants

BACKGROUND: After monoclonal antibody or donor-specific transfusion treatment, infectious tolerance to rat or mouse heart or skin transplants can be passed on to naive recipients by adoptive transfer of tolerant lymphocytes. We examined whether similar regulatory cells develop after the spontaneous acceptance of Lewis-to-Dark Agouti (DA) rat liver transplants without immunomodulating agents. METHODS: After Lewis-to-DA rat liver transplantation, 100 x 10(6) splenocytes were harvested and adoptively transferred into a 450 rad-irradiated naive DA rat 24 hours before Lewis heart transplantation. Adoptive transfer of CD4+ or CD8+ T cells was also examined. In some experiments, splenocytes from recipients with long-term accepted Lewis hearts induced by adoptive transfer were serially transferred to multiple generations of recipients before Lewis rat heart transplantation. In vitro mixed lymphocyte culture response and cytotoxic T lymphocyte generation were measured. RESULTS: When splenocytes from a DA rat recipient >60 days after Lewis rat liver acceptance were transferred into irradiated DA rat recipients, all Lewis rat hearts were accepted, whereas third-party Brown-Norway rat hearts were rejected. However, splenocytes from DA rat recipients 30 days after liver transplantation did not prolong Lewis rat heart survival. Adoptive transfer of 40 x 10(6) CD4+, 10 x 10(6) CD4+ or 10 x 10(6) CD8+ cells from a DA rat bearing Lewis rat liver >60 days resulted in acceptance of 88%, 80%, or 57% acceptance of Lewis rat hearts, respectively. Serial second and third adoptive transfer of long-term survivor splenocytes resulted in the acceptance of all Lewis rat hearts. In mixed lymphocyte culture, splenocytes from a naive DA rat and a DA rat accepting a Lewis rat liver transplant for >60 days showed similar proliferative responses to both Lewis and Brown-Norway rat stimulators. An equivalent level of indirect cytotoxic T lymphocyte activity was exhibited by splenocytes from both a naive DA and a DA rat accepting a Lewis rat liver transplant for >60 days. CONCLUSIONS: Regulatory cells developing after the spontaneous acceptance of a Lewis to DA liver transplant can serially adoptively transfer the acceptance of a Lewis rat cardiac graft in spite of the presence of in vitro antidonor reactivity. Both CD4+ and CD8+ populations have this regulatory activity, although the CD4+ population plays the dominant role.     

Allograft acceptance and rejection, mediated by a liver suppressor factor, LSF-1, purified from serum of liver transplanted rats

In certain rat strain combinations liver allografts are spontaneously accepted without immunosuppression and induce donor-specific tolerance to further skin and heart grafts in the recipient. Such an effect is also transferrable using serum from orthotopically liver transplanted rats (OLT serum). In the OLT serum of one such combination. DA (RT1^a) donor into PVG (RT1^c) recipient, a 40 kDa protein (liver suppressor factor, LSF-1) has been identified and shown to be immunosupressive in vitro. The aim of the present study is to investigate the immunological effect of LSF-1 and a polyclonal antibody (anti-LSF-1) against this molecule, in a rat heterotopic heart transplant (HHT) model and OLT model, respectively. Intramuscular injection of 300 μg of LSF-1, 1 h postoperatively, into a PVG recipient of either a DA or BN (RT1ⁿ) cardiac allograft caused significant prolongation of graft survival. Intravenous injection of polyclonal rabbit sera raised against an N-terminal peptide of LSF-1 (anti-LSF-1), within 1 h postoperatively, had variable effects on the survival of DA liver grafts in PVG recipients. In cases injection of between 1 and 2 ml of anti-LSF-1 resulted in death of the recipient. Histological examination of the liver showed severe rejection with lymphoid cell infiltration of the portal tract and sinusoids and extensive damage to the parenchyma. All control rats survived for more than 60 days without any signs of rejection. The anti-LSF-1 polyclonal antibody prevented the induction of tolerance in the normally tolerogenic model (DA into PVG). This, together with the in vivo results, suggests a role for LSF-1 in the induction of tolerance.     

T lymphocytes mediate leaflet destruction and allograft aortic valve failure in rats

BACKGROUND: Allograft heart valves are commonly used in cardiac surgery but ultimately fail. This situation is most acute in children. This study addresses the role of T cell-mediated immune damage in allograft valve failure. METHODS: Syngeneic (Lewis to Lewis) or allogeneic (Brown Norway to Lewis) aortic valve grafts were implanted infrarenally into Lewis rat recipients (n = 24). Allogeneic valve grafts were also implanted into T cell-deficient rats (nude; n = 12). At 7, 14, and 28 days the valves were explanted and examined for structural integrity and cellular infiltration. RESULTS: Syngeneic grafts maintained normal leaflet structure with little leaflet immune infiltration. Allografts showed leaflet infiltration (7 days), significant leaflet thickening, progressively decreased cellularity (14 days), and leaflet destruction (28 days). Infiltrates contained CD43+, CD3+, and CD8+ cells. Allografts in T cell-deficient rats showed none of the above changes and maintained normal structural integrity. CONCLUSIONS: Allograft heart valves in the rat model undergo T cell-mediated immune rejection, resulting in structural failure.     

Blockade of CD27/CD70 pathway to reduce the generation of memory T cells and markedly prolong the survival of heart allografts in presensitized mice

BackgroundAlloreactive memory T cells are a major obstacle to transplantation acceptance due to their capacity for accelerated rejection.MethodsC57BL/6 mice that had rejected BALB/c skin grafts 4 weeks earlier were used as recipients. The recipient mice were treated with anti-CD154/LFA-1 with or without anti-CD70 during the primary skin transplantation and anti-CD154/LFA-1 or not during the secondary transplantation of BALB/c heart. We evaluated the impact of combinations of antibody-mediated blockade on the generation of memory T cells and graft survival after fully MHC-mismatched transplantations.ResultsOne month after the primary skin transplantation, the proportions of CD4⁺ memory T cells/CD4⁺ T cells and CD8⁺memory T cells/CD8⁺ T cells in the anti-CD154/LFA-1 combination group were 47.32 ± 4.28% and 23.18 ± 2.77%, respectively. In the group that included anti-CD70 treatment, the proportions were reduced to 34.10 ± 2.71% and 12.19 ± 3.52% (P < 0.05 when comparing the proportion of memory T cells between the two groups). The addition of anti-CD70 to the treatment regimen prolonged the mean survival time following secondary heart transplantation from 10 days to more than 90 days (P < 0.001). Furthermore, allogenic proliferation of recipient splenic T cells and graft-infiltrating lymphocytes were significantly decreased. Meanwhile, the proportion of regulatory T cells was increased to 9.46 ± 1.48% on day 100 post-transplantation (P < 0.05).ConclusionsThe addition of anti-CD70 to the anti-CD154/LFA-1 combination given during the primary transplantation reduced the generation of memory T cells. This therapy regimen provided a potential means to alleviate the accelerated rejection mediated by memory T cells during secondary heart transplantation and markedly prolong the survival of heart allografts.     

Effect of elimination of OX-19+ and OX-8+ T-cell subsets upon pancreatic allograft survival

Depletion of T cell subsets with monoclonal antibody (mAb) permits analysis of cellular events mediating allograft destruction. Mab OX-19 and mAb OX-8 were used singly and in combination together with a short pretransplant course of cyclosporine A (CsA) to deplete OX-19+ cells (all T cells) and OX-8+ cells (cytotoxic/suppressor and NK cells), respectively, in diabetic Lewis (Lew) recipients of a Wistar Furth (WF) pancreatic allograft. Depletion of lymph node T cell subsets was assessed at rejection (blood sugar greater than 250 mg/dl) by flow cytometry. Untreated Lew recipients (Group 1) rapidly rejected their allograft (11.5 +/- 2.5 days). MAb OX-19 administration on the day prior to surgery (Day -1), on the day of surgery (Day 0), and alternate days thereafter until rejection (Group 2) prolonged graft survival (15.0 +/- 1.6 days, P less than 0.05). MAb OX-19 administration on alternate days beginning 14 days prior to transplantation (Day -14) until rejection (Group 3) further prolonged graft survival (22.6 +/- 3.4 days, P less than 0.01). At rejection large numbers of OX-19+ cells were present in both groups. Administration of mAb OX-8 alone (Group 4) failed to prolong graft survival despite marked depletion of OX-8+ cells at rejection. Administration of mAb OX-19 from Day -14 together with CsA (15 mg/kg) from Days -14 to -8 inclusive (Group 5) resulted in a marked and sustained depletion of OX-19+ cells at rejection but only a modest prolongation of graft survival (27.6 +/- 6.0 days, P = 0.11). CsA alone from Days -14 to -8 failed to prolong graft survival.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic T-cell elimination during anti-CD4-induced rat liver acceptance and rapid replacement of functional graft antigen-presenting cells.

In previous studies, we showed that primed T cells were eliminated in long-term survival Wistar Furth (WF) recipient rats with spontaneously accepted Lewis (LEW) liver graft and that the grafted liver lost the ability to elicit rejection reaction early after liver transplantation. We hypothesized that the same phenomenon may be observed in tolerant animals after immunosuppression in a rejector rat strain combination (WF-->LEW). Furthermore, we proposed the repopulation of liver allograft with host antigen-presenting cells rapidly after transplantation. Recipient LEW rats that underwent anti-CD4 therapy accepted the WF liver allografts after a transient rejection reaction. In tolerant animals, alloreactive CD8 T cell precursors were present, but primed T cells were absent. Intraperitoneal challenge with grafted WF liver homogenates obtained from recipient LEW rats on day 4 after transplantation did not induce transient rejection responses in long-term survival recipient LEW rats, a finding that differed from the results of experiments using normal WF liver homogenates. However, challenge with grafted WF liver homogenates, similar to those of normal LEW liver homogenates, induced rejection responses in long-term survival recipient WF rats with LEW liver allograft. Flow cytometric analysis confirmed that most of nonparenchymal cells in the grafted WF liver were recipient (LEW) genotype. These observations showed that the deletional mechanism of effector T cells also is observed in this setting, and professional donor antigen-presenting cells are replaced by those of recipient genotype within the graft during the early phase of transplantation.     

In vitro and in vivo effects of monoclonal antibodies against T cell subsets on allogeneic and xenogeneic responses in the rat

The Syrian hamster-to-rat represents an example of a concordant species difference, and therefore organ transplants using the hamster as the donor and the rat as the recipient are not rejected hyperacutely, as in discordant species combinations. Cellular mechanisms of xenogeneic rejection of hamster hearts by rats were studied both in vitro and in vivo, using monoclonal antibodies to rat T cell antigens. The results of this study reveal that CD4-positive cells of rats proliferated in vitro to both allogeneic stimulators and xenogeneic stimulators from a concordant strain, but required accessory cells of the responder phenotype to proliferate to discordant human stimulators. Monoclonal antibody therapy was used to prevent graft rejection in allogeneic and xenogeneic species combinations, using the rat as the recipient. Treatment with anti-CD4 antibodies was effective in prolonging allograft survival across a full MHC mismatch. No rejection occurred during antibody therapy, and long-term graft survival was achieved in 1/3 of transplanted grafts. The same monoclonal antibody therapy led to increased survival of grafts from hamster donors, but all of these grafts were rejected during therapy, and no long-term graft survival was achieved. Anti-CD8 antibody therapy, combined with anti-CD4 did not improve survival of hamster hearts in rats. Addition of cyclosporine to the anti-CD4 regimen also did not improve graft survival. Injection of an anti-T cell receptor antibody was no better than the anti-CD4 antibody in prolonging the survival times of heart grafts from the concordant xenogeneic species. These data suggest that the rejection of concordant xenogeneic tissue is not wholly a T cell-dependent phenomenon.     

Roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection

An essential role of murine CD4+ T cells in immune reactivity and skin graft rejection in discordant xenogeneic combinations have been reported. Our study was conducted to further clarify the roles of CD4+ and CD8+ T cells in discordant skin xenograft rejection, by using CD4 and CD8 knockout [C57BL/6 Cr Slc (B6; H-2b) background] mice. When human skins were grafted on CD8 knockout mice or B6 mice, both hosts rejected human skin grafts within 12 days after grafting. By contrast, survival of human skin grafts was significantly prolonged in CD4 knockout mice (mean survival times=19.3+/-(SD) 1.6 days; median 19 days). Fully allogeneic C3H/He Slc (H-2k) skin grafts were rejected within 14 days in CD4 knockout mice, suggesting that non-CD4+ T cells in CD4 knockout mice were immunocompetent for allograft rejection. In spleens of these recipient mice, CD8+ T cells seemed to be activated 10 days after human skin grafting. Immunohistological analysis revealed the infiltration of CD8+ T cells at the site of transplanted human skin on CD4 knockout mice. To further examine the role of CD8+ T cells in CD4 knockout mice, human skin grafting was performed on day 0 followed by administration of anti-CD8 monoclonal antibody on days 0, 5, and 14. The administration of anti-CD8 monoclonal antibodies caused the significant prolongation of human skin graft survival. These results indicate the following two conclusions: (1) CD4+ T cells have an essential role in rejecting discordant human skin xenografts rapidly and (2) however, CD8+ T cells also are capable of rejecting discordant human skin xenografts.     

Xenoreactive CD4+ memory T cells resist inhibition by anti-CD44 mAb and reject islet grafts via a Th2-dependent pathway

Peng YZ, Chen JB, Shao W, Wang FY, Dai HL, Cheng PP, Xia JJ, Wang F, Huang R, Zhu Q, Qi Z. Xenoreactive CD4⁺ memory T cells resist inhibition by anti‐CD44 mAb and reject islet grafts via a Th2‐dependent pathway. Xenotransplantation 2011; 18: 252–261. © 2011 John Wiley & Sons A/S. Abstract: Background: Memory T cells are a significant barrier to the induction of transplant tolerance. Our previous study demonstrated that multiple applications of anti‐CD44 monoclonal antibody (mAb) could significantly inhibit CD4⁺ memory T cells from mediating rejection of cardiac allografts. Now, we sought to explore the effect and mechanism of anti‐CD44 mAb on the rejection of islet allografts and xenografts mediated by CD4⁺ memory T cells. Methods: In this study, we first engrafted skin grafts of C57BL/6 (B6) mice or Dark Agouti (DA) rats onto BALB/c mice to induce donor‐reactive memory T cells. We adoptively transferred purified CD4⁺ memory T cells to BALB/c origin nude mice and then transplanted islet allografts and xenografts to produce the Allo‐Tx and Xeno‐Tx models, respectively. We subsequently administered multiple anti‐CD44 mAb and observed changes in the survival times of the islet grafts. Results: In the Allo‐Tx model, the mean survival time (MST) of the grafts was 7.7 days in the isotype group, and 20.3 days in the anti‐CD44 group. In the Xeno‐Tx model, the MST of the grafts was 7.2 days in the isotype group and 8.2 days in the anti‐CD44 group. Compared with the isotype group, CD4⁺ T cells on the grafts in the anti‐CD44 group were significantly decreased in both the Allo‐Tx and Xeno‐Tx models, but the proportion of CD4⁺ memory T cells in the spleens and draining lymph nodes of the recipient nude mice in the anti‐CD44 group was significantly decreased in the Allo‐Tx model, while it was increased in the Xeno‐Tx model. The production of donor‐specific IgG antibody in the anti‐CD44 group did not vary in the Allo‐Tx model, while it was markedly elevated in the Xeno‐Tx model. Furthermore, the expression of interferon gamma in the anti‐CD44 group was markedly decreased in both the Allo‐Tx and Xeno‐Tx models, while the expression of IL‐4 in the anti‐CD44 group was significantly increased only in the Xeno‐Tx model. Conclusion: Multiple applications of the anti‐CD44 mAb could significantly inhibit donor‐reactive CD4⁺ memory T cells from rejecting grafts via a Th1‐dependent pathway, but xenoreactive CD4⁺ memory T cells can avoid the effects of anti‐CD44 mAb to reject islet xenografts via a Th2‐dependent pathway.     

CTLA4-Ig abrogates the anti-globulin response and prolongs cardiac allograft survival after anti-CD2 treatment

CD2 is expressed on T cells and NK cells and is important in T cell activation, making it a potential target for immune intervention. Here, we report a series of experiments aimed at defining the ability of mAbs directed against the CD2 molecule to prevent cardiac allograft rejection in low and high responder rat strain combinations. Administration of the mouse anti-rat CD2 mAbs OX34 or OX55 around the time of transplantation prolonged survival of fully allogeneic Lewis (RT1l) cardiac allografts in low responder DA (RT1a) recipients (MST 14 days for OX55 and >100 days for OX34). Treatment with OX34 prolonged graft survival in the reciprocal high responder DA to Lewis rat strain combination (MST 19 days) and when combined with CTLA4-Ig resulted in long-term graft survival (MST>100 days). Despite these in vivo effects, OX34 had little effect on in vitro assays of lymphocyte activation. Instead, the ability of OX34 to extend allograft survival correlated with T cell depletion. Administration of OX34 induced a similar degree of CD4 T cell depletion in DA and Lewis recipients, but the CD4 depletion observed was more transient in Lewis recipients. Lewis, but not DA strain rats, developed an anti-murine Ig response. Combined treatment with CTLA4-Ig abolished the anti-globulin response to OX34 in Lewis recipients, prolonged circulation of OX34 and increased the extent and duration of CD4 depletion. We conclude that anti-CD2 treatment effectively prolongs cardiac allograft survival and addition of CTLA4-Ig increases its efficacy by abrogating the production of neutralising antibodies.     

Infectious tolerance develops after intrathymic alloantigen-induced acceptance of rat heart allografts can be adoptively transferred

BACKGROUND: We have shown that intrathymic (IT) injection of alloantigen with antirat lymphocyte serum (ALS) treatment can induce donor-specific allograft acceptance. The purpose of this study was to investigate whether T-regulatory (T-reg) cells play a role in the maintenance of donor-specific heart graft tolerance that develops after IT injection of Lewis (LEW, RT1(l)) alloantigen into a Dark Agouti (DA, RT1(a)). METHODS: Na?ve DA rats were injected IT with 2.5 x10(7) LEW donor splenocytes and injected intraperitoneally with 1 mL ALS. Twenty-one days after pretreatment, a LEW or Brown Norway (BN, RT1(n)) heart was transplanted into a treated DA recipient. Splenocytes (1 x 10(8) or 5 x 10(7)) from a LEW heart-tolerant long-term survivor (LTS; >60 days) DA recipient were harvested and adoptively transferred (AT) into an irradiated (450 rad) na?ve DA rat 24 hours before transplanting a LEW heart. RESULTS: All LEW heart allografts were rejected by untreated DA rats in a mean survival time (MST) of 7.4 +/- 1.7 days (n=7). In contrast, 66.7% of LEW heart grafts into IT+ALS-pretreated DA recipients were accepted indefinitely (n=24). When either 1 x 10(8) (n=5) or 5 x 10(7) (n=5) splenocytes from a LEW heart graft-tolerant LTS (>60 days) DA recipient were AT into a new na?ve DA rat, all new LEW heart grafts were accepted indefinitely. CONCLUSIONS: The donor-specific tolerance that develops after IT+ALS-induced LEW heart acceptance by DA recipients can be transferred adoptively to new na?ve DA recipients, thus indicating that it is infectious tolerance.     

Apoptosis and allograft rejection in the absence of CD8+ T cells.

BACKGROUND: The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells. METHODS: Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant. RESULTS: Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts. CONCLUSION: These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.     

Critical Role for CD8+ T Cells in Allograft Acceptance Induced by DST and CD40/CD154 Costimulatory Blockade

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.     

The role of Foxp3+ regulatory T cells in liver transplant tolerance

The liver has long been considered a tolerogenic organ that favors the induction of peripheral tolerance. The mechanisms underlying liver tolerogenicity remain largely undefined. In this study, we characterized Foxp3-expressing CD4+ CD25+ regulatory T cells (Treg) in liver allograft recipients and examined the role of Treg in inherent liver tolerogenicity by employing the mouse spontaneous liver transplant tolerance model. Orthotopic liver transplantation was performed from C57BL/10 (H2b) to C3H/HeJ (H2k) mice. The percentage of CD4+ CD25+ Treg was expanded in the liver grafts and recipient spleens from day 5 up to day 100 posttransplantation, associated with high intracellular Foxp3 and CTLA4 expression. Immunohistochemistry further demonstrated significant numbers of Foxp3+ cells in the liver grafts and recipient spleens and increased transforming growth factor beta expression in the recipient spleens throughout the time courses. Adoptive transfer of spleen cells from the long-term liver allograft survivors significantly prolonged donor heart graft survival. Depletion of recipient CD4+ CD25+ Treg using anti-CD25 monoclonal antibody (250 microg/d) induced acute liver allograft rejection, associated with elevated anti-donor T-cell proliferative responses, CTL and natural killer activities, enhanced interleukin (IL)-2, interferon-gamma, IL-10, and decreased IL-4 production, and decreased T-cell apoptotic activity in anti-CD25-treated recipients. Moreover, CTLA4 blockade by anti-CTLA4 monoclonal antibody administration exacerbated liver graft rejection when combined with anti-CD25 monoclonal antibody. Thus, Foxp3+ CD4+ CD25+ Treg appear to underpin spontaneous acceptance of major histocompatability complex- mismatched liver allografts in mice. CTLA4, IL-4, and apoptosis of alloreactive T cells appear to contribute to the function of Treg and regulation of graft outcome.     

Induction of Rejection After Small-for-Size Liver Transplantation: Size Matters

Background: Reduced-size liver transplantation is associated with liver regeneration. This study was designed to analyze the influence of graft size on liver rejection and liver regeneration. Methods: Reduced-size liver transplantations were performed in the rejecting ACI to Lewis and the graft acceptance BN to Lewis strain combination. The BN to Lewis control group was treated with the immunosuppressive drug FK506. Results: An accelerated liver rejection in the ACI to Lewis strain combination was found in small-for-size partial liver grafts. Graft weight to recipient liver weight ratio (GW/RLW) showed a positive correlation with survival time. In the BN to Lewis strain combination, lethal rejection was seen in small-for-size partial liver grafts. A critical immunologic GW/RLW of 33% was calculated. In rats dying from lethal rejection, GW/RLW and survival time showed a positive correlation. However, GW/RLW showed a negative correlation with hepatocellular proliferation. In regenerating livers, MHC II upregulation was also observed in the control group. All control animals survived small-for-size liver transplantation. Conclusions: The relative graft size seems to be a decisive factor influencing the kinetic of liver rejection and the induction of liver rejection. Relative critical immunologic liver mass was determined to be 33%.