Cytoprotective gene HO-1 and chronic rejection in heart transplantation

Chronic rejection in transplanted hearts or cardiac allograft vasculopathy (CAV) is the leading cause of late death among heart transplant recipients. We hypothesized that induction of HO-1 by D4-F, an apoA-I mimetic peptide with potent antiinflammatory/antioxidant properties, attenuated CAV. We utilized a previously characterized murine model of CAV. B6.C-H2(bml2) hearts were heterotopically transplanted into C57BL/6 mice. In the control group, recipient mice were treated with 20 microg of saline daily. In experimental group I, mice were treated daily with 20 microg of D4-F. In experimental group II, mice were treated daily with 20 microg of D4-F daily, plus CuPP, which does not have any effect on HO-1 activity. In experimental group III, recipient mice were treated with 20 mug of D4-F daily, plus SnPP, which is a competitive inhibitor of HO-1. Donor hearts were harvested on day 24 after transplantation. The donor hearts in the control group developed severe intimal lesions. In experimental group I, treatment with D4-F was associated with upregulation of HO-1 and a marked reduction in intimal lesions, which was consistent in experimental group II. In experimental group III, inhibition of HO-1 was associated with partial restoration of intimal lesions. Induction of HO-1 by an apoA-1 mimetic peptide was effective to control CAV. This class of antiinflammatory peptides, which show an ability to induce HO-1, provides a novel strategy for the treatment of CAV.     

CD8+ lymphocytes augment chronic rejection in a MHC class II mismatched model

Chronic rejection, or cardiac allograft vasculopathy (CAV), remains the leading cause of late death in heart transplant recipients. The precise role and contributions of T lymphocyte subsets to CAV development remains unknown. METHODS: Donor hearts from B6.C-H2bm12 mice were transplanted into T lymphocyte subset knockout recipients and T lymphocyte-reconstituted nude recipients. No immunosuppression was used. Intimal proliferation was measured morphometrically. In vitro studies were performed to analyze the donor-specific activation status of recipient CD8+ lymphocytes by examining cellular proliferation, interleukin-2 secretion, and interleukin-2Ralpha expression. Intracellular cytokine staining assay was performed to determine both the profile and source of intragraft cytokines. RESULTS: Hearts transplanted into wild-type recipients developed severe CAV by 24 days. Intimal lesions were absent in the hearts that were transplanted into nude and CD4-/- knockout mice (containing CD8+ lymphocytes). In contrast, the donor hearts in CD8-/- knockout recipients (containing CD4+ lymphocytes) developed CAV, but significantly less than in wildtype. Adoptive transfer of T lymphocyte subset populations into nude recipients confirmed that CAV was absolutely contingent on CD4+ lymphocytes, and that CD8+ lymphocytes played an additive role in intimal lesion progression in the presence of CD4+ lymphocytes. Although CD8+ lymphocytes alone did not cause CAV in vivo, we demonstrated that MHC class II disparate alloantigens activated CD8+ lymphocytes both in vivo and in vitro. Finally, both CD4+ and CD8+ lymphocytes contributed to the intragraft IL-2 and IFN-gamma production. CONCLUSIONS: In this MHC class II mismatched murine model, CAV is a T lymphocyte dependent event, and absolutely contingent on the presence of CD4+ lymphocytes. Furthermore, CD8+ lymphocytes (1) are activated by MHC class II disparate antigens and (2) play a significant role in the progression of lesion development. Finally, both CD4+ and CD8+ lymphocytes contribute to CAV development via secretion of IFN-gamma, a known mediator of CAV in this model.     

Indirect alloreactivity and chronic rejection

BACKGROUND: The relative contribution of the direct versus indirect pathway of T-lymphocyte alloreactivity to the development of chronic rejection is incompletely understood. Utilizing a murine model of cardiac allograft vasculopathy (CAV) and a recipient strain with markedly reduced capacity for indirect alloreactivity, we sought to define the importance of indirect allorecognition in CAV. METHODS: The cells from H2-M mutant mice are unable to present intact protein antigens via class II molecules and have a markedly reduced capacity to present exogenous peptides. B6C.H-2(bm12) strain donor hearts were transplanted into either C57Bl/6 wild-type (WT) or H2-M mutant mice (on C57Bl/6 background). Recipients were killed on day 24. T lymphocyte and macrophage infiltration were graded immunohistochemically. Intimal lesions were measured morphometrically. RESULTS: Donor hearts in WT recipients developed significant intimal lesions, as expected (50+/-7%). Moreover, the donor hearts in H2-M mutant mice also developed comparable intimal lesions (52+/-9%, P=NS vs. WT). Furthermore, the extent of T lymphocyte and macrophage infiltration was similar in both groups. CONCLUSIONS: This study demonstrates that a markedly reduced capacity for indirect alloreactivity does not affect the severity of intimal lesions in this model of CAV. The findings of this study question the role of indirect alloreactivity as the sole pathway of allorecognition leading to chronic rejection.     

Role of CD8+ lymphocytes in chronic rejection of transplanted hearts

BACKGROUND: The contribution of CD8(+) lymphocytes to the pathogenesis of cardiac allograft vasculopathy, or chronic rejection in heart transplants, remains undefined. We used both major histocompatibility complex class I mismatched and major histocompatibility complex class II mismatched models of cardiac allograft vasculopathy to characterize the role of CD8(+) lymphocytes in the development of cardiac allograft vasculopathy. METHODS: Donor hearts from B10.A mice were transplanted into B10.BR recipients (major histocompatibility complex class I mismatched). Donor hearts were harvested at 1, 7, 14, and 30 days after transplantation and (1) quantitated morphometrically for lesion development, (2) stained immunohistochemically, or (3) digested for isolation of graft-infiltrating cells. The cytotoxic phenotype of graft-infiltrating CD8(+) lymphocytes was determined with flow cytometry. Intracellular cytokine staining of CD8(+) and CD4(+) lymphocytes for interleukin 2, interferon g, interleukin 4, and interleukin 10 was performed with 2-color flow cytometry. Finally, B6.C-H2(bm12) donor hearts were transplanted into either C57BL/6 wild-type (major histocompatibility complex class II mismatched) or CD8 -/- knockout recipients and examined for the development of cardiac allograft vasculopathy. RESULTS: In the major histocompatibility complex class I mismatched model, CD8(+) lymphocytes were the predominant T-lymphocyte subset that infiltrated the allografts and demonstrated markers of activation. The intracellular cytokine-staining assay demonstrated that CD8(+) lymphocytes were the primary sources of allograft interleukin 2 and interferon gamma. Intimal lesions developed in the allografts by day 14 (12.0% +/- 4.0%) and further increased by day 30 (44.0% +/- 5.0%). In the major histocompatibility complex class II mismatched model, the donor hearts in the CD8 -/- knockout recipients had substantially less severe intimal lesions when compared with the donor hearts in wild-type recipients (19.0% +/- 6.0% vs 50.0% +/- 7.0%, respectively; P <.05). CONCLUSIONS: In both major histocompatibility complex class I and II mismatched models, CD8(+) lymphocytes contribute significantly to chronic rejection. The findings of this study suggest that control of chronic rejection requires interventions directed at CD8(+) lymphocytes.     

Depletion of recipient CD4+ but not CD8+ T lymphocytes prevents the development of cardiac allograft vasculopathy

BACKGROUND: We have described that chimeric rat hearts bearing recipient-type antigen-presenting cells (APCs) do not reject acutely, but develop cardiac allograft vasculopathy (CAV) in untreated recipients. This suggests that CAV is triggered either by CD8+ direct allorecognition or by CD4+ indirect allorecognition. To determine the allorecognition pathway responsible for CAV in this model, recipients of chimeric hearts underwent either CD8+ or CD4+ T cell depletion. METHODS: Chimeric hearts were created via bone marrow transplantation in two fully major histocompatibility-mismatched rat strain combinations. DA recipients were thymectomized and treated with Ox8 and Ox38 murine monoclonal antibodies, which deplete CD8+ and CD4+ T cells, respectively. Chimeric PVG hearts bearing DA APCs, abbreviated PVG(DA), were heterotopically transplanted into recipients undergoing thymectomy alone or recipients undergoing thymectomy plus either CD4+ or CD8+ T cell depletion. RESULTS: PVG(DA) allografts survived 100 days, but developed CAV in thymectomized recipients and in those permanently depleted of CD8+ T cells. In contrast, chimeric hearts transplanted into permanently CD4+ T cell-depleted recipients survived 100 days and demonstrated no evidence of CAV. CONCLUSIONS: In this specific strain combination, recipient CD8+ T cells are neither necessary nor sufficient for the development of CAV, whereas recipient CD4+ T cells are required for the development of CAV. These findings suggest that CAV is dependent on CD4+ indirect allorecognition and that CD8+ direct allorecognition stimulated by nonprofessional APCs plays a minor role.     

CD8 lymphocytes are sufficient for the development of chronic rejection

BACKGROUND: The role of CD8 lymphocytes, in chronic rejection or cardiac allograft vasculopathy (CAV), is incompletely understood. The purposes of this study were to determine whether CD8 lymphocytes, in the absence of CD4 lymphocytes, are capable of causing the intimal lesions of CAV; and if so, to define the effector mechanism(s) of CD8 lymphocytes. METHODS: We modified a previously characterized major histocompatibility complex class II mismatched murine model of CAV. Wild-type CD8 lymphocytes were transferred to nude mice followed by heterotopic heart transplantation. Recipient mice were then treated with a CD40 activating antibody, which is known to provide help for CD8 lymphocyte activation, in the absence of CD4 lymphocytes. Donor hearts were harvested on day 40 posttransplantation and analyzed for cellular infiltrates and intimal thickening. In separate experiments, isolated perforin -/-, Fas ligand (FasL) -/-, and interferon (IFN)-gamma -/- CD8 lymphocytes were transferred to nude mice followed by identical experimented protocol. RESULTS: With adaptive transfer of wild-type CD8 lymphocytes, the donor hearts were infiltrated with activated CD8 lymphocytes and displayed significant intimal lesions. Adoptive transfer of perforin -/- and FasL -/- CD8 lymphocytes to nude mice resulted in similar patterns of CD8 lymphocyte infiltration and similar severity of intimal lesions. The donor hearts from IFN-gamma -/- CD8 lymphocyte reconstituted recipients displayed minimal intimal lesions, although CD8 lymphocytes were present in the allografts. CONCLUSIONS: Unprimed CD8 lymphocytes in the absence of CD4 lymphocytes can cause intimal lesions of CAV. CD8 lymphocytes production of IFN-gamma, but not the perforin or the FasL-mediated cytotoxicity, is the critical step in the development of intimal lesions.     

Gene transfer of immunomodulatory peptides correlates with heme oxygenase-1 induction and enhanced allograft survival

BACKGROUND: Decapeptides derived from human HLA class I sequences have been shown to prolong allograft survival. The mechanism of action of these peptides has been uncertain, because they act in an MHC unrestricted manner. Recently, it was found that these peptides bind heme oxygenase 1 (HO-1). In the present study, we sought to determine whether local delivery of these peptides through gene transfer could extend allograft survival, and to explore the underlying mechanisms. METHODS: C57BL/6 neonatal hearts were transplanted to CBA/J recipients and the peptide, or plasmid DNA encoding the peptide, was injected directly into the allograft at the time of the transplant. RESULTS: Direct injection of 1 microg of the B2702 peptide into the allograft did not prolong survival (13.3+/-0.8 vs. 13.4+/-0.8 days for untreated controls), but injection of 400 microg of peptide did extend survival (22.0+/-0.6). Injection of plasmid DNA encoding the B2702 peptide was superior to peptide delivery, extending graft survival to 30.8+/-1.5 days. Similar results were obtained using another plasmid encoding the rationally designed peptide BC1 (28.5+/-1.7), whereas no significant prolongation was observed using a plasmid encoding the control peptide B2705 (16.5+/-1.0). To explore the hypothesis that these peptides exert their immunosuppressive effect by altering HO-1 activity, animals were treated with iron protoporphyrin, an inducer of HO-1 activity, or tin protoporphyrin, an inhibitor of HO-1. Treatment with iron protoporphyrin alone extended graft survival (24.5+/-1.6) and did not alter the benefit in survival seen with BC1 gene transfer (28.0+/-0.8). In contrast, treatment with tin protoporphyrin abolished the benefit of BC1 gene transfer (17.0+/-0.6). CONCLUSIONS: These results demonstrate that plasmid mediated gene transfer is an effective means for delivering immunosuppressive peptides to extend allograft survival. The experiments suggest that these peptides may act by increasing HO-1 activity and support a role for HO-1 in immune regulation and allograft survival.     

Attenuation of acute xenograft rejection by short-term treatment with LF15-0195 and monoclonal antibody against CD45RB in a rat-to-mouse cardiac transplantation model

BACKGROUND: We previously reported that Lewis rat hearts transplanted into BALB/c mice developed typical acute vascular rejection (AVR). The present study was undertaken to determine the efficacy of LF15-0195, a new analogue of 15-deoxyspergualin, in the prevention of AVR and to determine whether a combination of LF15-0195 and CD45RB monoclonal antibody (mAb) would have a synergistic effect in prolonging xenograft survival. METHODS: We transplanted 2-week-old Lewis rat hearts into BALB/c mice, followed by experimental immunosuppressive regimens. Control groups were either untreated or treated with mAb monotherapy (100 microg/mouse, days -1 to 7, intravenously). Experimental groups were either treated with LF15-0195 (2 mg/kg, days -1 to 14, subcutaneously) or with LF15-0195 combined with mAb at monotherapeutic doses. RESULTS: Heart xenografts in both untreated and mAb-treated BALB/c recipients were rejected at 6.0+/-0.7 days and 8.5+/-1.3 days, respectively, with typical features of AVR, characterized by hemorrhage, fibrin deposition, thrombosis, and massive accumulations of anti-rat IgG and IgM. Serum xenoreactive antibodies (xAbs) were also markedly elevated in these animals. In contrast, LF15-0195 monotherapy significantly prolonged graft survival to 19.3+/-0.7 days. Notably, xAbs were significantly decreased and graft rejection was of a cell-mediated nature instead of AVR. When mAb was combined with LF15-0195, graft survival was further increased to 65.2+/-9.1 days. Antibody production and T-cell infiltration were significantly inhibited at terminal stages of graft survival. Sequential studies on days 6 and 14 demonstrated that LF15-0195, either alone or combined with mAb, completely inhibited antibody production. However, intragraft infiltration by Mac-1+ cells in LF15-0195-treated recipients was similar to that of untreated recipients. CONCLUSIONS: LF15-0195 effectively attenuated AVR by markedly inhibiting antidonor xAb production. Treatment with a combination of LF15-0195 and CD45RB mAb also significantly reduced T-cell infiltration and should be studied further to evaluate its efficacy in nonhuman primate subjects.     

Rantes production during development of cardiac allograft vasculopathy.

BACKGROUND: RANTES (regulated on activation, normal T cell expressed and secreted) production has been shown to correlate with mononuclear cell recruitment and precede intimal thickening in cardiac allograft vasculopathy (CAV). However, the cells that produce RANTES in CAV are undefined. Therefore, in an MHC II-mismatched murine model of CAV, we sought to (1) define the cellular sources of RANTES and (2) determine the role of CD4+ lymphocytes in RANTES production during CAV development. METHODS: B6.CH-2bm12 strain donor hearts were transplanted heterotopically into wild-type (WT) or CD4 knockout (CD4KO) C57BL/6 mice (MHC II mismatch). No immunosuppression was used. Recipients were sacrificed at 7, 14, and 24 days. Intragraft RANTES gene expression and protein levels were determined with ribonuclease protection assay and ELISA, respectively. At days 7 and 24, RANTES production by graft-infiltrating cells was defined with intracellular RANTES staining and multicolor FACS analysis. Intimal thickening was quantitated morphometrically. In murine hearts and in six explanted human hearts with advanced CAV, RANTES was also localized immunohistochemically. RESULTS: NK, NKT, and gammadelta+ cells, in addition to CD4+, CD8+ lymphocytes, and CD11b+ macrophages, produced RANTES in early and late stages of CAV. RANTES-producing NK, NKT, and gammadelta+ cells tripled in number during CAV development; by day 24, NK and gammadelta+ cells each outnumbered CD4+ lymphocytes and CD11b+ macrophages. The presence of CD4+ lymphocytes was required for sustained RANTES production in allografts, which correlated with mononuclear cell recruitment and preceded intimal thickening. In murine and explanted human hearts with advanced CAV, RANTES immunolocalized with graft-infiltrating mononuclear cells and vessel wall cells. CONCLUSIONS: We present evidence that other cell types in addition to CD4+, CD8+ T lymphocytes, and CD11b+ macrophages contribute significantly to RANTES production in CAV. In this MHC II-mismatched murine model of CAV, sustained RANTES production requires CD4+ lymphocytes, correlates with mononuclear cell recruitment, and precedes intimal thickening. In experimental and human CAV, vessel wall cells may also produce RANTES. Interventions aimed at inhibiting RANTES production in CAV may need to target several types of cells, and neutralization of RANTES bioactivity may reduce mononuclear cell recruitment and CAV development.     

Acute rejection and cardiac graft vasculopathy in the absence of donor-derived ICAM-1 or P-selectin.

BACKGROUND: ICAM-1 and P-selectin are molecules that facilitate adhesion of circulating leukocytes to vessel walls. We have investigated the role of donor-derived ICAM-1 and P-selectin in acute and chronic cardiac allograft rejection. METHODS: C57BL/6J (H-2(b)) mice were used as donors for heterotopic heart transplantation into CBA/Ca (H-2(k)) recipients. The donors were wild-type or homozygous for gene mutations of ICAM-1 or P-selectin. We measured acute rejection in non-immunosuppressed recipients by daily palpation and sacrificed mice at Days 2, 4, and 6 for immunohistochemical analysis. For chronic rejection, recipients received monoclonal antibody against CD4+ T cells. We removed hearts at Days 60 to 62 for histologic assessment of vasculopathy using quantitative morphometry to measure intimal thickening. RESULTS: Time (days) to rejection was 7.1 +/- 0.57 for wild-type (n = 10), 7.0 +/- 0.71 for ICAM-1 -/- (not significantly different, n = 7) and 6.1 +/- 0.33 (p = 0.001) for P-selectin -/- donors. ICAM-1 deficiency was associated with delayed infiltrate at Day 4 compared with wild-type. In the model of chronic rejection, elastin-positive vessels showed a mean occlusion of 34% +/- 3% in transplanted wild-type hearts; vessels were divided into those showing 0% to 20%, 20% to 50%, and 50% to 100% occlusion. We observed no difference in the number of affected vessels or the amount of vascular thickening in donors lacking ICAM-1 or P-selectin compared with wild-type controls. CONCLUSIONS: The absence of ICAM-1 or P-selectin in donor tissues neither lengthens the time of allograft survival nor inhibits the vascular lesions associated with chronic rejection. Indeed, the absence of P-selectin may exacerbate alloimmune injury.     

The effect of thymectomy on tolerance induction and cardiac allograft vasculopathy in a miniature swine heart/kidney transplantation model.

BACKGROUND: We have previously demonstrated that MHC class I disparate hearts transplanted into miniature swine treated with a short course of cyclosporine developed florid cardiac allograft vasculopathy (CAV) and were rejected within 55 days. However, when a donor-specific kidney is cotransplanted with the heart allograft, recipients become tolerant to donor antigen and accept both allografts long-term without the development of CAV. In the present study, we have investigated the role of the host thymus in the induction of tolerance and prevention of CAV in heart/kidney recipients. METHODS: Total thymectomies were performed in six animals (postoperative day [POD]-21), which on day 0 received either an isolated MHC class I disparate heart allograft (n=3) or combined class I disparate heart and kidney allografts (n=3), followed in both cases by a 12-day course of cyclosporine (POD 0-11). Graft survival and the development of CAV in these thymectomized recipients were compared to the same parameters in non-thymectomized, cyclosporine-treated recipients bearing either class I disparate heart allografts (n=5) or heart and kidney allografts (n=4). RESULTS: In the group of animals bearing isolated class I disparate heart allografts, the thymectomized recipients rejected their allografts earlier (POD 8, 22, 27) than the non-thymectomized recipients (POD 33,35,45,47,55). The donor hearts in both the thymectomized and non-thymectomized animals developed florid CAV. In the group of animals bearing combined class I disparate heart and kidney allografts, the nonthymectomized recipients accepted both donor organs long term with no evidence of CAV. In contrast, none of the thymectomized heart/kidney recipients survived >100 days, and they all developed the intimal proliferation of CAV. CONCLUSION: Thymic-dependent mechanisms are necessary for the induction of acquired tolerance and prevention of CAV in porcine heart/kidney recipients.     

Regulated interleukin-10 expression prevents chronic rejection of transplanted hearts

OBJECTIVE: Interleukin-10 is a pleiotrophic cytokine with variable effects on the alloimmune response, depending on the experimental model system. The purpose of this study was to determine the role of regulated interleukin-10 expression on the development of chronic rejection in heart transplantation, or cardiac allograft vasculopathy. METHODS: Donor hearts from B6.C-H2(bm12) mice were transplanted into wild-type and interleukin-10 transgenic recipients. In interleukin-10 transgenic recipients, murine interleukin-10 cytokine is produced under the control of human interleukin-2 promoter. Donor hearts were sacrificed at days 7 and 24. No immunosuppression was used. Intimal proliferation was measured morphometrically. Intragraft cellular infiltrate was defined by both immunohistochemistry and flow cytometry. Intracellular cytokine staining assay was performed to determine both the type and source of intragraft cytokines. RESULTS: Hearts transplanted into wild-type recipients developed severe cardiac allograft vasculopathy by 24 days. Intimal lesions were absent in the donor hearts transplanted into interleukin-10 transgenic recipients. The number of graft-infiltrating T lymphocytes and the percentage of interleukin-2/interferon-gamma producing T lymphocytes were markedly reduced in interleukin-10 transgenic recipients. Finally, the overexpression of interleukin-10 resulted in the decline of graft-infiltrating macrophages at all time points. CONCLUSIONS: Regulated expression of interleukin-10 inhibits cardiac allograft vasculopathy development via reduction of mononuclear cell recruitment and alteration of their cytokine profile. This strategy may prove beneficial in controlling the alloimmune response in solid organ transplants.     

Peritransplant treatment with cobalt protoporphyrin attenuates chronic renal allograft rejection

Allogen-independent injury contributes to chronic rejection in renal allografts and heme oxygenase-1 (HO-1) has been shown to be protective in a number of settings. This study evaluated the effect of renal allograft recipient HO-1 up-regulation on chronic rejection in a rat model. Rat (F344 to Lewis) renal transplantation recipients were grouped: (i) cyclosporine (CsA) alone (0.75 mg/kg s.c. x 10 day; n = 5); (ii) CsA + low dose cobalt protoporphyrin (CoPP) an HO-1 inducer (0.5 mg/kg i.p. on days -5,0,5; n = 13) and (iii) CsA + high dose CoPP (5.0 mg/kg i.p. on days -5,0,5; n = 8). Renal function was assessed by serum creatinine levels on day 140. Histopathologic changes in allografts were graded. Morphometric analyses performed to objectively quantify the vascular changes and glomerulosclerosis. HO-1 expression quantified by Western blot and both HO-1 and endothelin (ET-1) localized using immunohistochemistry. Recipients treated with CsA + high dose CoPP had significantly decreased cortical scarring, vascular hyalinization and intimal thickness. They also had a significant, dose-dependent, reduction in luminal obliteration and glomerulosclerosis by morphometric analyses. This freedom from chronic rejection in recipients treated with CoPP translated into quiescent grafts at postoperative day 140 with immunostaining and Western blot demonstrating decreased level of HO-1 versus controls (P = 0.012). In summary, the peritransplant up-regulation of HO-1 in renal allograft recipients significantly attenuates chronic rejection in rat renal allografts by inhibiting transplant vasculopathy.     

Overexpression of heme oxygenase-1 protects allogeneic thyroid grafts from rejection in naive mice

BACKGROUND: Endocrine allografts are an option for the treatment of endocrine failure. METHODS: One lobe of the thyroid was transplanted under the kidney capsule. RESULTS: C57BL/10 (H2(b)) thyroids were rejected in naive CBA (H2(k)) mice within 14 days after transplantation. When mice were treated with anti-CD4 monoclonal antibodies (mAb), all grafts survived for more than 60 days. The first grafts still survived after second C57BL/10 or Balb/c (H2(d)) thyroid grafts that were transplanted into the same recipients were rejected acutely, which suggests that the primary grafts were modified under anti-CD4 mAb treatment. To confirm this hypothesis, C57BL/10 thyroid grafts from anti-CD4 mAb-treated mice were retransplanted. All grafts survived in naive mice; this correlated with the overexpression of heme oxygenase-1 (HO-1) in the grafts. Next, an inhibitor of HO-1 (zinc protoporphyrin) or control compound (copper protoporphyrin) was injected intraperitoneally after transplantation of C57BL/10 thyroid grafts into the primary CBA recipients that had been treated with anti-CD4 mAb. The grafts in mice that had been treated with zinc protoporphyrin, but not copper protoporphyrin, were rejected when retransplanted to naive recipients. CONCLUSIONS: Overexpression of HO-1 correlated with the protection of fully allogeneic thyroid grafts from rejection when retransplanted into naive recipients.     

Inhibition of chronic rejection and development of tolerogenic T cells after ICOS-ICOSL and CD40-CD40L co-stimulation blockade

BACKGROUND.: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. METHODS.: The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. RESULTS.: Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. CONCLUSIONS.: These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.     

Graft vasculopathy and tolerance: does the balance of Th cells contribute to graft vasculopathy?

BACKGROUND: Cardiac allograft vasculopathy (CAV) is sometimes observed even though the allograft may survive indefinitely. In this study, we examined whether or not the preferential activation of Th2-type cells prevents the development of CAV. METHODS: Hearts from C57BL/10 mice were transplanted heterotopically into the abdominal cavities of C3H.He recipient mice, and monoclonal antibodies (mAbs) to T-cell receptor (TCR) alphabeta or CD80/CD86 were administered after transplantation. The incidence of CAV was then examined histologically. To investigate the relative Th1/Th2 balance, the levels of IFNgamma and IL4 in the transplanted hearts were measured. RESULTS: Indefinite heart graft survival was observed in mice treated with either the anti-TCRalphabeta or anti-CD80/CD86 mAbs and these mice accepted donor-type (C57BL/10) skin grafts but rejected those from a third party (BALB/c). Evidence of CAV was found in the mice treated with the anti-TCRalphabeta mAb, but CAV did not develop in the mice treated with anti-CD80/CD86 mAbs. Preferential activation of Th2-type cells was dominant in the tolerant mice treated with anti-TCRalphabeta mAb, but it was not dominant in the tolerant mice treated with anti-CD80/CD86 mAbs. CONCLUSION: These findings suggest that the dominance of Th2-type cells does not prevent allograft vasculopathy.     

Reepithelialized orthotopic tracheal allografts expand memory cytotoxic T lymphocytes but show no evidence of chronic rejection

BACKGROUND: Acute rejection of mouse tracheal allografts is characterized by infiltration of the lamina propria with CD4+/CD8+ T cells that leads to the destruction of the epithelium and luminal obliteration. The donor epithelium is progressively replaced by recipient-derived epithelium. Once allograft reepithelialization has occurred, immunosuppression can be withdrawn without inciting acute rejection. We hypothesize that reepithelialization will also prevent chronic rejection of the trachea after withdrawal of immunosuppression. METHODS: BALB/c tracheal grafts were transplanted orthotopically into allogeneic C57BL/6 recipients. Allografted mice were nonimmunosuppressed for 10 or 100 days or immunosuppressed with cyclosporine A continuously for 50 days and then withdrawn from immunosuppression for an additional 50 days. In addition, grafts from this group were then heterotopically retransplanted into isogenic C57BL/6 or allogeneic BALB/c recipients to assess their immunogenicity. RESULTS: Cyclosporine A-treated mice showed no signs of chronic rejection or priming of cellular immunity as measured by proliferation and cytokine secretion in a mixed leukocyte reaction. However, there was a notable expansion of memory CD8+ T cells specific for donor major histocompatibility complex. When these tracheal allografts were retransplanted heterotopically into C57BL/6 or BALB/c, they demonstrated reduced responses toward BALB/c and primed responses toward C57BL/6, respectively. These results suggest that the grafts express a chimeric phenotype consisting of both BALB/c and C57BL/6 antigens. CONCLUSION: These observations suggest that long-term withdrawal of immunosuppression does not lead to chronic tracheal rejection even in the presence of alloantigen specific cytotoxic T-lymphocyte responses and that the reepithelialized grafts may contain donor elements that impact the generation of immunity.     

Suppression of cardiac allograft vasculopathy in mice by inhibition of CC-motif chemokine receptor 5

Cardiac allograft vasculopathy (CAV) is the leading cause of late morbidity and mortality in heart-transplant patients. Increasing evidences support the important role of chemokines and their receptors in transplant immunology. Chemokine-chemokine receptor interaction and subsequent recruitment of T-lymphocytes to the graft are early events in the development of chronic rejection of transplanted hearts. In this study, we first inhibited CC-motif chemokine receptor 5 (CCR5) expression by using lentiviral-mediated gene transfer of an anti-CCR5 siRNA, which introduced through CD34(+) hematopoietic stem/progenitor cell transplantation. Stably marked lymphocytes expressing siRNA and consistent downregulation of CCR5 expression were detected. Our results showed that survival was significantly prolonged in CCR5 knock-down mice and donor hearts from siRNA-treated mice developed markedly less CAV. Infiltration of CD4(+) and CD8(+) T-lymphocytes into transplanted hearts was also markedly decreased. These findings suggest that CCR5 plays an important role in CAV development and inhibition of this chemokine could improve long-term survival after cardiac transplantation.     

Inhibition of chronic rejection and development of tolerogenic T cells after ICOS-ICOSL and CD40-CD40L co-stimulation blockade

BACKGROUND: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. METHODS: The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and the fusion molecule CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. RESULTS: Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. CONCLUSIONS. These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.