Allogeneic Heart Research Articles

Further evidence that NK cells may contribute to the development of cardiac allograft vasculopathy

In prior experiments, we found that recipients, even though specifically tolerant of donor antigens, will develop striking cardiac allograft vasculopathy (CAV) in allogeneic mouse heart transplants. This suggested that innate immune responses, in addition to conventional adaptive immunity, may be involved in the development of CAV. We accordingly performed transplants in the parental-to-F1 combination seeking supportive evidence of NK cell activity directed against the vessels of parental donor transplants as a manifestation of “hybrid resistance.” When such lesions were indeed found, we investigated their pathogenesis employing immunopathological analysis, in vitro measurements of NK cytotoxicity, and donor-specific T-cell activity in F1 recipients of parental donor hearts. We present evidence that NK cells can promote cardiac allograft vasculopathy. Since NK cell activity is not well targeted by current immunosuppressive therapy, its control may offer a valuable new possibility for improving the long-term outcome of transplanted organs.

Activation of splenic dendritic cells by bacterial and allogeneic antigens

Allograft ischemia and cellular degradation accompanying rejection favor graft colonization by translocated microorganisms. Bacterial colonization adds to the graft destruction. The dendritic cells (DC) of allograft recipients engage in allogeneic and antibacterial reactions; they process and present to lymphocytes 2 types of antigens. This may lead to overstimulation of DCs that may nonspecifically intensify the rejection process. We investigated the effects of allogeneic and bacterial antigens on splenic DCs phenotypes. In vitro stimulation of a spleen DC–enriched population by E. coli, LPS, and CpG DNA brought about an increase in expression of OX6 + (MHC class II) from 47.4% in the control cells to 65% in the E. coli-stimulated group ( P < .05) and 85% in the LPS and CpGDNA groups ( P < .05). Interestingly, a significant drop in the frequency of OX62 + DC was observed after incubation with LPS. Allogeneic heart transplants brought about an increase of OX6 + in DCs to 100% and a decrease of ED1 + monocyte frequency. Simultaneously, an increase in expression of W3/13 + T cells in DC-enriched splenic cells was observed. There was no significant change in the frequency of OX62 + expression. Both types of antigens evoked splenic DC response; however, there were differences in the frequency of phenotype expression. Allogeneic but not bacterial antigens increased W3/13 antigen expression; the frequency of OX62 + in cells decreased after LPS but not after bacterial stimulation.

CCR4‐deficient mice show prolonged graft survival in a chronic cardiac transplant rejection model

Chronic graft rejection mediated by cellular immune responses still poses a serious clinical problem in transplant surgery. Chemokines coordinate the recruitment of leukocytes in inflammatory and immune responses. Their precise functions in the rejection of allografts are still ill defined. This study investigates the role of chemokine receptor 4 (CCR4) in acute and chronic cardiac allograft rejection in mice. Allogeneic hearts were transplanted into CCR4 deficient (CCR4(-/-)) and control recipients. Reverse transcription-PCR showed transcription of macrophage-derived chemokine and thymus and activation-regulated chemokine, the cognate chemokine ligands of CCR4, within the graft. Compared to wild-type controls, acute allograft rejection in CCR4(-/-) recipients was only slightly prolonged. In contrast, in a gallium nitrate chronic cardiac allograft rejection model, cardiac graft survival was significantly prolonged in CCR4(-/-) recipients. A relative increase in the percentage of graft infiltrating CD8(+) T cells in CCR4(-/-) recipients was observed 30 days after transplantation and was accompanied by a decrease in CD4(+) T cells. Moreover, the percentage of NK1.1(+)CD3(+) graft-infiltrating cells was significantly reduced on day 5 and day 30 post transplantation. These findings indicate that CCR4 is involved in the recruitment of NK1.1(+)CD3(+) cells into cardiac allografts and clearly establish an important and novel role for CCR4 in chronic graft rejection.

Role of CD4+ and CD8+ T cells in murine skin and heart allograft rejection across different antigenic desparities

The factors that influence the relative contribution of the T cell subsets to allograft rejection remain unclear. We compared skin and heart rejection in CD4 Knockout (KO), and CD8 KO mice across full-, minor-, and class II histocompatibility antigen (HA) mismatches. Skin allografts were rejected by either CD4+ or CD8+ T cells alone at any degree of antigenic mismatch. However, either the absence of CD4+ cells or a lesser degree of HA mismatch resulted in prolongation of graft survival. In contrast, fully allogeneic heart grafts were accepted in CD4 KO recipients, and minor HA mismatched heart grafts were accepted by both CD4 KO and CD8 KO mice. Thus, the T cell subsets required for allograft rejection are determined by the immunogenicity of the tissue transplanted. In the absence of CD8+ T cells, perforin and Fas ligand (FasL) but not granzyme B mRNA were detected in rejecting grafts. Thus, granzyme B is a CD8+ cytotoxic T lymphocyte (CTL)-specific effector molecule.

MHC class II isotype- and allele-specific attenuation of experimental autoimmune encephalomyelitis.

Most autoimmune diseases are associated with certain MHC class II haplotypes. Autoantigen-based specific immune therapy can lead either to beneficial or, in the context of inflammatory conditions, detrimental outcomes. Therefore, we designed a platform of peptides by combinatorial chemistry selected in a nonbiased Ag-independent approach for strong binding to the rat MHC class II isotype RT1.D(n) allelic product of the RT1(n) haplotype that is presenting autoantigen in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in LEW.1N rats. Peptide p17 (Ac-FWFLDNAPL-NH(2)) was capable of suppressing the induction of and also ameliorated established experimental autoimmune encephalomyelitis. MHC class II isotype and allele specificity of the therapeutic principle were demonstrated in myelin basic protein-induced experimental autoimmune encephalomyelitis in LEW rats bearing the RT1(l) haplotype. A general immunosuppressive effect of the treatment was excluded by allogeneic heart transplantation studies. In vitro studies demonstrated the blocking effect of p17 on autoantigenic T cell responses. We thus demonstrate a rational design of strong MHC class II-binding peptides with absolute isotype and allele specificity able to compete for autoantigenic sequences presented on disease-associated MHC class II molecules.

INFECTIOUS TOLERANCE MEDIATED BY DENDRITIC CELLS: A NOVEL MECHANISM OF ALLOGRAFT ACCEPTANCE THROUGH ACTIVATION OF T REGULATORY CELLS

O177* Purpose: It is established that transplant tolerance can be adoptively transferred from tolerant recipients to a naive recipient by T cells, a phenomena termed ,,infectious tolerance“. To date, the ability of other cells to transfer tolerance has not been demonstrated. The present study aims to determine whether tolerogenic dendritic cells (Tol-DC) isolated from tolerant mice can transfer graft-specific tolerance to naive recipients, and to dissect the mechanisms underlining infectious tolerance induced by Tol-DC. Methods: BALB/c mice receiving C57BL/6 cardiac allografts were treated with LF 15-0195, a novel analogue of 15-deoxyspergualine at a dose of 2 mg/kg for 20 days. Long-term survivors (>100 days) were defined as tolerant recipients. Tol-DC were isolated from tolerant recipients by anti-CD11c mAb-conjugated MACS beads. Tol-DC were intravenously injected into syngeneic (BALB/c) recipients followed by allogeneic (C57/BL6>Balb/c) heterotopic heart transplantation. Results: The Tol-DC isolated from tolerant recipients demonstrated an immature phenotype as exemplified by diminished expression of MHC II, CD40, and CD86. Functionally, Tol-DC failed to stimulate allogeneic T cell responses in MLR. Furthermore, the addition of Tol-DC isolated from tolerant recipients inhibited ongoing MLR, suggesting Tol-DC play a regulatory role. To further investigate the in vivo regulatory function of Tol-DC, ex vivo isolated Tol-DC (5x106 cells/mouse) from tolerant recipients were adoptively transferred into naive syngeneic recipients. While transferring control DC resulted no protection of allografts (MST 13 ± 1 day), transferring Tol-DC significantly prolonged allograft survival (MST 45 ± 8 days) in the absence of immune suppressants, suggesting an “infectious tolerance” occurred after adoptive transfer of Tol-DC. We next investigated whether transfer of Tol-DC was associated with generation of Treg. Indeed, subsequent to Tol-DC adoptive transfer, an increase in the CD4+CD25+ Treg subset was observed. Furthermore, CD4+CD25+CTLA4+ cells were detected at 31% after Tol-DC transfer compared to 6.1% in controls. Functionally, these Treg inhibited ongoing MLR in vitro. Finally, we examined the expression of indolamine-2,3-deoxygenase (IDO), an novel DC-derived T cell inhibiting enzyme, was remarkably up-regulated in the spleens of recipients after adoptive transfer of Tol-DC but not control DC. Conclusions: This study is the first demonstration of infectious tolerance by DC. The transplant tolerance can be adoptively transferred from a tolerant recipients to a naïve mouse. Mechanisms involved include generation of Treg in vivo and upregulation of IDO.

FURTHER EVIDENCE THAT NK CELLS MAY CONTRIBUTE TO THE DEVELOPMENT OF CARDIAC ALLOGRAFT VASCULOPATHY.

Abstract In prior experiments, we found that recipients, even though specifically tolerant of donor antigens, will develop striking cardiac allograft vasculopathy (CAV) in allogeneic mouse heart transplants. This suggested that innate immune responses, in addition to conventional adaptive immunity, may be involved in the development of CAV. We accordingly performed transplants in the parental-to-F1 combination seeking supportive evidence of NK cell activity directed against the vessels of parental donor transplants as a manifestation of “hybrid resistance.” When such lesions were indeed found, we investigated their pathogenesis employing immunopathological analysis, in vitro measurements of NK cytotoxicity, and donor-specific T-cell activity in F1 recipients of parental donor hearts. We present evidence that NK cells can promote cardiac allograft vasculopathy. Since NK cell activity is not well targeted by current immunosuppressive therapy, its control may offer a valuable new possibility for improving the long-term outcome of transplanted organs.

RECIPIENT IMMATURE DENDRITIC CELLS DO NOT PROLONG ALLOGRAFT SURVIVAL

P969 Introduction: According to some reports, immature donor derived myeloid (DC) prolong heart allograft survival. We reported previously that depletion of mature DC in donor heart and replacing them with recipient bone marrow derived DC did not prolong survival of heart allograft but to the contrary induced rejection of allogeneic and even syngeneic grafts. In this study we investigated influence of recipient immature BM-derived DC on heart allograft survival. Material and methods: Depopulation of prospective recipient was achieved by total body irradiation. Next, repopulation by syngeneic immature DC (BN to BN) from vascularised BM transplant followed. Three days after repopulation from transplanted BM, rats received allogeneic heart transplant from LEW rats. We compared heart allograft survival in recipients repopulated with non-repopulated (irradiated), non-depopulated (naive) recipients, and repopulated with syngeneic granulocytes. Immunohistological study of allograft recipients graft, spleen, lymph nodes and BM using mAb against OX 27 (specific for donor only) and OX6, OX62, ED1, W3/13 was performed after depopulation, repopulation and rejection. Double staining by OX27 and the above listed antibodies was carried out using flow cytometry. A low level of OX27/ OX6 and OX27/OX62 expression (fluorescence) reflected immaturity of DC. Results: Allograft survival in recipients repopulated with immature BM-derived DC was - 14±2 days, in non-repopulated (irradiated) recipient - 13±1 days, in non-depopulated (naive) recipient - 7±1 days, in irradiated and repopulated with syngeneic granulocytes - 15±2 days. There was low antigen expression of OX27/OX6 and OX27/OX62 antigens in repopulated hearts before transplantation and similar density of infiltrating OX6+ and OX27+ cells in rejecting allografts. Conclusion: In this model with donor heart repopulated by immature recipient DC no biologically significant prolongation of allograft survival was observed.

TOLERANCE INDUCTION BY UPREGULATION OF REGULATORY T-CELLS (EXPRESSING FOXP3 AND CTLA-4) BY COCULTURE WITH IN VITRO MODIFIED HUMAN MONOCYTES BEARING REGULATORY FUNCTION

P915 Aims: We identified a subpopulation of in vitro modified human monocytes bearing regulatory function and expressing specific proteins known to be expressed only by regulatory T-cells. In vivo rat derived modified monocytes induce tolerance to second set allogeneic heart transplantation and are therefore termed TAIC (Transplant acceptance inducing cells). In vitro TAIC induce a conversion of CD4+/CD25- cells into regulatory T-cells (Treg, CD4+/CD25+) and act synergistically in suppressing donor specific immune responses in human settings. Methods: We cultured human peripheral blood monocytes with MCSF and y-interferon (TAIC) for 5 days and cocultured them with lymphocytes (L) from a different donor for subsequent 4 days. Cells were purified after coculture and expression of FoxP3 and CTLA-4 was analyzed by PCR. Suppression of donor specific immune response was evaluated in a mixed lymphocyte reaction (MLR) analyzing each cell population separately and together. To exemplify the in vivo effect in a human setting we generated TAIC from a split-liver donor (father), whose recipient (child) is chronically rejecting the transplanted organ and cocultured TAIC from the father with recipients lymphocytes for subsequent functional analysis in an MLR. Results: After purification cocultured lymphocytes as well as TAIC upregulate FoxP3 and CTLA-4 expression unlike freshly isolated lymphocytes or those cultured without TAIC. In vitro functional assays repetitively showed a donor specific suppression of activated autologous lymphocytes in a MLR by TAIC and cocultured lymphocytes. In the above mentioned clinical setting we could show an upregulation of Treg from the recipient child after coculture with donor derived TAIC. Functional analysis showed a remarkable downregulation of the childs immune response after stimulation with APC cells from the father by TAIC cells and cocultured lymphocytes acting synergistically. Conclusions: Postulating that the expression of FoxP3 and CTLA-4 is in concordance with immunoregulatory function, we could herein show that the suppressive potential of TAIC paralells with an upregulation of regulatory genes which were up to here only described for lymphocytes. We here describe a new cell of monocytic origin capable of immunregulation by its own action and by inducing regulatory T-cells after coculture with allogeneic lymphocytes. The exemplarily demonstrated donor specific immunmodulation in a clinical setting in which rejection is taking place holds promise for future therapeutic applications of TAIC in the field of tolerance induction.

Gene therapy for organ grafts using rapid injection of naked DNA: application to the rat liver.

We developed a nonviral gene transfer method using rapid injection of naked DNA targeting the liver and applied it in a rat model of liver transplantation. Inbred Dark Agouti and Lewis rats were used. To test the efficacy and adverse effects of systemic or local (catheter-based) injection, different volumes of phosphate-buffered saline containing naked DNA encoding beta-galactosidase (lacZ) were injected. Luciferase expression was followed by non-invasive imaging, and a cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4Ig) protein was tested functionally by allogenic heart transplantation. Gene transfer was then tested in rat auxiliary liver transplantation (ALT) and orthotopic liver transplantation (OLT). The timing of gene transfer was evaluated in the auxiliary liver transplantation model, and OLT was performed using a liver graft to which luciferase or the CTLA4Ig gene was transferred 2 days before. LacZ was expressed extensively in a volume-dependent manner; however, a large volume often induced recipient death. After local delivery of CTLA4Ig cDNA to the liver, survival of Dark Agouti heart grafts lengthened with increased CTLA4Ig serum levels. Liver grafts injected with naked DNA at the time of donation did not survive, but livers grafted 2 days after gene transfer survived. Successful expression of luciferase and production of CTLA4Ig were finally confirmed in the rat that underwent OLT. We successfully applied a nonviral hydrodynamic gene transfer method to the rat liver and showed its potential in liver grafting. The high incidence of graft failure when this procedure is performed on the day of organ donation is a potential limitation that needs to be overcome in clinical application.

Monoclonal Antibody Specific for TIRC7 Induces Donor-specific Anergy and Prevents Rejection of Cardiac Allografts in Mice

T cell immune response c-DNA (TIRC7) is up-regulated during the early stages of T-cell activation in response to alloantigens. In this study, we analyzed the effects of newly developed monoclonal antibodies (mAb) against TIRC7 in acute cardiac allograft rejection. Fully vascularized heterotopic allogeneic heart transplantation was performed in mice across a full-mismatch barrier (C57Bl/10 into CBA). Recipients received seven injections (day 0–7) of a novel anti-TIRC7 mAb or remained untreated. Graft survival, histology and ex vivo lymphocyte functions were tested. Targeting of TIRC7 with an anti-TIRC7 mAb diminishes lymphocyte infiltration into grafts resulting in delay of morphological graft damage and prolongation of allograft survival. The lymphocytes from anti-TIRC7 mAb-treated animals exhibit hypo-responsiveness without evidence of lymphocyte depletion against the donor allo-antigens. Proliferation and expression of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were down-regulated while interleukin-4 (IL-4) and IL-10 expression were spared. Moreover, anti-TIRC7 mAb enhanced up-regulation of CTLA-4 expression but suppressed up-regulation of CD25 on stimulated lymphocytes in vitro and in vivo. Ligation of TIRC7 has important effects on the regulation of co-stimulatory signaling pathways associated with suppressing of T-cell activation. Targeting of TIRC7 may therefore provide a novel therapeutic approach for modulating T cell immune responses during organ transplantation.

New therapeutic option for cardiac ischemia in transplant vasculopathy: spinal cord stimulation

The main limitation to long-term survival for allogeneic heart transplant recipients is the onset of “accelerated allograft coronary artery disease” (ACAD). Over the years, different treatment options for the management of ACAD-induced critical ischemia have been taken into consideration. The use of percutaneous transluminal coronary angioplasty (PTCA) is still controversial, performed mostly for Type B/C and Type A lesions measuring 2.5 mm. According to Musci et al, there is no difference between PTCA-treated and non–PTCAtreated patients with ACAD. Myocardial re-vascularization surgery is burdened with high peri-operative mortality and poor long-term results. Transmyocardial laser re-vascularization initially provided encouraging short-term results, but these were not borne out by long-term outcome. In consideration of the increasing number of potential heart transplant recipients and the limited donors pool, which is not increasing proportionally, re-transplantation does not appear to be an ethically sufficient option, because a second transplantation entails a far higher short-term mortality than a first transplantation and is accompanied by a high probability of coronary artery disease (CAD) recurrence. The good outcome reported in the literature and our own experience since 1986 in the management of refractory angina with spinal cord stimulation (SCS) has led us to use this technique in posttransplantation CAD as well. To our knowledge SCS treatment for cardiac transplant recipients has not been reported to date. The Brussels Consensus Statement on SCS in 1998 stated that “to be accepted for SCS treatment, angina pectoris must be refractory both to drug therapy (beta-blockers, calcium channel antagonists, long action nitrates, aspirin) and revascularization methods.” We report a 32-year-old man (weight 58 kg, height 168 cm) who underwent orthotopic cardiac transplantation in February 1992 because of dilated cardiomyopathy, which was diagnosed 2 years earlier and probably had a viral etiology. Post-transplantation follow-up revealed hyperlipoproteinemia, hyperuricemia with sporadic episodes of gout, and obesity. During the 4 years after surgery the patient’s tolerance to the allograft was good. Following the onset of diastolic hypertension and atypical chest pain, left cardiac catheterization with coronary angiography was performed in July 1996. Hemodynamic study showed lesion-free coronary arteries. In January 2000 the patient’s weight increased to 97 kg. Also, he complained of chest pain, typical of exercise-induced angina. The pain was “band-like,” retrosternal, with short duration, and decreased at rest (CCS Functional Class II to III). One month From the Divisions of Pain Therapy and Palliative Care, and Cardiac Surgery, University of Turin, Turin, Italy. Submitted June 11, 2002; revised February 13, 2003; accepted February 13, 2003. Reprint requests: Paolo Centofanti, MD, Divisione di Cardiochirurgia, Ospedale Molinette, C.so A.M. Dogliotti 14, 10126 Turin, Italy. E-mail: paolocentofanti@tiscalinet.it Copyright © 2004 by the International Society for Heart and Lung Transplantation. 1053-2498/04/$–see front matter doi:10.1016/S1053-2498(03)00151-7

Viral interleukin-10 gene therapy to induce tolerance to solid organ transplants in mice

In this study, a novel gene therapy approach to prolong allograft survival was designed. Autologous (syngeneic) hematopoietic stem cell–enriched bone marrow cells (HSC; lin −) engineered with the vIL-10 gene (vIL-10-HSC) were injected (4 to 6 × 10 6 cells, iv) into lethally (9.5 Gy) or sublethally (4 Gy) irradiated CBA/J mice 6 weeks prior to allogeneic heart (C57BL/6) transplantation (Tx). Cardiac allograft survival was significantly ( P < .004) prolonged in lethally (71 ± 40 days) and sublethally (114 ± 15 days) irradiated mice that received vIL-10-HSC compared to controls that received no HSC (11 ± 1 days), unengineered HSC, or vector-DNA–engineered HSC (12 to 16 days). Tolerant graft histopathology demonstrated mild arteritis/venulitis (grade 0.7) and rejection (grade 1.0). Intragraft expression of costimulatory molecules (B7.1, B7.2), cytokines (IL-2, IL-4, mIL-10, IFN-γ), and iNOS molecules were markedly lower in tolerant grafts that survived for >100 days; recipient T lymphocytes demonstrated hyporeactivity to donor and third-party antigens in mixed lymphocyte cultures. These findings have important implications and potential therapeutic applications in transplantation and autoimmune diseases.

Myocardial tissue engineering and regeneration as a therapeutic alternative to transplantation.

Ischemic cardiomyopathy leading to congestive heart failure remains the leading source of morbidity and mortality in Western society and medical management of this condition offers only palliative treatment. While allogeneic heart transplantation can both extend and improve the quality of life for patients with end-stage heart failure, this therapeutic option is limited by donor organ shortage. Even after successful transplantation, chronic cardiac rejection in the form of cardiac allograft vasculopathy can severely limit the lifespan of the transplanted organ. Current experimental efforts focus on cellular cardiomyoplasty, myocardial tissue engineering, and myocardial regeneration as alternative approaches to whole organ transplantation. Such strategies may offer novel forms of therapy to patients with end-stage heart failure within the near future.

Rho kinase promotes alloimmune responses by regulating the proliferation and structure of T cells.

Coordinated rearrangements of the actin-myosin cytoskeleton facilitate early and late events in T cell activation and signal transduction. As many important features of cell shape rearrangement involve small GTP-binding proteins, we examined the contribution of Rho kinase to the functions of mature T cells. Inhibitors of the Rho kinase pathway all had similar actions to inhibit the proliferation of primary lymphocyte cultures. Likewise, transfection of the human Jurkat T cell line with a dominant negative, kinase-defective mutant of Rho kinase diminished Jurkat cell proliferation. Furthermore, inhibition of Rho kinase substantially attenuated the program of cytokine gene expression that characterizes T cell activation, blocked actomyosin polymerization, and prevented aggregation of the TCR/CD3 complex colocalized with lipid rafts. These actions are relevant to immune responses in vivo, as treatment with a Rho kinase inhibitor considerably prolonged the survival of fully allogeneic heart transplants in mice and diminished intragraft expression of cytokine mRNAs. Thus, Rho GTPases acting through Rho kinase play a unique role in T cell activation during cellular immune responses by promoting structural rearrangements that are critical for T cell signaling.

Generation of heme oxygenase-1-transgenic rats.

Heme oxygenase-1 (HO-1) expression protects cells from a variety of cellular insults and inhibits inflammation. However, its role in the regulation of immune responses has not yet been clearly established. We generated HO-1 transgenic rats to directly test the impact of HO-1 on the different immune mechanisms. To temporally control the expression of HO-1, we used a one-plasmid tetracycline (tet)-inducible system. This plasmid contains the H-2K(b) promoter, which transcribes the tet transactivator (tTA) and expression of a human HO-1 cDNA is obtained in the absence of tetracycline. The DNA construct was microinjected into one-cell rat embryos and mothers and pups were maintained with tetracycline. Eight transgenic founders were obtained. Analysis of transgene expression in the absence of tet showed that 2 lines (12.4 and 12.6) expressed HO-1 mRNA in several organs (as detected by reverse transcription polymerase chain reaction) and at the protein level only in the thymus. Expression levels of transgene-derived HO-1 increased after withdrawal of tet compared with transgenic rats maintained with tet, as detected by analysis of mRNA levels by quantitative real-time reverse transcription polymerase chain reaction. Gross examination and histopathological analysis of several organs in both lines showed no anomalies. Thymocytes and splenocytes of both lines showed normal cell subpopulations and allogeneic proliferation compared with controls. Systemic immune responses against cognate antigens were normal in both lines, as evaluated by the proliferation of lymph node cells and the production of antibodies against keyhole limpet hemocyanin after immunization. Animals from line 12.6 rejected transplanted allogeneic hearts with the same kinetics as controls. In conclusion, short-term induction of HO-1 overexpression did not modify immune responses compared to those of control non-transgenic animals.

Active suppression of allogeneic proliferative responses by dendritic cells after induction of long-term allograft survival by CTLA4Ig

AbstractCostimulatory blockade using cytotoxic T lymphocyte–associated antigen 4 immunoglobulin (CTLA4Ig) efficiently down-regulates immune responses in animal models and is currently used in autoimmune and transplantation clinical trials, but the precise cellular and molecular mechanisms involved remain unclear. Rats that received allogeneic heart transplants and were treated with adenoviruses coding for CTLA4Ig show long-term allograft survival. The immune mechanisms regulating induction of long-term allograft acceptance were analyzed in splenocytes using mixed leukocyte reactions (MLRs). MLRs of splenocytes but not purified T cells from CTLA4Ig-treated rats showed higher than 75% inhibition compared with controls. Splenocytes from CTLA4Ig-treated rats inhibited proliferation of naive and allogeneically primed splenocytes or T cells. MLR suppression was dependent on soluble secreted product(s). Production of soluble inhibitory product(s) was triggered by a donor antigen-specific stimulation and inhibited proliferation in an antigen-nonspecific manner. CTLA4Ig levels in the culture supernatant were undetectable and neither interleukin-10 (IL-10), transforming growth factor β1 (TGFβ1), IL-4, nor IL-13 were responsible for suppression of MLRs. Inhibition of nitrous oxide (NO) production or addition of IL-2 could not restore proliferation independently, but the combined treatment synergistically induced proliferation comparable with controls. Stimulation of APCs using tumor necrosis factor (TNF)–related activation-induced cytokine (TRANCE) or CD40L and addition of IL-2 normalized MLRs of CTLA4Ig-treated splenocytes. Finally, dendritic cells (DCs), but not T cells, from CTLA4Ig-treated rats inhibited naive MLRs. Altogether, these results provide evidence that after in vivo CTLA4Ig treatment, splenocytes, and in particular DCs, can inhibit alloantigen-induced proliferative responses through secretion of inhibitory products, thus promoting alloantigen-specific tolerance in vivo.

Synergistic tolerance induced by LF15-0195 and anti-CD45RB monoclonal antibody through suppressive dendritic cells.

LF 15-0195 (LF), a novel analogue of 15-deoxyspergualin (DSG), inhibits maturation of dendritic cells (DC). Anti-CD45RB is a monoclonal antibody (mAb) that blocks activation of T-helper (Th) 1 cells and generates T-regulatory cells. This study addressed whether these two reagents act synergistically to inducing tolerance, and investigated associated cellular mechanisms. BALB/c recipients were treated by a short course of mAb alone, LF alone, or the combination of both agents. Mice that accepted a C57BL/6 cardiac allograft for more than 100 days were considered tolerant. Splenic DC were purified using positive selection for CD11c. Bone marrow DC were generated by culture with interleukin-4 and granulocyte-macrophage colony-stimulating factor. Surface marker expression was determined by fluorescence-activated cell sorter analysis. DC function was assessed by the ability to stimulate or inhibit T cells in vitro. Although monotherapy with LF or mAb failed to induce tolerance, combination therapy resulted in long-lasting acceptance of allogeneic hearts (>200 days) and secondary donor skin grafts (>100 days). DC from tolerant recipients possessed lower major histocompatibility complex class II and CD40 expression, and were poorer co-stimulators for T-cell proliferation than control DC. Furthermore, DC from tolerant mice induced Th2 differentiation, suppressed overall T-cell proliferation, and were poor presenters of T cells specific for antigen to pigeon cytochrome c 81-104. The combination of LF and anti-CD45RB mAb induced stable tolerance. The synergy of these two approaches appears to be mediated through formation of tolerogenic DC and T-regulatory cells.

Chronic cardiac allograft rejection in a rat model disparate for one single class I MHC molecule is associated with indirect recognition by CD4 + T cells

T-cell mediated immune responses play a critical role in chronic allograft dysfunction. The complex nature of allograft rejection, particularly with respect to the vast repertoire of alloantigens and their mode of recognition by T cells, presents a major challenge for the design of well-controlled studies into the immunobiology of chronic rejection. The purpose of this study was to develop a rat model with restricted antigenic specificity that develops chronic rejection without any immunologic manipulation to study the T-cell response. PVG.1U allogeneic hearts disparate for one single class I antigen, RT.1A u, were transplanted into PVG.R8 rat recipients. Grafts from PVG.R8 were used as syngeneic controls. Chronic rejection was studied by histological analysis of the grafted hearts at various time points posttransplantation (20–100 days). Donor specific alloreactive response was studied in a mixed lymphocyte reaction assay. All allografts survived more than 90 days and showed extensive evidence of chronic rejection, which was characterized by interstitial fibrosis, vasculitis, and occlusive myointimal thickening. Chronic rejection was evident by day 20 and most extensive by day 100 posttransplantation. In marked contrast, syngeneic grafts remained free of chronic lesions. Lymphocytes harvested from graft recipients showed a more vigorous proliferative response to allogeneic splenocytes as compared with that of lymphocytes from naı̈ve animals. The proliferative response was primarily mediated by CD4 + T cells recognizing the RT1.A a molecule via the indirect pathway. A single class I disparity in this model generates chronic rejection associated with potent CD4 + T-cell responses induced by the indirect recognition pathway. The use of this antigenically restricted model may facilitate the design of well-controlled studies for the characterization of immune mechanisms responsible for chronic rejection.

Local antilaminin antibody treatment alters the rejection pattern of murine cardiac allografts: correlation between cellular infiltration and extracellular matrix.

Emerging data place extracellular matrix (ECM) proteins as important elements in lymphocyte positioning and effector function in alloreactive responses. Using a non-vascularized model of allogeneic heart transplantation in Swiss mice, we have observed a correlation between the cellular infiltration and ECM deposition towards the interior of the graft during the kinetics of rejection. To confirm the importance of ECM during the rejection process in this model, we treated the transplanted animals with local injections of antilaminin monoclonal antibody and analyzed, by histology and immunohistochemistry, the grafts on day 15, which corresponds to the peak of cellular infiltration and ECM deposition. The treatment with mAb antilaminin decreased the cellular infiltrate and ECM deposition within the grafts, as compared to controls. Moreover, we found a diminished IFN-gamma, TNF-alpha and IL-2 deposition in the transplant area, and a reduced co-localization of these cytokines with laminin. By contrast, the antilaminin treatment increased tenascin deposition, a molecule with immunosuppressive properties, and also caused an increase in apoptosis of the cellular infiltrate. These data hallmark the importance of laminin, in distinct aspects concerning the events leading to allograft rejection, and also reinforce this molecule as a potential target for immune intervention in organ transplantation.