Rat Heart Grafts Research Articles

Recipient natural killer cells alter the course of rejection of allogeneic heart grafts in rats.

Rejection of solid organ grafts is regarded to be dependent on T cell responses. Nonetheless, numerous studies have focused on the contribution of NK cells in this process, resulting in contradictory theories. While some conclude that there is no participation of NK cells, others found an inflammatory or regulative role of NK cells. However, the experimental settings are rarely comparable with regard to challenged species, strain combinations or the nature of the graft. Thus, clear definition of NK cell contribution might be impeded by these circumstances. In this study we performed heterotopic heart transplantation (HTx) in rats, choosing one donor-recipient-combination leading to a fast and a second leading to a prolonged course of graft rejection. We intervened in the rejection process, by depletion of recipient NK cells on the one hand and by injection of activated NK cells syngeneic to the recipients on the other. The fast course of rejection could not be influenced by any of the NK cell manipulative treatments. However, the more prolonged course of rejection was highly susceptible to depletion of NK cells, resulting in significant acceleration of rejection, while injection of NK cells induced acceptance of the grafts. We suggest that, depending on the specific setting, NK cells can attenuate the first trigger of immune response, which allows establishing the regulatory activity leading to tolerance of the graft.

Comparison of various methods of ischaemic cardioprotection on vitality of rat heart grafts

The aim of the study was to compare 4 modes of ischaemic cardioprotection using continuous prograde autologous blood perfusion of the coronary artery in two hypothermic modes (group A, B) or conventional protection by cooled Hartmann solution (group C) or cooled saline (group D) without perfusion of the graft. Male Wistar rats (n = 24) were divided into four groups (A–D). In groups A (22–25 °C) and B (4–8 °C), blood perfusion rate was 10 ml/h and the graft was placed in a water bath. Groups C, D were initially rinsed with cold (4–8 °C) Hartmann solution (C) and cold saline solution (D), next the graft was placed in a water bath of cold (4–8 °C) Hartmann solution (C) or saline solution (D). The observed time was 30 min after the implemented perfusion (A, B) or initial rinsing (C, D). At 30 min, hearts of all the groups were perfused for 10 min with prograde-autologous arterialized blood at room temperature. At perfusion minute 10, blood was collected for biochemical analysis (sample 1). Sample 2 involved blood from a portable syringe infusion pumps (in parallel with sample 1). Pairwise test differences between samples 1 and 2 were significant in all the groups as regards creatine kinase and lactate dehydrogenase values, sampling 1 values being always higher, while cardiac troponin I concentrations were non-significant in the same comparison. The heart rate during the final perfusion was identical in all the groups. Our study has demonstrated that all observed cardioprotection modes are useful for experimental heart grafting.

Pretreatment of the Donor with AICAR Significantly Protects Rat Heart Graft from Prolonged Cold Ischemia/Reperfusion Injury in an Isolated Model.

Purpose: To investigate whether pretreat donor rat with AMP-activated protein kinase (AMPK) activator AICAR can reduce heart graft injury after static, prolonged cold preservation and reperfusion in an isolated model. Methods: Eighteen Sprague-Dawley(SD) heart donor rats were equally divided into two groups: AICAR pretreatment group(AICAR group, n=9) and normal saline pretreatment group(Vehicle group, n=9). After the hearts were arrested with Histidine-Tryptophan-Ketoglutarate(HTK) solution, they were excised and preserved in HTK solution for 8 hours at 4 <sup style=“font-family: Arial, sans-serif;”>o[/sup]C. After cold preservation, hearts were reperfused for 1hr in an Langendorff model, concerned parameters were examined during and after reperfusion. Result: AICAR pretreated heart showed significantly higher left ventricular developing pressure(LVDP), heart rate(HR), coronary flow(CF) than vehicle pretreated group. Caspase 3 activity and percentage of TUNEL-positive cells were significantly reduced in AICAR pretreated group. Oxidative parameter 8-hydroxy-2′-deoxyguanosine(8-OHdG) and malondialdehyde (MDA) were also significantly decreased in AICAR pretreated group. Moreover, AICAR pretreatment significantly diminished cystolic cytochrome C release. Conclusion: Pretreatment of the donor with AICAR significantly protects heart grafts from prolonged cold ischemia/reperfusion injury. This protocol may be useful and feasible in clinical heart transplantation.

Recipient hyperbilirubinaemia protects cardiac graft in rat heterotopic heart transplantation

Since bilirubin is a known powerful antioxidant, this study examined whether recipient hyperbilirubinaemia protected heart grafts from ischaemia/reperfusion (I/R) injury and chronic rejection associated with rat cardiac transplantation. Heterotopic heart transplantation (HTx) was performed using congenitally hyperbilirubinaemic GUNN (j/j) and normobilirubinaemic GUNN (+/+) rats. Syngenic grafts from +/+ rats were transplanted into +/+ or j/j rats with 6 or 18 h cold storage in University of Wisconsin solution to study I/R injury. To evaluate the effect on chronic rejection, Brown Norway rat heart grafts were transplanted into +/+ or j/j rats under short-course tacrolimus immunosuppression. The +/+ grafts in j/j rats demonstrated significantly lower serum creatine phosphokinase and higher left ventricular developed pressures and had smaller infarct areas than +/+ rats at 3 h after reperfusion. Graft survival with 18 h cold storage increased from 0% in +/+ rats to 41.7% in j/j rats. Malondialdehyde (a marker of lipid peroxidation), mRNA of the inflammatory mediators and phosphorylation of ERK1/2 were significantly decreased in the grafts transplanted into j/j rats compared with those transplanted into +/+ rats 1-3 h after reperfusion. The mean allograft survival in j/j recipients was prolonged to a median survival of 150 days from 84 days in +/+ recipients and was associated with less macrophage infiltrates and less intragraft inflammatory cytokine mRNA at d60. In vitro T-cell proliferation was significantly inhibited in the presence of bilirubin. Recipient hyperbilirubinaemia ameliorated cardiac I/R injury, as well as chronic allograft rejection following HTx via regulation of inflammatory responses or T-cell proliferation.

Adoptive transfer of double negative T regulatory cells induces B‐cell death in vivo and alters rejection pattern of rat‐to‐mouse heart transplantation

Antibody-mediated hyperacute and acute graft rejection are major obstacles in achieving long-term graft survival in xenotransplantation. It is well documented that regulatory T (Treg) cells play a very important role in regulating immune responses to self and non-self antigens. Our previous studies have shown that TCRalphabeta+CD3+CD4-CD8- (double negative, DN)-Treg cells can suppress anti-donor T-cell responses and prolong graft survival in allo- and xenotransplantation models. We have demonstrated that DN-Treg cells can induce B-cell apoptosis in vitro through a perforin-dependent pathway. B6 mice received rat heart grafts, followed by 14 days of LF15-0195 treatment. Some mice received Lewis rat cell activated DN-Treg cells after LF treatment. DN-Treg cells, purified from perforin-/- mice and from B6 mice pre-immunized with third party rat cells, were used as controls. In this study, we investigated the possibility that adoptive transfer of xenoreactive DN-Treg cells could suppress B cells in vivo, thus prolonging xenograft survival. We found that apoptotic death of B cells significantly increased after adoptive transfer of DN-Treg cells. In addition, anti-donor IgG subtypes were significantly inhibited in the DN-Treg cell-treated group, in which the rejection pattern was altered towards cellular-mediated rejection rather than antibody-mediated acute vascular rejection. However, perforin-deficient DN-Treg cells failed to induce B-cell death and to prolong heart graft survival, indicating a perforin-dependent mechanism contributes to B-cell death in vivo. This study suggests that adoptive transfer of xenoreactive DN-Treg cells can inhibit B-cell responses in vivo. DN-Treg cells may be valuable in controlling B-cell responses in xenotransplantation.

Both Infiltrating Regulatory T Cells and Insufficient Antigen Presentation Are Involved in Long-Term Cardiac Xenograft Survival

We have previously shown that pretransplant donor lymphocyte infusion (DLI) together with transient depletion of CD4(+) T cells could induce permanent rat-to-mouse heart graft survival, whereas depleting CD4(+) T cells alone failed to do so. In this study, we investigated the mechanism leading to long-term xenograft survival. We found that peripheral CD4(+) T cells from DLI/anti-CD4-treated mice could mount rat heart graft rejection after adoptive transfer into B6 CD4(-/-) mice. Infusing donor-Ag-loaded mature dendritic cells (DCs) could break long-term cardiac xenograft survival in DLI/anti-CD4-treated mice. Interestingly, when the number and phenotype of graft-infiltrating cells were compared between anti-CD4- and DLI/anti-CD4-treated groups, we observed a significant increase in both the number and suppressive activity of alphabeta-TCR(+)CD3(+)CD4(-)CD8(-) double negative regulatory T cells and decrease in the numbers of CD4(+) and CD8(+) T cells in the xenografts of DLI/anti-CD4-treated mice. Moreover, there was a significant reduction in MHC class II-high DCs within the xenografts of DLI/anti-CD4-treated recipients. DCs isolated from the xenografts of anti-CD4- but not DLI/anti-CD4-treated recipients could stimulate CD4(+) T cell proliferation. Our data indicate that functional anti-donor T cells are present in the secondary lymphoid organs of the mice that permanently accepted cardiac xenografts. Their failure to reject xenografts is associated with an increase in double negative regulatory T cells as well as a reduction in Ag stimulation by DCs found within grafts. These findings suggest that local regulatory mechanisms need to be taken into account to control anti-xenograft T cell responses.

Double-Negative T Cells, Activated by Xenoantigen, Lyse Autologous B and T Cells Using a Perforin/Granzyme-Dependent, Fas-Fas Ligand-Independent Pathway

The ability to control the response of B cells is of particular interest in xenotransplantation as Ab-mediated hyperacute and acute xenograft rejection are major obstacles in achieving long-term graft survival. Regulatory T cells have been proven to play a very important role in the regulation of immune responses to self or non-self Ags. Previous studies have shown that TCRalphabeta+CD3+CD4-CD8- (double-negative (DN)) T cells possess an immune regulatory function, capable of controlling antidonor T cell responses in allo- and xenotransplantation through Fas-Fas ligand interaction. In this study, we investigated the possibility that xenoreactive DNT cells suppress B cells. We found that DNT cells generated from wild-type C57BL/6 mice expressed B220 and CD25 after rat Ag stimulation. These xenoreactive B220+CD25+ DNT cells lysed activated, but not naive, B and T cells. This killing, which took place through cell-cell contact, required participation of adhesion molecules. Our results indicate that Fas ligand, TGF-beta, TNF-alpha, and TCR-MHC recognition was not involved in DNT cell-mediated syngenic cell killing, but instead this killing was mediated by perforin and granzymes. The xenoreactive DNT cells expressed high levels of granzymes in comparison to allo- or xenoreactive CD8+ T cells. Adoptive transfer of DNT cells in combination with early immune suppression by immunosuppressive analog of 15-deoxyspergualin, LF15-0195, significantly prolonged rat heart graft survival to 62.1 +/- 13.9 days in mice recipients. In conclusion, this study suggests that xenoreactive DNT cells can control B and T cell responses in perforin/granzyme-dependent mechanisms. DNT cells may be valuable in controlling B and T cell responses in xenotransplantation.

Long-Term Survival of Xenogeneic Heart Grafts Achieved by Costimulatory Blockade and Transient Mixed Chimerism

Xenotransplantation holds great promise in clinical medicine, but is limited by the vigorous rejection response elicited against solid organs transplanted across species barriers. In this study, we investigated the role of anti-CD40L monoclonal antibody (mAb) in inducing xenogeneic mixed chimerism and donor-specific heart transplantation tolerance. One day before heart transplantation, mice were injected intraperitoneally with anti-mouse CD8/NK1.1/Thy1.2 mAbs. On day 0, the mice received 3 Gy total body irradiation (TBI), an intravenous injection of unseparated bone marrow (BM) harvested from F344 rats, and an intraperitoneal injection of hamster antimouse CD40L mAb, MR1. Heart grafts from F344 rats were heterotopically transplanted into the abdomen of B6 mouse recipients. Using flow cytometric analysis of peripheral white blood cells, we assessed donor hematopoiesis at various times after bone marrow transplantation (BMT). Chimerism subsided gradually and disappeared completely 18 weeks after BMT. The cardiac graft survived permanently, even after the mixed chimerism disappeared. To determine if the mice acquired donor-specific tolerance, second rat heart grafts were transplanted 120 days after the first heart transplantation. The second transplanted hearts were also accepted over 60 days. Histological analysis revealed no remarkable vasculopathy in the coronary vessels at any stage. These findings clearly show that costimulatory blockade plays an important role in inducing xenochimerism, and that transient mixed chimerism can induce permanent acceptance of rat to mouse cardiac xenografts. Transplantation of xenogeneic bone marrow cells under costimulatory blockade at the time of heart transplantation may induce transplantation tolerance.

Serial donor aloantigen stimulation can maintain the donor specific intrathymic induced tolerance to rat heart transplants

Background: We have shown that intrathymic (IT) injection of alloantigen (Ag) plus anti-rat lymphocyte serum (ALS) induces donor specific heart graft acceptance. However the mechanisms of the donor-specific tolerance are unclear. This study investigates whether serial donor Ag stimulation can maintain donor-specific tolerance after excision of the accepted heart allograft. Methods: Donor Lewis (LEW, RT1l) rat heart grafts were transplanted (TX) into recipient Dark Agouti (DA, RT1a) rats pretreated with IT plus ALS.

Tolerance to Rat Heart Grafts Induced by Intrathymic Immunomodulation Is Mediated by Indirect Recognition Primed CD4+CD25+ Treg Cells

In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.

Efficiency of adenovirus-mediated gene transduction in heart grafts in rats

Aim We determined the characteristics of transgene expression of heart grafts following ex vivo gene transfer using an adenovirus vector. Transgene expression was assessed periodically in the same animals by a non-invasive bioimaging system. Methods Rat heterotopic heart transplantation was performed in a syngenic combination. We infused 1 × 10 9 plaque-forming units of adenovirus vectors containing firefly luciferase gene into the heart graft via the coronary artery, with preservation at 4 °C and transplanted into the cervix of the recipient. Transgene expression was periodically visualized and quantified by a noninvasive bioimaging system without sacrificing experimental animals. Results Transgene expression in the graft peaked at day 7 and then fell gradually. Transgene expression was also observed in the recipient liver. Conclusions We have determined the time course of transgene expression in the heart graft. This constitutes important information about ex vivo gene therapy for heart grafts.

Adenovirus-Mediated Gene Transfer to the Rat Heart Graft

Adenovirus-Mediated Gene Transfer to the Rat Heart Graft

Response: Adenovirus-Mediated Gene Transfer to the Rat Heart Graft

Ogata et al. (1) demonstrate interesting findings regarding the expression of the luciferase gene after adenoviral transfection in rat hearts. The fact that gene expression was detected supports our technique of retrograde aortic injection at the time of donor heart procurement as a method of selective gene manipulation in the perioperative period (2). It is true that adenoviral vectors can cause an inflammatory response that could confound the results of any study using this vector. This is the reason that we selected null adenovirus controls for our experiments. Because of the use of these controls, we believe that the effects we demonstrated on reperfusion injury and graft coronary artery disease were due to the single altered variable, that is, the upregulation of bcl-2. Regarding our use of the syngeneic-allogeneic retransplantation technique, the main purpose of this method is to allow for maximal translation of the bcl-2 gene product at the time of allogeneic transplant reperfusion. As Kita et al. (3) demonstrated, this gene expression occurs at detectable levels over a course of approximately 7 days, and according to our unpublished preliminary data, this expression was optimal at 4 days after the syngeneic transplant. Another way of conceptualizing this double-transplant technique is that the syngeneic recipient essentially acts as a stable environment for bcl-2 gene upregulation, or a relatively inert means by which to house the genetically altered graft until the appropriate time for retransplantation. We did not observe any episodes of cardiac arrest attributed to adenoviral therapy. Regarding the concern that our model is not immediately clinically applicable, we agree. Our purpose was to demonstrate the potential importance of bcl-2 in the short and long-term function of heart allografts, and our model happened to incorporate the use of adenovirus to prove our hypothesis. The issues involved with the use of adenovirus in humans are many, and we do not expect to see adenoviral vectors utilized in the human transplant setting in the near future. However, a variety of pharmacological agents are utilized during the organ procurement process and, considering our data, it would be of interest to see how these agents affect the expression of bcl-2. Douglas N. Miniati Robert C. Robbins Department of Cardiothoracic Surgery Stanford University Stanford, CA

ADOPTIVE TRANSFER OF SPECIFIC SUBSETS OF DENDRITIC CELLS CHANGES THE XENOGRAFT SURVIVAL AND PATTERN OF REJECTION IN A RAT-TO-MOUSE CARDIAC TRANSPLANTATION MODEL

O53* Aims: Dendritic cells (DCs) are major regulators of both T cell and B cell immune responses. While we have previously demonstrated that Th2 cytokines are detrimental and Th1 cytokines are beneficial to xenograft survival (Nature Medicine 2000), the immunological factors controlling cytokine profiles of anti-xeno responses remain unknown. We previously reported that CD11c+CD8α+ cells (DC1-like) were predominant in C57BL/6 recipients, in which Lewis heart grafts were slowly rejected with typical cell-mediated rejection (CMR) in 21 days. In contrast, CD11c+CD8α −(DC2-like) were predominant in BALB/c mice, in which the Lewis heart grafts were rapidly rejected with typical acute vascular rejection (AVR) in 6 days. These data suggest that DCs may play an important role in navigating xenograft immune responses. The present study was undertaken to elucidate the role of distinct subsets of DCs in controlling xenograft rejection. Methods: Purified CD8α+ or CD8α− DCs isolated from wild type (WT) BALB/c, WT C57BL/6 and IL-12−/−C57BL/6 mice were adoptively transferred into syngenic BALB/c and IL-12−/− or WT C57BL/6 mice, and recipients subsequently received Lewis rat heart grafts heterotopically 2 days after DC transfer. Intragraft cytokine expression and serum levels of anti-donor antibodies were measured by RT-PCR and flow cytometry, respectively. Results: Adoptive transfer of CD8α+ DCs into BALB/c mice shifted the immune response to Th1 with increased ratios of IFN-γ/IL-4 and IL-12/IL-4 mRNA in the grafts, and increased level of Th1 mediated anti-donor IgG2a in the circulation, thereby altering the pattern of rejection from AVR to CMR and significantly prolonging xenograft survival to 14.2 ± 0.8 days, while transfer of CD8α − DCs did not change the pattern of rejection and the grafts were rejected at 6 days. In addition, transfer of exogenous CD8α+ DCs rather than CD8α − DCs from WT C57BL/6 mice into IL-12−/− C57BL/6 mice prevented AVR and prolonged xenograft survival to 16.4 ± 0.9 days, while IL-12−/− C57BL/6 mice without transfer rapidly rejected the xenografts with AVR in 6 days. Furthermore, adoptive transfer of CD8α − DCs from IL-12−/− mice into WT C57BL/6 recipients shifted the immune response to Th2 with decreased ratios of intragraft IFN-γ/IL-4 and IL-12/IL-4 mRNA, but increased level of Th2 mediated anti-donor IgG1, thereby altering the pattern of rejection from CMR to AVR and the grafts were rapidly rejected in 9.5 ± 0.6 days. Conclusions: Transfer of CD8α+ DCs (DC1-like) induces Th1-mediated CMR and prolongs xenograft survival, whereas administration of CD8α − DCs (DC2-like) facilitates Th2-mediated AVR and accelerates xenograft rejection. These data suggest that distinct DC subclasses differentially affect the mechanism of xenograft rejection and ultimately affect graft survival.

Cytomegalovirus-mediated upregulation of chemokine expression correlates with the acceleration of chronic rejection in rat heart transplants.

Cytomegalovirus (CMV) infections have been shown to dramatically affect solid organ transplant graft survival in both human and animal models. Recently, it was demonstrated that rat CMV (RCMV) infection accelerates the development of transplant vascular sclerosis (TVS) in both rat heart and small bowel graft transplants. However, the mechanisms involved in this process are still unclear. In the present study, we determined the kinetics of RCMV-accelerated TVS in a rat heart transplant model. Acute RCMV infection enhances the development of TVS in rat heart allografts, and this process is initiated between 21 and 24 days posttransplantation. The virus is consistently detected in the heart grafts from day 7 until day 35 posttransplantation but is rarely found at the time of graft rejection (day 45 posttransplantation). Grafts from RCMV-infected recipients had upregulation of chemokine expression compared to uninfected controls, and the timing of this increased expression paralleled that of RCMV-accelerated neointimal formation. In addition, graft vessels from RCMV-infected grafts demonstrate the increased infiltration of T cells and macrophages during periods of highest chemokine expression. These results suggest that CMV-induced acceleration of TVS involves the increased graft vascular infiltration of inflammatory cells through enhanced chemokine expression.

Elimination of donor-specific alloreactivity prevents cytomegalovirus-accelerated chronic rejection in rat small bowel and heart transplants.

The primary cause for late failure of vascularized allografts is chronic rejection (CR) characterized by transplant vascular sclerosis (TVS). Cytomegalovirus (CMV) infection accelerates TVS and CR by unclear mechanisms involving direct effects of CMV, indirect effects of the recipient's immune response to CMV, or interactions between CMV and the recipient's alloreactivity. This study examined the role of CMV and the alloreactive response in the development of TVS using bone marrow chimerism (BMC) in rat small bowel (SB) and heart transplantation models. Fisher 344 (F344) rat heart or SB grafts were transplanted into F344/Lewis bone marrow chimera. F344 heart or SB grafts transplanted into Lewis recipients (low-dose cyclosporine) were positive controls for the development of TVS. Lewis heart or SB grafts transplanted into Lewis recipients (+/-cyclosporine) were transplantation controls. The effect of rat CMV (RCMV) (5x105 plaque-forming units) on TVS (neointimal index, NI) and graft survival was studied in these groups. RCMV infection was assessed by serologic analysis and quantitative polymerase chain reaction techniques (TaqMan). RCMV infection accelerated the time to graft CR (SB 70-38 days; hearts 90-45 days) and increased the severity of TVS in both the SB allografts (day 38, NI=27 vs. 52) and the heart allografts (day 45, NI=43 vs. 83). Grafts from CMV-infected syngeneic recipients failed to develop TVS and CR. Donor-specific tolerance induced by BMC prevented allograft TVS and CR in both transplant models. In contrast to naïve Lewis recipients, RMCV infection failed to cause allograft TVS and CR in bone marrow (BM) chimeras. The events in CMV-induced acceleration of TVS involve a crucial interplay between CMV infection and the recipient's alloreactive immune response.

Allopeptide-mediated expression of endothelial Fas-Ligand (CD95L) in long-term tolerized heterotopic rat heart grafts

Allopeptide-mediated expression of endothelial Fas-Ligand (CD95L) in long-term tolerized heterotopic rat heart grafts

Induction of tolerance with nondepleting anti-CD4 monoclonal antibodies is associated with down-regulation of TH2 cytokines.

Induction of tolerance with anti-CD4 has mainly focused on monoclonal antibodies (mAbs) that deplete CD4+ T cells. In this study, the mechanisms by which nondepleting anti-CD4 mAbs induce tolerance in the Dark Agouti to PVG rat heart graft model were examined. Five anti-CD4 mAbs were tested. Immunohistology and cytokine mRNA profiles were analyzed within grafts. Effects of combining anti-CD4 therapy with alloantibody (alloAb), interleukin (IL)-4, and anti-IL-4 mAb were also examined. All mAbs tested induced indefinite graft survival (>150 days), with blocking of alloAb production. Exogenous alloAb did not restore rejection. Similar T cell receptor alphabeta+, CD8+, IL-2 receptor+ T cell, macrophage, and natural killer cell infiltration and comparable MHC II and intercellular adhesion molecule-1 levels were seen in rejecting and tolerant grafts. mRNA for IL-2, interferon-gamma, lymphotoxin, tumor necrosis factor-alpha, transforming growth factor-beta, cytolysin, and granzyme-A/B was comparable, although inducible nitric oxide synthase was slightly reduced in tolerant grafts. IL-4 and IL-5 were significantly reduced in tolerant grafts, although IL-6, IL-10, and IL-13 levels were similar; this was consistent with partial T helper (Th)2 response inhibition, which was also manifested by inhibited alloAb. The combination of alloAb, IL-4, or anti-IL-4 mAb with anti-CD4 did not prevent tolerance induction. This study demonstrated that anti-CD4 mAb therapy did not inhibit activation and infiltration of Th1 and CD8+ effector T cells. Preferential induction of Th2 responses, especially IL-4, was not essential for the induction of tolerance. Our studies also found no evidence to support induction of anergy or transforming growth factor-beta as mechanisms of tolerance induction. These results question whether IL-4 is required for induction of transplantation tolerance.

Suppressed alloreactivity and mixed chimerism in rats with accepted cardiac allografts by intrathymic injection of donor bone marrow cells.

Intrathymic injection of donor bone marrow cells (ITBMCs) at the time of transplantation and treatment with antilymphocyte serum (ALS) permitted the indefinite survival of Brown Norway (BN, RT1n) rat heart grafts in 6 out of 8 Lewis (LEW, RT1l) rat recipients, LEW recipients with long-surviving BN heart grafts (LSGs) also accepted additional BN heart grafts without further immunosuppression, though they rejected Piebald Virol Glaxo (PVG, RT1c) rat heart grafts in the usual fashion. In the in vitro study, the proliferative response of the lymphocytes from LEW recipients with LSGs remained suppressed when they were stimulated by BN spleen cells, but not when stimulated by PVG cells. Bone marrow cells (BMCs) from LEW rats with LSGs showed strong, nonspecific, suppressive effects on the proliferative response in the mixed lymphocyte culture reaction, suggesting that one of the possible explanations for tolerance might be the involvement of a suppressor mechanism.

Suppressed alloreactivity and mixed chimerism in rats with accepted cardiac allografts by intrathymic injection of donor bone marrow cells

Abstract. Intrathymic injection of donor bone marrow cells (ITBMCs) at the time of transplantation and treatment with antilymphocyte serum (ALS) permitted the indefinite survival of Brown Norway (BN, RTln) rat heart grafts in 6 out of 8 Lewis (LEW, RT11) rat recipients. LEW recipients with long-surviving BN heart grafts (LSGs) also accepted additional BN heart grafts without further immunosuppression, though they rejected Piebald Virol Glaxo (PVG, RT1C) rat heart grafts in the usual fashion. In the in vitro study, the proliferative response of the lymphocytes from LEW recipients with LSGs remained suppressed when they were stimulated by BN spleen cells, but not when stimulated by PVG cells. Bone marrow cells (BMCs) from LEW rats with LSGs showed strong, nonspecific, suppressive effects on the proliferative response in the mixed lymphocyte culture reaction, suggesting that one of the possible explanations for tolerance might be the involvement of a suppressor mechanism.