MHC Mismatch Research Articles

Regulatory T Cell Reconstitution is Delayed Following Allogeneic Bone Marrow Transplantation

Delayed T cell reconstitution following allogeneic bone marrow transplantation (aBMT) has been implicated in post‐transplant mortality due to infection and relapse. CD4+CD25+FoxP3+ regulatory T cells (Tregs) play a role in the dampening of graft‐vs‐host‐disease (GVHD). However, little is known about Treg reconstitution in the absence of GVHD. BALB/c mice were lethally irradiated and reconstituted with T cell depleted B6.SJL bone marrow in a full MHC mismatch model of aBMT. When analyzing thymic Tregs as a percentage of CD4+ T cells, host Treg proportions were maintained early but rose at later time points while donor proportions decreased early and approached untransplanted levels over time. Conversely, splenic Treg proportions (donor and host) were increased at D14 but returned to pretransplant levels. Host Treg numbers remained constant while thymic and splenic donor Treg numbers increased, albeit at a slower rate than conventional T cells. Previous studies have shown that proliferating Tregs were less suppressive especially following lymphopenia. Using Treg suppression assays, we observed that the suppressive ability of Tregs after BMT was maintained and remained at similar levels to Tregs isolated from untransplanted mice (of either donor or host stains). In conclusion, we have shown that splenic donor Treg reconstitution is delayed post aBMT but those Tregs that are produced are functional.

Impact of MHC mismatch and freezing on bone graft incorporation: An experimental study in rats

Cortical bone graft failure develops for poorly defined reasons, and the effects of the immune responses on the incorporation of an allograft are less clear. In a rat model of tibial allotransplantation, we have studied biometric and histological changes of the graft and the humoral immune response against it. We have also compared fresh with prefrozen grafts to study putative effects of freezing on the healing of the graft and the immune response against it. Fresh and frozen cortical bone grafts matched or mismatched for major histocompatibility complex antigens (syngeneic and allogeneic grafts) were implanted in an 8-mm segmental defect in the tibia. The construct was stabilized with intramedullary nailing. Incorporation of the graft was assessed with use of conventional radiography, micro computed tomography (CT(, biomechanical testing and histological examination. The immune response was evaluated by monitoring distribution of leukocytes in the blood and by measuring antibodies in a tailor-made fluorescence activating cell scanning (FACS( analysis. We found that the fresh syngeneic grafts were well integrated after 8 weeks with intact bone cells. In the fresh allogeneic grafts, all cells were dead with radiological signs of resorption, and mechanical testing indicated failure of incorporation. The frozen grafts showed poorer overall reconstruction than the fresh syngeneic grafts, but the incorporation was better than the fresh allogeneic grafts. A measurable alloantibody response was only detected after fresh allografting. The combined results suggest that freezing of bone allograft impedes the antibody response against major histocompatibility complex (MHC( antigens and improves incorporation, but frozen allografts still perform poorer than do frozen syngeneic grafts.

Vascularized composite islet-kidney transplantation in a miniature swine model

Previous work from this laboratory has demonstrated that transplantation of allogeneic thymic tissue as part of a composite vascularized graft is far more successful in terms of both engraftment and long-term survival than transplantation of thymic tissue or cells alone. We have subsequently extended this concept to transplantation of allogeneic islets, comparing survival of islet cell suspensions to that of vascularized composite islet-kidneys (IK), prepared by injection of autologous islets underneath the renal capsule 2-3 months prior to allogeneic transplantation of the composite organ. We have utilized partially inbred miniature swine with defined MHC loci as the experimental large animals for this study, permitting reproducible transplantation across specific MHC barriers. Composite IK have been transplanted successfully across minor and full MHC mismatch barriers, using treatment regimens previously demonstrated to induce long-term tolerance of kidney allografts across these barriers. IK allografts containing > or =5000 islet equivalents (IE)/kg recipient body weight were found capable of reversing surgically induced diabetes, while injection of comparable numbers of purified islets via the portal vein or under the renal capsule did not. Studies are also being directed toward preparation of autologous "thymo-isletkidneys" (TIK), for potential use as xenografts, in which the thymic component is intended to induce tolerance and the islets to reverse diabetic hyperglycemia. The use of both types of composite organ transplants may eventually be applicable to the treatment of type I diabetic patients suffering from end-stage diabetic nephropathy.

Prolonged Survival of Vascularized Skin Grafts Across a Full MHC Mismatch Using Donor Antigen-Pulsed Tolerogenic GM+Rapa Dendritic Cells (102.22)

Abstract We compared the tolerogeneic properties of BM-derived GM+IL-4 and GM+Rapa host DCs and their potential to induce long-term acceptance of vascularized skin allograft in rats across a full MHC mismatch with transient immunosuppression. Results show that both DCs were CD11b+ and expressed low levels of CD86 and produced low levels of IL-12p70 following TLR activation. GM+Rapa DC produced lower levels of anti- and pro-inflammatory cytokines in response to LPS. Both DCs had low T cell stimulatory capacity in vitro. Interestingly, donor Ag-pulsed GM+Rapa DC (GM+Rapa DCp) induced long-term survival of the vascularized skin grafts and this was significant compare to GM+IL-4 DC and control groups. PBMC from long-term graft survivors that were previously treated with donor Ag-pulsed GM+Rapa DC were hyporesponsive to donor Ag, but proliferated in response to the third-party Ag and produced IL-10 in addition to IFN-gamma. Recipients of the long-term surviving allografts were challenged with full-thickness tail skin grafts. While the third-party grafts were rejected fast the donor graft had delayed rejection, suggesting that this treatment induced a regulatory mechanism that prevented the acute rejection of donor skin grafts. Further experiments demonstrated presence of CD4+ Foxp3+ T cells in the vascularized skin and secondary lymphoid tissues. Taken together, these results demonstrate that the donor Ag-pulsed tolerogenic GM+Rapa DC have ability to induce regulatory T cells and prevent vascularized skin graft rejection across a full MHC mismatch.

Conditioning with Treosulfan and Cyclophosphamide without Application of Antibodies in MHC Mismatch Transplantations in Mice.

BACKGROUND: Treosulfan is increasingly used in clinical conditioning regimens because of its myeloablative and immunosuppressive effects and the low hepatotoxicity compared to busulfane. The myeloablative and immunosuppressive characteristics of treosulfane was investigated in a murine MHC mismatch transplantation model.METHODS: C57BL/10 (H-2Db) female mice were treated with treosulfan (2000 mg/kg) on day -3 to -1 and increasing doses of cyclophosphamide (without, 100 or 200mg/kg) at day -1, or 3000 mg/kg treosulfan with and without 200 mg/kg cyclophosphamide at day -1. Some mice were not transplanted to examine the myeloablative effect of the regimens, the rest of mice were transplanted with 10 × 10^6 marrow cells from Balb/c mice (H-2Dd) donors and monitored for donor-type chimerism determined by flow cytometry.RESULTS: Mice treated with treosulfan and cyclophosphamid without transplantation died, indicating the myeloablative effect of the regimens. Ca. 50% of mice treated with 3000 mg/kg treosulfan and 200 mg/kg cyclophosphamide at day -1 died. Analysis of bone marrow cells in the remaining mice showed a complete rejection at day 55. Approximately 90% of the recipients of three daily doses of treosulfan and increasing doses of cyclophosphamide survived until the end of experiment (day +100). All hosts engrafted when 200 mg/kg cyclophosphamide was applied. 85% of mice engrafted after 100mg/kg cyclophosphamide. 100% of grafts were rejected in the group with treosulfan alone (2000 mg/kg).CONCLUSION: A chemotherapeutic regimen for achieving a long term survival in a MHC-mismatch situation without additional need of antibodies was established in mice. While investigated for the practicability in an animal model as alternative to TBI, the high rate of engraftments in a MHC mismatch situation makes the chemotherapeutic regimen suitable for clinical situations.

Early CD30 Signaling Is Critical for Adoptively Transferred CD4+CD25+ Regulatory T Cells in Prevention of Acute Graft Versus Host Disease.

CD4+CD25+ regulatory T cells (Treg) have been demonstrated to reduce the severity of acute graft-versus-host disease (aGvHD) in murine models of bone marrow transplantation. However, the surface molecules that are critical for suppression are unclear. The TNF-R superfamily member CD30 has been shown to be expressed on regulatory T cells that down-modulate nickel specific immune responses and to be relevant for Treg mediated protection from allograft rejection. Deficiency of the CD30 molecule (CD30−/−) is associated with impaired thymic negative selection and augmented T cell autoreactivity. Therefore, we investigated the role of CD30 signaling in Treg function in an aGvHD model. Treg derived from CD30−/ − animals were significantly less effective in preventing aGvHD lethality (wt vs. CD30−/ − p=0.002). Signal intensity derived from expanding luciferase expressing alloreactive conventional T cells (Tconv) was significantly higher if CD30−/ − Treg as compared to wt Treg (p=0.007) were transferred as assessed by bioluminescencent based imaging. Blockade of the CD30/CD153 pathway with a neutralizing anti-CD153 mAb during the early (days −2 to +4) but not late (days +4 to +10) phase of adoptive Treg transfer reduced Treg mediated protection from proinflammatory cytokine accumulation and apoptosis of donor-type CD4 and CD8 T cells. In vivo bioluminescence imaging demonstrated intact Treg homing, but reduced expansion when CD153 was blocked during the early phase after adoptive transfer. CD30 surface expression on Treg increased with alloantigen exposure and CD153 expression on recipient-type dendritic cells increased in the presence of an irradiation induced proinflammatory environment but not when T cell depleted bone marrow and Tconv were transferred into non-irradiated Rag 2−/ −γc−/ − recipients. These data are the first to demonstrate that early CD30 signaling is relevant for Treg mediated aGvHD protection after major MHC mismatch bone marrow transplantation.

Identification of the Immunodominant HY H2-Dk Epitope and Evaluation of the Role of Direct and Indirect Antigen Presentation in HY Responses

Minor histocompatibility Ags derive from self-proteins and provoke allograft rejection and graft-vs-host disease in MHC-matched donor-recipient combinations. In this study, we define the HYD(k) epitope of the HY minor histocompatibility Ag as the 8mer peptide RRLRKTLL derived from the Smcy gene. Using HY tetramers, the response to this peptide was found to be immunodominant among the four characterized MHC class I-restricted HY epitopes (HYD(k)Smcy (defined here), HYK(k)Smcy, HYD(b)Uty, and HYD(b)Smcy). Indirect presentation stimulated a robust primary HYD(k)Smcy response. Indirect presentation and priming of HY-specific CD8+ T cells is also operative in the presence of a full MHC mismatch. To determine whether the indirect route of Ag presentation is required for HY priming, female parent into F1 (H2bxk) female recipient bone marrow chimeras were immunized with male cells of the other parental haplotype, limiting presentation to the direct pathway. The dominant H2b HY response (HYD(b)Uty) was dependent on indirect presentation. However, the dominant H2k HY response (HYD(k)Smcy) could be stimulated efficiently by the direct pathway. In contrast, secondary expansion of both HYD(k)Smcy and HYD(b)Uty-specific CD8+ T cells was effective only when Ag was presented by the direct route. Transgenic overproduction of Smcy mRNA within the immunizing cells resulted in a corresponding increase in the HYD(k)Smcy, HYD(b)Smcy, and HYK(k)Smcy-specific CD8+ T cell responses when presented via the direct pathway but did not enhance indirect presentation demonstrating the independent regulation of MHC class I-peptide occupancy in the two Ag-processing pathways.

Prevention of Acute Graft-Versus-Host Disease by CD4+CD25+ Regulatory T Cells Is Dependent on the CD30/CD153 Pathway.

CD4+CD25+ regulatory T (Treg) cells suppress acute graft versus host disease (aGVHD), prevent autoimmunity and delay allograft rejection. CD30 and other TNF-R family members have been demonstrated to be expressed by Treg and to function as alternative costimulatory pathways for T cell activation. In this study we assessed the significance of the CD30/CD153 pathway in Tregs suppression of aGVHD in a murine major MHC mismatch BMT model. Using bioluminescence imaging proliferation of donor derived luciferase-labeled CD4+ and CD8+ T cells was quantified at serial time points after transplantation. Treg suppressed the early expansion of alloreactive T-cells. Immunofluorescence microscopy revealed a predominant infiltration of donor derived Treg in CD153 positive regions of secondary lymphoid organs, namely parafollicular T cell zones of lymph nodes and the subepithelial dome regions of Peyers Patches. In vivo blockade of the CD30/CD153 pathway with anti CD153 Ab did not alter Treg migration to secondary lymphoid organs but reduced their suppressive effect. Proliferation of donor T cells as measured in photons/second/mouse was significantly higher in animals receiving Treg and CD153 blocking antibodies as compared to recipients of Treg only (p=0.0038). Gene expression profiling of Treg with DNA microarrays indicated a Treg signature that was consistently found in different mouse strains. This Treg signature was altered after CD153 blockade in vitro. Importantly, aGVHD lethality was significantly increased (p=0.021) when CD30-CD153 interaction was blocked during Treg transfer. This study provides direct evidence that the TNF-R family member CD30 is critical for Treg cell function in the regulation of pathological T cell responses that lead to aGVHD.

Correction of factor IX deficiency in mice by embryonic stem cells differentiated in vitro

Murine embryonic stem (ES) cells are pluripotent, but significant functional engraftment does not occur when they are introduced into the liver. However, here we demonstrate that functional liver engraftment does occur if the ES cells (from strain 129 mice) are first differentiated in vitro for 7 days in the presence of FGF. Strikingly, when these differentiated cells, termed putative endodermal precursors (PEPs), were injected into their livers, two of six C57BL/6 and four of eight BALB/c factor IX (F-IX)-deficient mice survived for >7 days, even though the recipients were of a different strain and, in the case of the BALB/c recipients, had a complete MHC mismatch. F-IX was detected in all six of the PEP-injected survivors. Two mice subsequently died of causes unrelated to F-IX; the others survived until death at 38 or 115 days after the transplantation. No uninjected control F-IX-deficient mice survived for >7 days. Large confluent regions of sinusoidal PEP engraftment were demonstrated by immunofluorescence in the long-term BALB/c survivors. The PEP engraftment was not associated with detectable cell fusion, and the transplantation was accompanied with only a low incidence of teratoma formation.

Control of intestinal allograft rejection by FTY720 and costimulation blockade

Introduction The clinical application of small bowel transplantation (SBTx) is hampered byits pronounced immunogenicity. In this study we examined the effects of the novelimmunosuppressant FTY720 and costimulation blockade by an anti-CD40L mAb (MR-1) ina stringent mouse model of SBTx. Methods SBTx was performed in mice with a full MHC mismatch (donors: C3H=H-2 k;recipients: C57BL/6=H-2 b). Recipients were divided into four groups: 1, untreated group; 2,MR1 monotherapy (500 μg IV on days 0, 2, 4, and 7); 3, FTY720 monotherapy (1 mg/kg bodyweight PO for 14 consecutive days after transplantation); 4, FTY720 plus MR1–treated group.Graft rejection grades were assessed by H&E staining. Graft mesenteric lymph nodes (MLNs),Peyer's patches (PPs), as well as intraepithelial lymphocytes (IELs) and lamina proprialymphocytes (LPLs) were analyzed by flow cytometry and three-color immunofluorescencestaining. Results Neither FTY720 nor MR1 monotherapy was efficient in preventing the rejection ofmouse intestinal allografts, whereas FTY720 plus MR1 profoundly inhibited the rejectionresponse at the 14th posttransplant day. The infiltration of host lymphocytes was reduced ingraft MLNs, PPs, IELs, and LPLs by FTY720 therapy. FTY720 plus MR1 inhibited hostCD8 + T-cell infiltration in graft LPLs when compared with grafts treated with FTY720 only.Additionally, two subpopulations, CD11b +high Gr1 − and CD11b +intermediate Gr1 + cells, weredecreased in FTY720-treated grafts. Conclusions FTY720 plus MR1 efficiently delayed intestinal allograft rejection in a mousemodel by preventing the infiltration of host lymphocytes, particularly of CD8 + cells.

Risk Factors for the Development of Post-Transplant Lymphoproliferative Disorder in a Large Animal Model

A high incidence of a post-transplant lymphoproliferative disorder (PTLD) is observed in miniature swine conditioned for allogeneic hematopoietic cell transplantation using a protocol involving T-cell depletion and cyclosporine therapy. This study was designed to assess contributing factors to disease development. Forty-six animals were studied including 12 (26%) that developed PTLD. A number of risk factors for PTLD were examined, including degree of immunosuppression, degree of MHC mismatch and infection by a porcine lymphotrophic herpesvirus (PLHV-1). Flow cytometry was used to measure host and donor T- and B-cell levels in the peripheral blood. Porcine lymphotrophic herpesvirus viral load was determined by quantitative PCR. Animals developing PTLD had significantly lower levels of T cells on the day of transplant. Cyclosporine levels did not differ significantly between animals with and without PTLD. Animals receiving transplants across a two-haplotype mismatch barrier showed an increased incidence of PTLD. All animals with PTLD had significant increases in PLHV-1 viral loads. Porcine lymphotrophic herpesvirus viral copy numbers remained at low levels in the absence of disease. The availability of a preclinical large-animal model with similarities to PTLD of humans may allow studies of the pathogenesis and treatment of that disorder.

CO-STIMULATORY INHIBITION THROUGH CD134 (OX40) BLOCKADE PROMOTES MURINE HEART ALLOGRAFT SURVIVAL

P1031 Aims: CD134 is a co-stimulatory molecule that is expressed as a late event in T cell activation and contributes to the generation of immunological memory. CD134 blockade effectively ameliorates inflammation in models of autoimmune disease but its role in transplantation has hitherto been little studied. Methods: An OX40-Ig fusion protein was used to investigate the role of this co-stimulatory molecule in alloimmunity both in vitro, through addition of the fusion protein to allogeneic lymphocyte co-cultures, and in vivo through administration of OX40-Ig to mouse recipients of vascularised heart allografts. The role of CD134 was studied in mouse strain combinations representing both major histocompatibility (MHC, BALB/c to CBA/Ca) and minor histocompatibility (mHC, B10.BR to CBA/Ca) antigen mismatched tissues. Results: In vitro T cell activation following encounter with alloantigen resulted in delayed expression of CD134, as has been reported previously for T cells activated by nominal protein antigens. Alloantigen-stimulated T cells expressed increasing levels of CD25 and CD69 until 72 hours post stimulation, but similar levels of CD134 expression were not seen until 96 hours. Addition of OX40-Ig to lymphocyte co-cultures inhibited proliferation in a dose-dependent manner by up to 83% in both MHC and mHC mismatched strain combinations. Interestingly, CD134 blockade was most effective when addition of the OX40-Ig fusion protein was delayed until day 4 of co-culture, corresponding to maximum CD134 expression on activated responder cells. MHC mismatch-induced proliferation was inhibited by 84% when addition was delayed for 96 hours, compared with 0% inhibition when OX40-Ig was added at 24 hours. In the minor mismatch strain combination, inhibition of proliferation was 86% at 96 hours compared with 42% at 24 hours. Similarly, cytokine production (IFN-γ, IL-2 and IL-10) in lymphocyte co-cultures was markedly inhibited in a time-dependent manner following addition of OX40-Ig. When administered in vivo, daily for 15 days, OX40-Ig failed to extend graft survival in the major mismatch strain combination (median survival time [MST] 8 days in both treated and non-treated groups). In contrast, OX40-Ig was highly effective at prolonging the survival of B10.BR heart allografts in CBA/Ca recipients (mHC mismatch, MST extended from 14 [n=7] to >100 days [n=11]). Immunohistological analysis of the transplanted hearts revealed that both rejecting and long-surviving heart grafts had similar degrees of mononuclear cell infiltration but that OX40-Ig treatment appears to delay the cellular infiltration. Conclusions: We conclude that CD134/CD134L interaction plays an important role in the co-stimulatory cascade and that blockade of this molecular interaction may be of therapeutic value in helping to prevent allograft rejection.

SIROLIMUS CONTROLS GVH REACTIVITY IN ANTI-CD45 MAB CONDITIONED ALLOGENEIC BMT IN ORDER TO ACHIEVE TOLERANCE BY MIXED CHIMERISM

O271 Aims: We could recently introduce in a rat system less-toxic BMT conditioning using only an anti-CD45 mAb. As severe side effect in fully MHC mismatch setting risk of aplasia was observed and most likely provoked by an atypical GvH reaction on bone marrow level. In this study, unspecific immunosuppression (IS) was tested as addition to anti-CD45 mAb conditioning for establishment of a safe and tolerizing BMT protocol. Methods: The model consisted of LEW.1W (RT1u, RT7a) recipients, LEW.7B (RT1l,RT7b) BM donors, a single injection of anti-CD45 mAb (7.5 mg/kg BW) detecting RT7a as conditioning and orally applicated cyclosporine A (CsA 5 or 15 mg/kg BW) or sirolimus (1 or 2 mg/kg BW) as IS for 14 days. Animals were monitored for clinical status and haematological parameters. They were tested for multilineage chimerism at day 20 and day 100 by FACS. Long-term survivors received skin grafts of BM donor- and 3rd party-type on day 100. Results: Only animals of high-dosage sirolimus (2 mg/kg BW) treated group showed reproducible stable chimerism (9 of 12) compared to all other groups (no IS: 1 of 9, CsA 5: none of 9, CsA 15: 1 of 9, sirolimus 1: 1 of 9). Thereby, initial leukocyte depletion by anti-CD45 mAb as parameter for conditioning extent was highly critical for BMT success. Animals, which did not became stable chimeras, showed transient chimerism but suffered mainly from severe aplasia syndrome and died between days 29 to 70 (no IS: 7 of 9, CsA 5: 9 of 9, CsA 15: 6 of 9, sirolimus 1: 5 of 9, sirolimus 2: 3 of 12). GvHD signs were only seen in high-dosage CsA treated animals (2 of 7). The attempt to stabilize BM engraftment by prolongation of high-dosage CsA treatment (28 days) even aggravated GvHD symptoms (4 of 6) like thymic selection disturbance by thymic irradiation did in high-dosage sirolimus group (4 of 4). Moreover, analysis of persisting anti-CD45 mAb coated thymocytes suggested reduced thymocyte selection progress. Long-term lineage-typing of stable chimeras from high-dosage sirolimus group revealed mainly high-graded mixed chimerism in lymphoid and fully chimerism in myeloid lineages. Stable chimeras tolerated skin grafts only of donor-type (> 100 days), while acutely rejecting 3rd party control grafts like none chimeric survivors rejecting both graft-types. Conclusions: Addition of high-dosage sirolimus to isolated anti-CD45 mAb BMT conditioning reduced significantly risk of fatal aplasia in allogeneic BMT. This is in contrast to CsA, where the combination of intrathymically persisting anti-CD45 mAb and CsA seems to disturb TC selection processes leading to severe GvHD without stabilizing HSC engraftment. Without any clinical signs for GvHD the combination of anti-CD45 mAb and sirolimus in allogeneic BMT is promising for generation of stable mixed chimerism and tolerance induction.

The origin of cynomolgus monkey affects the outcome of kidney allografts under neoral immunosuppression

YNOMOLGUS monkeys are found in various sources around the world, and the influence of the origin of these monkeys on the outcome of transplantation (TX) experiments is not clear. This study aimed at comparing the effects of cyclosporine (CsA) on kidney allograft survival in cynomolgus monkeys from two distinct locations: from the Philippines (Siconbrec Ltd) and from Mauritius (CRP Ltd). METHODS The study was performed in accordance with the Swiss Animal Welfare regulations. Donor-recipient combinations were selected according to ABO blood group compatibility and MHC mismatch as assessed by in vitro mixed lymphocyte reaction (MLR stimulation index 10). Life-supporting kidney allograft TX were performed within these two populations. Recipients were treated post-TX with Neoral daily orally using two different regimens: R1, 150 mg/kg/d for 15 days followed by 100 or 75 mg/kg/d; R2, 100 mg/kg/d for 15 days followed by 30 mg/kg/d. 1,2 Graft function was followed by monitoring changes in serum creatinine (Screa). Screa levels 500 mol/L were considered indicative of rejection, which was confirmed histologically. RESULTS

Analysis of robust innate immune response after transplantation in the absence of adaptive immunity.

Both animal models and clinical outcomes studies of transplantation suggest that antigen-independent mechanisms can alter graft survival and function. It has been suggested that antigen-independent processes interact with alloantigen-specific responses to augment the rejection reaction. A major link between antigen-specific adaptive immunity and pro-inflammatory stimuli is innate immunity. During transplantation, innate immunity may be stimulated by multiple factors, including ischemia, reperfusion, sterile injury, systemic stress, and cell death. We investigated the hypothesis that transplantation induces a potent innate immune response in a murine model of vascularized solid organ transplantation. In our studies, we analyzed three experimental groups: (a) alymphoid group in which both the donor and recipients strains lacked an adaptive immune response due to deletion of the recombinase activating gene, thus blocking production of both T cell and B cell antigen receptors; (b) syngeneic group in which the donors and recipients were genetically identical; and (c) allogeneic group in which the donors and recipients had a complete MHC mismatch. To analyze a large number of parameters we determined the level of expression of a panel of cytokines, chemokines, receptors, and cell surface markers by RNase protection assays. In addition, serum cytokines were determined by ELISA and the infiltration of inflammatory cells was assessed by histology. Our results showed macrophage infiltration and up-regulation of multiple cytokines, chemokines, and chemokine receptors within the first day after transplantation in all groups, including the syngeneic and alymphoid recipients. Our study demonstrated a robust innate immune response that is independent of adaptive immunity and natural killer cell responses.

Spontaneous acceptance of liver transplants in rodents: evidence that liver leucocytes induce recipient T-cell death by neglect.

In many animal models transplanted livers are not rejected, even when there is a complete MHC mismatch between the donor and recipient and the recipient is not immunosuppressed. This distinguishes liver transplants from other organs, such as kidneys and hearts, which are rapidly rejected in mismatched individuals. Acceptance of transplanted livers in a rat model is not due to the absence of an immune response to the liver and there is a rapid, abortive response that is ultimately exhausted. Donor leucocytes transferred with the liver appear to be responsible for both liver acceptance and the abortive activation of the recipient's T cells. The immune mechanism of liver transplant acceptance appears to be due to 'death by neglect' in which T cells are activated to express IL-2 and IFN-gamma mRNA in the recipient lymphoid tissues, but not at adequate levels within the graft. Subsequently the activated T cells die leading to specific clonal deletion of liver donor-reactive T cells. These findings have important implications for liver transplant patients as immunosuppressive drugs that are given to prevent rejection can also interfere with this form of tolerance. In addition, it might be possible to modify the immunosuppressive drug treatment of transplant patients to promote the process of death by neglect of recipient alloreactive T cells.

Mixed allogeneic chimerism as a reliable model for composite tissue allograft tolerance induction across major and minor histocompatibility barriers.

Although prolonged composite tissue allograft (CTA) survival is achievable in animals using immunosuppressive drugs, long-term immunosuppression of CTAs in the clinical setting may be unacceptable for most patients. The purpose of this study was to develop a model for reliable CTA tolerance induction in the adult rat across a major MHC mismatch without the need for long-term immunosuppression. Mixed allogeneic chimeras were prepared by using rat strains with strong MHC incompatibility [WF (RT1Au), ACI (RT1Aa)] WF + ACI-->WF, n=23. The bone marrow (BM) of recipient animals was pretreated with low-dose irradiation (500-700 cGy), followed by reconstitution with a mixture of T cell-depleted syngeneic (WF) and allogeneic (ACI) cells. Additionally, the recipient animals received a single dose of anti-lymphocyte serum (10 mg) 5 days before bone marrow transplantation (BMT) and tacrolimus (1 mg/kg/day) from the day before BMT to 10 days post-BMT. Hindlimb transplants were performed 12 months after BMT. Five animals received a limb allograft irradiated (1000 cGy) just before transplantation. Rat chimeras were characterized (percentage of donor cells present within the bloodstream) by flow cytometry at 3 and 12 months after BM reconstitution and after hindlimb transplantation. Peripheral blood lymphocyte chimerism (WF/ACI) remained stable >12 months after BM reconstitution in 18/23 animals. Multi-lineage chimerism of both lymphoid and myeloid lineages was present, suggesting that engraftment of the pluripotent rat stem cell had occurred. In animals with donor chimerism >60% (n=18) no sign of limb rejection was present for the duration of the study. All animals with chimerism <20% (n=5) developed moderate signs of rejection clinically and histologically. Gross motor and sensory reinnervation (weight bearing, toe spread) developed at >60 days in 14/21 rats. Postoperative flow cytometry studies demonstrated stable chimerism in all animals studied (n=10). Five out of five animals with irradiated limb transplants showed no sign of GVHD at >100 days. Stable mixed allogeneic chimerism can be achieved in a rat hindlimb model of composite tissue allotransplantation. Hindlimb allografts to mixed allogeneic chimeras exhibit prolonged, rejection-free survival. Partial functional return should be expected. The BM transplanted as part of the hindlimb allograft plays a role in the etiology of GVHD. Manipulating that BM before transplantation may influence the incidence of GVHD. This represents the first reliable rat hindlimb model demonstrating rejection-free CTA survival in an adult animal across a major MHC mismatch without the long-term need for immunosuppressive agents.

Turnover of Rat Brain Perivascular Cells

Brain perivascular spaces harbor a population of cells which exhibit high phagocytic capacity. Therefore, these cells can be labeled by intraventricular injection of tracers. Such perivascular cells at the interface between blood and brain are believed to belong to the monocyte/macrophage lineage and to be involved in antigen presentation. Currently, it is unclear whether these cells undergo a continuous turnover by entering and leaving the bloodstream. Using bone-marrow-chimeric animals, migration of donor macrophages into brain perivascular spaces has been reported. On the other hand, following intracerebral injection of india ink into nontransplanted animals, ink-labeled perivascular cells were still found 2 years after injection, suggesting a high stability of this cell pool. Thus, the turnover of perivascular cells observed in chimeras might be a result of bone marrow transplantation rather than a physiological occurrence. To address this issue, we monitored de novo invasion of macrophages into perivascular spaces of apparently healthy adult rats by applying techniques other than bone marrow transplantation, (i) consecutive injections of different tracers and (ii) ex vivo isolation of macrophages from the blood, cell labeling, and reinjection into the same animal to avoid MHC mismatch. Both approaches revealed vivid de novo invasion of macrophages into perivascular spaces, but not into brain parenchyma, rendering untenable the concept of perivascular cells forming a stable population of macrophages in the brain. Thus, brain perivascular spaces are under permanent immune surveillance of blood borne macrophages in normal adult rats.

Analysis of cellular events in hepatic allografts: Donor progenitors induce intragraft chimerism

Abstract Long-term graft acceptance and tolerance induction after allogeneic rat liver transplantation are well described. However, the underlying mechanisms remain unclear. In this study we investigated the cellular events within the liver graft during initial immunosuppression and long-term acceptance. Orthotopic liver transplantation was performed in the Dark Agouti (DA)-to-Lewis (LEW) and LEW-to-DA rat strain combination. In order to achieve long-term acceptance, LEW recipients of DA livers were treated with two different short-term therapies. Non-parenchymal cells (NPC) were isolated from liver allografts on days + 10 and + 100 after transplantation and donor-specific leukocytes were immunophenotyped by flow cytometry. Both the monotherapy and triple therapy prolonged graft survival (> 100 days). Liver allografts from LEW donors into DA recipients were spontaneously accepted across a complete MHC mismatch without immunosuppression. Liver allograft rejection was induced by infiltrating alloreactive immunocompetent cells. But the intensities of cell infiltration in the early and late phases after transplantation did not correlate with eventual outcome. Donor-specific NPC decreased to 18-25% on day + 10 in both therapeutic groups, but had rebounded to up to 40% by day + 100. Recurrence of donor-specific cells was caused almost exclusively by rising T cell counts. The persistence of dendritic cells in the late phase after transplantation could be clearly demonstrated. Repopulation by donor-specific T lymphocytes was observed in long-term accepted liver grafts. This recurrence may be based on the differentiation of liver-derived progenitor cells. The persistent coexistence of donor and recipient cells within the liver allograft (intrahepatic chimerism) appears to be characteristic and may be important for long-term acceptance.

The relationship between acute rejection and chronic rejection is highly dependent on specific MHC matching: a multi-strain rat heterotopic heart transplant study.

The impact of acute rejection, immunosuppression, and infection, specifically cytomegalovirus infection, on the development of chronic rejection in the cardiac allograft, has been the subject of a large number of investigations. One of the difficulties in finding associations has been the marked immunologic heterogeneity of the patient population coupled with the lack of the ability to HLA match. Furthermore, the ideal animal model, which duplicates as well as controls for this immunologic heterogeneity, is lacking. To try to simulate differences in HLA matching, immunosuppression regiments and cytomegalovirus infection, heterotopic heart transplantation was performed in two separate, complete MHC mismatch, rat strain combinations (WF-LEW, BN-LEW) requiring chronic immunosuppression and employing four separate immunosuppression/infection protocols. Animals were followed for 6 months, killed, and rejection and vascular changes were scored blinded to the group. The mean vascular and acute rejection scores were not significantly different between treatment regiments for either specific strain combination. There was a trend for the subtherapeutic groups to have higher vascular scores. Overall, there were no significant differences in vascular scores between the WF-LEW and BN-LEW groups (1.25+/-0.18 vs. 1.13+/-0.20, P=NS). Similar numbers of WF-LEW and BN-LEW exhibited cellular infiltration and necrosis of the allograft, but the intensity of the response (rejection score) was more severe in the WF-LEW combination (4.54+/-0.22 vs. 3.92+/-0.21, P=0.052) when limiting the analysis to those with myocyte necrosis. There was no significant correlation between acute rejection and vascular lesion severity in the WF-LEW combination (r=0.22, P=NS) but a high correlation between these parameters in the BN-LEW combination (r=0.74, P<0.0001). These data suggest that, although acute rejection and chronic rejection are related, MHC matching may influence their interdependence. These data also may explain why the clinical association between acute and chronic rejection is difficult to demonstrate.