Cardiac Transplantation Model Research Articles

Establishment of mice heterotopic cardiac transplantation and observation between different strains

Objective To establish the mice heterotopic cardiac transplantation as well as to observe the different graft reaction between different strains.Methods According to the surgical procedures of Ono's pattern in mice cardiac heterotopic transplantation model,we did allograft cardiac transplantation using same (BALB/c→BALB/c ; CBA→CBA; C57BL/6j→C57BL/6j) or different (BALB/c→C57BL/6j ; C57BL/6j→BALB/c; C57BL/6j→CBA; CBA→C57BL/6j)strains male mice.Survival time and pathological examination were observed after transplantation Results The operative survival was 88.6％ (62/70).Mean donor graft harvest time of was 4.8 min,mean anatomic time was (28.70 ± 3.56) min (BALB/c→BALB/c),(20.70±4.22) min (CBA→CBA) and (27.90±2.51) min (C57BL/6j→C57BL/6j) in same strains while (28.00 ± 2.71) min (BALB/c→C57BL/6j),(22.10 ± 4.33) min (C57BL/6j→CBA),(29.90±3.14) min (C57BL/6j→BALB/c) and (30.33 ±23.00) min (CBA→C57BL/6j) in different strains.The survival time was over 100 days in same strains and (7.10 ±0.74) d (C57BL/6j→BALB/c) ; (7.40 ± 0.82) d (BALB/c→ C57 BL/6j) ; (8.90 ± 1.10) d (C57 BL/6j→ CBA) ; (8.50 ± 0.85) d (CBA→C57BL/6j) d in different strains.It was confirmed with pathological examination.Conclusion On the base of mastering the microsurgical techniques,we can establish the mice cardiac heterotopic transplantation model.The immune response in C57BL/6j to BALB/c model was very strong,while weaker in C57BL/6j to CBA models.The different transplant immune response can be used in the transplantation research. Key words: Mice; Cardiac transplantation; Models ; Strains

HMGB1 Is Involved in Chronic Rejection of Cardiac Allograft via Promoting Inflammatory-Like mDCs

Chronic rejection that leads to diffuse narrowing and occlusion of graft vessels is the most important cause of morbidity and mortality following cardiac transplantation. The role and underlying mechanism of high-mobility group box 1 (HMGB1), as an established inflammatory mediator in acute rejection, remains poorly understood in chronic rejection. Here, we assessed the effects and mechanisms of HMGB1 on the chronic rejection using single MHC Class II-mismatched mouse cardiac transplantation model. It was found that HMGB1 was increased accompanying with the development of chronic rejection, while blockade of HMGB1 with specific neutralizing mAb substantially ameliorated chronic rejection-mediated vasculopathy and fibrosis of allograft, as well as markedly decreased T cell infiltration and production of IL-17A and interferon-gamma in allograft and recipient's spleen. Further, anti-HMGB1 antibody treatment significantly declined the number and frequency of mature dendritic cells (DCs) in allograft and recipient's spleen, especially CD11b(+) Ly6C(high) matured DCs that share the phenotypes with inflammatory-DCs. These findings indicate that HMGB1 contributes to chronic rejection, and HMGB1 blockade may be a novel mean to disrupt the proinflammatory loop after heart transplantation.

The Role of CX3CR1 in dDC (Donor Dendritic Cells) Trafficking in Cardiac Transplant Model.

The Role of CX3CR1 in dDC (Donor Dendritic Cells) Trafficking in Cardiac Transplant Model.

Combination of Anti-BTLA Antibody (6B2) and Anti-PD-1 Antibody (PIM-2) Induced Indefinite Survival of Fully MHC-Mismatched Murine Cardiac Allograft.

Background: Programmed death (PD)-1 and B and T lymphocyte attenuator (BTLA) had been implicated in the regulation of autoimmune and may potentially play an important role in alloimmune responses. In this study, we investigated the effect of anti-PD-1 monoclonal antibody (mAb) (PIM-2) and anti-BTLA mAb (6B2) in fully MHC-mismatched murine model of cardiac allograft transplantation. Methods: CBA mice underwent transplantation of C57BL/6 hearts and received a single dose (100μg) of 6B2 on the day of transplantation (day 0) or four doses on day 0, 3, 6 and 9. Moreover, CBA recipients were also given one dose of combination of 6B2 and PIM-2 on day 0. Adoptive transfer was performed to determine whether regulatory cells were generated. Cell-proliferation and cytokine assessments were also performed. Result: Untreated CBA mice rejected C57BL/6 allografts acutely (median survival time [MST], 7 days). When CBA recipients were given one and four doses of 6B2, the allograft survivals were significantly prolonged (MSTs, 46 and >100 days, respectively). Moreover, when CBA mice were given with combination for one dose of 6B2 and PIM-2, the allograft survival was indefinitely prolonged (MST, >100 days). Secondary CBA recipients given whole splenocytes from primary combination-treated CBA recipients with C57BL/6 cardiac allografts 30 days after grafting had prolonged C57BL/6 allograft survival (MST, >30 days). Proliferation of splenocytes and interferon-γ production were suppressed and interleukin-4 production was increased in combination-treated mice. Conclusion: Combination of anti-BTLA mAb (6B2) and anti-PD-1 mAb (PIM-2) could induce hyporesponsiveness of fully MHC-mismatched cardiac allografts and may generate regulatory cells.

Preemptive Donor Apoptotic Cell Infusions Induce IFN-γ-Producing Myeloid Derived Suppressor Cells for Cardiac Allograft Protection.

We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid derived suppressor cells (MDSCs) in the graft protection provided by infusion of such apoptotic donor cells is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b+ cells in the spleen that phenotypically resemble monocytic-like (CD11b+Ly6CHI) and granulocytic-like (CD11b+Gr1HI) MDSCs. Both populations suppress T cell proliferation in vitro, and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b+Gr1HI cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ dependent mechanisms. These findings reveal a novel link between apoptotic donor cell infusions and induction of MDSCs as a mechanism for transplant graft protection, and provide several targets that are amenable to therapeutic manipulations for tolerance induction for cardiac transplantation.

B Cells and CD40/CD154 Interactions Are Required for CD8 T Cell Recovery Following Antibody-Mediated Lymphoablation.

Antibody-meditaed lymphoablation is commonly used as an induction therapy in transplant patients. However, recipients eventually reconstitute their T cell repertoire, develop anti-donor immune responses and reject transplanted organ. The goal of this study was to investigate the requirements for T cell recovery following lymphoablation in a mouse model of heterotopic cardiac transplantation. We previously reported that memory CD4 T cells are less susceptible to depletion by murine antithymocyte globulin (mATG) than CD8 T cells. Additional CD4 T cell depletion impaired CD8 T cell recovery after mATG treatment in B6 recipients of BALB/c heart allografts (0.2% vs 3-5% of spleen CD8 T cells recovered by d. 10 posttransplant) and in non-transplanted B6 mice. Analogous findings were made in recipients treated with anti-CD8 ± anti-CD4 mAbs demonstrating that the requirement for CD4 cell help is a common feature of antibody-mediated lymphoablation. In addition, memory CD8 T cell reconstitution depended on CD4 T cell help in non-transplanted B6 mice and in B6 isograft recipients treated with mATG. Treatment with blocking anti-CD154 mAb plus mATG inhibited memory CD8 T cell recovery in B6 heart allograft recipients similar to the levels observed after CD4 T cell depletion. Conversely, agonistic anti-CD40 mAb restored expansion of memory CD8 T cells following mATG treatment of CD4 T cell depleted heart allograft recipients. To test whether B cells are required for CD8 T cell recovery, we used B6.huCD20tg mice. Treatment with anti-human CD20 mAb depleted >90% of peripheral B cells and markedly inhibited CD8 T cell expansion in huCD20tg recipients compared to B cell-sufficient mice. Next, B cells were isolated from mATG treated recipients with or without CD4 depletion. In the absence of CD4 T cells, B cells expressed lower levels of MHC class I, CD80, CD86, and TNFa suggesting that both cognate interactions and cytokines promote CD8 T cell expansion. Our results show that CD4 T cell help mediated through B cells and CD40/CD154 interactions drives CD8 T cell reconstitution following antibody-mediated depletion. These findings may be used to improve the efficiency of lymphoablation in sensitized transplant recipients.

High Percentage of PD-1+ CD4+ T Cells in a Subset of Tolerant Pediatric Liver Recipients.

BACKGROUND: Programmed death-1 (PD-1) is a co-inhibitory receptor up-regulated on activated and functionally exhausted T cells that may limit immune responses to persistent antigenic stimulation. PD-1 inhibits alloimmune responses in skin and cardiac transplant (tx) models, contributing to allograft tolerance. We studied PD-1+ CD4+ T cells at study entry / baseline (BL) and during followup of participants (pts) in ITN029, a multi-center trial of pediatric living donor liver tx recipients undergoing immunosuppression (IS) withdrawal. ITN029 identified 12 tolerant (TOL) pts whose liver tests and biopsies remain stable off IS for 6.5 (5.2-6.9) yrs and 7 non-tolerant (NONTOL) pts. METHODS: Banked PBMCs from ITN029 were stained with directly-labeled antibodies: CD3, CD4, CD8, CD25, CD28, CD2, Ki67, CD45RO, HLA-DR, CD57 and PD-1. Live/Dead Blue was used for viability gating with CD14 and CD19 as dump markers. All individual pt samples were run on the same day on a BD Fortessa and analyzed using FlowJo software. RESULTS: At BL, prior to IS withdrawal, the 12 TOL pts have significantly higher % of PD-1+ CD4+ T cells (Fig) but not CD8+ T cells (data not shown) compared to the 7 NONTOL pts (p=0.021). Among the 12 TOL pts, the % of PD-1+ CD4+ T cell is distinctively higher in 6; the remaining 6 TOL pts are similar to the NONTOL pts. The elevated PD-1+ CD4+ T cell % in the TOL subgroup persists for more than 5 yrs after IS withdrawal (Fig). TOL pts with high vs low PD-1+ CD4+ T cell % had shorter interval between tx and enrollment (7.3±2.18 vs 9.2±2.60 yrs) although small numbers preclude definitive correlation (Fig). CONCLUSIONS: The 12 TOL pts segregate into 2 groups based on BL / pre-withdrawal % of PD1+ CD4+ T cells that persists for more than 5 yrs off IS. CD4+ T cell exhaustion may represent a tolerance mechanism that emerges earlier after liver tx than other mechanisms although larger pt numbers are needed to confirm these preliminary findings.Figure: No Caption available.DISCLOSURES:Turka, L.: Employee, Novartis, Stockholder, Novartis.

Monocyte Recruitment to HLA Class I Antibody-Activated Endothelial Cells Is Dependent Upon mTOR.

Aim: A hallmark of antibody (Ab)-mediated rejection is the presence of intravascular monocytes in the allograft. The purpose of this study was to identify the role of the mTOR signaling pathway in HLA class I Ab-mediated monocyte recruitment to endothelial cells (EC). Methods: Static and flow-based (ibidi) adhesion assays were used to assess monocyte (MM6) recruitment to HLA-activated primary human aortic endothelial cells. mTOR inhibition was characterized using pharmacological inhibitors (KU 0063794 and sirolimus) at different time points and doses in endothelial cells and monocytes. Results were verified using siRNA knock-down (ko) of rictor and raptor, components of the mTOR pathway. Cell-based ELISA was performed to quantitate Weibel-Palade Body exocytosis; protein phosphorylation was measured by Western blot and quantitated by densitometry. An established murine cardiac transplant model was used to determine the effect of sirolimus treatment on acute AMR. Briefly, Rag1 ko animals were transplanted with an H-2d mismatched heart transplant and received passive transfer of anti-donor Dd+Kd IgG antibodies. Results: Pretreatment of EC for 24 hours with sirolimus or Ku reduced monocyte adherence to EC by > 80%, whereas 2- hour mTOR inhibition resulted in partial inhibition of monocyte binding. siRNA ko of mTOR using siRNA yielded comparable results significantly decreasing monocyte adherence. siRNA against raptor and rictor revealed that both mTORC1 and mTORC2 are involved in HLA-mediated monocyte recruitment. Inhibition of mTOR had no effect on P-selectin induction. Instead, flow-based adhesion assays revealed that mTOR inhibition impairs the ability of EC to support firm adhesion of tethered monocytes by affecting late stage ICAM-1 clustering and its downstream signaling effects. mTOR inhibition in monocytes using sirolimus and Ku significantly impaired monocyte binding to activated endothelium. A reduction in endothelial damage and infiltrating mononuclear cells was observed in vivo in recipients of heart allografts treated with sirolimus. Conclusion: We found that mTOR blockade prevents monocyte recruitment to HLA-activated EC through mTORC1 and mTORC2 signaling pathways. In EC, mTOR inhibition prevents ICAM-1 clustering and blunts firm adhesion of monocytes. Our results provide novel insights into the mechanisms underlying monocyte recruitment in the setting of AMR. DISCLOSURE:Salehi, S.: Grant/Research Support, Pfizer. Reed, E.: Grant/Research Support, Pfizer.

Costimulation Blockade With Anti CD40 Antibody Maintains CD4+ and Treg Cell Numbers in Pig To Baboon Cardiac Transplantation Model.

Costimulation Blockade With Anti CD40 Antibody Maintains CD4+ and Treg Cell Numbers in Pig To Baboon Cardiac Transplantation Model.

Preemptive donor apoptotic cell infusions induce IFN-γ-producing myeloid-derived suppressor cells for cardiac allograft protection.

We have previously shown that preemptive infusion of apoptotic donor splenocytes treated with the chemical cross-linker ethylcarbodiimide (ECDI-SPs) induces long-term allograft survival in full MHC-mismatched models of allogeneic islet and cardiac transplantation. The role of myeloid-derived suppressor cells (MDSCs) in the graft protection provided by ECDI-SPs is unclear. In this study, we demonstrate that infusions of ECDI-SPs increase two populations of CD11b(+) cells in the spleen that phenotypically resemble monocytic-like (CD11b(+)Ly6C(high)) and granulocytic-like (CD11b(+)Gr1(high)) MDSCs. Both populations suppress T cell proliferation in vitro and traffic to the cardiac allografts in vivo to mediate their protection via inhibition of local CD8 T cell accumulation and potentially also via induction and homing of regulatory T cells. Importantly, repeated treatments with ECDI-SPs induce the CD11b(+)Gr1(high) cells to produce a high level of IFN-γ and to exhibit an enhanced responsiveness to IFN-γ by expressing higher levels of downstream effector molecules ido and nos2. Consequently, neutralization of IFN-γ completely abolishes the suppressive capacity of this population. We conclude that donor ECDI-SPs induce the expansion of two populations of MDSCs important for allograft protection mediated in part by intrinsic IFN-γ-dependent mechanisms. This form of preemptive donor apoptotic cell infusions has significant potential for the therapeutic manipulation of MDSCs for transplant tolerance induction.

MiRNA-181a is critical for the tolerogenic properties of plasmacytoid dendritic cells (TRAN3P.886)

Abstract Liver allografts are well tolerated and other solid organ allografts, when transplanted concurrently with livers, show improved graft outcomes. However, the mechanisms underlying “hepatic tolerance” have yet to be elucidated. Previous data show that hepatic dendritic cells (HDC) have diminished antigen presenting and immune stimulatory function compared with DC in lymphoid tissue. It has been hypothesized that immature plasmacytoid (p)DC are inherently tolerogenic with studies supporting this hypothesis. Our work confirms that HpDC prolong cardiac allograft survival (p<0.01) in a murine model compared to bulk and myeloid (m)DC. Micro-ribonucleic acid (miR) are short (15-22 nucleotides) RNA molecules which can post-transcriptionally regulate messenger RNA transcripts, resulting in translational repression or upregulation. We hypothesize that miR-181a, which we found is highly expressed in HpDC, plays a critical role in the tolerogenic properties of pDC. TLR 9 stimulation of pDC from miR-181a-/- mice exhibited higher levels of maturation markers and differential cytokine production as compared to wild-type C57BL/6 mice. More, there is altered phosphorylation of key members of signal transduction pathways, such as ERK1/2. Finally, in the same murine model of cardiac allograft transplantation, use of pDC from miR-181a-/- mice abrograted the tolerogenic properties seen with HpDC. Together, our data shows a critical role for miR-181a in the tolerance-inducing properties of pDC.

Effect of 34 Kinds of Traditional Japanese Herbal Medicines on Prolongation of Cardiac Allograft Survival

Herbal medicines have been used for over 3,000 years in Asian as alternative therapy for their variety effects and have recently become popular in Europe and the United States. In the last 30 years, Japanese herbal medicines were widely used for treatment of diseases after been recognized officially by Japanese government. In this study, we investigated the effect of 34 kinds of traditional Japanese herbal medicines on alloimmune responses in a murine model of cardiac allograft transplantation. CBA mice (H2k) underwent transplantation of a C57BL/6 (H2b) heart and received oral administration of 2 g/kg/d of the 34 kinds of herbal medicines from the day of transplantation until 7 days afterward. Naïve CBA mice rejected B6 cardiac grafts acutely (median survival time [MST], 7 days). CBA transplant recipients given 2 g/kg/d of Sairei-to (TJ-114) and Tokishakuyaku-san (TJ-23) had prolonged C57BL/6 allograft survival indefinitely (both MSTs > 100 days). Moreover, CBA transplant recipients given Seisinrensiin (TJ-111), Tokishigyakukagoshuyushokyoto (TJ-38), Rikkunshito (TJ-43), Maobushisaishinto (TJ-127), Ninjin-yoei-to (TJ-108), Ryokan-kyomi-shinge-nin-to (TJ-119), Inchingorei-san (TJ-117), Hochuekkito (TJ-41), Kihi-to (TJ-65), and Sinbu-to (TJ-30) had also prolonged C57BL/6 allograft survival significantly (MSTs of 28, 22, 16, 14, 14, 13, 12, 9.5, 9 and 9 days, respectively). However, none of other 22 kinds of herbal medicines could prolong the allograft survival. Furthermore, oral administration of 2 g/kg/d of Daikenchuto (TJ-100) induced sudden death (within 1 minute) in CBA mice. In conclusion, 12 kinds of Japanese herbal medicines prolonged allograft survival and one showed toxic effect in mice.

Characteristics of cardiac allograft vasculopathy induced by immunomodulation in the miniature Swine.

We aimed to develop swine cardiac transplantation model for study of cardiac allograft vasculopathy (CAV) and to characterize the mechanisms of its formation. Heterotropic cardiac transplantation was performed in swine leukocyte antigen mismatched miniature swine, and CAV was induced by immunomodulation by cyclosporine A (CyA). Histology and immunohistochemistry were performed to identify cellular components of CAV. Fluorescence in situ hybridization (FISH) was developed for detection of 1 and Y-chromosome for identification of cell origin in the female donor to the male recipient heart transplantation model. CAV was successfully developed by immunomodulation of CyA. Severity of CAV revealed more prominent in the distal epicardial coronary arteries than proximal coronary arteries. Phenotype of the SMCs proliferated in the intimal thickening of CAV were mostly embryonal/secretory type. Our new chromosome specific probes for FISH method were useful for discrimination of sex of each cell, and proliferated SMCs were revealed to be mainly donor origin. CAV mimicking human heart transplantation can be developed by appropriate immunomodulation in the swine. In swine CAV, proliferated SMCs seen in the intimal thickening were demonstrated to be from the donor origin.

Role of myeloid-derived suppressor cells in mouse pre-sensitized cardiac transplant model

Harness of sensitized transplantation remains a clinical challenge particularly in parallel with prolonged cold ischemia time (PCI)-mediated injury. Our present study was to test the role of myeloid-derived suppressor cells (MDSCs) in mouse pre-sensitized transplantation. Our findings revealed that CD11b+Gr1(low) MDSC was shown to have strong suppressive activity. MDSCs subsets from the tolerated mice exhibited higher suppressive capacities compared with counterparts from naive (untreated) mice. Depletion of Tregs could not affect splenic CD11b+Gr1(-low) MDSC frequency, but increase peripheral and intragraft CD11b+Gr1(-low) frequency. Intriguingly, boost of Tregs remarkably caused an increase of CD11b+Gr1(-low) frequency in the graft, peripheral blood, and spleen. Furthermore, peripheral CD11b+Gr1(-low) cells were massively accumulated at the early stage when allogeneic immune response was enhanced. Taken together, MDSCs could prevent grafts from PCI-mediated injury independent on Tregs in the pre-sensitized transplant recipients. Utilization of MDSC subset particularly CD11b+Gr1(-low) might provide a novel insight into improving graft outcome under such clinical scenarios.

Regulation of T Cell Differentiation and Alloimmunity by the Cyclin-Dependent Kinase Inhibitor p18ink4c

Cellular proliferation in response to mitogenic stimuli is negatively regulated by the Cip/Kip and the Ink4 families of cyclin-dependent kinase (CDK) inhibitors. Several of these proteins are elevated in anergic T cells, suggesting a potential role in the induction or maintenance of tolerance. Our previous studies showed that p27kip1 is required for the induction of T cell anergy and transplantation tolerance by costimulatory blockade, but a role for Ink4 proteins in these processes has not been established. Here we show that CD4+ T cells from mice genetically deficient for p18ink4c divide more rapidly than wild-type cells in response to antigenic, costimulatory and growth factor signals. However, this gain of proliferative function was accompanied by a moderate increase in the rate of cell death, and was accompanied by an overall defect in the generation of alloreactive IFNγ-producing effector cells. Consistent with this, p18ink4c-deficient T cells were unable to induce graft-vs-host disease in vivo, and p18ink4c deficiency cooperated with costimulatory blockade to significantly increase the survival of fully mismatched allografts in a cardiac transplantation model. While both p18ink4c and p27kip1 act to restrict T cell proliferation, p18ink4c exerts an opposite effect from p27kip1 on alloimmunity and organ transplant rejection, most likely by sustaining T cell survival and the development of effector function. Our studies point to additional important links between the cell cycle machinery and the processes of T cell differentiation, survival and tolerance.

Suppressor of cytokine signaling 3 (SOCS3) gene transfer prolongs the survival of the murine cardiac allograft by attenuating interleukin-17-producing alloreactive T-cell responses

Suppressor of cytokine signaling 3 (SOCS3) is the main negative feedback regulator of cytokine signals. We investigated the hypothesis that overexpression of SOCS3 in a murine cardiac allograft transplantation model may result in a survival advantage of the allograft by attenuating alloreactive T-cell responses. With the use of BALB/c (H-2(d) ) donor mice and C57Bl/6j (H-2(b) ) recipient mice, the murine cardiac transplantation model was established. Recipient mice received a tail intravenous injection with eukaryotic expression plasmid pEF-FLAG-I/mSOCS3 before and after transplantation. The heart beat of the grafts and immune responses were monitored. Overexpression of SOCS3 within grafts and spleens can prolong the survival time of cardiac allografts by attenuating infiltration of inflammatory cells such as T cells and macrophages in the grafts, decreasing the number of CD4(+) IL-17(+) cells and CD8(+) IL-17(+) cells in spleens, as well as reducing the expression of STAT3 in grafts and phosphorylation of STAT3 in both grafts and spleens. Overexpression of SOCS3 significantly delays cardiac allograft acute rejection, which is associated with reduced allograft proinflammatory leukocyte infiltration and impaired alloreactive IL-17(+) T cell immunity.

Prevention of Allogeneic Cardiac Graft Rejection by Transfer of Ex Vivo Expanded Antigen-Specific Regulatory T-Cells

The rate of graft survival has dramatically increased using calcineurin inhibitors, however chronic graft rejection and risk of infection are difficult to manage. Induction of allograft-specific regulatory T-cells (Tregs) is considered an ideal way to achieve long-term tolerance for allografts. However, efficient in vitro methods for developing allograft-specific Tregs which is applicable to MHC full-mismatched cardiac transplant models have not been established. We compared antigen-nonspecific polyclonal-induced Tregs (iTregs) as well as antigen-specific iTregs and thymus-derived Tregs (nTregs) that were expanded via direct and indirect pathways. We found that iTregs induced via the indirect pathway had the greatest ability to prolong graft survival and suppress angiitis. Antigen-specific iTregs generated ex vivo via both direct and indirect pathways using dendritic cells from F1 mice also induced long-term engraftment without using MHC peptides. In antigen-specific Treg transferred models, activation of dendritic cells and allograft-specific CTL generation were suppressed. The present study demonstrated the potential of ex vivo antigen-specific Treg expansion for clinical cell-based therapeutic approaches to induce lifelong immunological tolerance for allogeneic cardiac transplants.

High-mobility group box 1 accelerates early acute allograft rejection via enhancing IL-17+γδ T-cell response

Th17 and γδ T cells are the dominant IL-17-producing cell. We previously reported that high-mobility group box 1 (HMGB1) is critical in inducing IL-17-producing alloreactive T cells during early stage of acute allograft rejection. However, the role of γδ T cells during this process and its implication in HMGB1-mediated allograft rejection are not fully understood. Here, we use a murine model of cardiac allograft transplantation to further study the role of HMGB1 and IL-17-producing γδ T cells in acute allograft rejection. It was found that the expression of HMGB1 was increased in allograft, while blockade of HMGB1 suppressed IL-17(+) γδ T-cell response and inhibited the gene transcription of IL-23 and IL-1β. Furthermore, in vitro HMGB1 indirectly promoted the development of IL-17(+) γδ T cells by stimulating dendritic cells to produce IL-23 and IL-1β, meanwhile depletion of γδ T cells in vivo prolonged allograft survival and reduced the level of IL-17 in serum. In conclusion, our findings inferred that increased HMGB1 expression could enhance IL-17(+) γδ T-cell response by promoting the secretion of IL-23 and IL-1β, while IL-17(+) γδ T cells contribute to the early stage of acute allograft rejection.

Combination of paeoniae radix and cnidii rhizoma prolonged survival of fully mismatched cardiac allografts and generated regulatory cells in mice.

In previous studies, we have demonstrated that Tokishakuyakusan (TJ-23) can prolong the survival of allogeneic cardiac grafts and induce regulatory T cells. In this study we investigated the effects of Paeoniae radix and Cnidii rhizoma, two components of TJ-23, on alloimmune responses in a murine cardiac transplantation model and whether the two agents have synergistic effect. CBA mice underwent transplantation of a C57BL/6 heart and received oral administration of 2 g/kg/day of Paeoniae radix, Cnidii rhizoma, or the mixture of two agents from the day of transplantation until 7 days afterward. Naïve CBA mice rejected C57BL/6 cardiac graft acutely (median survival time (MST): 7 days). Paeoniae radix and Cnidii rhizoma prolonged C57BL/6 allograft survival (MSTs: 13.5 and 15.5 days, resp.). However, the mixture of two agents prolonged C57BL/6 allograft survival indefinitely (MST > 100 days). Secondary CBA recipients given whole splenocytes from primary combination-treated CBA recipients with B6 cardiac allografts 30 days after grafting had prolonged survival of B6 hearts (MST: 33 days). Flow cytometry studies showed that the CD4+CD25+Foxp3+ regulatory cell population was increased in combination-treated recipients. Combination of Paeoniae radix and Cnidii rhizoma induced hyporesponsiveness to fully allogeneic cardiac allografts and may generate CD4+CD25+Foxp3+ regulatory cells in our model.

Confined ischemia may improve remote myocardial outcome after rat cardiac arrest

Background. Confined ongoing ischemia after ischemia-reperfusion injury (IRI) may alter myocardial recovery. We evaluated in a rat cardiac transplantation model whether distal persistent myocardial ischemia (dMI) and remote preconditioning (RPreC) have a remote myocardial impact after IRI. Material and methods. Syngeneic heterotopic cardiac transplantation was performed on 29 Fischer344 rats to induce IRI, including nine rats which underwent distal ligation of the left anterior coronary artery (LAD) to yield distal MI (IRI+ dMI). RPreC was applied by occluding the left renal artery 5 min prior to reperfusion in six rats with IRI (IRI+ RPreC) as well as in seven with distal MI (IRI+ dMI+ RPreC). Microdialysis, histology and qRT-PCR for inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) were performed after graft harvesting. Results. In contrast to IRI + dMI + RPreC (39 ± 7 μmol), glutamate decreased in IRI + RPreC and IRI + dMI as compared with IRI (26 ± 3 and 31 ± 8 vs 91 ± 20, μmol respectively, p < 0.007). The relative number of vacuolated intramyocardial artery nuclei decreased in IRI + dMI as compared with IRI (0.02 ± 0.01, range 0–12 vs. 0.42 ± 0.31, range 0–3.25 PSU respectively, p < 0.04). iNOS expression decreased in IRI + RPreC as compared with IRI (p < 0.04), and eNOS expression decreased in IRI + dMI + RPreC as compared with IRI + dMI (p < 0.006) along with increased glycerol release. Conclusions. dMI after IRI has a potentially beneficial myocardial impact after cardiac arrest, which is hampered by RPreC.