Mouse Heart Transplantation Research Articles

Cervical heterotopic heart transplantation in mice using a novel suture technique

BackgroundVascularized transplantation models in mice are critical to understand mechanisms that mediate rejection and to develop new therapeutics. Standard abdominal heterotopic heart transplantation techniques employ an end-to-side suture technique and are the workhouse of transplant immunology research laboratories. Recently, cervical heterotopic heart transplantation in mice has emerged as an alternative due to several advantages but is conventionally performed by suture or cuff techniques in an end-to-end fashion. Therefore, we introduce an end-to-side anastomosis technique. MethodsThe donor pulmonary artery is end-to-side anastomosed to the recipient right external jugular vein, using a continuous 10-0 nylon suture. Vascular suturing is accomplished inside the vessel on the posterior wall, and then outside the vessel on the anterior wall. Finally, the donor ascending aorta is end-to-side anastomosed to the recipient common carotid artery with an identical suture technique. ResultsThe median times for the donor heart harvest, recipient preparation, anastomoses of the pulmonary artery to the external jugular vein, and the ascending aorta to the common carotid artery were 12, 10, 12 and 11 min, respectively. The survival rate was 100% (n=20). ConclusionsWe provide a detailed description of how to perform end-to-side anastomoses using a suture technique in the mouse cervical heart transplantation model. This procedure reconstitutes coronary blood flow in the heart graft with minimal interruption to recipient anatomy and provides an experimental platform to study transplant immunology.

Early graft-infiltrating lymphocytes are not associated with graft rejection in a mouse model of skin transplantation.

Graft-infiltrating lymphocytes (GILs) play an important role in promoting rejection after organ transplantation. We recently reported that GILs that accumulated up to 3 days post-transplantation did not promote rejection, whereas GILs present 3-5 days post-transplantation promoted rejection in a mouse heart transplantation model. However, the immunological behaviour of GILs in murine skin transplantation remains unclear. GILs were isolated on days 3, 5 or 7 post-transplantation from C57BL/6 (B6) allogeneic skin grafts transplanted onto BALB/c mice. BALB/c Rag2-/- γc-/- mice (BRGs) underwent B6 skin graft transplantation 10 weeks after adoptive transfer of day 3, 5, or 7 GILs. BRGs reconstituted with day 5 or 7 GILs completely rejected B6 grafts. However, when B6 grafts harvested from recipient BALB/c mice on day 5 or 7 were re-transplanted into BRGs, half of the re-transplanted day 5 grafts established long-term survival, although all re-transplanted day 7 grafts were rejected. BRGs reconstituted with day 3 GILs did not reject B6 grafts. Consistently, re-transplantation using day 3 skin grafts resulted in no rejection. Administration of anti-CD25 antibodies did not prevent the phenomenon observed for the day 3 skin grafts. Furthermore, BRGs reconstituted with splenocytes from naïve BALB/c mice immediately rejected the naïve B6 skin grafts and the re-transplanted day 3 B6 grafts, suggesting that day 3 GILs were unable to induce allograft rejection during the rejection process. In conclusion, the immunological role of GILs depends on the time since transplantation. Day 3 GILs had neither protective nor alloreactive effects in the skin transplant model.

Siglec-E is a novel co-inhibitory receptor in the modulation of innate activation after transplantation

Abstract Early after transplantation, inflammation and tissue injury are associated with significant activation of the innate immune cells, such as DCs. This activation drives adaptive immune responses, triggering acute rejection while hindering the development of transplant tolerance. Current immunosuppressive drugs primarily target T cells, while innate immunity is not efficiently inhibited in transplantation. The sialic acid–binding immunoglobulin-like lectin E (Siglec-E, or SigE) is an innate receptor that binds to ligands carrying sialic acids and inhibits TLRs-triggered inflammatory responses. Here, we demonstrate the role of SigE in experimental solid organ transplantation mouse models. Upon heart transplantation in mice, SigE is upregulated in graft-infiltrating APCs, including DCs. In humans, Siglec-7 and -9, the human homologs of SigE, are also upregulated in human heart biopsies following transplantation. Genetic ablation of recipient SigE leads to accelerated rejection of heart, kidney, and skin allografts. Mechanistically, SigE-deficient DCs are more susceptible to activation by DAMPS with an enhanced NF-kB activation and production of TNF-a. Moreover, DCs lacking SigE have a higher ability to induce allo-T cell responses in vitro and in vivo. In contrast, overexpression of SigE on DCs decreases their activation by DAMPs and reduces their T cell allostimulatory capacity. Thus, Siglec-E is a major inhibitory receptor in transplantation by controlling APC activation.

Optimal conditions for adenoviral transduction of immature dendritic cells without affecting the tolerogenic activity of DC-based immunotherapy

Dendritic cells (DCs) play a pivotal role in maintaining immune tolerance. Using recombinant adenovirus (rAd) to deliver vectors to immature dendritic cells (imDCs) is an important method for studying the tolerogenic function of DCs. We found that using RPMI medium and a higher MOI during transduction increased the expression of CD80, CD86, and MHC-II on the surface of imDCs. Our data reveal a significant increase in the secretion of the pro-inflammatory cytokine IL-6 in the group showing the most pronounced phenotypic changes. In the mouse heart transplant model, imDCs with unstable phenotype and function due to adenoviral transduction resulted in an increased proportion of Th1 and Th17 cells in recipients. However, these effects can be managed, and our proposed optimized transduction strategy significantly minimizes these adverse effects. Our study holds significant implications for the development and optimization of immunotherapy utilizing tolerogenic dendritic cells.

Human amniotic mesenchymal stem cells inhibit immune rejection injury from allogeneic mouse heart transplantation: A preliminary study on the microRNA expression

BackgroundMesenchymal stem cell therapy is a new treatment for immune rejection in heart transplantation. The aim of this paper is to investigate the effect of human amniotic mesenchymal stem cells (hAMSCs) on alleviating immune rejection of allogeneic heart transplantation in mice and its possible underlying mechanism. MethodsWe injected hAMSCs into cervical ectopic heart transplantation model mice via tail vein to observe the survival time, the pathological changes of donor myocardium, and the fluorescent distribution of hAMSCs after the transplantation. MicroRNAs (miRs) with significantly differential expression were obtained by RNA sequencing and bioinformatic analysis, and a dual luciferase reporter gene assay together with real-time quantitative PCR (qRT-PCR) was performed to verify the relationship between miRs and their targeting genes. ResultsThe intervention of hAMSCs prolonged the graft survival time and alleviated the pathological damage of the donor heart. The injected hAMSCs were distributed mainly in the liver, spleen, and kidney, only a very small portion in the donor and recipient hearts. In the allogeneic transplantation models, the expression of miR-34b-5p significantly increased after hAMSC treatment. MiR-34b-5p showed a knockdown effect on gene Fc gamma receptor 2B (FCGR2B). ConclusionshAMSCs can reduce the immune rejection injury after allogeneic heart transplantation. This effect may be associated with the upregulation of miR-34b-5p expression to knock down its targeting gene FCGR2B.

Homoharringtonine promotes heart allograft acceptance by enhancing regulatory T cells induction in a mouse model.

Homoharringtonine (HHT) is an effective anti-inflammatory, anti-viral, and anti-tumor protein synthesis inhibitor that has been applied clinically. Here, we explored the therapeutic effects of HHT in a mouse heart transplant model. Healthy C57BL/6 mice were used to observe the toxicity of HHT in the liver, kidney, and hematology. A mouse heart transplantation model was constructed, and the potential mechanism of HHT prolonging allograft survival was evaluated using Kaplan-Meier analysis, immunostaining, and bulk RNA sequencing analysis. The HHT-T cell crosstalk was modeled ex vivo to further verify the molecular mechanism of HHT-induced regulatory T cells (Tregs) differentiation. HHT inhibited the activation and proliferation of T cells and promoted their apoptosis ex vivo . Treatment of 0.5 mg/kg HHT for 10 days significantly prolonged the mean graft survival time of the allografts from 7 days to 48 days ( P <0.001) without non-immune toxicity. The allografts had long-term survival after continuous HHT treatment for 28 days. HHT significantly reduced lymphocyte infiltration in the graft, and interferon-γ-secreting CD4 + and CD8 + T cells in the spleen ( P <0.01). HHT significantly increased the number of peripheral Tregs (about 20%, P <0.001) and serum interleukin (IL)-10 levels. HHT downregulated the expression of T cell receptor (TCR) signaling pathway-related genes ( CD4 , H2-Eb1 , TRAT1 , and CD74 ) and upregulated the expression of IL-10 and transforming growth factor (TGF)-β pathway-related genes and Treg signature genes ( CTLA4 , Foxp3 , CD74 , and ICOS ). HHT increased CD4 + Foxp3 + cells and Foxp3 expression ex vivo , and it enhanced the inhibitory function of inducible Tregs. HHT promotes Treg cell differentiation and enhances Treg suppressive function by attenuating the TCR signaling pathway and upregulating the expression of Treg signature genes and IL-10 levels, thereby promoting mouse heart allograft acceptance. These findings may have therapeutic implications for organ transplant recipients, particularly those with viral infections and malignancies, which require a more suitable anti-rejection medication.

Circular RNA MAP2K2-modified immunosuppressive dendritic cells for preventing alloimmune rejection in organ transplantation.

Long-term patient and graft survival has been achieved in organ transplantation but at the expense of toxic side effects that are associated with long-term use of nonspecific immunosuppressive drugs. Discovering new regulators of dendritic cells is the key for development of an ideal treatment to prevent immune rejection. We hypothesized that knockdown of circMAP2K2 induces immunosuppressive DCs and that treatment with circMAP2K2 silenced-DCs can prevent alloimmune rejection. DCs were cultured and transfected with siRNA for circMAP2K2. circMAP2K2 levels were measured by qRT-PCR. DC's maturation and immune function were assessed by flow cytometry and mixed lymphocyte reactions. The function of circMAP2K2 was illustrated by a series of RIP and IP. The therapeutics of engineered DCs was tested in a mouse heart transplantation model. We found that circMAP2K2 was highly expressed in mature DCs. Knockdown of circMAP2K2 reduced expression of MHCII, CD40 and CD80, attenuated the ability of DCs to activate allogeneic naïve T cells, and enhanced CD4+CD25+FOXP3+ regulatory T cells (Treg). circMAP2K2-induced immunosuppressive DCs by interacting with SENP3. Treatment with circMAP2K2-knockdown DCs attenuated alloimmune rejection and prolonged allograft survival in a murine heart transplantation model. The immune suppression induced in vivo was donor-antigen specific. In conclusion, knockdown of circMAP2K2 can induce immunosuppressive DCs which are able to inhibit overactive immune response, highlighting a new promising therapeutic approach for immune disorder diseases.

Novel ceRNA network construction associated with programmed cell death in acute rejection of heart allograft in mice.

T cell-mediated acute rejection(AR) after heart transplantation(HT) ultimately results in graft failure and is a common indication for secondary transplantation. It's a serious threat to heart transplant recipients. This study aimed to explore the novel lncRNA-miRNA-mRNA networks that contributed to AR in a mouse heart transplantation model. The donor heart from Babl/C mice was transplanted to C57BL/6 mice with heterotopic implantation to the abdominal cavity. The control group was syngeneic heart transplantation with the same kind of mice donor. The whole-transcriptome sequencing was performed to obtain differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs) and lncRNAs (DElncRNAs) in mouse heart allograft. The biological functions of ceRNA networks was analyzed by GO and KEGG enrichment. Differentially expressed ceRNA involved in programmed cell death were further verified with qRT-PCR testing. Lots of DEmRNAs, DEmiRNAs and DElncRNAs were identified in acute rejection and control after heart transplantation, including up-regulated 4754 DEmRNAs, 1634 DElncRNAs, 182 DEmiRNAs, and down-regulated 4365 DEmRNAs, 1761 DElncRNAs, 132 DEmiRNAs. Based on the ceRNA theory, lncRNA-miRNA-mRNA regulatory networks were constructed in allograft acute rejection response. The functional enrichment analysis indicate that the down-regulated mRNAs are mainly involved in cardiac muscle cell contraction, potassium channel activity, etc. and the up-regulated mRNAs are mainly involved in T cell differentiation and mononuclear cell migration, etc. The KEGG pathway enrichment analysis showed that the down-regulated DEmRNAs were mainly enriched in adrenergic signaling, axon guidance, calcium signaling pathway, etc. The up-regulated DEmRNAs were enriched in the adhesion function, chemokine signaling pathway, apoptosis, etc. Four lncRNA-mediated ceRNA regulatory pathways, Pvt1/miR-30c-5p/Pdgfc, 1700071M16Rik/miR-145a-3p/Pdgfc, 1700071M16Rik/miR-145a-3p/Tox, 1700071M16Rik/miR-145a-3p/Themis2, were finally validated. In addition, increased expression of PVT1, 1700071M16Rik, Tox and Themis2 may be considered as potential diagnostic gene biomarkers in AR. We speculated that Pvt1/miR-30c-5p/Pdgfc, 1700071M16Rik/miR-145a-3p/Pdgfc, 1700071M16Rik/miR-145a-3p/Tox and 1700071M16Rik/miR-145a-3p/Themis2 interaction pairs may serve as potential biomarkers in AR after HT.

MTHFD2 ablation in T cells protects against heart transplant rejection by perturbing IRF4/PD-1 pathway through the metabolic-epigenetic nexus

One-carbon metabolism supports the activation, proliferation, and function of multiple immune cells. However, researchers have not clearly determined whether and how one-carbon metabolic enzymes contribute to heart transplant rejection. We investigated the dynamic metabolic adaptation in grafts during heart transplant rejection by conducting transcriptomics, metabolomics and single-cell RNA sequencing studies of cardiac tissue from human and mouse heart transplant recipients. We also assessed the expression of the one-carbon metabolic enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) in cardiac grafts by immunofluorescence and flow cytometry assays. Then we constructed a murine heart transplant model with T cell-specific Mthfd2 knockout mice, analyzed T cells function by flow cytometry assays and enzyme-linked immunospot assays, and studied the mechanism by Cleavage Under Targets and Tagmentation assays. Finally, we studied the effect of a pharmacological inhibitor of MTHFD2 in humanized skin transplant model. We revealed that the one-carbon metabolism enzyme MTHFD2 was a hallmark of alloreactive T cells and was linked to T cell proliferation and function after exposure to alloantigen. And, Mthfd2 ablation prevented murine heart transplant rejection. Mechanistically, we found Mthfd2 ablation affected the interferon regulatory factor 4/programmed death-1 pathway through a metabolic-epigenetic mechanism involving H3K4me3. Furthermore, we found that inhibiting MTHFD2 attenuated human allograft rejection in a humanized skin transplant model. These data show that the one-carbon metabolic enzyme MTHFD2 serves as a metabolic checkpoint of alloreactive T cells and suggest that it may be a potential therapeutic target for heart transplant rejection.

IL-35 Stabilizes Treg Phenotype to Protect Cardiac Allografts in Mice.

Interleukin-35 (IL-35), secreted by regulatory T cells (Treg) and B cells, is immunosuppressive under both physiological and pathological conditions. However, the role of IL-35 in all responses has yet to be investigated. Here, we demonstrate that IL-35 protects allografts by stabilizing the Treg phenotype and suppressing CD8 + T-cell activation in a mouse heart transplantation model. The effect of IL-35 on immune cell infiltration in grafts and secondary lymphoid organs was examined using mass cytometry, flow cytometry, and immunofluorescence. Moreover, using quantitative real-time polymerase chain reaction, flow cytometry, and phospho-flow assays, we demonstrated that IL-35 maintains Treg phenotypes to restrain CD8 + T cells via the gp130/signal transducer and activator of transcription 1 pathway. Mass cytometry analysis of intragraft immune cells showed that IL-35 decreased CD8 + T-cell infiltration and increased Foxp3 and IL-35 expressions in Treg. In vitro, we demonstrated that IL-35 directly promoted Treg phenotypic and functional stability and its IL-35 secretion, generating a positive feedback loop. However, Treg are required for IL-35 to exert its suppressive effect on CD8 + T cells in vitro. After depleting Treg in the recipient, IL-35 did not prolong graft survival or decrease CD8 + T-cell infiltration. Mechanistically, we found that IL-35 sustained Treg stability via the gp130/signal transducer and activator of transcription 1 signaling pathway. Our findings highlight that IL-35 stabilizes the Treg phenotype to ameliorate CD8 + T-cell infiltration in the allograft, which has never been described in the transplanted immunological milieu.

#3211 INHIBITION OF TRAINED INNATE IMMUNITY BY MYELOID CD40-TRAF6 BLOCKADE PROMOTES ALLOGRAFT TOLERANCE

Abstract Background and Aims Allograft rejection is largely mediated by adaptive immune cells. Although innate immune cells are also involved, their role is less clear. We hypothesize that suppression of innate immune responses, particularly trained innate immunity, can contribute to graft tolerance in transplantation. Trained immunity refers to the long-term epigenetic and metabolic reprogramming of innate immune cells, which potentiates responses to secondary stimuli. Here, we studied the effect of blocking CD40-TRAF6 signaling in myeloid cells on trained immunity induction using in vitro stimulations, ChIP-sequencing and metabolic analyses. We evaluated the effect of myeloid CD40-TRAF6 inhibition on T cell responses in mixed lymphocyte reactions (MLR) and the effect of CD40-TRAF6 inhibitor (TRAF6i) nanobiologic (NB) treatment on allograft rejection in a murine heterotopic heart transplantation model. Method Human peripheral blood mononuclear cells (PBMCs) were stimulated for 24 h with heat-killed Candida albicans (HKCA), a well-described inducer of trained immunity, in presence or absence of TRAF6i, or RPMI medium (control) followed by 5 days of rest. Interleukin-6 (IL-6) and tumor necrosis factor (TNF) production upon restimulation with lipopolysaccharide (LPS) was assessed using ELISA. We performed ChIP-sequencing and metabolic analyses with Seahorse assays at day 6. To investigate the effect of TRAF6i on T cell responses to allogeneic stimuli, we performed 7-day MLR with HKCA- or HKCA+TRAF6i trained monocytes and CellTrace Violet-labeled naïve T cells. Proliferation and FOXP3 expression of naïve T cells was measured by flow cytometry. The effect of TRAF6i-NB on graft survival was assessed by injection of myeloid-directed TRAF6i-NB 0, 2 and 5 days post-transplantation in C57BL/6J mice heterotopically transplanted with BALB/c hearts, that did or did not receive pre-operative CTLA4-Ig treatment. Results TRAF6i treatment inhibited IL-6 and TNF production upon LPS restimulation of HKCA-treated PBMCs in vitro (Fig. 1A). ChIP-sequencing analysis and Seahorse technology respectively revealed that the epigenetic changes (Fig. 1B) and metabolic alterations (Fig. 1C) underlying trained immunity were prevented by TRAF6i. Induction of trained immunity in monocytes reduced differentiation of naïve T cells to FOXP3+CD4+ T cells upon allogeneic stimulation, which was partially reversed by TRAF6i treatment of monocytes (Fig. 1D). Treatment with TRAF6i-NB, combined with CTLA4-Ig, induced &amp;gt;100 days graft survival in 5 out of 6 C57BL/6J mice heterotopically transplanted with BALB/c hearts, while one graft was rejected at 99 days post-transplantation (Fig. 1E). Conclusion Inhibition of TRAF6 prevents trained immunity in monocytes at the functional, epigenetic and metabolic level. We show in vitro that inhibiting trained immunity has the effect of modulating T cell responses to allogeneic stimuli towards FOXP3+CD4+ T cell differentiation. Using a mouse heart transplant model, we show that the combination of TRAF6i and CTLA4-Ig treatment prevents allograft rejection in vivo. This study identifies CD40-TRAF6 signaling as a novel target to inhibit trained immunity and to promote allograft tolerance.

Modified Heterotopic Abdominal Heart Transplantation and a Novel Aortic Regurgitation Model in Rats.

Over the past 50 years, many researchers have reported heterotopic abdominal heart transplantation in mice and rats, with some variations in the surgical technique. Modifying the transplantation procedure to strengthen the myocardial protection could prolong the ischemia time while preserving the donor's cardiac function. This technique's key points are as follows: transecting the donor's abdominal aorta before harvesting to unload the donor's heart; perfusing the donor's coronary arteries with a cold cardioplegic solution; and topical cooling of the donor's heart during the anastomosis procedure. Consequently, since this procedure prolongs the acceptable ischemia time, beginners can easily perform it and achieve a high success rate. Moreover, a new aortic regurgitation (AR) model was established in this work using a technique different from the existing one,which is created by inserting a catheter from the right carotid artery and puncturing the native aortic valve under continuous echocardiographic guidance. A heterotopic abdominal heart transplantation was performed using the novel AR model. In the protocol, after the donor's heart is harvested, a stiff guidewire is inserted into the donor's brachiocephalic artery and advanced toward the aortic root. The aortic valve is punctured by pushing the guidewire further even after the resistance is felt, thus inducing AR. It is easier to damage the aortic valve using this method than with the procedure described in the conventional AR model. Additionally, this novel AR model does not contribute to the recipient's circulation; therefore, this method is expected to produce a more severe AR model than the conventional procedure.

T cell specific deletion of IRF4 with Ox40-Cre impairs effector and memory T cell responses in heart transplantation

BackgroundIRF4 is the pioneer factor for effector T cell maturation. Here we investigated the function of IRF4 in maintaining OX40-related T cell responses following alloantigen activation in a mouse heart transplantation model. MethodsIrf4flox/flox mice were bred with Ox40cre/+ mice to generate Irf4flox/floxOx40cre/+ mice. Wild type C57BL/6, Irf4flox/floxOx40cre/+ mice were transplanted with BALB/c heart allografts, with or without BALB/c skin-sensitization. CD4+ TEa T cells co-transfer experiments and flow cytometric analysis were conducted to investigate the amount of CD4+ T cells and the percentage of the T effector subset. ResultsIrf4flox/floxOx40cre/+ and Irf4flox/floxOx40cre/+ TEa mice were constructed successfully. IRF4 ablation in activated OX40-mediated alloantigen specific CD4+ TEa T cells reduced effector T cell differentiation (CD44hiCD62Llo, Ki67, IFN-γ), which caused long-term allograft survival (> 100 d) in the chronic rejection model. In the donor skin-sensitized heart transplantation model, the formation and function of alloantigen-specific memory CD4+ TEa cells were also impaired in Irf4flox/floxOx40cre/+ mice. Additionally, deletion of IRF4 after T cell activation in Irf4flox/floxOx40cre/+ mice reduced T cell reactivation in vitro. ConclusionsIRF4 ablation after OX40-related T cell activation could reduce effector and memory T cell formation and inhibit their function in response to alloantigen stimulation. These findings could have significant implications in targeting activated T cells to induce transplant tolerance.

Monomethyl fumarate prevents alloimmune rejection in mouse heart transplantation by inducing tolerogenic dendritic cells.

Dendritic cells (DCs) are important targets for eliciting allograft rejection after transplantation. Previous studies have demonstrated that metabolic reprogramming of DCs can transform their immune functions and induce their differentiation into tolerogenic DCs. In this study, we aim to investigate the protective effects and mechanisms of monomethyl fumarate (MMF), a bioactive metabolite of fumaric acid esters, in a mouse model of allogeneic heart transplantation. Bone marrow-derived DCs are harvested and treated with MMF to determine the impact of MMF on the phenotype and immunosuppressive function of DCs by flow cytometry and T-cell proliferation assays. RNA sequencing and Seahorse analyses are performed for mature DCs and MMF-treated DCs (MMF-DCs) to investigate the underlying mechanism. Our results show that MMF prolongs the survival time of heart grafts and inhibits the activation of DCs in vivo. MMF-DCs exhibit a tolerogenic phenotype and function in vitro. RNA sequencing and Seahorse analyses reveal that MMF activates the Nrf2 pathway and mediates metabolic reprogramming. Additionally, MMF-DC infusion prolongs cardiac allograft survival, induces regulatory T cells, and inhibits T-cell activation. MMF prevents allograft rejection in mouse heart transplantation by inducing tolerogenic DCs.

Solid organ and vascularized composite allotransplantation have distinct requirements for localized delivery of enhanced costimulation blockadeto control rejection

Abstract We recently demonstrated, in a mouse heart transplant model, the therapeutic effect of Enhanced Costimulation Blockade: the transient combination of CTLA4-Ig with the JAK inhibitor Tofacitinib (Tofa). In the same model, we also proved the efficacy of a graft-localized delivery by means of a Tofa releasing hydrogel (Tofa-Hydro). In this study, we implemented a mouse orthotopic hind-limb VCA model to delineate any differential drug delivery requirement. Moreover, we explored the potential of Tofa-containing lipid nanoparticles (LNp) in achieving selective targeting of immune cells and its therapeutic effect. Local injections of Tofa-Hydro combined with CTLA4-Ig promoted VCA survival, although with a smaller therapeutic benefit (MST=23 days vs CTLA4-Ig-only MST=14 days). We identified a LNp formulation with negligible toxicity and favorable Tofa encapsulation efficiency. Live animal imaging and flow analysis indicated these particles have the unique property of accumulating in draining lymphoid tissues, with minimal distribution to other tissues, and deliver their cargo to multiple immune cells. Four post-transplant SQ injections of Tofa-LNp (with CTLA4-Ig) promoted graft survival longer than Tofa-Hydro (MST&amp;gt;60 days). Our results reveal that SOT and VCA differ in the topography of inflammatory events that contribute to rejection. This novel observation suggests that different activatory mechanisms contribute to allopriming in the two systems, although they share utilization of JAK signaling pathways. Nevertheless, our study demonstrates the efficacy of localized actuation of enhanced costimulation blockade in achieving safer regulation of rejection. DOD Award # W81XWH-18-1-0789

Granzyme B-responsive fluorescent probe for non-invasive early diagnosis of transplant rejection

Allograft rejection has always been a major obstacle in organ transplantation. The current clinical diagnostic gold standard for allograft rejection is an invasive biopsy. However, biopsy has some limitations, such as sampling errors, risk of serious complications, and high cost. In this study, we have rationally developed an activatable fluorescent probe CYGB for imaging of granzyme B, which is a biomarker released by CD8+T cells attacking the graft. Moreover, the ability of CYGB to detect rejection early in mouse heart and skin transplantation models was evaluated. The probe CYGB consists of a caged hemicyanine-based fluorophore and a GzmB-specifically cleaved peptide substrate linked via a self-immolating spacer, p-aminobenzyl alcohol. Endogenous GzmB in CD8+ T cells specifically activated the near-infrared fluorescence (NIRF) signal of CYGB. In vivo imaging in mice skin and heart graft models, showed that CYGB preferentially accumulates in grafts, enabling early diagnosis of rejection. Moreover, CYGB enables non-invasive assessment of the level of immunosuppression in allogeneic mice treated with FK506. This study provides an alternative method for monitoring the status of allografts without biopsy.

Cardiomyocytic cyclic GMP-AMP synthase is critical for the induction of experimental cardiac graft rejection

ObjectiveDuring cardiac transplantation, cellular injury and DNA damage can result in the accumulation of cytosolic double-stranded DNA (dsDNA), which can activate the cyclic GMP-AMP synthase (cGAS)/stimulator of interferon gene (STING) signaling pathway and thus induce multiple proinflammatory responses. However, the role of the cGAS-STING pathway in cardiac transplantation remains unclear. This study explored the role of cardiomyocytic cGAS in mouse heart transplantation during the ischemia/reperfusion and rejection processes. Methods and ResultsCytosolic dsDNA accumulation and cGAS-STING signaling pathway component upregulation were observed in the grafts posttransplantation. The use of cGAS-deficient donor tissues led to significantly prolonged graft survival. The underlying mechanisms involved decreased expression and phosphorylation of downstream proteins, including TANK binding kinase 1 and nuclear factor κB. In parallel, notably diminished expression levels of various proinflammatory cytokines were observed. Accordingly, substantially decreased proportions of macrophages (CD11b+F4/80+) and CD8+ T cells were observed in the spleen. The activation of CD8+ T cells (CD8+CD69+) within the graft and the proportion of effector memory (CD44highCD62Llow) lymphocytes in the spleen were notably decreased. Treatment with the cGAS inhibitor Ru.521 led to significantly prolonged graft survival. ConclusionsCardiomyocytic cGAS plays a critical role by sensing cytosolic dsDNA during cardiac transplantation and could serve as a potential therapeutic target to prevent graft rejection.

Adenovirus-IL-10 relieves chronic rejection after mouse heart transplantation by inhibiting miR-155 and activating SOCS5.

Objective: Chronic rejection remains the main factor that influence long-term survival of patients after heart transplantation. Interleukin-10 (IL-10) play critical role in macrophages-mediated transplant immune responses. We investigated the mechanism of IL-10 in macrophage related chronic rejection after mouse heart transplantation. Methods: Mouse heart transplant chronic rejection model was established to evaluate pathological changes in the allograft. Myocardial interstitial fibrosis, apoptosis, and inflammatory factor levels were detected in ad-IL-10-treated mice. The positive iNOS+ and Arg-1+ expressions, macrophage subset changes, and the proportion of regulatory T-cells (Tregs) and TIGIT+ Tregs were quantified by flow. In in vitro experiments, ad-IL-10 was transfected into macrophages followed by detection of apoptosis, phagocytosis, and CD163, CD16/32, and CD206 expression. The expression and relationships between IL-10, miR-155, and SOCS5 were also detected and verified. A rescue experiment was performed to evaluate macrophage function through the combined treatment of ad-IL-10 and overexpression of miR-155. Results: Significantly decreased IL-10 expression in chronic rejection during mouse heart transplantation was observed. Ad-IL-10-treated mice showed decreased pathological injury, perivascular fibrosis, apoptosis, inflammation, and iNOS+ and CD16/32+ expression, and increased Treg/TIGIT+ Treg cell, Arg-1+ and CD206+ cell proportion. Ad-IL-10-treated macrophages in vitro showed reduced apoptosis, improved phagocytosis, and M2 polarization. Mechanically, IL-10 negatively regulated miR-155 to activate SOCS5. Overexpression of miR-155 reversed IL-10 mediated-positive regulation of macrophage function. Conclusion: IL-10 downregulated miR-155 and activated SOCS5, thereby promoting macrophage M2 polarization to relieve chronic rejection after heart transplantation.

Transport-Associated Vibrational Stress Triggers Drug-Reversible Apoptosis and Cardiac Allograft Failure in Mice.

Increasingly complex and long-range donor organ allocation routes coupled with implementation of unmanned aerial vehicles (UAVs) have prompted investigations of the conditions affecting organs once packaged for shipment. Our group has previously demonstrated that different modes of organ transport exert unique environmental stressors, in particular vibration. Using a mouse heart transplant model, we demonstrated that vibrational forces exert tangible, cellular effects in the form of cardiomyocyte apoptosis and cytoskeletal derangement. Functionally, these changes translated into accelerated allograft loss. Notably, administration of an apoptosis inhibitor, Z-VAD-FMK, helped to ameliorate the detrimental cellular and functional effects of mechanical vibration in a dose-dependent manner. These findings constitute one of the first reports of the negative impact of transit environment on transplant outcomes, a contributing mechanism underpinning this effect, and a potential agent to prophylax against this process. Given current limitations in measuring donor organ transit environments in situ, further study is required to better characterize the impact of transport environment and to potentially improve the care of donor organs during shipment. Clinical and Translational Impact Statement: We show that apoptosis inhibitor, Z-VAD-FMK, ameliorated transport-related vibrational stress in murine heart transplants, which presents a potential therapeutic or preservation solution additive for future use in transporting donor organs.

ZBTB20 regulates cardiac allograft rejection through NFкB-mediated inflammation in mouse heart transplantation

Allograft rejection is a major obstacle for the long-term survival of heart transplantation (Htx) patients. The cardiac allograft rejection requires the activation of macrophages and effector T cells. In this study, we explored the role of zinc-finger and BTB domain containing protein 20 (ZBTB20) in the regulation of heart allograft rejection. Flow cytometry analysis of the spleen cells from mice undergoing an acute cardiac rejection revealed that the ZBTB20 protein expression was upregulated in both T and B cells(n = 4，P < 0.01). In addition, ZBTB20 gene knockdown significantly prolonged the survival of heart allografts in mice(n = 4，P < 0.01). Lack of ZBTB20 increased the expression of Foxp3 and limited the response of T helper 1 (Th1) cells(n = 4，P < 0.01). The ZBTB20-related regulation occurred through the activation of the NFкB pathway. In conclusion, our data suggest that ZBTB20 is involved in the regulation of T cells involved in acute heart allograft rejection. Hence, downregulation of ZBTB20 expression may inhibit T cells to prolong heart transplant survival.