BACKGROUND: T cell dysfunction, which includes exhaustion, anergy, and senescence, is a distinct T cell differentiation state that occurs after antigen exposure. Although T cell dysfunction has been a cornerstone of cancer immunotherapy, its potential in transplant research, while not yet as extensively explored, is attracting growing interest. Interferon regulatory factor 4 (IRF4) has been shown to play a pivotal role in inducing T cell dysfunction. METHODS: A novel ultra-low-dose combination of Trametinib and Rapamycin, targeting IRF4 inhibition, was employed to investigate T cell proliferation, apoptosis, cytokine secretion, expression of T-cell dysfunction-associated molecules, effects of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways, and allograft survival in both in vitro and BALB/c to C57BL/6 mouse cardiac transplantation models. RESULTS: In vitro , blockade of IRF4 in T cells effectively inhibited T cell proliferation, increased apoptosis, and significantly upregulated the expression of programmed cell death protein 1 (PD-1), Helios, CD160, and cytotoxic T lymphocyte-associated antigen (CTLA-4), markers of T cell dysfunction. Furthermore, it suppressed the secretion of pro-inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17. Combining ultra-low-dose Trametinib (0.1 mg·kg -1 ·day -1 ) and Rapamycin (0.1 mg·kg -1 ·day -1 ) demonstrably extended graft survival, with 4 out of 5 mice exceeding 100 days post-transplantation. Moreover, analysis of grafts at day 7 confirmed sustained IFN regulatory factor 4 (IRF4) inhibition, enhanced PD-1 expression, and suppressed IFN-γ secretion, reinforcing the in vivo efficacy of this IRF4-targeting approach. The combination of Trametinib and Rapamycin synergistically inhibited the MAPK and mTOR signaling network, leading to a more pronounced suppression of IRF4 expression. CONCLUSIONS Targeting IRF4, a key regulator of T cell dysfunction, presents a promising avenue for inducing transplant immune tolerance. In this study, we demonstrate that a novel ultra-low-dose combination of Trametinib and Rapamycin synergistically suppresses the MAPK and mTOR signaling network, leading to profound IRF4 inhibition, promoting allograft acceptance, and offering a potential new therapeutic strategy for improved transplant outcomes. However, further research is necessary to elucidate the underlying pharmacological mechanisms and facilitate translation to clinical practice.
Animals ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Interferon Regulatory Factors/metabolism* ; Heart Transplantation/methods* ; T-Lymphocytes/immunology* ; Sirolimus/therapeutic use* ; Pyridones/therapeutic use* ; Graft Survival/drug effects* ; Pyrimidinones/therapeutic use* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Male ; Signal Transduction/drug effects*

Heart transplantation is the last treatment option in refractory end stage heart failure, which can prolong survival. The number of heart transplantations has increased and the survival rate has improved during the last few decades which was contributed by advanced understanding of immunologic mechanism of rejection, pharmaceutical development and clinical management of donors and recipients. However, only a fraction of patients can be offered to transplantation due to shortage of donor heart and many patients suffer high mortality while waiting. Meanwhile, technical advancement of mechanical assist device in recent years enabled long term implantable left ventricular assist devices (LVAD) to bridge the patients with high mortality in the waiting list to transplantation and to assist as a long term destination therapy for patients who are not eligible for transplantation. Development of solid phase assay increased the sensitivity and the specificity of detection of anti-human leukocyte antigen (HLA) antibodies in the recipient. It enabled identifying unacceptable HLA antigens, acquire calculated Panel Reactive Antibodies and perform virtual cross match that can enhance the efficacy of donor allocation system to decrease the waiting time, obviate prospective cross match to decrease ischemic time and to assess the risk of rejection in presensitized patients. Antibody mediated rejection is a challenging entity in diagnosis and management. However, standardized classification of histology and immunology of endomyocardial biopsies was made recently and immunotherapy is moving toward targeted therapies directed at antibody production and function. This review focuses on those major changes in the heart transplantation field in the last decade.
Allergy and Immunology ; Antibodies ; Antibody Formation ; Biopsy ; Classification ; Diagnosis ; Graft Rejection ; Heart ; Heart Failure ; Heart Transplantation* ; Heart-Assist Devices* ; HLA Antigens ; Humans ; Immunotherapy ; Leukocytes ; Mortality ; Sensitivity and Specificity ; Survival Rate ; Tissue Donors ; Waiting Lists

Recent data suggest that activation of aryl hydrocarbon receptor (AhR) by its high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in expansion of regulatory T (Treg) cells and suppresses the development of autoimmune and allergic diseases in several models. Treg cells have been increasingly documented to suppress allograft rejection and even to establish stable long-term graft acceptance. However, the involvement of TCDD in the regulation of solid organ transplantation rejection is largely unknown. Here, we examined whether activation of AhR with TCDD altered cardiac allograft rejection in an allogeneic heart transplant model. Recipient C57BL/6 (H-2b) mice were administrated with a single intraperitoneal injection of TCDD, and the murine cardiac transplant models from BALB/c (H-2d) to C57BL/6 (H-2b) were built 24 h later. The complete cessation of cardiac contractility was defined as the observation endpoint. The effect of TCDD on T-cell proliferation was assessed by mixed lymphocyte reaction (MLR). Histological and immunohistochemical analyses were performed to estimate the severity of rejection. The phenotype and cytokine profile of lymphocytes were analyzed by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Activation of AhR remarkably prolonged the survival of cardiac allografts to more than 20 days. In vitro, TCDD ugregulated the frequency of CD4+CD25+Foxp3+ Treg cells and suppressed the proliferation of T lymphocytes. In vivo, the prolonged survival time was associated with increased number of Treg cells in allografts and spleens. Furthermore, the secretion of interferon-γ (IFN-γ) and interleukin-17 (IL-17) was reduced to less than 50% of that of the PBS treatment control group by TCDD treatment, whereas IL-10 was elevated to 10-fold of that of the PBS treatment control group. Collectively, our data indicate that activation of AhR with a single dose of TCDD significantly prolonged the survival of fully allogeneic cardiac grafts, and the mechanism underlying this effect might be involved in the induction of Treg cells.
Animals ; Graft Rejection ; immunology ; pathology ; prevention & control ; Graft Survival ; drug effects ; immunology ; Heart Transplantation ; adverse effects ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Polychlorinated Dibenzodioxins ; pharmacology ; Receptors, Aryl Hydrocarbon ; immunology ; T-Lymphocytes ; immunology ; pathology ; T-Lymphocytes, Regulatory ; immunology ; pathology

BACKGROUNDAlveolar echinococcosis (AE) is caused by the metacestode stage of Echinococcus multilocularis (E. multilocularis) and is a rare but life-threatening disease. This disease commonly is characterized by an infiltrative, tumor-like growth of the E. multilocularis metacestode in the liver of human. Liver transplantation is an effective therapy for end-stage of hepatic AE, but the characteristics of host immunity associated with E. multilocularis infection with organ transplantation are poorly defined. We hereby aimed to study the immunological status and allograft heart survival in inbred rats with E. multilocularis infection.

METHODSRat models of AE were established by injecting the E. multilocularis suspension made from E. multilocularis infected tissues into the abdomen of Lewis (LEW) rats. Three months later, in the experimental group, allograft heart transplantation was performed from Brown-Norway (BN) rats to the E. multilocularis infected LEW rats. In the control group, we transplanted hearts from BN rats to healthy LEW rats. The influence of the disturbed immune system in E. multilocularis infected rats on the heart transplantation was assessed, including observation of allograft heart survival time, histopathological examination of grafts and immunohistochemical examination of infiltrating cells (CD4(+) T cells, CD8(+) T cells and eosinophile granulocytes), measurement of interleukin (IL)-4 and interferon (IFN)-γ in the serum by enzyme-linked immunosorbent assay (ELISA) and analysis of CD4(+)CD25(+) regulatory T cells in peripheral blood by fluorescence activated cell sorting (FACS) flow cytometric analysis.

RESULTSThe survival time of recipients in the experimental group was prolonged compared with those in the control group. The numbers of graft infiltrating CD8(+) T cells were decreased whereas the graft infiltrating eosinophil granulocytes (CD15(+)) were increased in grafts in the experimental group (P < 0.05). Furthermore, the proportion of CD4(+)CD25(+) regulatory T cells in the peripheral blood was 10.8% on average in the experimental group, which was significantly higher than that in the control group (6.1%). In addition, the level of serum IL-4 in E. multilocularis infected rats was higher than that in the control group rats, whereas the level of serum IFN-γ in experimental group was lower than that in the control group when graft rejection occurred (P < 0.05).

CONCLUSIONSThis study suggests that E. multilocularis infection could prolong the allograft survival time through the polarization of Th1/Th2-type cells and induction of CD4(+)CD25(+) regulatory T cells. This strategy may provide a new idea for establishing transplantation tolerance.

Animals ; Echinococcosis ; blood ; immunology ; Echinococcus multilocularis ; immunology ; pathogenicity ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Gerbillinae ; Heart Transplantation ; Immunohistochemistry ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Male ; Rats

BACKGROUNDDonor organ rejection continues to be a significant problem for patients receiving transplants. We therefore tested whether transferring a donor's major histocompatibility complex (MHC) gene to the recipient would mitigate the rejection of transplanted hearts in mice.

METHODSH-2K(k) gene from donor mice was amplified using nested polymerase chain reaction (PCR) and ligated into a mammalian expression vector, which was then transfected into thymus ground mass cells collected from the recipients. Clones stably expressing the transgene were then injected into the recipients' thymus visualized using ultrasound. Control mice were administered cells previously transfected with empty vector. Following heart transplantation, cardiac activity was monitored electrocardiographically. Recipient thymus cells were tested for MHC antigenicity using flow cytometry and spleen cells were subjected to mixed lymphocyte culture tests. Finally, the transplanted hearts were sectioned, stained and examined under light microscopy.

RESULTSSouthern analysis following nested PCR revealed clear expression of H-2K(k) gene. Following transplantation, electrocardiosignals were detectable highly significantly longer in recipients administered thymal cells expressing donor H-2K(k) than in those receiving control cells. Flow cytometric analysis using an anti-H-2K(k) antibody confirmed its expression in H-2K(k) treated recipients but not in control mice. Mixed lymphocyte cultures containing H-2K(k) treated cells showed significantly less proliferation than those containing control cells. Hearts from control mice showed substantially greater lymphocyte infiltration than those from H-2K(k) treated mice and large areas of necrosis.

CONCLUSIONRejection of transplanted hearts can be mitigated substantially by introducing the donor's MHC into the recipient.

Animals ; Blotting, Southern ; Electrocardiography ; Female ; Flow Cytometry ; Graft Rejection ; genetics ; immunology ; Heart Transplantation ; immunology ; methods ; Major Histocompatibility Complex ; genetics ; immunology ; Male ; Mice ; Polymerase Chain Reaction

OBJECTIVE: To investigate the role of T helper cell 17(Th17) cells and their cytokines IL-17 in heart allograft rejection in mice. METHODS: The heart transplantation models were randomly divided into 2 groups: a allograft group (n=12) and an isograft group (n=12).On the post-operative day (POD) 3 and 7, serum IL-17 level was tested by enzyme-linked immunosorbent assay, and Th17 cells in heart grafts were measured by flow cytometry. The heart grafts were harvested and saved in 10% formalin, and then embedded in paraffin. RESULTS: Compared with the iso-graft group, the allograft group had a higher serum level of IL-17 on POD3 and POD7 (P<0.05), and the level of IL-17 was higher on POD7 than that on POD3 (P<0.05). The allograft group had more Th17 cells infiltrating in grafts on POD3 and POD7 (P<0.05) and there were more Th17 cells infiltrating on POD7 than that on POD3 (P<0.05). Histological examination showed that the prolongation of post transplantation time resulted in more rejection pathological changes. CONCLUSION Th17 cells may play an important role in the development of heart transplant rejection. IL-17 may serve as a predictive parameter for allograft rejection in the future.
Animals ; Graft Rejection ; blood ; diagnosis ; immunology ; Heart Transplantation ; adverse effects ; immunology ; Interleukin-17 ; blood ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Random Allocation ; Th17 Cells ; immunology

OBJECTIVETo investigate the feasibility of strategy of allograft tolerance induction by injection of FasL-decorated donor splenocytes.

METHODSChimeric FasL with core streptavidin (SA-FasL) was efficiently displayed on the surface of splenocytes by the technology of ProtEx™. Heterotopic heart transplant procedures were performed from donor WF rats to recipient ACI rats, F344 rats were used as third-party. Intraperitoneal injection of ACI rats with "decorated" WF splenocytes was used as the approach to induce tolerance in this study. According to different therapeutic strategies, three groups were set up: SA-FasL group (n = 23), SA group (n = 20) and naive splenocytes only group (n = 8). No treatment group was regarded as control (n = 10). Adoptive transfer was underwent with injection of splenocytes from tolerant recipients into naive ACI followed by heart transplant procedures. Mixed lymphocyte reaction (MLR) and third party transplantation were performed to detect allogenic tolerance.

RESULTSThe injection of ACI rats with WF rat splenocytes displaying SA-FasL on their surface resulted in tolerance to donor, but not F344 third-party cardiac allografts. There were 70% cardiac allografts in SA-FasL group achieved long term survival, and it was significantly higher than the rats in other groups (P < 0.05). Adoptive transfer of splenocytes from long-term graft recipients into naive unmanipulated ACI rats resulted in indefinite survival of secondary WF grafts. Donor specific tolerance was identified by MLR and third-party transplant.

CONCLUSIONThe direct display of SA-FasL on the cell membrane in a rapid and efficient manner provides a practical and clinically applicable means of immunomodulation for tolerance induction with considerable therapeutic potential for transplantation.

Animals ; Fas Ligand Protein ; genetics ; immunology ; Heart Transplantation ; immunology ; Male ; Rats ; Rats, Inbred ACI ; Rats, Inbred F344 ; Rats, Inbred WF ; Spleen ; cytology ; metabolism ; Tissue Donors ; Transplantation Tolerance ; immunology

BACKGROUNDCelecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is a non-steroidal anti-inflammatory drug used as an adjuvant to sensitize cancer cells to apoptosis. However, in rats suffering from acute rejection, celecoxib reduced apoptosis of myocardial cells. We hypothesize that celecoxib reduces myocardial apoptosis either by inducing apoptosis in peripheral blood lymphocytes (PBLs) or by altering the percentage of CD4(+) and CD8(+) lymphocytes.

METHODSAfter cardiac transplantation, rats were administered intragastrically with celecoxib (50 mg/kg per day) for 3, 5 or 7 days, at which time the graft was excised and evaluated for organ rejection. In addition, PBLs were isolated from the blood to determine PBLs apoptosis, and the percentage of CD4(+) and CD8(+) lymphocytes.

RESULTSCelecoxib induced PBLs apoptosis in 3 days, but protected the cells from apoptosis at 5 and 7 days. Also, the percentage of CD4(+) lymphocytes decreased only at 3 days, but a reduction in the percentage of CD8(+) lymphocytes was not seen until 7 days after the transplant surgery. Celecoxib only decreased acute rejection at 5 days, with no discernible difference in rejection after 3 and 7 days.

CONCLUSIONSThe results suggested that celecoxib displayed a multiple physiological function in a time-dependent manner.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Apoptosis ; drug effects ; Celecoxib ; Cells, Cultured ; Graft Rejection ; immunology ; prevention & control ; Heart Transplantation ; immunology ; Lymphocytes ; cytology ; drug effects ; immunology ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Pyrazoles ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Rats, Wistar ; Sulfonamides ; pharmacology ; Transplantation, Homologous ; immunology

BACKGROUNDAcute allograft rejection in heart transplantation remains as one of the major complications. Obligatory graft surveillance is still achieved with the invasive and expensive endomyocardial biopsy (EMB). Our study aimed to study the use of intramyocardial electrograms combined with other noninvasive methods for the monitoring of acute rejection after human heart transplantation.

METHODSPermanent pacemakers were implanted in 58 patients undergoing heart transplantations. Intramyocardial electrograms (IMEG) were recorded periodically and the results were compared with those from EMBs. The R wave amplitude of the IMEG was used as the index value, the average R wave amplitude at the third week following transplantation was considered as the baseline, and a reduction of > 20% compared with the baseline was regarded as a positive result. EMB was performed in cases of positive IMEG results and also at other times. Other noninvasive methods were used to help the diagnosis. Acute rejection (AR) was defined as International Society of Heart-Lung Transplantation grade IIIA or higher.

RESULTSWe obtained 1231 IMEG records and 127 EMBs. Of the total 127 EMBs, 53 were positive, in which there were 42 IMEG positive results and 11 negative, while in the rest 74 negative EMBs, there were 9 IMEG positive results and 65 negative. The sensitivity of IMEG for the diagnosis of AR was 79.2%, and the specificity was 87.8%. The positive predictive value was 82.4% and the negative predictive value was 85.5%. Of the total of 1231 IMEG records, 51 were positive and 1180 were negative. Excluding 11 proved by EMB to be false negative, if the other 1169 were considered as no evidence of rejection, through the other noninvasive methods, AR diagnosed by this noninvasive monitoring strategy, the sensitivity was 79.2%, and the specificity was 99.2%. The positive predictive value was 82.4% and the negative predictive value was 99.1%.

CONCLUSIONSIMEG can be used as a noninvasive method for monitoring AR following heart transplantation. It is a continuous, safe and inexpensive method, and could reduce the need for EMB combined with other noninvasive methods, without reducing the detection of rejection.

Adolescent ; Adult ; Electrocardiography ; instrumentation ; methods ; Electrodes, Implanted ; Female ; Graft Rejection ; immunology ; Heart Transplantation ; immunology ; Humans ; Male ; Middle Aged ; Pacemaker, Artificial ; Predictive Value of Tests ; Retrospective Studies ; Young Adult

It has been reported that the immune response due to alpha-Gal epitopes is an important factor in tissue valve failure. The elimination of the interaction between the natural anti-Gal antibodies and alpha-gal epitopes on the xenografts is a prerequisite to the success of xenografts in humans. Previously, we reported that the green coffee bean alpha-galactosidase could remove all alpha-Gal epitopes from cell surface of porcine aortic valve and pericardial tissue, but it has limitations on cost effectiveness. In this study we wanted to know whether the recently produced recombinant human alpha-galactosidase A has the same effective enzymatic activity as green coffee bean alpha-galactosidase in removing alpha-Gal epitopes from the same tissues. After treating fresh porcine aortic valve and pericardial tissue with recombinant alpha-galactosidase A, each sample was stained with Griffonia simplicifolia type I isolectin B4 indirect immunoperoxidase avidin-biotin technique. We then examined whether the alpha-Gal epitopes were reduced or abolished in each consecutive concentration of recombinant alpha-galactosidase A by comparing the degree of the Griffonia simplicifolia isolectin B4 staining. As a result, the recombinant alpha-galactosidase A could remove cell surface alpha-Gals on porcine aortic valve and pericardial tissue as effectively as green coffee bean alpha-galactosidase.
Adolescent ; Animals ; Aortic Valve/chemistry/cytology/*immunology ; Child ; Coffea/enzymology ; Epitopes/*immunology ; Heart Valve Prosthesis Implantation ; Humans ; Pericardium/chemistry/cytology/*immunology ; Recombinant Proteins/genetics/*immunology ; Swine ; Transplantation, Heterologous/immunology ; alpha-Galactosidase/genetics/*immunology