Heart Allograft Rejection Research Articles

Pharmacological inhibition of epsilon PKC suppresses chronic inflammation in murine cardiac transplantation model

Epsilon protein kinase C (εPKC) plays pivotal roles in myocardial infarction and in heart failure. Although cardiac transplantation is a well-established therapy for severe heart failure, allograft rejection and host inflammatory responses limit graft function and reduce life expectancy. Here we determined whether sustained εPKC inhibition beginning 3 days after transplantation suppress allograft rejection and improve cardiac transplantation using a murine heterotopic transplantation model. Hearts of FVB mice (H-2 q) were transplanted into C57BL/6 mice (H-2 b). Delivery of the εPKC inhibitor, TAT 47–57–εV1-2 (εV1-2, n = 9, 20 mg/kg/day), or the carrier control peptide, TAT 47–57 (TAT, n = 8), by osmotic pump began 3 days after transplantation and continued for the remaining 4 weeks. εV1-2 treatment significantly improved the beating score throughout the treatment. Infiltration of macrophages and T cells into the cardiac grafts was significantly reduced and parenchymal fibrosis was decreased in animals treated with εV1-2 as compared with control treatment. Finally, the rise in pro-fibrotic cytokine, TGF-β and monocyte recruiting chemokine MCP-1 levels was almost abolished by εV1-2 treatment, whereas the rise in PDGF-BB level was unaffected. These data suggest that εPKC activity contributes to the chronic immune response in cardiac allograft and that an εPKC-selective inhibitor, such as εV1-2, could augment current therapeutic strategies to suppress inflammation and prolong graft survival in humans.

Cardiovascular molecular imaging of apoptosis

IntroductionMolecular imaging strives to visualise processes at the molecular and cellular level in vivo. Understanding these processes supports diagnosis and evaluation of therapeutic efficacy on an individual basis and thereby makes personalised medicine possible.Apoptosis and molecular imagingApoptosis is a well-organised mode of cell suicide that plays a role in cardiovascular diseases (CVD). Apoptosis is associated with loss of cardiomyocytes following myocardial infarction, atherosclerotic plaque instability, congestive heart failure and allograft rejection of the transplanted heart. Thus, apoptosis constitutes an attractive target for molecular imaging of CVD. Our current knowledge about the molecular players and mechanisms underlying apoptosis offers a rich palette of potential molecular targets for molecular imaging. However, only a few have been successfully developed so far.AimsThis review highlights aspects of the molecular machinery and biochemistry of apoptosis relevant to the development of molecular imaging probes. It surveys the role of apoptosis in four major areas of CVD and portrays the importance and future perspectives of apoptosis imaging. The annexin A5 imaging protocol is emphasised since it is the most advanced protocol to measure apoptosis in both preclinical and clinical studies.

Sanglifehrin A Blocks Key Dendritic Cell Functions In Vivo and Promotes Long-Term Allograft Survival Together with Low-Dose CsA

Sanglifehrin A (SFA) is a novel compound with unsurpassed cyclophilin-binding affinity, but relatively low direct antilymphocytic activity. Here, we report the capacity of SFA to selectively inhibit key dendritic cell (DC) functions in vivo and to suppress acute and chronic heart allograft rejection. We show that in vivo, SFA profoundly decreases DC receptor-mediated endocytosis and macropinocytosis and DC-T-cell allostimulatory activity. Furthermore, SFA abrogates >90% of IL-12p70 production in vivo while having no significant effect on IL-10 and TNF-alpha production. In a rat vascularized heart transplant model, SFA alone did not prevent graft rejection and rejection occurred within 23 days after low-dose CsA, whereas addition of SFA to low-dose CsA promoted long-term graft survival (median survival time >100 days; p = 0.0007). With respect to chronic rejection, SFA + CsA almost completely prevented graft arteriosclerosis compared to animals treated with CsA alone and controls. We propose that SFA represents a novel class of immunophilin-binding immunosuppressants with high activity against DCs and the development of graft vasculopathy in CsA-treated recipients.

Effect of Baohuoside‐1 aglycone and tacrolimus monotherapy and combination therapy on prevention of acute heart allograft rejection in the rat

Baohuoside-1 (B-1), a recently introduced novel immunosuppressant that was proved to be potent in inhibition of T and B cell proliferation and B-1, also prevents cardiac allograft rejection in rodents. The present study further proved that monotherapy of B-1's analogue B-1 aglycone effectively prolongs cardiac allograft survival and combination therapy of B-1 aglycone with tacrolimus (FK506) produces synergistic effect in prevention acute cardiac allograft rejection in the rat. .

Targeting Acute Allograft Rejection by Immunotherapy With Ex Vivo-Expanded Natural CD4+CD25+ Regulatory T Cells

Natural CD4CD25 regulatory T (Treg) cells have been implicated in suppressing alloreactivity in vitro and in vivo. We hypothesized that immunotherapy using ex vivo-expanded natural Treg could prevent acute allograft rejection in mice. Natural CD4+ CD25+ Treg were freshly purified from naive mice via automated magnetic cell sorter and expanded ex vivo by anti-CD3/CD28 monoclonal antibody (mAb)-coated Dynabeads. Suppression was assayed in vitro by mixed lymphocyte reaction and in vivo by targeting cardiac allograft rejection. Survival of Treg or effector T (Teff) cells after adoptive transfer in vivo was tracked by flow cytometry and all allografts were examined by histology and immunohistochemistry. By day nine in culture, 26.6+/-5.3-fold of expansion was achieved by co-culture of fresh natural Treg with anti-CD3/CD28 mAb-coated Dynabeads and interleukin-2. Ex vivo-expanded Treg exerted stronger suppression than fresh ones towards alloantigens in vitro and prevented CD4 Teff-mediated but only delayed CD4+/CD8+ Teff-mediated heart allograft rejection in Rag-/- mice. Long-term surviving allografts showed no signs of acute or chronic rejection with graft-infiltrating Treg expressing CD25 and FoxP3. Infused Treg persisted and expanded long-term in vivo and trafficked through the peripheral lymphoid tissues. CD25 expression was dynamic in vivo: maintained CD25 expression on Treg was indicative for the preservation of allosuppression, while significantly enhanced CD25 expression on CD4+ effector T cells was most likely associated with T-cell expansion and graft rejection. Therapeutic use of ex vivo-expanded natural CD4+ CD25+ Treg may be a feasible and nontoxic modality for controlling allograft rejection or perhaps inducing allograft tolerance.

Tacrolimus for the treatment of rheumatoid arthritis: are broad-based immunosuppressants still valid?

Tacrolimus is an immunosuppressive drug that is approved and currently used primarily for the prophylaxis of liver, heart and kidney allograft rejection. Recently, oral tacrolimus 1.5–3 mg/day has been approved in Japan and Canada for the treatment of rheumatoid arthritis in patients who respond insufficiently to other therapies. Tacrolimus potently inhibits calcineurin and, consequently, T-cell activation and proliferation with a mechanism similar to that of cyclosporine. Clinical studies of the efficacy and safety of tacrolimus as monotherapy and in combination with methotrexate have shown it to be less effective than many of the biologics, but similar to cyclosporine for symptomatic relief. Unlike the biologics and other immunosuppressants, it has not been shown to retard progression of joint damage on x-ray. However, clinical response, when it does occur, can be robust and appears to be maintained for at least 18 months. Longer-term safety and efficacy in rheumatoid arthritis have not been studied. In...

A Negative Regulatory Role in Mouse Cardiac Transplantation for a Splice Variant of CD80

Members of the B7 costimulatory protein family (CD80 and CD86) play a determining role in allograft rejection. Both CD80 and CD86 have naturally occurring splice variants whose roles in transplantation are unknown. Full length CD80 has two immunoglobulin (Ig)-like domains in the extracellular portion, IgC and IgV. In mouse, the isoform IgV-CD80 lacks the IgC-like domain. Here we analyzed the role of mouse IgV-CD80 in heart allograft rejection and search for equivalent splice variants in human. Mice made deficient for full-length CD80 but which retain expression of the shorter IgV-CD80 (CD80 mice) were used as donor or recipient of a heart allograft. Recipient animals were untreated or pretreated with alloantigen expressing cells and/or treated with CD80 and CTLA4 monoclonal antibodies (mAbs). Recipients expressing IgV-CD80 but not full length CD80 exhibited a slight prolongation in survival of either wild-type (Wt) or CD80 grafts. More dramatically, CD80 animals pretreated with donor alloantigen exhibited permanent graft survival, whereas their Wt counterparts rejected their grafts with a median survival of 24 days. This prolonged survival was due to the expression of IgV-CD80 in recipients since treatment with CD80 mAb abrogated the beneficial effect observed. We identified and report here a similar isoform of CD80 from human cDNA encoding a putative soluble, IgV-containing protein. IgV-CD80 bearing recipients show enhanced allograft survival especially after donor alloantigen pretreatment. This together with data from other species suggests that regulation delivered by splice variants of CD80 significantly modulates immunity and may be common across the species.

Evaluation of C4d Staining in Liver and Small Intestine Allografts

Antibody-mediated humoral rejection in kidney and heart allografts is well recognized and is often associated with poor outcome. C4d deposition in allograft biopsy specimens occurs at sites of antibody-mediated complement activation and has become one of the histopathologic criteria for diagnosis of humoral rejection in the kidney and the heart. To study immunohistochemical C4d staining as a potential diagnostic marker in liver and small intestine allograft biopsy specimens. Thirty-six small intestine and 71 liver specimens, including native specimens, allografts with and without histologic features of acute cellular rejection, and explants, were stained with antisera to C4d using an immunohistochemical method on formalin-fixed, paraffin-embedded tissue. In small intestine, C4d labeled capillaries in 27% of cases with no evidence of rejection, 36% of cases with evidence of acute rejection, and 2 (28%) of 7 specimens of native normal small intestine. In liver allograft biopsy specimens, C4d stained endothelium of veins, arteries, and/or sinusoids in 2 (8%) of 25 cases of acute rejection with central vein involvement; C4d staining was negative in biopsy specimens with no evidence of rejection. C4d stained the endothelium in a subset of explanted liver allografts with ductopenic rejection or chronic vascular rejection and strongly stained 1 explant with features of hyperacute rejection. The clinical utility of C4d staining in solid organ transplantation may vary by organ. Our data show C4d is unlikely to have utility in small intestine allograft biopsy specimens; however, further study in liver allografts, in conjunction with donor-specific antibody testing, is warranted.

The Predictive Value of Soluble Major Histocompatibility Complex Class I Chain-Related Molecule A (MICA) Levels on Heart Allograft Rejection

Recently the presence of a soluble form of major histocompatibility complex class I chain-related molecule A (sMICA) has been detected in the sera of patients with tumors. Shedding of sMICA by tumor cells downregulates NKG2D-mediated antitumor immunity. The aim of this investigation was to study the possible involvement of sMICA in the allograft acceptance after heart transplantation (HTX). We monitored the levels of sMICA by specific enzyme-linked immunosorbent assay (ELISA) in a total of 146 serum samples obtained from 34 heart transplantation patients followed up during the first year post-HTX. The persistence of sMICA expression was correlated with the clinical evolution of these patients. sMICA was detected in the serum of 21 of 34 patients (61.70%) between 15 and 20 days after implantation and was practically absent in pretransplant serum samples. Twenty of these 21 patients (95.24%) with sMICA did not experience episodes of severe rejection during this period (P = 0.0001), whereas sMICA was practically absent in patients with manifestations of severe acute rejection. The longitudinal study of these patients revealed that the presence of sMICA was consistently maintained in 75% of the patients with good graft status during the period of observation. This has led us to believe that the presence of levels of sMICA during the first year post-HTX may contribute to allograft acceptance. Additionally, functional studies indicate that sMICA downregulates NKG2D surface expression, which may lead to a functional impairment of cell-mediated cytolysis. These data suggest a significant correlation between the presence of sMICA and a lower incidence of rejection.

Phosphorylated S6 Ribosomal Protein: A Novel Biomarker of Antibody-Mediated Rejection in Heart Allografts

We tested the hypothesis that phosphorylation of S6 ribosomal protein (S6RP), a downstream target of the PI3K/Akt/mTOR pathway, is a biomarker of antibody-mediated rejection (AMR) in heart allografts. Primary cultures of human aortic and microvascular endothelial cells (EC) were treated with anti-HLA class I and class II antibodies (Ab) and cell lysates were studied for phosphorylation of S6 ribosmal protein at Serine235/236 (p-S6RP). Treatment of cultured EC with anti-class I and class II Ab stimulated S6RP phosphorylation. Immunohistochemical techniques were used to detect the level of p-S6RP in endomyocardial biopsies (n = 131) from 46 heart transplant recipients and the results were correlated with histopathological diagnosis of rejection, C4d staining, production of posttransplant anti-HLA Ab and clinical outcome. Increased phosphorylation of S6RP in endomyocardial biopsies was significantly associated with the diagnosis of AMR (p < 0.0001). No significant association between acute cellular rejection (ACR) and p-S6RP was observed. C4d staining was positively associated with both AMR and p-S6RP. Posttransplant anti-HLA class II Ab production was also significantly associated with a positive p-S6RP status in cardiac biopsies. These results indicate that p-S6RP is a useful biomarker for the diagnosis of AMR.

Genetic Polymorphism in Platelet-derived Growth Factor and Vascular Endothelial Growth Factor Are Significantly Associated With Cardiac Allograft Vasculopathy

The role of cytokine gene polymorphism and its association with acute heart allograft rejection and cardiac allograft vasculopathy (CAV) is controversial. The role of growth factor gene polymorphism has never been investigated in heart allograft recipients. Seventy heart transplant recipients were studied. Mean age at transplant was 50.4 +/- 12.4 years (73% white, 91% male). Patients were followed for an average of 28 +/- 12 months. Cellular rejection episodes were determined based on criteria established by the International Society of Heart and Lung Transplantation. Angiography and intravascular ultrasound (IVUS) were performed annually. Cytokine and growth factor polymorphism data were analyzed using the single-nucleotide polymorphism polymerase chain reaction (SNP PCR) approach. Patients who developed early CAV, documented by angiography, had increased frequency of the interferon-gamma high-producer phenotype, increased frequency of PDGF-286 AA, and decreased frequency of PDGF-1135 CC (p < 0.03, p < 0.03 and p = 0.01, respectively). Platelet-derived growth factor (PDGF) associations with early CAV were substantiated when vasculopathy was determined by IVUS. Additional associations were identified with vascular endothelial growth factor (VEGF) polymorphisms-1154 GG and -2578 AC (p < 0.03 and p = 0.01, respectively). Some of these associations translated to decreased patient survival, as indicated by Kaplan-Meier analysis. No significant association was identified between cytokine gene polymorphism (tumor necrosis factor-alpha, transforming growth factor-beta, interferon-gamma, interleukin-6 and interleukin-10) and acute cellular rejection episodes. These data suggest an association between PDGF and VEGF polymorphism and CAV. It is essential, however, due to the redundancy of the immune system and other confounding factors, that future studies be centrally conducted and include multiple programs, large cohorts of patients and properly chosen controls. Only then will we be able to identify the true association between cytokine and growth factor polymorphism and heart transplant outcome.

Effects of the alkaloid sinomenine on COX-2 activity and acute rejection in heart allografts: an experiment with rats

To evaluate the effects of the alkaloid sinomenine (SIN), a COX-2 inhibitor, on the acute rejection in heart allografts. Forty Wistar rats received the allograft of the hearts of 40 SD rats into the peritoneum. Then the recipients were randomly divided into 2 groups: SIN group, injected with SIN within 24 hours after the operation; and control group, injected with normal saline. The survival time was observed. The heartbeat was examined every day. The Wistar rats were killed 3 and 5 days after the operation respectively and the left ventricular tissues were taken to undergo pathological examination to detect the acute rejection and cell apoptosis. Immunochemistry and Western blotting were used to detect the COX-2 protein, and RT-RCR was used to detect the COX-2 mRNA. The mean numbers of apoptotic cardiomyocytes were determined with the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) technique. The survival time of the SIN group was 12.5 +/- 2.6 days, significantly longer than that of the control group (6.8 +/- 0.5 days, P = 0.001). Examination 3 and 5 days after the treatment of SIN, the extents of inflammatory reaction, endovasculites, myocardial edema, and cardiomyocyte damage in the allografts of the SIN group were all significantly less, the mean numbers of apoptotic cardiomyocyte was significantly smaller compared with the control group (all P < 0.05). At day 5, the levels of COX-2 protein and mRNA of the SIN group were both significantly lower than those of the control group. Inhibition of COX-2 prolongs the allograft survival and reduces the myocardial damage and inflammation during acute cardiac allograft rejection.

Tissue inhibitor of metalloproteinases 4 (TIMP4) is involved in inflammatory processes of human cardiovascular pathology

Tissue inhibitors of matrix metalloproteinases (TIMPs) comprise a family of four members, of which TIMP4 is characterized by being primarily restricted to cardiovascular structures. We demonstrate with immunohistochemical analysis of healthy human tissue that TIMP4 is present in medial smooth muscle cells and adventitial capillaries of arteries as well as in cardiomyocytes. Animal studies have suggested a role for TIMP4 in several inflammatory diseases and cardiovascular pathologies. We therefore examined whether TIMP4 is involved in human inflammatory cardiovascular disorders, specifically atherosclerosis, giant cell arteritis and chronic rejection of heart allografts. TIMP4 was most clearly visible in cardiovascular tissue areas populated by abundant inflammatory cells, mainly macrophages and CD3+ T cells. Using western blotting and immunocytochemistry, human blood derived lymphocytes, monocytes/macrophages and mast cells were shown to produce TIMP4. In advanced atherosclerotic lesions, TIMP4 was detected around necrotic lipid cores, whereas TIMP3 and caspase 3 resided within and around the core regions, indicating different roles for TIMP3 and TIMP4 in inflammation-induced apoptosis and in matrix turnover. In conclusion, the data demonstrate upregulation of TIMP4 in human cardiovascular disorders exhibiting inflammation, suggesting its future use as a novel systemic marker for vascular inflammation.

Allograft Rejection Requires STAT5a/b-Regulated Antiapoptotic Activity in T Cells but Not B Cells

STATs play key roles in immune function. We examined the role of STAT5a/b in allograft rejection. STAT5a/b-deficient mice showed a 4-fold increased survival time of heart allografts (p < 0.01). Unlike wild type, purified STAT5a/b-/- T cells transferred to Rag1-/- recipients failed to mediate heart allograft rejection until supplemented with STAT5a/b-/- B cells. In vitro, STAT5a/b-/- T cells did not proliferate in response to Con A or alloantigens but entered apoptosis within 48 h (95%). Activated STAT5a/b-/- T cells showed increased expression of proapoptotic (caspases, DNA repair genes, TNF/TNFR-associated factor family genes) and decreased antiapoptotic mRNAs in microarrays, while Western blots confirmed reduced antiapoptotic Bcl-2 and elevated proapoptotic Bax protein expression. Interestingly, at 24 h postactivation, STAT5a/b+/+ and STAT5a/b-/- T cells produced similar levels of IL-2, IL-4, IL-10, and IFN-gamma mRNA; ELISPOT assay showed an equivalent number of IL-4- and IFN-gamma-producing T cells in both STAT5a/b+/+ and STAT5a/b-/- splenic populations. Sera from STAT5a/b+/+ and STAT5a/b-/- rejectors had donor-specific IgM, IgG1, IgG2a, and IgG2b Ab, while STAT5a/b deficiency had no impact on B cell survival or proliferation in response to LPS. Compared with allografts from STAT5a/b+/+ recipients, heart allografts from STAT5a/b-/- recipients had markedly reduced infiltration by CD4 and CD8 T cells but increased infiltration by B cells and dense endothelial deposition of C4d, a marker of humoral rejection. Thus, activated STAT5a/b-/- T cells produce cytokines prior to entering apoptosis, thereby promoting differentiation of B cells yielding donor-specific IgM and IgG Ab that mediate allograft rejection.

Isolation and Characterization of Human Antigen-Specific TCRαβ+ CD4−CD8− Double Negative Regulatory T Cells.

Compelling evidence indicate that regulatory T (Treg) cells play an important role in the maintenance of immune tolerance to self and foreign antigens (Ag). Various subsets of T lymphocytes have been isolated in mice and humans that have the ability to down-regulate the proliferation of autoimmune effector cells. Recently, a novel subset of Ag-specific T-cell receptor (TCR)αβ+ CD4−CD8− (double negative, DN) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of anti-graft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naïve and Ag-experienced cells. Furthermore, analysis of T-cell receptor excision circles (TRECs) indicate that DN T cells are not recent thymic emigrants, but rather an expanded T-cell subset. MHC multimer staining revelaed a distinct population of DN T cells recognizing common MHC class I-restricted CMV and EBV antigens. DN T cells exhibited a strong proliferative response upon stimulation with allogeneic antigen presenting cells (APC) and secreted high amounts of IFN-γ but no IL-2, with some IL-5, and marginal levels of IL-4 and IL-10. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide-HLA-A2 complexes from APCs by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2-restricted self peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A-HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizeable population of peripheral DN Treg cells exists in humans that are able to suppress Ag-specific T cells. DN Treg cells may serve to limit clonal expansion of allo-Ag-specific T cells after transplantation.

Inhibition of Chronic Rejection and Development of Tolerogenic T Cells after ICOS-ICOSL and CD40-CD40L Co-stimulation Blockade

: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. : The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. : Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. : These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.

The Prognostic Validity of the Ventricular Evoked Response (VER) Signals in Cardiac Transplantation

Computerized heart allograft rejection monitoring (CHARM) used for non-invasive rejection monitoring in heart transplant recipients is based on the analysis of ventricular evoked response (VER) signals. The aim of this study was to evaluate the prognostic validity of the Tslew(C), a parameter calculated from the VER. During orthotopic heart transplantation, 2 unipolar fractally coated screw-in leads were implanted epimyocardially, one to the left and one to the right ventricle, and connected to a telemetric pacemaker. Recordings of intramyocardial electrograms were performed routinely at the hospital. Data processing was done, artifacts were eliminated, and trend curves of the patients were displayed. Tslew(C) was calculated from the tangent of VER and evaluated from 105 patients. The patients were divided into survivor and non-survivor groups. For statistical analysis, the 2-tailed t-test was performed. Patients in the non-survivors group compared with the control group showed a significantly lower Tslew(C) in the final follow-up (p < 0.001). The differences were even more impressive in patients with recorded intramyocardial electrograms 30 days before death or with a cardiac reason for death. Tests were performed to find an optimal prognostic threshold of the Tslew(C), and the value of 26 mV was finally found. Tslew(C) can function as prognostic factor after orthotopic heart transplantation. Further studies should be initiated to provide the prognostic threshold so that patients would not be forced to show up routinely every 4 weeks for follow-up visits. Patients would only have to be admitted to the hospital if the Tslew(C) were less than this prognostic threshold.

Baohuoside-1 inhibits activated T cell proliferation at G 1–S phase transition

Background The effect of baohuoside-1 (B1), a novel flavonoid, on cell proliferation and the cell cycle was evaluated in this study. Methods The antiproliferative properties of B1 were evaluated by proliferation assay. Western blotting and flow cytometric analysis were employed to investigate the expression of cyclins and cyclin-dependent kinase proteins. Results The major findings were (1) B1 effectively inhibited the cell proliferation activated by mitogenic antigen, with a 50% inhibitory concentration in low μM and in a dose- and time-dependent manner. (2) B1 resulted in G 1–S phase cells arrest. (3) It down-regulated the expression of cyclin A, D and p33 cyclin-dependent kinase-2 (p33cdk2) proteins. (4) B1 suppressed the growth of several tumor cell lines. (5) B1 prevented rat heart allograft rejection in vivo. Conclusions B1 immunosuppression of mitogen-activated T cell proliferation occurs in G 1–S transition. It may be associated with the expression of cyclin A, D and p33cdk2 proteins. B1 prevents rat heart allograft rejection in vivo. The mechanism of B1 is different from tacrolimus and sirolimus.

Analysis of the Role of Negative T Cell Costimulatory Pathways in CD4 and CD8 T Cell-Mediated Alloimmune Responses In Vivo

Negative costimulatory signals mediated via cell surface molecules such as CTLA-4 and programmed death 1 (PD-1) play a critical role in down-modulating immune responses and maintaining peripheral tolerance. However, their role in alloimmune responses remains unclear. This study examined the role of these inhibitory pathways in regulating CD28-dependent and CD28-independent CD4 and CD8 alloreactive T cells in vivo. CTLA-4 blockade accelerated graft rejection in C57BL/6 wild-type recipients and in a proportion of CD4(-/-) but not CD8(-/-) recipients of BALB/c hearts. The same treatment led to prompt rejection in CD28(-/-) and a smaller proportion of CD4(-/-)CD28(-/-) mice with no effect in CD8(-/-)CD28(-/-) recipients. These results indicate that the CTLA-4:B7 pathway provides a negative signal to alloreactive CD8(+) T cells, particularly in the presence of CD28 costimulation. In contrast, PD-1 blockade led to accelerated rejection of heart allografts only in CD28(-/-) and CD8(-/-)CD28(-/-) recipients. Interestingly, PD-1 ligand (PD-L1) blockade led to accelerated rejection in wild-type mice and in all recipients lacking CD28 costimulation. This effect was accompanied by expansion of IFN-gamma-producing alloreactive T cells and enhanced generation of effector T cells in rejecting allograft recipients. Thus, the PD-1:PD-L1 pathway down-regulates alloreactive CD4 T cells, particularly in the absence of CD28 costimulation. The differential effects of PD-1 vs PD-L1 blockade support the possible existence of a new receptor other than PD-1 for negative signaling through PD-L1. Furthermore, PD-1:PD-L1 pathway can regulate alloimmune responses independent of an intact CD28/CTLA-4:B7 pathway. Harnessing physiological mechanisms that regulate alloimmunity should lead to development of novel strategies to induce durable and reproducible transplantation tolerance.

Isolation and characterization of human antigen-specific TCRαβ+ CD4-CD8- double-negative regulatory T cells

AbstractDown-regulation of immune responses by regulatory T (Treg) cells is an important mechanism involved in the induction of tolerance to allo-antigens (Ags). Recently, a novel subset of Ag-specific T-cell receptor (TCR)αβ+ CD4-CD8- (double-negative [DN]) Treg cells has been found to be able to prevent the rejection of skin and heart allografts by specifically inhibiting the function of antigraft-specific CD8+ T cells. Here we demonstrate that peripheral DN Treg cells are present in humans, where they constitute about 1% of total CD3+ T cells, and consist of both naïve and Ag-experienced cells. Similar to murine DN Treg cells, human DN Treg cells are able to acquire peptide–HLA-A2 complexes from antigen-presenting cells by cell contact-dependent mechanisms. Furthermore, such acquired peptide-HLA complexes appear to be functionally active, in that CD8+ T cells specific for the HLA-A2–restricted self-peptide, Melan-A, became sensitive to apoptosis by neighboring DN T cells after acquisition of Melan-A–HLA-A2 complexes and revealed a reduced proliferative response. These results demonstrate for the first time that a sizable population of peripheral DN Treg cells, which are able to suppress Ag-specific T cells, exists in humans. DN Treg cells may serve to limit clonal expansion of allo-Ag–specific T cells after transplantation.