Long-term Heart Allograft Survival Research Articles

Fibroblastic reticular cells orchestrate long-term graft survival following recipient treatment with CD40 ligand–targeted costimulatory blockade

Fibroblastic reticular cells (FRCs) maintain the architecture of secondary lymphoid organs, which optimize interactions between antigen-presenting dendritic cells and reactive naive T cells. In this issue of the JCI, Zhao, Jung, and colleagues investigated CD4+FoxP3+ regulatory T cell development and long-term heart allograft survival in recipients treated with peritransplant costimulatory blockade to inhibit CD40/CD40 ligand (CD40L) signaling. Treatment with an anti-CD40L monoclonal antibody (mAb) increased the lymph node (LN) population of Madcam1+ FRCs and altered their transcription profile to express immunoregulatory mediators. Administration of nanoparticles, containing the anti-CD40L mAb and a targeting antibody against high endothelial venules, delivered the treatment into LNs of allograft recipients. Direct LN delivery of the costimulatory blockade allowed decreased dosing and increased the efficacy in extending graft survival. The results provide insights into mechanisms by which FRCs can promote donor-reactive tolerance, and establish a strategy for administering costimulation-blocking reagents that circumvent systemic effects and improve allograft outcomes.

Genetically targeting the BATF family transcription factors BATF and BATF3 in the mouse abrogates effector T cell activities and enables long-term heart allograft survival

T cells must be activated and become effectors first before executing allograft rejection, a process that is regulated by diverse signals and transcription factors. In this study, we studied the basic leucine zipper ATF-like transcription factor (BATF) family members in regulating T cell activities in a heart transplant model and found that mice deficient for both BATF and BATF3 (Batf-/- Batf3-/- mice) spontaneously accept the heart allografts long-term without tolerizing therapies. Similarly, adoptive transfer of wild type T cells into Rag1-/- hosts induced prompt rejection of heart and skin allografts, whereas the Batf-/- Batf3-/- T cells failed to do so. Analyses of graft-infiltrating cells showed that Batf-/- Batf3-/- T cells infiltrate the graft but fail to acquire an effector phenotype (CD44high KLRG1+ ). Co-transfer experiments in a T cell receptor transgenic TEa model revealed that the Batf-/- Batf3-/- T cells fail to expand in vivo, retain a quiescent phenotype (CD62L+ CD127+ ), and unable to produce effector cytokines to alloantigen stimulation, which contrasted sharply to that of wild type T cells. Together, our data demonstrate that the BATF and BATF3 are critical regulators of T effector functions, thus making them attractive targets for therapeutic interventions in transplant settings.

A short course of tofacitinib sustains the immunoregulatory effect of CTLA4-Ig in the presence of inflammatory cytokines and promotes long-term survival of murine cardiac allografts

Costimulation blockade-based regimens are a promising strategy for management of transplant recipients. However, maintenance immunosuppression via CTLA4-Ig monotherapy is characterized by high frequency of rejection episodes. Recent evidence suggests that inflammatory cytokines contribute to alloreactive T cell activation in a CD28-independent manner, a reasonable contributor to the limited efficacy of CTLA4-Ig. In this study, we investigated the possible synergism of a combined short-term inhibition of cytokine signaling and CD28 engagement on the modulation of rejection. Our results demonstrate that the JAK/STAT inhibitor tofacitinib restored the immunomodulatory effect of CTLA4-Ig on mouse alloreactive T cells in the presence of inflammatory cytokines. Tofacitinib exposure conferred dendritic cells with a tolerogenic phenotype reducing their cytokine secretion and costimulatory molecules expression. JAK inhibition also directly affected T cell activation. In vivo, the combination of CTLA4-Ig and tofacitinib induced long-term survival of heart allografts and, importantly, it was equally effective when using grafts subjected to prolonged ischemia. Transplant survival correlated with a reduction in effector T cells and intragraft accumulation of regulatory T cells. Collectively, our studies demonstrate a powerful synergism between CTLA4-Ig and tofacitinib and suggest their combined use is a promising strategy for improved management of transplanted patients.

ACTH treatment promotes murine cardiac allograft acceptance.

Heart transplantation is the optimal therapy for patients with end-stage heart disease, but its long-term outcome remains inadequate. Recent studies have highlighted the importance of the melanocortin receptors (MCRs) in inflammation, but how MCRs regulate the balance between alloreactive T cells and Tregs, and whether they impact chronic heart transplant rejection, is unknown. Here, we found that Tregs express MC2R, and MC2R expression was highest among all MCRs by Tregs. Our data indicate that adrenocorticotropic hormone (ACTH), the sole ligand for MC2R, promoted the formation of Tregs by increasing the expression of IL-2Rα (CD25) in CD4+ T cells and activation of STAT5 in CD4+CD25+ T cells. ACTH treatment also improved the survival of heart allografts and increased the formation of Tregs in CD28KO mice. ACTH treatment synergized with the tolerogenic effect of CTLA-4–Ig, resulting in long-term survival of heart allografts and an increase in intragraft Tregs. ACTH administration also demonstrated higher prolongation of heart allograft survival in transgenic mouse recipients with both complete KO and conditional KO of PI3Kγ in T cells. Finally, ACTH treatment reduced chronic rejection markedly. These data demonstrate that ACTH treatment improved heart transplant outcomes, and this effect correlated with an increase in Tregs.

Immunotherapeutic strategy based on anti-OX40L and low dose of IL-2 to prolong graft survival in sensitized mice by inducing the generation of CD4+ and CD8+ Tregs

Alloreactive memory cells play a critical role after a second transplant and are difficult to suppress. This study investigated the effect of an immunotherapeutic strategy that combines anti-OX40L, rapamycin (Rapa), and a low dose of IL-2 in a memory cell-based adoptive model. In this model, the median survival time (MST) of the grafts of the combined treatment group was significantly extended compared to that of the control group and other treatment groups. A similar effect was observed regarding a reduction in memory T cells (Tm) and inflammatory cytokines production. Also, the percentages of Foxp3+ regulatory T cells (Tregs) increased in our model. In addition, mounting evidence has shown CD8+CD122+ T cells are also Tregs. We found that the group of CD8+CD122+PD1+ T cells was markedly increased in the combined treatment group, especially in the graft. We further demonstrated that CD8+CD122+PD1+ T cells could suppress activated T cells. Our data suggest that anti-OX40L combined with Rapa and a low dose of IL-2 can suppress Tm, modulate CD4 and CD8 Tregs, and induce long-term heart allograft survival in sensitized mice.

T follicular helper and memory cell responses and the mTOR pathway in murine heart transplantation

The mammalian target of rapamycin (mTOR) inhibitors are valuable immunosuppressants in clinical transplantation; however, the mTOR regulation of allogeneic T-cell responses is not fully understood yet. Therefore, the objective of this study is to investigate the effects of T-cell-specific mTOR deletion on the allogeneic T-cell responses and heart transplant survival. BALB/c heart allografts, with or without BALB/c skin sensitization, were transplanted in the wild-type C57BL/6, Mtorfl/flCd4-Cre, Stat3fl/flCd4-Cre, and Mtorfl/flStat3fl/flCd4-Cre mice. Graft survival and histology, as well as T-cell frequencies and phenotypes, were evaluated after transplantation. In the absence of donor skin sensitization, long-term heart allograft survival was achieved in the Mtorfl/flCd4-Cre recipients, which was associated with significantly decreased frequencies of CD62L-CD44+ effector T cells and BCL-6+CXCR5+ T follicular helper (Tfh) cells in the periphery. Long-term heart allograft survival was also achieved in the donor skin-sensitized Mtorfl/flStat3fl/flCd4-Cre mice, whereas the heart allograft survival was prolonged in the donor skin-sensitized Mtorfl/flCd4-Cre and Stat3fl/flCd4-Cre mice. mTOR is required for Tfh cell response in murine heart transplantation. T-cell-specific deletion of both mTOR and Stat3 abrogates the memory response to heart transplants. These findings help us to better understand the molecular mechanisms underlying the T cell immunity to transplanted organs.

Characterization of Early Indicators of Cardiac Allograft Vasculopathy Lesions in a Rat Model Using Non-Invasive Cellular MR

Background: Cardiac allograft vasculopathy (CAV) remains a major obstacle to long-term heart allograft survival. A number of studies show that immune mechanisms involved in CAV. Using non-invasive cellular MRI (CMRI) to explore indicators of CAV lesions and characterize its development could provide new insights into the target immune cells that are responsible for the progression of CAV and reveal early markers of the disease before irreversible changes occur. Methods and Results: Rat heart transplant allografts (n = 88) and isografts (n = 22) were employed in this study. CMRI at 4.7-Tesla revealed a few hypointense foci in the graft early on during CAV with more regions becoming involved as the disease progressed. The areas with hypointensity corresponded with infiltrating ED1+ cells labeled with micrometer-sized superparamagnetic iron oxide (MPIO), confirmed by MR microscopy (MRM) at 11.7-Tesla and pathology. MRI abnormalities of the transplanted hearts correlate well with the histopathological findings. MPIO-labeled cells counted by a computer algorithm that analyzed 3D MRM volumes exhibited a strong correlation with those counted by immunohistochemical inspection of ED1+ cell infiltration (R2 = 0.8075). Histology found that in the early phases of CAV, lesions were focal and mostly began in the adventitia. Immunohistochemistry indicated that the infiltrates were mostly ED1+ cells and that their density was significantly correlated with the severity of CAV (p < 0.05). Conclusions: This study illustrates the feasibility of monitoring MPIO-labeled ED1+ cells as an early indicator for CAV before vessel wall changes using non-invasive CMRI, which provides information on the entire heart.

Role of Macrophage Subsets in Chronic Allograft Rejection and Novel Strategies to Target Them.

chronic rejection or transplant vasculopathy is a major problem in the clinic hindering long-term transplant survival. Chronic rejection primarily targets the graft vasculature, but the exact mechanism underlying the slow and progressive vasculature damage in transplanted organs remains poorly defined. In the present study, we addressed the role of macrophages in chronic allograft rejection in a mouse heart transplant model. We found that treatment of B6 recipients with a single dose of CTLA-4Ig prolonged the survival of Balb/c heart allografts(MST=35d) as compared to untreated controls (MST=8 days). Histologically, the CTLA-4Ig treated grafts showed signs of extensive chronic rejection characterized by heavy leukocyte infiltration and prominent neointima formation. Phenotypically, macrophages were a key infiltrating cell type in the vascular lesions in the grafts. Further studies revealed a M2 (F4/80+CD206+) phenotype for grafting infiltrating macrophages. An increased number of M2 macrophages were also detected in the spleen of recipient mice that experienced chronic rejection. We polarized macrophages in vitro to M1 and M2 subsets and systemically screened for differences and specific pathways that could be targeted for inhibition of M1 and M2 macrophages. We showed that when macrophages were polarized to a M2 phenotype, they preferentially expressedP2x7 receptor, a member of P2x family receptors that sense metabolites. We also found that oxidized ATP (P2x7R inhibitor)could effectively inhibit M2 induction in vitro. Importantly, in the heart transplant model, CTLA-4Ig plus oxidized ATP induced long-term heart allograft survival(MST>100 days), which was associated with markedly reduced neointima generation in the grafts, and prevention of chronic rejection was associated with significant inhibition of M2 macrophages in situ. Our studies identified a previously unknown mechanism of transplant vasculopathy and pinpointed as a molecular target in the inhibition of infiltrating M2 macrophages and chronic rejection.

Identification of Novel Mechanisms of Tolerance Resistance Induced By OX40.

Induction of donor specific tolerance remains an important goal in transplantation and to identify and overcome barriers to tolerance will significantly improve transplant outcomes in the clinic. In the present study, we examined the effector programs that evade tolerance induction by costimulatory blockade in a mouse heart transplant model. Blocking CD40-CD40L costimulation using an anti-CD40L monoclonal antibody (MR1) induced prolonged survival of C57BL/6 heart allografts in BALB/c recipients (MST>60 days). However, we found that MR1 completely failed to prolong survival of heart allografts that express the OX40L transgene (OX40Ltg grafts) (MST=9 days), indicating that OX40 signaling evades tolerance induction by costimulatory blockade. Histologically, rejection of OX40Ltg heart allografts was associated with extensive lymphocyte infiltration, and a significant proportion of graft infiltrating T cells produced IL-9 (40%), while few of them produced IL-4 or IFN-γ, suggesting that OX40 may drive a new mechanism of tolerance resistance. In an in vitro T cell polarization assay, we showed that OX40 engagement inhibited the induction of TH17 cells by TGF-β/IL-6 and that of inducible Treg cells by TGF- β/IL-2. Instead, OX40 diverted CD4+ T cells to IL-9 producing TH9 cells. Mechanistically, OX40 signaling mediated the induction of TH9 cells by triggering noncanonical NF-κB activation in activated T cells. In an in vivo CFSE model in which CD4+ T cells were stimulated by alloantigens, blocking CD40-CD40L costimulation induced TH9 cells only in the presence of OX40 signaling. These data suggest that induction of TH9 cells may constitute a novel mechanism of tolerance resistance. Interestingly, blocking OX40 costimulation induced long-term heart allograft survival (MST>100 days) and markedly reduced transplant vasculopathy (chronic allograft rejection) in CD40L-/- recipient mice. In conclusion, we identified that OX40 dramatically induces the generation of TH9 cells both in vitro and in vivo, and TH9 cells are closely involved in tolerance resistance. These findings may lead to the development of new therapeutics to promote graft tolerance in transplantation.

Adoptive transfer of DNT cells induces long-term cardiac allograft survival and augments recipient CD4+Foxp3+ Treg cell accumulation

Regulatory T (Treg) cells play an important role in the regulation of immune responses but whether Treg will induce tolerance in transplant recipients in the clinic remains unknown. Our previous studies have shown that TCRαβ+CD3+CD4−CD8−NK1.1− (double negative, DN) T cells suppress T cell responses and prolong allograft survival in a single locus MHC-mismatched mouse model. In this study, we investigated the role of DNT cells in a more robust, fully MHC-mismatched BALB/c to C57BL/6 transplantation model, which may be more clinically relevant. Adoptive transfer of DNT cells in combination with short-term rapamycin treatment (days 1–9) induced long-term heart allograft survival (101±31 vs. 39±13days rapamycin alone, p<0.01). Furthermore adoptive transfer DNT cells augmented CD4+Foxp3+ Treg cells accumulation in transplant recipients while depletion of CD4+ Treg cells by anti-CD25 inhibited the effect of DNT cells on long-term graft survival (48±12days vs. 101±31days, p<0.001). In conclusion, DNT cells combined with short-term immunosuppression can prolong allograft survival, which may be through the accumulation of CD4+Foxp3+ Treg cells in the recipient. Our result suggests that allograft tolerance may require the co-existence of different type Treg cell phenotypes which are affected by current immunosuppression.

HIF-1α accumulation upregulates MICA and MICB expression on human cardiomyocytes and enhances NK cell cytotoxicity during hypoxia-reoxygenation

Aims To investigate whether hypoxia-inducible factor (HIF) 1α and cyclosporin A (CsA) can regulate MICA/B expression and affect NK cytotoxicity during ischemia/reperfusion (I/R) injury. Main methods We generated an HIF-1αΔODD-expressing adenovirus which can functionally and steadily express HIF-1α during normoxia and transfected human cardiomyocytes (HCMs) to investigate whether HIF-1α, as a single factor, can upregulate MICA/B expression. Alternatively, HCMs were treated with HIF-1α RNAi or CsA, and then cultured under hypoxia/reoxygenation (H/R) condition to simulate I/R injury in vitro. Cells were collected at different time points and used for studies of gene expression and NK cytotoxicity. Key findings Expression of MICA/B in HCMs is upregulated through HIF-1α overexpression in normoxia, and inhibited by HIF-1α RNAi treatment during hypoxia-reoxygenation (H/R). NK cytotoxicity towards HCMs shows a positive correlation with HIF-1α expression. Moreover, CsA can inhibit HIF-1α and MICB expression but upregulates MICA expression during H/R. Significance These findings suggest that proper control of HIF-1α expression via CsA dose may be a potential therapeutic approach for avoiding MIC expression, and improving the function and long-term survival of heart allografts.

Endoplasmic Reticulum Stress-Mediated Apoptosis Involved in Indirect Recognition Pathway Blockade Induces Long-Term Heart Allograft Survival

Implementation of dendritic cell- (DC-) based therapies in organ transplantation can reduce dependency on nonspecific immunosuppression. Despite extensive research, mechanisms of equipped DCs inducing transplant tolerance remain incomplete. Here, we applied RNA interference technique to inhibit CD80 and CD86 expression in host bone marrow-derived DCs. This approach could specifically and effectively knock down CD80 and CD86 expression. T cells primed by these DCs inhibited allogeneic responses. Administration of recipient DCs loaded with alloantigen after CD80 and CD86 blockade prolonged cardiac allograft survival. We also found a higher percentage of apoptotic T cells in lymph tissues and grafts than that detected in control group. In addition, these T cells expressed high expression of GRP78 than controls, indicating activation of unfolded protein responses. Upregulation of CHOP expression among these cells suggested that the endoplasmic reticulum stress (ERS) response switched to a proapoptotic response. Our results indicated that ERS-induced apoptosis may be involved in allogeneic T-cell apoptosis, and the ERS-mediated apoptosis pathway may be a novel target in clinical prevention and therapy of allograft rejection.

Alloantibodies in heart transplantation

The presence of complement fixing anti–human leukocyte antigen (HLA) antibodies in the circulation of organ transplant recipients may result in heart allograft rejection. Here, we assessed the clinical impact of pre- and post-transplantation allosensitization on long-term survival of heart allografts. Sequential samples of sera from heart allograft recipients were screened pretransplantation for panel reactive antibodies using the complement-dependent cytotoxicity test. Patients were monitored post-transplantation for donor specific anti-HLA class I and class II antibodies. Kaplan-Meier graft survival plots were generated to analyze the effect of anti-HLA antibodies on transplantation outcomes. Statistical analysis showed that the post-transplantation development of alloantibodies was a significant risk factor that was associated with low long-term survival rates; in contrast, recipients' gender, age, previous transplantations, and degree of HLA matching with the donor had no effect on long-term survival. The presence in pretransplantation sera of antibodies against more than 10% of the HLA reference panel (PRA >10%) was associated with AMR and with a relatively lower rate of graft survival after 1 year but did not affect 10-year survival. The present data underline the importance of monitoring the development of anti-HLA antibodies as a tool for early diagnosis and treatment of AMR.

A Blocking Anti-CD28-Specific Antibody Induces Long-Term Heart Allograft Survival by Suppression of the PKCθ-JNK Signal Pathway

A Blocking Anti-CD28-Specific Antibody Induces Long-Term Heart Allograft Survival by Suppression of the PKCθ-JNK Signal Pathway

A Blocking Anti-CD28-Specific Antibody Induces Long-Term Heart Allograft Survival by Suppression of the PKCθ-JNK Signal Pathway

A Blocking Anti-CD28-Specific Antibody Induces Long-Term Heart Allograft Survival by Suppression of the PKCθ-JNK Signal Pathway

A Blocking Anti-CD28-Specific Antibody Induces Long-Term Heart Allograft Survival by Suppression of the PKCθ-JNK Signal Pathway

This study investigated the effects of a blocking anti-CD28 antibody (Anti-CD28-PV1-IgG3) in vitro and in vivo. Anti-CD28-PV1-IgG3, a hamster-mouse chimeric antibody against murine CD28, which does not provide CD28-positive signaling during TCR-driven T cell activation, enabled long-term survival of heart allografts across a complete mismatch of the MHC in rats. Among the T cell signaling proteins tested in the spleens from recipients, we found that recipients treated with anti-CD28-PV1-IgG3 exhibited suppression of alloantigen-initiated proximal TCR signaling events, including Lck, Zap70, Vav, and PI3K expression, and their PKC theta- and JNK-regulated expression/activation. This leads to attenuation of intragraft T cell infiltration and expression of T cell effector molecules. These results indicate that targeting the CD28 receptor with a blocking antibody leads to long-term allograft survival by reducing activation of alloantigen-mediated key signaling events in T cells that might be crucial for full T cell activation.

Promotion of Long-Term Heart Allograft Survival by Combination of Mobilized Donor Plasmacytoid Dendritic Cells and Anti-CD154 Monoclonal Antibody

Infusion of donor immature dendritic cells (DC) can significantly prolong survival of organ allografts, and this is believed to be due to antigen recognition by T cells in the absence of co-stimulation. In this study we report that a single pre-operative infusion of donor-mobilized immature plasmacytoid dendritic cells (pDCs) is superior to that of other DC sub-sets in suppressing allograft rejection. The combination of pDC infusion with injection of anti-CD154 monoclonal antibody further inhibited graft rejection and, in 50% of the mice, led to indefinite graft survival. This finding suggests a role for the plasmacytoid DC sub-set in facilitating organ transplant survival and also in the treatment of autoimmune disorders.

Induction of Donor‐Specific Allograft Tolerance by Short‐Term Treatment with LF15‐0195 After Transplantation. Evidence for a Direct Effect on T‐Cell Differentiation

A 20-day treatment with LF15-0195, a deoxyspergualine analog, induced long-term heart allograft survival in the rat without signs of chronic rejection. LF15-0195-treated recipients did not develop an anti-donor alloantibody response. Analysis of graft-infiltrating cells, IL10, TNFalpha, IFNgamma mRNA and iNOS protein expression in allografts, 5 days after transplantation, showed that they were markedly decreased in allografts from LF15-0195-treated recipients compared with allografts from untreated recipients. Surprisingly, spleen T cells from LF15-0195 recipients, 5days after grafting, were able to proliferate strongly in vitro, when stimulated with donor cells, but had reduced mRNA expression for IFNy compared with spleen T cells from untreated graft recipients. Furthermore, when T cells from naive animals were stimulated in vitro, using anti-CD3 and anti-CD28, LF15-0195 also increased T-cell proliferation in a dose-dependent fashion: however, these cells expressed less of the Th1 -related cytokines, IFNgamma and IL2, compared with untreated cells, suggesting that LF15-0195 could act on T-cell differentiation. In conclusion, we show here that a short-term treatment with LF15-0195 induced long-term allograft tolerance, decreasing the in situ anti-donor response, and we illustrate evidence for the development of regulatory mechanisms.

Prolonged blockade of CD40-CD40 ligand interactions by gene transfer of CD40Ig results in long-term heart allograft survival and donor-specific hyporesponsiveness, but does not prevent chronic rejection.

Previous work on blockade of CD40-CD40 ligand interaction in mice and primates with anti-CD40 ligand mAbs has resulted in a moderate prolongation of allograft survival without the development of true allograft tolerance. In this study, we show in rats that adenovirus-mediated gene transfer of CD40Ig sequences into the graft resulted in prolonged (>200 days) expression of CD40Ig and in long-term (>300 days) survival. Recipients expressing CD40Ig displayed strongly (>90%) inhibited mixed leukocyte reactions and alloantibody production at early (days 5 and 17) and late time points (>100 day) after transplantation, but showed limited inhibition of leukocyte infiltration and cytokine production as evaluated by immunohistology at early time points (day 5). Recipients of long-surviving hearts showed donor-specific hyporesponsiveness since acceptance of second cardiac allografts was donor specific. Nevertheless, long-term allografts (>100 days) displayed signs of chronic rejection vasculopathy. Occluded vessels showed leukocyte infiltration, mainly composed of CD4(+) and CD8(+) cells, macrophages, and mast cells. These recipients also showed antidonor CTL activity. Recipients expressing CD40Ig did not show nonspecific immunosuppression, as they were able to mount anticognate immune responses that were partially inhibited at early time points and were normal thereafter. We conclude that gene transfer-mediated expression of CD40Ig resulted in a highly efficient inhibition of acute heart allograft rejection in rats. This treatment induced donor-specific inhibition of certain alloreactive mechanisms in the short-, but not the long-term, which resulted in long-term survival of allografts concomitant with the development of chronic rejection.

A short-term combination therapy with cyclosporine and rapamycin or leflunomide induces long-term heart allograft survival in a strongly immunogenic strain combination in rats

Abstract Synergism between cyclosporine (CsA) and rapamycin (RAPA) or leflunomide (LF) was studied in a strongly immunogenic cardiac allograft model in rats. In the absence of immunosuppression, PVG recipients rejected WKAH heart grafts after a mean survival time (MST) of 5.2 ± 1.1 days. A dose of 7.5 mg/kg per day CsA did not prolong graft survival (MST 5.6 ± 1.2 days). CsA given at 10 mg/ kg per day for 30 days extended MST of the grafts to 48 ± 7 days. A short course of combination therapy consisting of adding a non-therapeutic dose of RAPA or a subthera-peutic dose of LF to a 1-month course of CsA resulted in permanent graft survival. These data suggest that RAPA and LF synergize with CsA enabling not only the lowering of the dose of CsA, but also inducing transplantation tolerance.