Mayuko Uehara

Donor exosomes rather than passenger leukocytes initiate alloreactive T cell responses after transplantation

Early T cell activation by recipient antigen-presenting cells cross-dressed with donor MHC antigens may initiate acute allograft rejection. Transplant ride along Despite the successful use of organ transplantation in the clinic, the mechanisms behind early rejection of transplants remain unclear. The passenger leukocyte theory suggests that graft leukocytes that express donor major histocompatibility complexes (MHCs) migrate to recipient lymphoid organs, where they activate host T cells. In multiple mouse models of transplantation, Marino et al. found few donor leukocytes in lymph nodes; rather high numbers of recipient antigen-presenting cells (APCs) were present that were cross-dressed with donor MHC. The donor MHC was derived from allogeneic exosomes, which could induce proinflammatory alloimmune responses even without transplantation. These data suggest that cross-dressed recipient APCs rather than passenger leukocytes may contribute to early T cell activation and transplant rejection. Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host’s lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation.

Brief treatment with a highly selective immunoproteasome inhibitor promotes long-term cardiac allograft acceptance in mice

Significance The potential of proteasome inhibitors to prevent transplant rejection and to treat other immune disorders is hindered by mechanism-based toxicity from inhibition of constitutive proteasomes. Here, we demonstrate that briefly, reversibly, and selectively inhibiting the immunoproteasome prolonged the survival of transplanted hearts in mice and allowed long-term survival when combined with single-dose CTLA4-Ig. Immunoproteasome inhibition noncytotoxically reduced T-cell proliferation and the numbers of effector T cells in the allograft and draining nodes while increasing T-cell expression of exhaustion markers. The immunoproteasome thus appears to play a role in suppressing induction of T-cell exhaustion. Selective inhibition of the immunoproteasome may be a potential treatment option for the management of transplant rejection. Constitutive proteasomes (c-20S) are ubiquitously expressed cellular proteases that degrade polyubiquitinated proteins and regulate cell functions. An isoform of proteasome, the immunoproteasome (i-20S), is highly expressed in human T cells, dendritic cells (DCs), and B cells, suggesting that it could be a potential target for inflammatory diseases, including those involving autoimmunity and alloimmunity. Here, we describe DPLG3, a rationally designed, noncovalent inhibitor of the immunoproteasome chymotryptic subunit β5i that has thousands-fold selectivity over constitutive β5c. DPLG3 suppressed cytokine release from blood mononuclear cells and the activation of DCs and T cells, diminished accumulation of effector T cells, promoted expression of exhaustion and coinhibitory markers on T cells, and synergized with CTLA4-Ig to promote long-term acceptance of cardiac allografts across a major histocompatibility barrier. These findings demonstrate the potential value of using brief posttransplant immunoproteasome inhibition to entrain a long-term response favorable to allograft survival as part of an immunomodulatory regimen that is neither broadly immunosuppressive nor toxic.

Targeted Delivery of Immunomodulators to Lymph Nodes

SUMMARY Active-targeted delivery to lymph nodes represents a major advance toward more effective treatment of immune-mediated disease. The MECA79 antibody recognizes peripheral node address in molecules expressed by high endothelial venules of lymph nodes. By mimicking lymphocyte trafficking to the lymph nodes, we have engineered MECA79-coated microparticles containing an immunosuppressive medication, tacrolimus. Following intravenous administration, MECA79-bearing particles showed marked accumulation in the draining lymph nodes of transplanted animals. Using an allograft heart transplant model, we show that targeted lymph node delivery of microparticles containing tacrolimus can prolong heart allograft survival with negligible changes in tacrolimus serum level. Using MECA79 conjugation, we have demonstrated targeted delivery of tacrolimus to the lymph nodes following systemic administration, with the capacity for immune modulation in vivo.

ABCB5 Identifies Immunoregulatory Dermal Cells

Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly defined immunomodulatory cell populations poses a barrier to this field. Here, we show that ATP-binding cassette member B5 (ABCB5) identifies dermal immunoregulatory cells (DIRCs) capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1). Purified Abcb5(+) DIRCs suppressed T cell proliferation, evaded immune rejection, homed to recipient immune tissues, and induced Tregs in vivo. In fully major-histocompatibility-complex-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5(+) DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy.

Salt Accelerates Allograft Rejection through Serum- and Glucocorticoid-Regulated Kinase-1–Dependent Inhibition of Regulatory T Cells

A high-salt diet (HSD) in humans is linked to a number of complications, including hypertension and cardiovascular events. Whether a HSD affects the immune response in transplantation is unknown. Using a murine transplantation model, we investigated the effect of NaCl on the alloimmune response in vitro and in vivo. Incremental NaCl concentrations in vitro augmented T cell proliferation in the settings of both polyclonal and allospecific stimulation. Feeding a HSD to C57BL/6 wild-type recipients of bm12 allografts led to accelerated cardiac allograft rejection, despite similar mean BP and serum sodium levels in HSD and normal salt diet (NSD) groups. The accelerated rejection was associated with a reduction in the proportion of CD4(+)Foxp3(+) regulatory T cells (Tregs) and a significant decrease in Treg proliferation, leading to an increased ratio of antigen-experienced CD4(+) T cells to Tregs in mice recipients of a HSD compared with mice recipients of a NSD. Because serum- and glucocorticoid-regulated kinase-1 (SGK1) has been proposed as a potential target of salt in immune cells, we fed a HSD to CD4(Cre)SGK1(fl/fl) B6-transplanted recipients and observed abrogation of the deleterious effect of a HSD in the absence of SGK1 on CD4(+) cells. In summary, we show that NaCl negatively affects the regulatory balance of T cells in transplantation and precipitates rejection in an SGK1-dependent manner.

Endovascular Stent-Graft Repair of Aortobronchial Fistulas

BACKGROUND Endovascular repair of the descending thoracic aorta has recently emerged as a feasible treatment option; however, little is known about its application for aortobronchial fistula (ABF). Experience with endovascular repair of the thoracic aorta and the outcome of patients with ABFs was reviewed to assess whether thoracic endovascular repair is a realistic option. METHODS From February 2001 to May 2011, 386 patients were successfully treated with endoluminal grafts to the distal arch or descending thoracic aorta. Among them, 26 patients with ABF underwent thoracic endovascular repair. These cases were reviewed and analyzed retrospectively. Follow-up was 100% complete (mean, 21 months). RESULTS The subjects included 26 patients (22 males, 85%; 4 females, 15%) with a median age of 71 years. Ten patients (38%) were diagnosed with atherosclerotic aneurysms, 13 (50%) had pseudoaneurysms associated with prior open surgical repair, 1 (4%) had rupture of dissecting aneurysm, and 2 (8%) had mycotic aneurysm. There were 4 (15%) in-hospital mortalities, in which the causes included bleeding owing to recurrence of hemoptysis (n=3, 11%) and multiple organ failure (n=1, 4%). None sustained postoperative stroke or paraplegia. During follow-up, ABFs recurred in 4 patients; of these, endograft explantation occurred in 3 patients and 1 patient required additional open surgery. No hospital mortality resulted among the 4 patients with ABF recurrence. CONCLUSIONS Endovascular management of ABFs appears to be safe and well tolerated with minimal risk, even in surgically high-risk patients. Endovascular stent-graft repair is likely the first choice for ABF presenting as hemoptysis.

Evaluation of gastroepiploic arterial grafts to right coronary artery using transit-time flow measurement

OBJECTIVES The objective of this study was to analyse the relationship between the intraoperative transit-time flow measurement (TTFM) parameter values and the postoperative angiographic results of gastroepiploic arterial (GEA) grafts to the right coronary artery (RCA). We investigated whether the intraoperative TTFM parameter values are reliable indicators of early patency in GEA grafts to the RCA. METHODS Patients undergoing off-pump coronary artery bypass surgery with GEA grafts were included in this study. Eighty-three GEA grafts were individually anastomosed and examined by angiography 1 week after surgery. The quality of each graft was graded using FitzGibbon grading (Study 1) and graft-flow grading (Study 2). RESULTS Study 1: Seventy-two grafts were determined as Grade A and 11 as Grades B or O. There were no significant differences in the average of mean graft flow (MGF), pulsatility index or diastolic filling percentage between Grade A and Grades B or O grafts. Study 2: Sixty-two grafts were graded as good-graft dominant, 16 as bidirectional and 5 as occlusion including string. The average of the MGF, pulsatility index and diastolic filling percentage in the grafts graded as bidirectional and occlusion including string were not significantly different from those of grafts graded as good-graft dominant. CONCLUSIONS Previously reported cut-off values for intraoperative TTFM parameters could not be adapted for the early patency of GEA grafts to the RCA. However, the smoothness of the graft-flow curve may be a reliable predictor of postoperative graft patency.

Serine Protease Inhibitor‐6 Differentially Affects the Survival of Effector and Memory Alloreactive CD8‐T Cells

The clonal expansion of effector T cells and subsequent generation of memory T cells are critical in determining the outcome of transplantation. While cytotoxic T lymphocytes induce direct cytolysis of target cells through secretion of Granzyme‐B (GrB), they also express cytoplasmic serine protease inhibitor‐6 (Spi6) to protect themselves from GrB that has leaked from granules. Here, we studied the role of GrB/Spi6 axis in determining clonal expansion of alloreactive CD8‐T cells and subsequent generation of memory CD8‐T cells in transplantation. CD8‐T cells from Spi6−/− mice underwent more GrB mediated apoptosis upon alloantigen stimulation in vitro and in vivo following adoptive transfer into an allogeneic host. Interestingly, while OT1.Spi6−/− CD8 T cells showed significantly lower clonal expansion following skin transplants from OVA mice, there was no difference in the size of the effector memory CD8‐T cells long after transplantation. Furthermore, lack of Spi6 resulted in a decrease of short‐lived‐effector‐CD8‐cells but did not impact the pool of memory‐precursor‐effector‐CD8‐cells. Similar results were found in heart transplant models. Our findings suggest that the final alloreactive CD8‐memory‐pool‐size is independent from the initial clonal‐proliferation as memory precursors express low levels of GrB and therefore are independent of Spi6 for survival. These data advance our understanding of memory T cells generation in transplantation and provide basis for Spi6 based strategies to target effector T cells.

Novel Application of Localized Nanodelivery of Anti–Interleukin-6 Protects Organ Transplant From Ischemia–Reperfusion Injuries

Ischemia–reperfusion injury (IRI) evokes intragraft inflammatory responses, which markedly augment alloimmune responses against the graft. Understanding the mechanisms underlying these responses is fundamental to develop therapeutic regimens to prevent/ameliorate organ IRI. Here, we demonstrate that IRI results in a marked increase in mitochondrial damage and autophagy in dendritic cells (DCs). While autophagy is a survival mechanism for ischemic DCs, it also augments their production of interleukin (IL)‐6. Allograft‐derived dendritic cells (ADDCs) lacking autophagy‐related gene 5 (Atg5) showed higher death rates posttransplantation. Transplanted ischemic hearts from CD11cCre/Atg5 conditional knockout mice showed marked reduction in intragraft expression of IL‐6 compared with controls. To antagonize the effect of IL‐6 locally in the heart, we synthesized novel anti–IL‐6 nanoparticles with capacity for controlled release of anti–IL‐6 over time. Compared with systemic delivery of anti–IL‐6, localized delivery of anti–IL‐6 significantly reduced chronic rejection with a markedly lower amount administered. Despite improved allograft histology, there were no changes to splenic T cell populations, illustrating the importance of local IL‐6 in driving chronic rejection after IRI. These data carry potential clinical significance by identifying an innovative, targeted strategy to manipulate organs before transplantation to diminish inflammation, leading to improved long‐term outcomes.

Surgical resection of primary cardiac lymphoma presenting as a huge mass: report of two cases.

Primary cardiac lymphoma (PCL) is a rare malignancy that is sometimes diagnosed at later stages because it is not associated with specific symptoms. Although chemotherapy is the standard for treating PCL, the value of surgical resection is controversial. We describe two patients who were treated by surgical resection and chemotherapy. Case 1 is a 37-year-old man with a history of acute lymphocytic leukemia and shortness of breath, and Case 2 is a 70-year-old woman with general fatigue. Both of them were presented at hospital. In Case 2, arrhythmic syncope occurred and direct current cardioversion was performed. Echocardiography showed a massive tumor in the right atrium and disturbed hemodynamics in both cases. The restricted venous return was alleviated by emergency surgery. The pathology report indicated primary cardiac lymphoma that was regressed by post-operative chemotherapy. A massive PCL should be surgically resected to prevent sudden death.