Use of Extracorporeal Membrane Oxygenation for Primary Graft Dysfunction After Cardiac Transplantation: Results of an A Priori Ventless Approach.

Primary graft dysfunction (PGD) after cardiac transplantation is a devastating complication with increasing frequency lately in the setting of donation after circulatory death (DCD). Severe PGD is commonly treated with extracorporeal membrane oxygenation (ECMO) using central or peripheral cannulation. We retrospectively reviewed the outcomes of PGD after cardiac transplantation requiring ECMO support at our center from 2015 to 2020, focused on our now preferential approach using peripheral cannulation without a priori venting. During the study period, 255 patients underwent heart transplantation at our center and 26 (10.2%) of them required ECMO for PGD. Of 24 patients cannulated peripherally 19 (79%) were alive at 30 days and 17 (71%) 1 year after transplant; two additional patients underwent central ECMO cannulation due to unfavorable size of femoral vessels and concern for limb ischemia. Successful decannulation with full graft function recovery occurred in 22 of 24 (92%) patients cannulated peripherally. Six of them had an indwelling intra-aortic balloon pump placed before the transplantation. None of the other 18 patients received a ventricular vent. In conclusion, the use of an a priori peripheral and ventless ECMO approach in patients with PGD after heart transplant is an effective strategy associated with high rates of graft recovery and survival.

Lactate Dehydrogenase and Hemorrhagic Stroke During Extracorporeal Membrane Oxygenation for COVID-19.

PURPOSE: Hemorrhagic stroke (HS) is a devastating complication during extracorporeal membrane oxygenation (ECMO) but markers of risk stratification during COVID-19 are unknown. Lactate dehydrogenase (LDH) is a readily available biomarker of cell injury and permeability. We sought to determine whether an elevated LDH before ECMO placement is related to the occurrence of HS during ECMO for COVID-19. METHODS: Adult patients with COVID-19 requiring ECMO between March 2020 and February 2022 were included. LDH values prior to ECMO placement were captured. Patients were categorized into high (> 750 U/L) or low (≤ 750 U/L) LDH groups. Multivariable regression modeling was used to determine the association between LDH and HS during ECMO. RESULTS: There were 520 patients that underwent ECMO placement in 17 centers and 384 had an available LDH. Of whom, 122 (32%) had a high LDH. The overall incidence of HS was 10.9%, and patients with high LDH had a higher incidence of HS than those with low LDH level (17% vs 8%, p = 0.007). At 100 days, the probability of a HS was 40% in the high LDH group and 23% in those with a low LDH, p = 0.002. After adjustment for clinical covariates, high LDH remained associated with subsequent HS (aHR: 2.64, 95% CI 1.39-4.92). Findings were similar when restricting to patients supported by venovenous ECMO only. CONCLUSION: Elevated LDH prior to ECMO cannulation is associated with a higher incidence of HS during device support. LDH can risk stratify cases for impending cerebral bleeding during ECMO.

Characterization of CD41+ cells in the lymph node.

Lymph nodes (LNs) are the critical sites of immunity, and the stromal cells of LNs are crucial to their function. Our understanding of the stromal compartment of the LN has deepened recently with the characterization of nontraditional stromal cells. CD41 (integrin αIIb) is known to be expressed by platelets and hematolymphoid cells. We identified two distinct populations of CD41+Lyve1+ and CD41+Lyve1- cells in the LNs. CD41+Lyve1- cells appear in the LN mostly at the later stages of the lives of mice. We identified CD41+ cells in human LNs as well. We demonstrated that murine CD41+ cells express mesodermal markers, such as Sca-1, CD105 and CD29, but lack platelet markers. We did not observe the presence of platelets around the HEVs or within proximity to fibroblastic reticular cells of the LN. Examination of thoracic duct lymph fluid showed the presence of CD41+Lyve1- cells, suggesting that these cells recirculate throughout the body. FTY720 reduced their trafficking to lymph fluid, suggesting that their egress is controlled by the S1P1 pathway. CD41+Lyve1- cells of the LNs were sensitive to radiation, suggestive of their replicative nature. Single cell RNA sequencing data showed that the CD41+ cell population in naïve mouse LNs expressed largely stromal cell markers. Further studies are required to examine more deeply the role of CD41+ cells in the function of LNs.

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.

miR-21 antagonism reprograms macrophage metabolism and abrogates chronic allograft vasculopathy.

Despite much progress in improving graft outcome during cardiac transplantation, chronic allograft vasculopathy (CAV) remains an impediment to long-term graft survival. MicroRNAs (miRNAs) emerged as regulators of the immune response. Here, we aimed to examine the miRNA network involved in CAV. miRNA profiling of heart samples obtained from a murine model of CAV and from cardiac-transplanted patients with CAV demonstrated that miR-21 was most significantly expressed and was primarily localized to macrophages. Interestingly, macrophage depletion with clodronate did not significantly prolong allograft survival in mice, while conditional deletion of miR-21 in macrophages or the use of a specific miR-21 antagomir resulted in indefinite cardiac allograft survival and abrogated CAV. The immunophenotype, secretome, ability to phagocytose, migration, and antigen presentation of macrophages were unaffected by miR-21 targeting, while macrophage metabolism was reprogrammed, with a shift toward oxidative phosphorylation in naïve macrophages and with an inhibition of glycolysis in pro-inflammatory macrophages. The aforementioned effects resulted in an increase in M2-like macrophages, which could be reverted by the addition of L-arginine. RNA-seq analysis confirmed alterations in arginase-associated pathways associated with miR-21 antagonism. In conclusion, miR-21 is overexpressed in murine and human CAV, and its targeting delays CAV onset by reprogramming macrophages metabolism.

Characterization of Leptin Receptor+ Stromal Cells in Lymph Node.

Lymph node (LN)-resident stromal cells play an essential role in the proper functioning of LNs. The stromal compartment of the LN undergoes significant compensatory changes to produce a milieu amenable for regulation of the immune response. We have identified a distinct population of leptin receptor-expressing (LepR+) stromal cells, located in the vicinity of the high endothelial venules (HEVs) and lymphatics. These LepR+ stromal cells expressed markers for fibroblastic reticular cells (FRCs), but they lacked markers for follicular dendritic cells (FDCs) and marginal reticular cells (MRCs). Leptin signaling deficiency led to heightened inflammatory responses within the LNs of db/db mice, leakiness of HEVs, and lymphatic fragmentation. Leptin signaling through the JAK/STAT pathway supported LN stromal cell survival and promoted the anti-inflammatory properties of these cells. Conditional knockout of the LepR+ stromal cells in LNs resulted in HEV and extracellular matrix (ECM) abnormalities. Treatment of ob/ob mice with an agonist leptin fusion protein restored the microarchitecture of LNs, reduced intra-LN inflammatory responses, and corrected metabolic abnormalities. Future studies are needed to study the importance of LN stomal cell dysfunction to the pathogenesis of inflammatory responses in type 2 diabetes (T2D) in humans.

Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner. Sb9-deficient mice exhibited protective T cell-based host immunity to tumors in association with a decline in GrB-expressing immunosuppressive cells within the tumor microenvironment (TME). Maximal protection against tumor development was observed when the tumor and host were deficient in Sb9. The therapeutic utility of Sb9 inhibition was demonstrated by the control of tumor growth, resulting in increased survival times in mice. Our studies describe a molecular target that permits a combination of tumor ablation, interference within the TME, and immunotherapy in one potential modality.

Selective Trafficking of Light Chain-Conjugated Nanoparticles to the Kidney and Renal Cell Carcinoma.

Specific delivery platforms for drugs to the kidney and diagnostic agents to renal cell carcinoma (RCC) constitute urgent but unfulfilled clinical needs. To address these challenges, we engineered nanocarriers that interact selectively for the first time with proximal tubule epithelial cells (PTECs) in the kidney and with RCC through the interplay between lambda light chains (LCs) attached to PEGylated polylactic-co-glycolic acid (PLGA) nanoparticles and the membrane protein megalin. Systemic administration of these light chain-conjugated nanoparticles (LC-NPs) to mice resulted in their specific retention by megalin-expressing PTECs for seven days. Repetitive dosing of LC-NPs demonstrated no renal toxicity. LC-NPs also localized selectively to megalin-expressing RCC tumors in mice. Moreover, we confirmed that both the primary tumor and lymph node metastases of human RCC express megalin, reinforcing the potential of LC-NPs for clinical use. Thus, LC-NPs can contribute potentially to improving the management of both non-oncologic and oncologic renal disorders.

Cellular Mechanisms of Rejection of Optic and Sciatic Nerve Transplants: An Observational Study.

BACKGROUND: Organ transplantation is a standard therapeutic strategy for irreversible organ damage, but the utility of nerve transplantation remains generally unexplored, despite its potential benefit to a large patient population. Here, we aimed to establish a feasible preclinical mouse model for understanding the cellular mechanisms behind the rejection of peripheral and optic nerves. METHODS: We performed syngenic and allogenic transplantation of optic and sciatic nerves in mice by inserting the nerve grafts inside the kidney capsule, and we assessed the allografts for signs of rejection through 14 d following transplantation. Then, we assessed the efficacy of CTLA4 Ig, Rapamycin, and anti-CD3 antibody in suppressing immune cell infiltration of the nerve allografts. RESULTS: By 3 d posttransplantation, both sciatic and optic nerves transplanted from BALB/c mice into C57BL/6J recipients contained immune cell infiltrates, which included more CD11b+ macrophages than CD3+ T cells or B220+ B cells. Ex vivo immunogenicity assays demonstrated that sciatic nerves demonstrated higher alloreactivity in comparison with optic nerves. Interestingly, optic nerves contained higher populations of anti-inflammatory PD-L1+ cells than sciatic nerves. Treatment with anti-CD3 antibody reduced immune cell infiltrates in the optic nerve allograft, but exerted no significant effect in the sciatic nerve allograft. CONCLUSIONS: These findings establish the feasibility of a preclinical allogenic nerve transplantation model and provide the basis for future testing of directed, high-intensity immunosuppression in these mice.

Lymph node fibroblastic reticular cells deposit fibrosis-associated collagen following organ transplantation.

Although the immune response within draining lymph nodes (DLNs) has been studied for decades, how their stromal compartment contributes to this process remains to be fully explored. Here, we show that donor mast cells were prominent activators of collagen I deposition by fibroblastic reticular cells (FRCs) in DLNs shortly following transplantation. Serial analysis of the DLN indicated that the LN stroma did not return to its baseline microarchitecture following organ rejection and that the DLN contained significant fibrosis following repetitive organ transplants. Using several FRC conditional-knockout mice, we show that induction of senescence in the FRCs of the DLN resulted in massive production of collagen I and a proinflammatory milieu within the DLN. Stimulation of herpes virus entry mediator (HVEM) on FRCs by its ligand LIGHT contributed chiefly to the induction of senescence in FRCs and overproduction of collagen I. Systemic administration of ex vivo-expanded FRCs to mice decreased DLN fibrosis and strengthened the effect of anti-CD40L in prolonging heart allograft survival. These data demonstrate that the transformation of FRCs into proinflammatory myofibroblasts is critically important for the maintenance of a proinflammatory milieu within a fibrotic DLN.

Nanodelivery of Mycophenolate Mofetil to the Organ Improves Transplant Vasculopathy.

Inflammation occurring within the transplanted organ from the time of harvest is an important stimulus of early alloimmune reactivity and promotes chronic allograft rejection. Chronic immune-mediated injury remains the primary obstacle to the long-term success of organ transplantation. However, organ transplantation represents a rare clinical setting in which the organ is accessible ex vivo, providing an opportunity to use nanotechnology to deliver therapeutics directly to the graft. This approach facilitates the directed delivery of immunosuppressive agents (ISA) to target local pathogenic immune responses prior to the transplantation. Here, we have developed a system of direct delivery and sustained release of mycophenolate mofetil (MMF) to treat the donor organ prior to transplantation. Perfusion of a donor mouse heart with MMF-loaded PEG-PLGA nanoparticles (MMF-NPs) prior to transplantation abrogated cardiac transplant vasculopathy by suppressing intragraft pro-inflammatory cytokines and chemokines. Our findings demonstrate that ex vivo delivery of an ISA to donor organs using a nanocarrier can serve as a clinically feasible approach to reduce transplant immunity.

Immune heterogeneity of head and tail pancreatic lymph nodes in non-obese diabetic mice.

The pancreatic lymph node is critical to the pathogenesis of autoimmune diabetes, as it constitutes the initial site for the priming of autoreactive T cells. In this study, we compared the histopathology of the head pancreatic lymph node (HPLN) to the tail pancreatic lymph node (TPLN) in NOD mice. HPLNs and TPLNs were harvested from 4 week-, 8 week-, and 12 week-old NOD mice, and their microvasculature, extracellular matrix, and immune cell subsets were characterized. The percentages of B cells and antigen-presenting cells (APCs) were much higher in the HPLN, as compared to the TPLN. Notably, the HPLNs of 12 week-old mice were characterized by greater expansion of high endothelial venules (HEVs) and lymphatic vessels in comparison to the TPLNs. Finally, we observed a higher density of extracellular matrix (ECM) fibers surrounding the lymphatic vasculature in the HPLNs than in the TPLNs. These data for the first time demonstrate that the HPLN possesses a different immune microanatomy and organization from the TPLN. These novel observations unveil a major phenotypic difference between two types of LNs from the same organ and may highlight an independent fundamental role played by each PLN during the establishment of T1D.

Anti-IL-6 eluting immunomodulatory biomaterials prolong skin allograft survival.

A primary goal in the management of burn wounds is early wound closure. The use of skin allografts represents a lifesaving strategy for severe burn patients, but their ultimate rejection limits their potential efficacy and utility. IL-6 is a major pleiotropic cytokine which critically links innate and adaptive immune responses. Here, we devised anti-IL-6 receptor eluting gelatin methacryloyl (GelMA) biomaterials (GelMA/anti-IL-6), which were implanted at the interface between the wound beds and skin allografts. Our visible light crosslinked GelMA/anti-IL-6 immunomodulatory biomaterial (IMB) demonstrated a stable kinetic release profile of anti-IL-6. In addition, the incorporation of anti-IL-6 within the GelMA hydrogel had no effect on the mechanical properties of the hydrogels. Using a highly stringent skin transplant model, the GelMA/anti-IL-6 IMB almost doubled the survival of skin allografts. The use of GelMA/anti-IL-6 IMB was far superior to systemic anti-IL-6 receptor treatment in prolonging skin allograft survival. As compared to the untreated control group, skin from the GelMA/anti-IL-6 IMB group contained significantly fewer alloreactive T cells and macrophages. Interestingly, the environmental milieu of the draining lymph nodes (DLNs) of the mice implanted with the GelMA/anti-IL-6 IMB was also considerably less pro-inflammatory. The percentage of CD4+ IFNγ+ cells was much lower in the DLNs of the GelMA/anti-IL-6 IMB group in comparison to the GelMA group. These data highlight the importance of localized immune delivery in prolonging skin allograft survival and its potential utility in treating patients with severe burns.

Urine podoplanin heralds the onset of ischemia-reperfusion injury of the kidney.

Ischemia-reperfusion injury represents one of the most common causes of acute kidney injury, a serious and often deadly condition that affects up to 20% of all hospitalized patients in the United States. However, the current standard assay used universally for the diagnosis of acute kidney injury, serum creatinine, does not detect renal damage early in its course. Serendipitously, we found that the immunofluorescent signal of the constitutive podocyte marker podoplanin fades in the glomerulus and intensifies in the tubulointerstitial compartment of the kidney shortly after ischemia-reperfusion injury in 8- to 10-wk-old male C57Bl/6j mice. Therefore, we sought to define the appearance and course of the podoplanin-positive signal in the kidney after ischemia-reperfusion injury. The tubulointerstitial podoplanin-positive signal increased as early as 2 h but persisted for 7 days after ischemia-reperfusion injury. In addition, the strength of this tubulointerstitial signal was directly proportional to the severity of ischemia, and its location shifted from the tubules to interstitial cells over time. Finally, we detected podoplanin in the urine of mice after ischemia, and we observed that an increase in the urine podoplanin-to-creatinine ratio correlated strongly with the onset of renal ischemia-reperfusion injury. Our findings indicate that the measurement of urine podoplanin harbors promising potential for use as a novel biomarker for the early detection of ischemia-reperfusion injury of the kidney.

The Role of Costimulatory Pathways in Transplant Tolerance.

Costimulation is a critical step in T-cell activation, and costimulatory blockade at the time of T cell activation leads to T-cell anergy and allograft tolerance in animal models of transplantation. CD28:B7 is the most important costimulatory pathway and the balance of signals between CD28 and cytotoxic T-lymphocyte associated protein 4 (CTLA-4) is a central determinant of transplant outcome. Form a clinical standpoint, CTLA-4 Ig is the only approved agent for costimulation blockade in transplantation. Advantages and disadvantages of its use are discussed. Progress in developing novel agents to target other pathways, including the promising CD40:CD154 pathway, is also discussed.

Activation of fibroblastic reticular cells in kidney lymph node during crescentic glomerulonephritis.

Crescentic glomerulonephritis is an inflammatory condition characterized by rapid deterioration of kidney function. Previous studies of crescentic glomerulonephritis have focused on immune activation in the kidney. However, the role of fibroblastic reticular cells, which reside in the stromal compartment of the kidney lymph node, has not been studied in this condition. We investigated the activation of kidney lymph node-resident fibroblastic reticular cells in nephrotoxic serum nephritis, a classic murine model of crescentic glomerulonephritis. We found that increased deposition of extracellular matrix fibers by fibroblastic reticular cells in the kidney lymph node was associated with the propagation of high endothelial venules, specialized blood vessels through which lymphocytes enter the lymph node, as well as with expansion of the lymphatic vasculature. The kidney lymph node also contained an expanding population of pro-inflammatory T cells. Removal of the kidney lymph node, depletion of fibroblastic reticular cells, and treatment with anti-podoplanin antibody each resulted in reduction of kidney injury. Our findings suggest that modulating the activity of fibroblastic reticular cells may be a novel therapeutic approach in crescentic glomerulonephritis.

Ectopic high endothelial venules in pancreatic ductal adenocarcinoma: A unique site for targeted delivery.

BACKGROUND: Nanomedicine offers an excellent opportunity to tackle treatment-refractory malignancies by enhancing the delivery of therapeutics to the tumor site. High endothelial venules (HEVs) are found primarily in lymph nodes or formed de novo in peripheral tissues during inflammatory responses. They express peripheral node addressin (PNAd), which is recognized by the monoclonal antibody MECA79. METHODS: Here, we demonstrated that HEVs form de novo in human pancreatic ductal adenocarcinoma (PDAC). We engineered MECA79 coated nanoparticles (MECA79-NPs) that recognize these ectopic HEVs in PDAC. FINDINGS: The trafficking of MECA79-NPs following intravenous delivery to human PDAC implanted in a humanized mouse model was more robust than non-conjugated NPs. Treatment with MECA79-Taxol-NPs augmented the delivery of Paclitaxel (Taxol) to the tumor site and significantly reduced the tumor size. This effect was associated with a higher apoptosis rate of PDAC cells and reduced vascularization within the tumor. INTERPRETATION: Targeting the HEVs of PDAC using MECA79-NPs could lay the ground for the localized delivery of a wide variety of drugs including chemotherapeutic agents. FUND: National Institutes of Health (NIH) grants: T32-EB016652 (B·B.), NIH Cancer Core Grant CA034196 (L.D.S.), National Institute of Allergy and Infectious Diseases grants R01-AI126596 and R01-HL141815 (R.A.).

Targeting antigen-presenting cells by anti-PD-1 nanoparticles augments antitumor immunity.

Recent studies in cancer research have focused intensely on the antineoplastic effects of immune checkpoint inhibitors. While the development of these inhibitors has progressed successfully, strategies to further improve their efficacy and reduce their toxicity are still needed. We hypothesized that the delivery of anti-PD-1 antibody encapsulated in PLGA nanoparticles (anti-PD-1 NPs) to the spleen would improve the antitumor effect of this agent. Unexpectedly, we found that mice treated with a high dose of anti-PD-1 NPs exhibited significantly higher mortality compared with those treated with free anti-PD-1 antibody, due to the overactivation of T cells. Administration of anti-PD-1 NPs to splenectomized LT-α-/- mice, which lack both lymph nodes and spleen, resulted in a complete reversal of this increased mortality and revealed the importance of secondary lymphoid tissues in mediating anti-PD-1-associated toxicity. Attenuation of the anti-PD-1 NPs dosage prevented toxicity and significantly improved its antitumor effect in the B16-F10 murine melanoma model. Furthermore, we found that anti-PD-1 NPs undergo internalization by DCs in the spleen, leading to their maturation and the subsequent activation of T cells. Our findings provide important clues that can lead to the development of strategies to enhance the efficacy of immune checkpoint inhibitors.