Extracellular Vesicles in Transplantation: Friend or Foe.

The long-term function of transplanted organs, even under immunosuppression, is hindered by rejection, especially chronic rejection. Chronic rejection occurs more frequently after lung transplantation, termed chronic lung allograft dysfunction (CLAD), than after transplantation of other solid organs. Pulmonary infection is a known risk factor for CLAD, as transplanted lungs are constantly exposed to the external environment; however, the mechanisms by which respiratory infections lead to CLAD are poorly understood. The role of extracellular vesicles (EVs) in transplantation remains largely unknown. Current evidence suggests that EVs released from transplanted organs can serve as friend and foe. EVs carry not only major histocompatibility complex antigens but also tissue-restricted self-antigens and various transcription factors, costimulatory molecules, and microRNAs capable of regulating alloimmune responses. EVs play an important role in antigen presentation by direct, indirect, and semidirect pathways in which CD8 and CD4 cells can be activated. During viral infections, exosomes (small EVs <200 nm in diameter) can express viral antigens and regulate immune responses. Circulating exosomes may also be a viable biomarker for other diseases and rejection after organ transplantation. Bioengineering the surface of exosomes has been proposed as a tool for targeted delivery of drugs and personalized medicine. This review focuses on recent studies demonstrating the role of EVs with a focus on exosomes and their dual role (immune activation or tolerance induction) after organ transplantation, more specifically, lung transplantation.

Immune responses of lung transplant recipients against SARS-CoV-2 and common respiratory coronaviruses: Evidence for pre-existing cross-reactive immunity.

Humoral and cellular immune responses to SARS-CoV-2 and other coronaviruses in lung transplant recipients are unknown. We measured antibodies and T cell responses against the SARS-CoV-2 spike S2 and nucleocapsid antigens and spike antigens from common respiratory coronaviruses (229E, NL63, OC43, and HKU1) after vaccination or infection of LTxRs. 148 LTxRs from single center were included in this study: 98 after vaccination and 50 following SARS-CoV-2 infection. Antibodies were quantified by enzyme-linked immunosorbent assay. The frequency of T cells secreting IL2, IL4, IL10, IL17, TNFα, and IFNγ were enumerated by enzyme-linked immunospot assay. Our results have shown the development of antibodies to SARS-CoV-2 spike protein in infected LTxRs (39/50) and vaccinated LTxRs (52/98). Vaccinated LTxRs had higher number of T cells producing TNFα but less cells producing IFNγ than infected LTxRs in response to the nucleocapsid antigen and other coronavirus spike antigens. We didn't find correlation between the development of antibodies and cellular immune responses against the SARS-CoV-2 spike protein after vaccination. Instead, LTxRs have pre-existing cellular immunity to common respiratory coronaviruses, leading to cross-reactive immunity against SARS-CoV-2 which likely will provide protection against SARS-Cov-2 infection.

Cytosolic EpCAM cooperates with H-Ras to regulate epithelial to mesenchymal transition through ZEB1.

Next generation sequencing of human cancer mutations has identified novel therapeutic targets. Activating Ras oncogene mutations play a central role in oncogenesis, and Ras-driven tumorigenesis upregulates an array of genes and signaling cascades that can transform normal cells into tumor cells. In this study, we investigated the role of altered localization of epithelial cell adhesion molecule (EpCAM) in Ras-expressing cells. Analysis of microarray data demonstrated that Ras expression induced EpCAM expression in normal breast epithelial cells. Fluorescent and confocal microscopy showed that H-Ras mediated transformation also promoted epithelial-to-mesenchymal transition (EMT) together with EpCAM. To consistently localize EpCAM in the cytosol, we generated a cancer-associated EpCAM mutant (EpCAM-L240A) that is retained in the cytosol compartment. Normal MCF-10A cells were transduced with H-Ras together with EpCAM wild-type (WT) or EpCAM-L240A. WT-EpCAM marginally effected invasion, proliferation, and soft agar growth. EpCAM-L240A, however, markedly altered cells and transformed to mesenchymal phenotype. Ras-EpCAM-L240A expression also promoted expression of EMT factors FRA1, ZEB1 with inflammatory cytokines IL-6, IL-8, and IL1. This altered morphology was reversed using MEK-specific inhibitors and to some extent JNK inhibition. Furthermore, these transformed cells were sensitized to apoptosis using paclitaxel and quercetin, but not other therapies. For the first time, we have demonstrated that EpCAM mutations can cooperate with H-Ras and promote EMT. Collectively, our results highlight future therapeutic opportunities in EpCAM and Ras mutated cancers.

Loss of IGFBP2 mediates alveolar type 2 cell senescence and promotes lung fibrosis.

Accumulation of senescent cells contributes to age-related diseases including idiopathic pulmonary fibrosis (IPF). Insulin-like growth factor binding proteins (IGFBPs) regulate many biological processes; however, the functional contributions of IGFBP2 in lung fibrosis remain largely unclear. Here, we report that intranasal delivery of recombinant IGFBP2 protects aged mice from weight loss and demonstrated antifibrotic effects after bleomycin lung injury. Notably, aged human-Igfbp2 transgenic mice reveal reduced senescence and senescent-associated secretory phenotype factors in alveolar epithelial type 2 (AEC2) cells and they ameliorated bleomycin-induced lung fibrosis. Finally, we demonstrate that IGFBP2 expression is significantly suppressed in AEC2 cells isolated from fibrotic lung regions of patients with IPF and/or pulmonary hypertension compared with patients with hypersensitivity pneumonitis and/or chronic obstructive pulmonary disease. Altogether, our study provides insights into how IGFBP2 regulates AEC2-cell-specific senescence and that restoring IGFBP2 levels in fibrotic lungs can prove effective for patients with IPF.

Downregulation of a tumor suppressor gene LKB1 in lung transplantation as a biomarker for chronic murine lung allograft rejection.

BACKGROUND: We recently demonstrated decreased tumor suppressor gene liver kinase B1 (LKB1) level in lung transplant recipients diagnosed with bronchiolitis obliterans syndrome. STE20-related adaptor alpha (STRADα) functions as a pseudokinase that binds and regulates LKB1 activity. METHODS: A murine model of chronic lung allograft rejection in which a single lung from a B6D2F1 mouse was orthotopically transplanted into a DBA/2J mouse was employed. We examined the effect of LKB1 knockdown using CRISPR-CAS9 in vitro culture system. RESULTS: Significant downregulation of LKB1 and STRADα expression was found in donor lung compared to recipient lung. STRADα knockdown significantly inhibited LKB1, pAMPK expression but induced phosphorylated mammalian target of rapamycin (mTOR), fibronectin, and Collagen-I, expression in BEAS-2B cells. LKB1 overexpression decreased fibronectin, Collagen-I, and phosphorylated mTOR expression in A549 cells. CONCLUSIONS: We demonstrated that downregulation of LKB1-STRADα pathway accompanied with increased fibrosis, results in development of chronic rejection following murine lung transplantation.

Extracellular vesicles: a potential new player in antibody-mediated rejection in lung allograft recipients.

Identification of recipients with pre-existing antibodies and cross-matching of recipient sera with donor lymphocytes have reduced the incidence of antibody-mediated rejection (AMR) after human lung transplantation. However, AMR is still common and requires not only immediate intervention but also has long-term consequences including an increased risk of chronic lung allograft dysfunction (CLAD). The mechanisms resulting in AMR remain largely unknown due to the variation in clinical and histopathological features among lung transplant recipients; however, several reports have demonstrated a strong association between the development of antibodies against mismatched donor human leucocyte antigens [donor-specific antibodies (DSAs)] and AMR. In addition, the development of antibodies against lung self-antigens (K alpha1 tubulin and collagen V) also plays a vital role in AMR pathogenesis, either alone or in combination with DSAs. In the current article, we will review the existing literature regarding the association of DSAs with AMR, along with clinical diagnostic features and current treatment options for AMR. We will also discuss the role of extracellular vesicles (EVs) in the immune-related pathogenesis of AMR, which can lead to CLAD.

Extracellular Vesicles Mediate Immune Responses to Tissue-Associated Self-Antigens: Role in Solid Organ Transplantations.

Transplantation is a treatment option for patients diagnosed with end-stage organ diseases; however, long-term graft survival is affected by rejection of the transplanted organ by immune and nonimmune responses. Several studies have demonstrated that both acute and chronic rejection can occur after transplantation of kidney, heart, and lungs. A strong correlation has been reported between de novo synthesis of donor-specific antibodies (HLA-DSAs) and development of both acute and chronic rejection; however, some transplant recipients with chronic rejection do not have detectable HLA-DSAs. Studies of sera from such patients demonstrate that immune responses to tissue-associated antigens (TaAgs) may also play an important role in the development of chronic rejection, either alone or in combination with HLA-DSAs. The synergistic effect between HLA-DSAs and antibodies to TaAgs is being established, but the underlying mechanism is yet to be defined. We hypothesize that HLA-DSAs damage the transplanted donor organ resulting in stress and leading to the release of extracellular vesicles, which contribute to chronic rejection. These vesicles express both donor human leukocyte antigen (HLA) and non-HLA TaAgs, which can activate antigen-presenting cells and lead to immune responses and development of antibodies to both donor HLA and non-HLA tissue-associated Ags. Extracellular vesicles (EVs) are released by cells under many circumstances due to both physiological and pathological conditions. Primarily employing clinical specimens obtained from human lung transplant recipients undergoing acute or chronic rejection, our group has demonstrated that circulating extracellular vesicles display both mismatched donor HLA molecules and lung-associated Ags (collagen-V and K-alpha 1 tubulin). This review focuses on recent studies demonstrating an important role of antibodies to tissue-associated Ags in the rejection of transplanted organs, particularly chronic rejection. We will also discuss the important role of extracellular vesicles released from transplanted organs in cross-talk between alloimmunity and autoimmunity to tissue-associated Ags after solid organ transplantation.

Low-dose IL-2 prevents murine chronic cardiac allograft rejection: Role for IL-2-induced T regulatory cells and exosomes with PD-L1 and CD73.

To determine the effects and immunological mechanisms of low-dose interleukin-2 (IL-2) in a murine model of chronic cardiac allograft rejection (BALB/c to C57BL/6) after costimulatory blockade consisting of MR1 (250 µg/ip day 0) and CTLA4-Ig (200 µg/ip day 2), we administered low-dose IL-2 (2000 IU/day) starting on posttransplant day 14 for 3 weeks. T regulatory (Treg) cell infiltration of the grafts was determined by immunohistochemistry; circulating exosomes by western blot and aldehyde bead flow cytometry; antibodies to donor MHC by immunofluorescent staining of donor cells; and antibodies to cardiac self-antigens (myosin, vimentin) by ELISA. We demonstrated that costimulation blockade after allogeneic heart transplantation induced circulating exosomes containing cardiac self-antigens and antibodies to both donor MHC and self-antigens, leading to chronic rejection by day 45. Treatment with low-dose IL-2 prolonged allograft survival (>100 days), prevented chronic rejection, and induced splenic and graft-infiltrating CD4+ CD25+ Foxp3 Treg cells by day 45 and circulating exosomes (Foxp3+) with PD-L1 and CD73. MicroRNA 142, associated with the TGFß pathway, was significantly downregulated in exosomes from IL-2-treated mice. In conclusion, low-dose IL-2 delays rejection in a murine model of chronic cardiac allograft rejection and also induces graft-infiltrating Tregs and circulating exosomes with immunoregulatory molecules.

Novel role for tumor suppressor gene, liver kinase B1, in epithelial-mesenchymal transition leading to chronic lung allograft dysfunction.

Epithelial-mesenchymal transition (EMT) has been implicated to play a role in chronic lung allograft dysfunction (CLAD). Liver kinase B1 (LKB1), a tumor suppressor gene, can regulate EMT. However, its role in CLAD development following lung transplantation remains unknown. Using qRT-PCR, biopsies from lung transplant recipients with bronchiolitis obliterans syndrome (BOS) demonstrated significant downregulation of LKB1 (p = .0001), compared to stable biopsies. To determine the role of LKB1 in EMT development, we analyzed EMT in human bronchial epithelial cell line BEAS-2B. Knockdown of LKB1 by siRNA significantly dysregulated mesenchymal markers expression in BEAS-2B cells. Following incubation of human primary bronchial epithelial cell or BEAS-2B cells with exosomes isolated from BOS or stable lung transplant recipients, LKB1 expression was inhibited when incubated with BOS-exosome. Incubation with BOS-exosomes also decreased LKB1 expression and induced EMT markers in air-liquid interface culture method. Our results provide novel evidence that exosomes released from transplanted lungs undergoing chronic rejection are associated with inactivated tumor suppressor gene LKB1 and this loss induces EMT leading to the pathogenesis of CLAD following human lung transplantation.

Cutting Edge: Circulating Exosomes with COVID Spike Protein Are Induced by BNT162b2 (Pfizer-BioNTech) Vaccination prior to Development of Antibodies: A Novel Mechanism for Immune Activation by mRNA Vaccines.

Severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) causes severe acute respiratory syndrome. mRNA vaccines directed at the SARS-CoV-2 spike protein resulted in development of Abs and protective immunity. To determine the mechanism, we analyzed the kinetics of induction of circulating exosomes with SARS-CoV-2 spike protein and Ab following vaccination of healthy individuals. Results demonstrated induction of circulating exosomes expressing spike protein on day 14 after vaccination followed by Abs 14 d after the second dose. Exosomes with spike protein, Abs to SARS-CoV-2 spike, and T cells secreting IFN-γ and TNF-α increased following the booster dose. Transmission electron microscopy of exosomes also demonstrated spike protein Ags on their surface. Exosomes with spike protein and Abs decreased in parallel after four months. These results demonstrate an important role of circulating exosomes with spike protein for effective immunization following mRNA-based vaccination. This is further documented by induction of humoral and cellular immune responses in mice immunized with exosomes carrying spike protein.

A decline in club cell secretory proteins in lung transplantation is associated with release of natural killer cells exosomes leading to chronic rejection.

BACKGROUND: In human lung transplant recipients, a decline in club cell secretory protein (CCSP) in bronchoalveolar lavage fluid has been associated with chronic lung allograft dysfunction (CLAD) as well as the induction of exosomes and immune responses that lead to CLAD. However, the mechanisms by which CCSP decline contributes to CLAD remain unknown. METHODS: To define the mechanisms leading to CCSP decline and chronic rejection, we employed two mouse models: 1) chronic rejection after orthotopic single lung transplantation and 2) anti-major histocompatibility complex (MHC) class I-induced obliterative airway disease. RESULTS: In the chronic rejection mouse model, we detected circulating exosomes with donor MHC (H2b) and lung self-antigens and also development of antibodies to H2b and lung self-antigens and then a decline in CCSP. Furthermore, DBA2 mice that received injections of these exosomes developed antibodies to donor MHC and lung self-antigens. In the chronic rejection mouse model, natural killer (NK) and CD8 T cells were the predominant graft-infiltrating cells on day 14 of rejection followed by exosomes containing NK cell-associated and cytotoxic molecules on day 14 and 28. When NK cells were depleted, exosomes with NK cell-associated and cytotoxic molecules as well as fibrosis decreased. CONCLUSIONS: Induction of exosomes led to immune responses to donor MHC and lung self-antigens, resulting in CCSP decline, leading to NK cell infiltration and release of exosomes from NK cells. These results suggest a novel role for exosomes derived from NK cells in the pathogenesis of chronic lung allograft rejection.

Circulating exosomes induced by respiratory viral infections in lung transplant recipients activate cellular stress, innate immune pathways and epithelial to mesenchymal transition.

BACKGROUND: Chronic lung transplant rejection occurs in over 50% of lung transplant recipients and mechanism of chronic rejection is unknown. Evaluation of potential mechanism of exosomes from lung transplant recipients diagnosed with respiratory viral infection (RVI) in inducing chronic lung allograft dysfunction (CLAD). METHOD: Exosomes were isolated from lung transplant recipients followed by DNA and RNA isolation from exosomes. Cell signaling mechanisms were studied by co-culturing exosomes with human epithelial cells. Mice were immunized with exosomes and lung homogenates were studied for immune signaling proteins. RESULTS: Exosomes from lung transplant recipients with RVI carry nucleic acids which are capable of inducing innate immune signaling, endoplasmic reticulum stress, and epithelial mesenchymal transition. CONCLUSION: Therefore, we propose that RVI can lead to induction of exosomes that initiate the process leading to CLAD in mice models. These novel findings identified the molecular mechanisms by which RVI increases the risk of CLAD.

Identification of Phospho-Tyrosine Targets as a Strategy for the Treatment of Esophageal Adenocarcinoma Cells.

INTRODUCTION: Esophageal cancer (EC) is an aggressive cancer type that is increasing at a high rate in the US and worldwide. Extensive sequencing of EC specimens has shown that there are no consistent driver mutations that can impact treatment strategies. The goal of this study was to identify activated tyrosine kinase receptors (TKRs) in EC samples as potential targets in the treatment of EC. METHODS: Activated tyrosine kinase receptors were detected using a dot-blot array for human TK receptors. Human esophageal cancer cell lines were transplanted into immunocompromised mice, and tumor xenografts were subjected to tyrosine kinase inhibitors based on the dot-blot array data. RESULTS: Using the OE33 esophageal cancer cell line, we identified activated EGF receptor (EGFR), as well as ErbB2 and ErbB3. Treatment of this cell line with erlotinib, a specific inhibitor of EGFR, did not impact the growth of this tumor cell line. Treating the OE33 cell line with afatinib, a pan-EGFR family inhibitor resulted in the growth inhibition of OE33, indicating that the ErbB2 and ErbB3 receptors were contributing to tumor cell proliferation. Afatinib treatment of mice growing OE33 tumors inhibited growth of the OE33 tumor cells. DISCUSSION: Activated tyrosine kinase receptors were readily detected in both cancer cell lines and human esophageal cancer samples. By identifying the activated receptors and then using the appropriate tyrosine kinase inhibitors, we can block tumor growth in vitro and in animal xenografts. We propose that identifying and targeting activated TKRs can be used as a personalized EC tumor treatment strategy.

Cancer-associated mutations reveal a novel role for EpCAM as an inhibitor of cathepsin-L and tumor cell invasion.

BACKGROUND: EpCAM (Epithelial cell adhesion molecule) is often dysregulated in epithelial cancers. Prior studies implicate EpCAM in the regulation of oncogenic signaling pathways and epithelial-to-mesenchymal transition. It was recently demonstrated that EpCAM contains a thyroglobulin type-1 (TY-1) domain. Multiple proteins with TY-1 domains are known to inhibit cathepsin-L (CTSL), a cysteine protease that promotes tumor cell invasion and metastasis. Analysis of human cancer sequencing studies reveals that somatic EpCAM mutations are present in up to 5.1% of tested tumors. METHODS: The Catalogue of Somatic Mutations in Cancer (COSMIC) database was queried to tabulate the position and amino acid changes of cancer associated EpCAM mutations. To determine how EpCAM mutations affect cancer biology we studied C66Y, a damaging TY-1 domain mutation identified in liver cancer, as well as 13 other cancer-associated EpCAM mutations. In vitro and in vivo models were used to determine the effect of wild type (WT) and mutant EpCAM on CTSL activity and invasion. Immunoprecipitation and localization studies tested EpCAM and CTSL protein binding and determined compartmental expression patterns of EpCAM mutants. RESULTS: We demonstrate that WT EpCAM, but not C66Y EpCAM, inhibits CTSL activity in vitro, and the TY-1 domain of EpCAM is responsible for this inhibition. WT EpCAM, but not C66Y EpCAM, inhibits tumor cell invasion in vitro and lung metastases in vivo. In an extended panel of human cancer cell lines, EpCAM expression is inversely correlated with CTSL activity. Previous studies have demonstrated that EpCAM germline mutations can prevent EpCAM from being expressed at the cell surface. We demonstrate that C66Y and multiple other EpCAM cancer-associated mutations prevent surface expression of EpCAM. Cancer-associated mutations that prevent EpCAM cell surface expression abrogate the ability of EpCAM to inhibit CTSL activity and tumor cell invasion. CONCLUSIONS: These studies reveal a novel role for EpCAM as a CTSL inhibitor, confirm the functional relevance of multiple cancer-associated EpCAM mutations, and suggest a therapeutic vulnerability in cancers harboring EpCAM mutations.

Optimized polyepitope neoantigen DNA vaccines elicit neoantigen-specific immune responses in preclinical models and in clinical translation.

BACKGROUND: Preclinical studies and early clinical trials have shown that targeting cancer neoantigens is a promising approach towards the development of personalized cancer immunotherapies. DNA vaccines can be rapidly and efficiently manufactured and can integrate multiple neoantigens simultaneously. We therefore sought to optimize the design of polyepitope DNA vaccines and test optimized polyepitope neoantigen DNA vaccines in preclinical models and in clinical translation. METHODS: We developed and optimized a DNA vaccine platform to target multiple neoantigens. The polyepitope DNA vaccine platform was first optimized using model antigens in vitro and in vivo. We then identified neoantigens in preclinical breast cancer models through genome sequencing and in silico neoantigen prediction pipelines. Optimized polyepitope neoantigen DNA vaccines specific for the murine breast tumor E0771 and 4T1 were designed and their immunogenicity was tested in vivo. We also tested an optimized polyepitope neoantigen DNA vaccine in a patient with metastatic pancreatic neuroendocrine tumor. RESULTS: Our data support an optimized polyepitope neoantigen DNA vaccine design encoding long (≥20-mer) epitopes with a mutant form of ubiquitin (Ubmut) fused to the N-terminus for antigen processing and presentation. Optimized polyepitope neoantigen DNA vaccines were immunogenic and generated robust neoantigen-specific immune responses in mice. The magnitude of immune responses generated by optimized polyepitope neoantigen DNA vaccines was similar to that of synthetic long peptide vaccines specific for the same neoantigens. When combined with immune checkpoint blockade therapy, optimized polyepitope neoantigen DNA vaccines were capable of inducing antitumor immunity in preclinical models. Immune monitoring data suggest that optimized polyepitope neoantigen DNA vaccines are capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine tumor. CONCLUSIONS: We have developed and optimized a novel polyepitope neoantigen DNA vaccine platform that can target multiple neoantigens and induce antitumor immune responses in preclinical models and neoantigen-specific responses in clinical translation.

Autoantibodies in lung transplantation.

Chronic lung allograft dysfunction (CLAD) comprises both bronchiolitis obliterans syndrome and restrictive allograft syndrome as subtypes. After lung transplantation, CLAD remains a major limitation for long-term survival, and lung transplant recipients therefore have poorer outcomes compared with recipients of other solid organ transplants. Although the number of lung transplants continues to increase globally, the field demands detailed understanding of immunoregulatory mechanisms and more effective individualized therapies to combat CLAD. Emerging evidence suggests that CLAD is multifactorial and involves a complex, delicate interplay of multiple factors, including perioperative donor characteristics, inflammation induced immediately following transplant, post-transplant infection and interplay between allo- and autoimmunity directed to donor antigens. Recently, identification of stress-induced exosome release from the transplanted organ has emerged as an underlying mechanism in the development of chronic rejection and promises to prompt novel strategies for future therapeutic interventions. In this review, we will discuss recent studies and ongoing research into the mechanisms for the development of CLAD, with emphasis on immune responses to lung-associated self-antigens-that is, autoimmunity.

Precision delivery of RAS-inhibiting siRNA to KRAS driven cancer via peptide-based nanoparticles.

Over 95% of pancreatic adenocarcinomas (PDACs), as well as a large fraction of other tumor types, such as colorectal adenocarcinoma, are driven by KRAS activation. However, no direct RAS inhibitors exist for cancer therapy. Furthermore, the delivery of therapeutic agents of any kind to PDAC in particular has been hindered by the extensive desmoplasia and resultant drug delivery challenges that accompanies these tumors. Small interfering RNA (siRNA) is a promising modality for anti-neoplastic therapy due to its precision and wide range of potential therapeutic targets. Unfortunately, siRNA therapy is limited by low serum half-life, vulnerability to intracellular digestion, and transient therapeutic effect. We assessed the ability of a peptide based, oligonucleotide condensing, endosomolytic nanoparticle (NP) system to deliver siRNA to KRAS-driven cancers. We show that this peptide-based NP is avidly taken up by cancer cells in vitro, can deliver KRAS-specific siRNA, inhibit KRAS expression, and reduce cell viability. We further demonstrate that this system can deliver siRNA to the tumor microenvironment, reduce KRAS expression, and inhibit pancreatic cancer growth in vivo. In a spontaneous KPPC model of PDAC, this system effectively delivers siRNA to stroma-rich tumors. This model has the potential for translational relevance for patients with KRAS driven solid tumors.