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Immune checkpoint inhibitor treatment has the potential to prolong survival in non-small cell lung cancer (NSCLC), however, some of the patients develop resistance following initial response. Here, we analyze the immune phenotype of matching tumor samples from a cohort of NSCLC patients showing good initial response to immune checkpoint inhibitors, followed by acquired resistance at later time points. By using imaging mass cytometry and whole exome and RNA sequencing, we detect two patterns of resistance¨: One group of patients is characterized by reduced numbers of tumor-infiltrating CD8 +  T cells and reduced expression of PD-L1 after development of resistance, whereas the other group shows high CD8 +  T cell infiltration and high expression of PD-L1 in addition to markedly elevated expression of other immune-inhibitory molecules. In two cases, we detect downregulation of type I and II IFN pathways following progression to resistance, which could lead to an impaired anti-tumor immune response. This study thus captures the development of immune checkpoint inhibitor resistance as it progresses and deepens our mechanistic understanding of immunotherapy response in NSCLC., (© 2023. Springer Nature Limited.)

Cell lines are extensively used to study cancer biology. However, the use of highly passaged commercial cell lines has to be questioned, as they do not closely resemble the originating tumor. To understand the reliability of preclinical models for Malignant pleural mesothelioma (MPM) studies, we have performed whole transcriptome and whole exome analyses of fresh frozen MPM tumors and compared them to cell lines generated from these tumors, as well as commercial cell lines and a preclinical MPM mouse model. Patient-derived cell lines were generated from digested fresh tumors and whole exome sequencing was performed on DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor samples, corresponding patient-derived cell lines, and normal tissue. RNA sequencing libraries were prepared from 10 fresh frozen tumor samples, the 10 corresponding patient-derived cell lines, and 7 commercial cell lines. Our results identified alterations in tumor suppressor genes such as FBXW7 , CDKN2A , CDKN2B , and MTAP , all known to drive MPM tumorigenesis. Patient-derived cell lines correlate to a high degree with their originating tumor. Gene expressions involved in multiple pathways such as EMT, apoptosis, myogenesis, and angiogenesis are upregulated in tumor samples when compared to patient-derived cell lines; however, they are downregulated in commercial cell lines compared to patient-derived cell lines, indicating significant differences between the two model systems. Our results show that the genome and transcriptome of tumors correlate to a higher degree with patient-derived cell lines rather than commercial cell lines. These results are of major relevance for the scientific community in regard to using cell lines as an appropriate model, resembling the pathway of interest to avoid misleading results for clinical applications.

Background: Malignant pleural and peritoneal mesotheliomas are rare malignancies with unacceptable poor prognoses and limited treatment options. The genomic landscape is mainly characterised by the loss of tumour suppressor genes and mutations in DNA repair genes. Currently, data from next-generation sequencing (NGS) of mesothelioma tumours is restricted to a limited number of cases; moreover, data comparing molecular features of mesothelioma from the pleural and peritoneal origin with NGS are lacking., Methods: We analysed 1113 pleural mesothelioma and 355 peritoneal mesothelioma samples. All tumours were sequenced with the FoundationOne® or FoundationOne®CDx assay for detection of substitutions, insertion-deletions, copy-number alterations and selected rearrangements in at least 324 cancer genes., Results: This analysis revealed alterations in 19 genes with an overall prevalence of at least 2%. Alterations in BAP1, CDKN2A, CDKN2B, NF2, MTAP, TP53 and SETD2 occurred with a prevalence of at least 10%. Peritoneal, compared to pleural mesothelioma, was characterised by a lower prevalence of alterations in CDKN2A, CDKN2B and MTAP. Moreover, we could define four distinct subgroups according to alterations in BAP1 and CDKN2A/B. Alterations in Hedgehog pathway-related genes (PTCH1/2 and SUFU) and Hippo pathway-related gene (NF2) as well as KRAS, EGFR, PDGFRA/B, ERBB2 and FGFR3 were detected in both cohorts., Conclusion: Here, we report the molecular aberrations from the largest cohort of patients with mesothelioma. This analysis identified a proportion of patients with targetable alterations and suggests that molecular profiling can identify new treatment options for patients with mesothelioma., (© 2022. The Author(s).)

Background: Mesenchymal-to-epithelial transition (MET) exon 14 skipping mutations and MET gene amplification occur in 3-5% of non-small cell lung cancer (NSCLC) patients. Tyrosine kinase inhibitors (TKIs) targeting MET alterations have shown promising results in these patients., Objective: The aim of this study was to describe the genomic profile, PD-L1 expression and clinicopathological features of MET dysregulated NSCLC., Patients and Methods: We identified 188 patients with advanced-stage NSCLC with data on MET expression by immunohistochemistry (IHC). IHC for PD-L1 expression was performed in 131 patient samples, and next-generation sequencing (NGS) analysis was performed in 109 patient samples., Results: MET exon 14 skipping alterations were identified in 16 (14.7%) samples, MET amplifications with cut-off ≥4 copy number variations were identified in 11 (10.1%) samples, and an oncogenic MET mutation (MET p.D1228N) was identified in 1 (0.9%) sample. 12/15 tumors (80.0%) harboring MET exon 14 alterations and 7/11 (63.6%) MET-amplified tumors expressed PD-L1 in ≥1% of tumor cells. Tumors harboring MET exon 14 skipping alterations expressed PD-L1 more frequently than MET wild-type IHC-positive tumors (p = 0.045). Twenty-five percent of MET exon 14-altered cases and 33% of MET-amplified cases harbored potentially targetable oncogenic co-mutations in KRAS, BRAF, and EGFR. The most frequent co-occurring mutations in all MET-altered tumors were TP53, KRAS, BRAF, and CDK4., Conclusions: We demonstrated that MET exon 14 skipping alterations and MET amplification are not mutually exclusive to other oncogenic co-mutations, and report the association of genomic MET alterations with PD-L1 expression. Since genomic MET alterations are emerging targets requiring upfront treatment, optimal understanding of the co-mutational landscape for this patient population is needed., (© 2022. The Author(s).)

Metastatic non-small cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (ICIs) may suffer from heavy side effects and not all patients benefit from the treatment. We conducted a comprehensive statistical analysis to identify promising (bio-)markers for treatment response. We analyzed retrospective data from NSCLC patients treated with ICIs in first- or further-line therapy settings at the University Hospital Zurich. We investigated 16 possible prognostic markers with respect to overall survival, tumor size reduction, and the development of an immune-related adverse event (irAE) and assessed the robustness of our results. For the further-line patient group, the most significant result was that increased basophil counts were associated with increased odds of tumor size reduction within three months and with the development of an irAE. For the first-line patient group, the most significant results were that increased lymphocyte counts, the histology of adenocarcinoma, and the intake of non-steroidal anti-rheumatic drugs (NSAR) were associated with decreased hazards of dying. Our study yielded new hypotheses for predictive (bio-)markers for response to ICIs in NSCLC patients. The possibly beneficial role of high basophil counts is a particularly interesting finding. Our results should be tested on independent data in a prospective fashion.

Invasive urothelial bladder cancer (UBC) has high recurrence rates even after radical cystectomy (RC). Exosomes are membrane-bound nanovesicles, which have been shown to contribute to carcinogenesis and metastasis. We previously showed that urinary exosomes display a malignant profile in UBC patients despite the absence of detectable tumour. Here, we investigated exosomes from sampling sites close to or distant from the former tumour, aiming to understand the effect of the tumour on the local milieu. Ten patients scheduled for cystectomy after transurethral bladder resection (TUR-B), without remaining detectable tumour, were included. Exosomes were isolated from tissue explants of both the previous tumour site and distant bladder tissue. Proteins were quantified by mass spectrometry in seven patients. Exosomes from the previous tumour site were enriched in inflammatory but not cancer-related pathways compared to distant tissue. However, the 69 most abundant proteins in tissue-derived exosomes regardless of site, 20 of which were also found in urinary exosomes from our previous study, were enriched for cancer-related metabolic pathways and associated with poor prognosis in an external mRNA dataset. The enrichment of cancer-related pathways in the most abundant proteins, regardless of sampling site, confirms our hypothesis that despite the absence of detectable tumour, the entire bladder releases exosomes that contribute to metastasis and highlights the need for early RC.

Malignant pleural mesothelioma (MPM) is a rare and fatal disease of the pleural lining. Up to 80% of the MPM cases are linked to asbestos exposure. Even though its use has been banned in the industrialized countries, the cases continue to increase. MPM is a lethal cancer, with very little survival improvements in the last years, mirroring very limited therapeutic advances. Platinum-based chemotherapy in combination with pemetrexed and surgery are the standard of care, but prognosis is still unacceptably poor with median overall survival of approximately 12 months. The genomic landscape of MPM has been widely characterized showing a low mutational burden and the impairment of tumor suppressor genes. Among them, BAP1 and BLM are present as a germline inactivation in a small subset of patients and increases predisposition to tumorigenesis. Other studies have demonstrated a high frequency of mutations in DNA repair genes. Many therapy approaches targeting these alterations have emerged and are under evaluation in the clinic. High-throughput technologies have allowed the detection of more complex molecular events, like chromotripsis and revealed different transcriptional programs for each histological subtype. Transcriptional analysis has also paved the way to the study of tumor-infiltrating cells, thus shedding lights on the crosstalk between tumor cells and the microenvironment. The tumor microenvironment of MPM is indeed crucial for the pathogenesis and outcome of this disease; it is characterized by an inflammatory response to asbestos exposure, involving a variety of chemokines and suppressive immune cells such as M2-like macrophages and regulatory T cells. Another important feature of MPM is the dysregulation of microRNA expression, being frequently linked to cancer development and drug resistance. This review will give a detailed overview of all the above mentioned features of MPM in order to improve the understanding of this disease and the development of new therapeutic strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Hiltbrunner, Mannarino, Kirschner, Opitz, Rigutto, Laure, Lia, Nozza, Maconi, Marchini, D’Incalci, Curioni-Fontecedro and Grosso.)

Some breast tumors metastasize aggressively whereas others remain dormant for years. The mechanism governing metastatic dormancy remains largely unknown. Through high-parametric single-cell mapping in mice, we identify a discrete population of CD39 + PD-1 + CD8 + T cells in primary tumors and in dormant metastasis, which is hardly found in aggressively metastasizing tumors. Using blocking antibodies, we find that dormancy depends on TNFα and IFNγ. Immunotherapy reduces the number of dormant cancer cells in the lungs. Adoptive transfer of purified CD39 + PD-1 + CD8 + T cells prevents metastatic outgrowth. In human breast cancer, the frequency of CD39 + PD-1 + CD8 + but not total CD8 + T cells correlates with delayed metastatic relapse after resection (disease-free survival), thus underlining the biological relevance of CD39 + PD-1 + CD8 + T cells for controlling experimental and human breast cancer. Thus, we suggest that a primary breast tumor could prime a systemic, CD39 + PD-1 + CD8 + T cell response that favors metastatic dormancy in the lungs.

Natural killer (NK) cells can kill target cells via the recognition of stress molecules and down-regulation of major histocompatibility complex class I (MHC-I). Some NK cells are educated to recognize and kill cells that have lost their MHC-I expression, e.g., tumor or virus-infected cells. A desired property of cancer immunotherapy is, therefore, to activate educated NK cells during anti-tumor responses in vivo. We here analyze NK cell responses to α-galactosylceramide (αGC), a potent activator of invariant NKT (iNKT) cells, or to exosomes loaded with αGC. In mouse strains which express different MHC-I alleles using an extended NK cell flow cytometry panel, we show that αGC induces a biased NK cell proliferation of educated NK cells. Importantly, iNKT cell-induced activation of NK cells selectively increased in vivo missing self-responses, leading to more effective rejection of tumor cells. Exosomes from antigen-presenting cells are attractive anti-cancer therapy tools as they may induce both innate and adaptive immune responses, thereby addressing the hurdle of tumor heterogeneity. Adding αGC to antigen-loaded dendritic-cell-derived exosomes also led to an increase in missing self-responses in addition to boosted T and B cell responses. This study manifests αGC as an attractive adjuvant in cancer immunotherapy, as it increases the functional capacity of educated NK cells and enhances the innate, missing self-based antitumor response.

Enhanced growth and proliferation of cancer cells are accompanied by profound changes in cellular metabolism. These metabolic changes are also common under physiological conditions, and include increased glucose fermentation accompanied by elevated cytosolic pH (pHc) 1,2 . However, how these changes contribute to enhanced cell growth and proliferation is unclear. Here, we show that elevated pHc specifically orchestrates an E2F-dependent transcriptional programme to drive cell proliferation by promoting cyclin D1 expression. pHc-dependent transcription of cyclin D1 requires the transcription factors CREB1, ATF1 and ETS1, and the histone acetyltransferases p300 and CBP. Biochemical characterization revealed that the CREB1-p300/CBP interaction acts as a pH sensor and coincidence detector, integrating different mitotic signals to regulate cyclin D1 transcription. We also show that elevated pHc contributes to increased cyclin D1 expression in malignant pleural mesotheliomas (MPMs), and renders these cells hypersensitive to pharmacological reduction of pHc. Taken together, these data demonstrate that elevated pHc is a critical cellular signal regulating G1 progression, and provide a mechanism linking elevated pHc to oncogenic activation of cyclin D1 in MPMs, and possibly other cyclin D1~dependent tumours. Thus, an increase of pHc may represent a functionally important, early event in the aetiology of cancer that is amenable to therapeutic intervention.

Invasive urinary bladder cancer shows high recurrence rates after cystectomy even with apparent complete downstaging at cystectomy. Exosomes are nano-sized vesicles important in cell-cell communication, which have been hypothesized to contribute to cancer dissemination and recurrence. The aim of this study was to investigate if pro-carcinogenic exosomes could be detected in urine from histologically downstaged bladder cancer patients. 13 Patients were included in this study. Paired ureter and urine samples from nine patients underwent mass spectrometry, while samples from the remaining patients were used for exosome characterization. At cystectomy, exosomes were isolated from bladder and ureter urine, whereafter quantitative proteome profiling was performed. Urinary exosomes clustered based on whether they came from the bladder, with tumour contact, or the ureters, without tumour contact, even though all came from completely downstaged patients. Proteins overexpressed in exosomes derived from bladder urine contained several oncogenes and were mainly associated with tumour metabolism pathways. Although patients were histologically tumour-free at cystectomy, the bladder urine contained exosomes with a carcinogenic metabolic profile. This suggests a continuous release of exosomes from the bladder, which may promote recurrence at distant sites through metabolic rewiring, even after apparent complete downstaging. These exosomes, coming from either undetected cancer cells or partly transformed cells, are likely to increase the risk of metastasis and encourages cystectomy even in completely downstaged patients.

Purpose: To evaluate whether F-fluoroethyltyrosine (FET) PET can discriminate progression from pseudoprogression of brain metastases in patients with non-small cell lung cancer undergoing immunotherapy and radiotherapy to the brain., Methods: Retrospective analysis of F-FET PET scans in cases with documented progression of brain metastases on MRI in a cohort of 53 patients with non-small cell lung cancer receiving immune-checkpoint inhibitors and radiotherapy of brain metastases at the University Hospital of Zürich from June 2015 until January 2019. Response to radiotherapy was assessed by MRI. In case of equivocal findings and/or radiological progression in clinically asymptomatic patients, further assessment with F-FET PET was performed., Results: From the cohort of 53 patients, the restaging MRI showed in 30 patients (56.6%) progression of at least 1 treated metastasis. Thereof, F-FET PET was performed in 11 patients, based on the absence of neurological symptoms or presence of systemic response and physicians' decision. F-FET PET correctly identified pseudoprogression in 9 of 11 patients (81.8%). In patients who did not undergo F-FET PET, 5 of 19 (26.3%) were diagnosed with pseudoprogression., Conclusions: Pseudoprogression of brain metastases occurred in 50% of patients diagnosed with progression on MRI. F-FET PET may help differentiate pseudoprogression from real progression in order to avoid discontinuation of effective therapy or unneeded interventions.

Background: Despite recent studies, the effect of chemotherapy on programmed death-ligand 1 (PD-L1) expression remains controversial. In this study, we investigated whether PD-L1 expression is affected by platinum-based chemotherapy. Furthermore, we evaluated correlation of PD-L1 expression with oncogenic driver alterations., Materials and Methods: We retrospectively evaluated changes in PD-L1 expression by immunohistochemical (IHC) analysis in resected specimens and in biopsies at non-small cell lung cancer recurrence in patients receiving or not adjuvant chemotherapy after surgical resection. Four IHC score groups were defined: TC0 < 1%, T ≥ 1% and < 5%, TC2 ≥ 5% and < 50%, and TC3 ≥ 50%., Results: Thirty-six patients with adenocarcinoma were included. Twenty (56%) patients underwent adjuvant chemotherapy, and 16 (44%) patients did not receive adjuvant chemotherapy. PD-L1 expression was present in 10 (28%) of 36 initial tumor specimens. From patients receiving adjuvant chemotherapy, 7 (35%) of 20 tumor biopsies showed significant upregulation in PD-L1 expression at recurrence. In contrast, from patients with no adjuvant therapy, only 2 (12.5%) of 16 showed a change in PD-L1 expression. Six (17%) of 36 patients were PD-L1-negative in the primary tumor and turned positive at recurrence. KRAS mutation was present in 70% of patients expressing PD-L1., Conclusion: PD-L1 expression in non-small cell lung cancer can change from primary to recurrence, implicating the need for re-biopsy at recurrence. Moreover, chemotherapy might increase expression of PD-L1, supporting a combinatorial therapy with chemotherapy and anti-PD(L)1 treatment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EV) are candidates for cancer immunotherapy because of their capacity to stimulate tumor-specific activity in vivo. However, clinical trials using peptide-loaded autologous EVs have so far only showed moderate T cell responses, suggesting a need for optimization of EV-induced immunity in humans. We previously demonstrated that induction of Ag-specific CD8 + T cells and antitumor responses to whole Ag were independent of MHC class I on EVs and hypothesized that multiple injections of allogeneic EVs could potentiate Ag-specific responses. In this study, we show that the allogeneic EV from mouse bone marrow-derived dendritic cells enhances Ag-specific CD8 + T cell, follicular helper T cell, and Ag-specific Ab responses. EV-injected mice demonstrated Ag-specific memory after 4 mo, with the highest Ab avidity in mice receiving double allogeneic EV injections. Reduced B16mOVA melanoma tumor growth was shown in all EV-injected groups. Our findings support the application of allogeneic EVs for therapeutic use in clinical studies in which an adaptive immune response is desired., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Purpose: Combination of immune checkpoint inhibitors with chemotherapy is under investigation for cancer treatment., Experimental Design: We studied the rationale of such a combination for treating mesothelioma, a disease with limited treatment options., Results: The combination of gemcitabine and immune checkpoint inhibitors outperformed immunotherapy alone with regard to tumor control and survival in a preclinical mesothelioma model; however, the addition of dexamethasone to gemcitabine and immune checkpoint inhibitors nullified the synergistic clinical response. Furthermore, treatment with gemcitabine plus anti-PD-1 resulted in an objective clinical response in two patients with mesothelioma, who were resistant to gemcitabine or anti-PD-1 as monotherapy., Conclusions: Thus, treatment of mesothelioma with a combination of gemcitabine with immune checkpoint inhibitors is feasible and results in synergistic clinical response compared with single treatment in the absence of steroids., (©2018 American Association for Cancer Research.)

Extracellular vesicles (EV), including exosomes and microvesicles (MV), represent a rapidly expanding field of research with diagnostic and therapeutic applications. Although many aspects of EV function remain to be revealed and broad investigations are warranted, most published findings focus on only one vesicle category or a non-separated mix of EVs. In this paper, we investigated both MVs and exosomes from Ovalbumin (OVA)-pulsed dendritic cells for their immunostimulatory potential side-by-side in vivo. Only exosomes induced antigen-specific CD8 + T-cells, and were more efficient than MVs in eliciting antigen-specific IgG production. Further, mainly exosome-primed mouse splenocytes showed significant ex vivo interferon gamma production in response to antigen restimulation. Exosomes carried high levels of OVA, while OVA in MVs was barely detectable, which could explain the more potent antigen-specific response induced by exosomes. Moreover, exosomes induced increased germinal center B cell proportions, whereas MVs had no such effect. Immunisation with both vesicle types combined showed neither inhibitory nor synergistic effects. We conclude that DC-derived MVs and exosomes differ in their capacity to incorporate antigen and induce immune responses. The results are of importance for understanding the role of EVs in vivo, and for future design of vesicle-based immunotherapies and vaccines.

Peptide-loaded exosomes are promising cancer treatment vehicles; however, moderate T cell responses in human clinical trials indicate a need to further understand exosome-induced immunity. We previously demonstrated that antigen-loaded exosomes carry whole protein antigens and require B cells for inducing antigen-specific T cells. Therefore, we investigated the relative importance of exosomal major histocompatibility complex (MHC) class I for the induction of antigen-specific T cell responses and tumour protection. We show that ovalbumin-loaded dendritic cell-derived exosomes from MHCI-/- mice induce antigen-specific T cells at the same magnitude as wild type exosomes. Furthermore, exosomes lacking MHC class I, as well as exosomes with both MHC class I and II mismatch, induced tumour infiltrating T cells and increased overall survival to the same extent as syngeneic exosomes in B16 melanoma. In conclusion, T cell responses are independent of exosomal MHC/peptide complexes if whole antigen is present. This establishes the prospective of using impersonalised exosomes, and will greatly increase the feasibility of designing exosome-based vaccines or therapeutic approaches in humans., Competing Interests: S.G. has a patent on B-cell exosomes for immune therapy, US patent no. US 8932855 B2.

Exosomes are small 40-120 nm vesicles secreted by nearly all cells and are an important form of intercellular communication. Exosomes are abundant, stable, and highly bioavailable to tissues in vivo. Increasingly, exosomes are being recognized as potential therapeutics as they have the ability to elicit potent cellular responses in vitro and in vivo. Patient-derived exosomes have been employed as a novel cancer immunotherapy in several clinical trials, but at this point lack sufficient efficacy. Still other researchers have focused on modifying the content and function of exosomes in various ways, toward the end-goal of specialized therapeutic exosomes. Here we highlight major advances in the use of exosomes for cancer immunotherapy and exosome bioengineering followed by a discussion of focus areas for future research to generate potent therapeutic exosomes. From the Clinical Editor: Exosomes are small vesicles used by cells for intercellular communication. In this short article, the authors described the current status and the potential use of exosomes in the clinical setting., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In recent years exosomes have emerged as potent stimulators of immune responses and as agents for cancer therapy. Exosomes can carry a broad variety of immunostimulatory molecules depending on the cell of origin and in vitro culture conditions. Dendritic cell-derived exosomes (dexosomes) have been shown to carry NK cell activating ligands and can be loaded with antigen to activate invariant NKT cells and to induce antigen-specific T and B cell responses. Dexosomes have been investigated as therapeutic agents against cancer in two phase I clinical trials, with a phase II clinical trial currently ongoing. Dexosomes were well tolerated but therapeutic success and immune activation were limited. Several reports suggest that multiple factors need to be considered in order to improve exosomal immunogenicity for cancer immunotherapy. These include antigen-loading strategies, exosome composition and exosomal trafficking in vivo. Hence, a better understanding of how to engineer and deliver exosomes to specific cells is crucial to generate strong immune responses and to improve the immunotherapeutic potential of exosomes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Anticancer immunotherapy is a promising treatment modality since it bears the potential of being highly specific, but effective clinical applications are still under development. We have recently described an exosome-based strategy for co-delivery of α-galactosylceramide and a tumor-associated antigen that synergistically potentiates tumor-specific adaptive immune responses while preventing the anergy of invariant natural killer T (iNKT) cells. We propose that the next generation of exosome-based immunotherapies should involve iNKT-cell ligands to induce a broad, amplified and sustainable antitumor immune response.

Exosomes and the invariant NKT (iNKT) immune cell ligand α-galactosylceramide (αGC) may offer novel tools for cancer immunotherapy. In this study, we investigated whether exosomes loaded with αGC can activate iNKT cells and potentiate a cancer-specific adaptive immune response. αGC loaded exosomes readily activated iNKT cells both in vitro and in vivo. Exosomes loaded with αGC plus the model antigen ovalbumin (OVA) induced potent NK and γδ T-cell innate immune responses, and they also synergistically amplified T- and B-cell responses that were OVA specific. In contrast to soluble αGC, which anergizes iNKT cells, we found that αGC/OVA-loaded exosomes did not induce iNKT cell anergy but were more potent than soluble αGC + OVA in inducing adaptive immune responses. In an OVA-expressing mouse model of melanoma, treatment of tumor-bearing mice with αGC/OVA-loaded exosomes decreased tumor growth, increased antigen-specific CD8(+) T-cell tumor infiltration, and increased median survival, relative to control mice immunized with soluble αGC + OVA alone. Notably, an additional injection of αGC/OVA-loaded exosomes further augmented the treatment effects. Our findings show that exosomes loaded with protein antigen and αGC will activate adaptive immunity in the absence of triggering iNKT-cell anergy, supporting their application in the design of a broad variety of cancer immunotherapy trials., (©2013 AACR.)

Background: Asthma is characterized by increased airway narrowing in response to nonspecific stimuli. The disorder is influenced by both environmental and genetic factors. Exosomes are nanosized vesicles of endosomal origin released from inflammatory and epithelial cells that have been implicated in asthma. In this study we characterized the microRNA (miRNA) content of exosomes in healthy control subjects and patients with mild intermittent asthma both at unprovoked baseline and in response to environmental challenge., Objective: To investigate alterations in bronchoalveolar lavage fluid (BALF) exosomal miRNA profiles due to asthma, and following subway air exposure., Methods: Exosomes were isolated from BALF from healthy control subjects (n = 10) and patients with mild intermittent asthma (n = 10) after subway and control exposures. Exosomal RNA was analyzed by using microarrays containing probes for 894 human miRNAs, and selected findings were validated with quantitative RT-PCR. Results were analyzed by using multivariate modeling., Results: The presence of miRNAs was confirmed in exosomes from BALF of both asthmatic patients and healthy control subjects. Significant differences in BALF exosomal miRNA was detected for 24 miRNAs with a subset of 16 miRNAs, including members of the let-7 and miRNA-200 families, providing robust classification of patients with mild nonsymptomatic asthma from healthy subjects with 72% cross-validated predictive power (Q(2) = 0.72). In contrast, subway exposure did not cause any significant alterations in miRNA profiles., Conclusion: These studies demonstrate substantial differences in exosomal miRNA profiles between healthy subjects and patients with unprovoked, mild, stable asthma. These changes might be important in the inflammatory response leading to bronchial hyperresponsiveness and asthma., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: Tumor necrosis factor super family (TNFSF) members regulate important processes involved in cell proliferation, survival and differentiation and are therefore crucial for the balance between homeostasis and inflammatory responses. Several members of the TNFSF are closely associated with inflammatory bowel disease (IBD). Thus, they represent interesting new targets for therapeutic treatment of IBD., Methodology/principal Findings: We have used mice deficient in TNFSF member HVEM in experimental models of IBD to investigate its role in the disease process. Two models of IBD were employed: i) chemical-induced colitis primarily mediated by innate immune cells; and ii) colitis initiated by CD4(+)CD45RB(high) T cells following their transfer into immuno-deficient RAG1(-/-) hosts. In both models of disease the absence of HVEM resulted in a significant reduction in colitis and inflammatory cytokine production., Conclusions: These data show that HVEM stimulatory signals promote experimental colitis driven by innate or adaptive immune cells.

Objective: RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation., Methods and Results: Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho-myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells., Conclusions: RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers.