Increased shedding of extracellular vesicles (EVs)—small, lipid bilayer-delimited particles with a role in paracrine signalling—has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a⁺ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a⁺ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a⁺ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. We identified circulating CD172a⁺ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients. Cermaides contained in CD172⁺ extracellular vesicles that are released by cardiomyocytes upon hypoxic stress may exert a positive inotropic effect on unstressed areas of the heart (top), and aortic stenosis patients with higher counts of these vesicles have better prognosis for valve replacement (bottom).

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis and it is characterized by predominant pro-tumor Th2-type inflammation. T follicular helper (Tfh) cells are relevant immunoregulators in cancer, and often correlate with better survival. How the Th2-skewed microenvironment in PDAC modulates the differentiation of Tfh cells and their immunoregulatory function is unknown. We carried out high-dimensional flow cytometry and T-cell receptor- and RNA-sequencing, as well as bioinformatics, immunohistochemistry and in vitro mechanistic studies. We identified Tfh1-, Tfh2-, and Tfh17-like cell clusters in the blood, tumors and tumor-draining lymph-nodes (TDLNs) of chemo-naïve PDAC patients and showed that high percentages of Tfh2 cells within the tumor tissue and TDLNs correlated with reduced patient survival. Moreover, only Tfh2 cells were highly activated and were reduced in frequency in patients who responded to neoadjuvant chemotherapy. RNA-sequencing analysis of immunoglobulin expression showed that tumor and TDLN samples expressed all immunoglobulin (IGH) isotypes apart from IGHE. Consistent with these findings, Tfh2 cells differentiated in vitro by tumor microenvironment-conditioned dendritic cells promoted the production of anti-inflammatory IgG4 antibodies by co-cultured B cells, dependent on IL-13. Moreover, unexpectedly, Tfh2 cells inhibited the secretion of pro-inflammatory IgE, dependent on prostaglandin E₂. Our results indicate that in PDAC, highly activated pro-tumor Tfh2 favor anti-inflammatory IgG4 production, while inhibit pro-inflammatory IgE. Thus, targeting the circuits that drive Tfh2 cells, in combination with chemotherapy, may re-establish beneficial anti-tumor Tfh–B cell interactions and facilitate more effective treatment. Research grants from the 10.13039/501100005010 Italian Association for Cancer Research (AIRC) IG-19119 to MPP and the AIRC Special Program in Metastatic disease: the key unmet need in oncology, 5 per Mille no. 22737 to CB, MF, CD, MR and MPP; the ERA-NET EuroNanoMed III (a collaborative european grant financed by the Italian Ministry of Health, Italy) project PANIPAC (JTC2018/041) to MPP; the Fondazione Valsecchi to SC.

Immunological consequences of endoscopic ultrasound (EUS)-local thermal ablation (LTA) for pancreatic ductal adenocarcinoma (PDAC) have not been extensively assessed. We aimed to explore EUS-LTA effects on the systemic immune response in PDAC. Peripheral blood was collected from 10 treatment-naïve patients with borderline resectable and locally advanced PDAC, randomly allocated to Nab-paclitaxel plus Gemcitabine chemotherapy (CT-arm, n = 5) or EUS-LTA with HybridTherm Probe plus CT (HTP + CT-arm, n = 5). Twenty healthy donors were included as controls. Flow-cytometry and multiplex assays were used to profile immune cell subsets and measure serum cytokines/chemokines, respectively. At baseline, PDAC patients showed increased circulating monocytes and lower circulating lymphocytes and CD19+ B cells counts compared to healthy controls. After 4 months, CT induced decrease of B regulatory cells, CD4+ cytotoxic T cells and IL-1β. The addition of EUS-HTP to CT selectively decreased the serum levels of APRIL/TNFSF13 as well as T regulatory cells, total, classic and inflammatory monocytes. Serum levels of APRIL/TNFSF13 and total, classic and inflammatory monocytes counts at baseline were associated with worse overall survival. EUS-HTP has the potential to selectively impact on immune cells and cytokines associated with poor outcomes in PDAC.

We describe a multi-step high-dimensional (HD) flow cytometry workflow for the deep phenotypic characterization of T cells infiltrating metastatic tumor lesions in the liver, particularly derived from colorectal cancer (CRC-LM). First, we applied a novel flow cytometer setting approach based on single positive cells rather than fluorescent beads, resulting in optimal sensitivity when compared with previously published protocols. Second, we set up a 26-color based antibody panel designed to assess the functional state of both conventional T-cell subsets and unconventional invariant natural killer T, mucosal associated invariant T, and gamma delta T (γδT)-cell populations, which are abundant in the liver. Third, the dissociation of the CRC-LM samples was accurately tuned to preserve both the viability and antigenic integrity of the stained cells. This combined procedure permitted the optimal capturing of the phenotypic complexity of T cells infiltrating CRC-LM. Hence, this study provides a robust tool for high-dimensional flow cytometry analysis of complex T-cell populations, which could be adapted to characterize other relevant pathological tissues.

Regulatory T-cells (Tregs) maintain immune homeostasis, but antigens activating adaptive Tregs in human pathologies are ill-defined. EOMES ⁺ type-1 regulatory (Tr1)-like T-cells had a dysregulated homeostasis in multiple sclerosis (MS), which was related to their activation in the central nervous system (CNS). EOMES ⁺ Tr1-like cells were enriched and clonally expanded in patient’s cerebrospinal fluid (CSF) and were the major IL-10-producing T-cell subset in the CNS. Regulatory T-cells from PwMS produced IL-10 and IFN-γ with antigens derived from Epstein-Barr Virus (EBV), but not from myelin. EOMES ⁺ Tr1-like cells responded selectively to the latency-associated antigen EBNA1, whereas FOXP3 ⁺ Tregs and Th1-cells responded also to lytic EBV antigens. EBNA1-specific EOMES ⁺ Tr1-like cells were present in patients carrying the HLA-DRB1*15:01 risk allele, were associated with anti-EBNA1 IgG and disappeared upon therapeutic B-cell depletion. IL-10 ⁺ EOMES ⁺ Tr1-like were present in MS brain lesions, and some were in the vicinity of EBV-infected B-cells and CD8 ⁺ T-cells. Notably, EOMES ⁺ Tr1-like cells suppressed CD8+T-cell activation by EBV-infected B-cells. We propose that the insufficient protective functions of Tregs in MS are due to their aberrant anti-viral specificities that promote immune escape of a disease-associated virus.