Thymic stromal lymphopoietin (TSLP) is a cytokine with pleiotropic functions in the immune system. It has been associated with allergic reactions in the skin and lungs but also homeostatic tolerogenic responses in the thymus and gut. In human subjects TSLP is present in 2 isoforms, short and long. Here we wanted to investigate the differential expression of the TSLP isoforms and discern their biological implications under homeostatic or inflammatory conditions. We evaluated the expression of TSLPs in tissues from healthy subjects, patients with ulcerative colitis, patients with celiac disease, and patients with atopic dermatitis and on epithelial cells and keratinocytes under steady-state conditions or after stimulation. We then tested the immune activity of TSLP isoforms both in vitro and in vivo. We showed that TSLP isoforms are responsible for 2 opposite immune functions. The short isoform is expressed under steady-state conditions and exerts anti-inflammatory activities by affecting the capacity of PBMCs and dendritic cells to produce inflammatory cytokines. Moreover, the short isoform TSLP ameliorates experimental colitis in mice and prevents endotoxin shock. The long isoform of TSLP is proinflammatory and is only expressed during inflammation. The isoforms are differentially regulated by pathogenic bacteria, such as Salmonella species and adhesive-invasive Escherichia coli. We have solved the dilemma of TSLP being both homeostatic and inflammatory. The TSLP isoform ratio is altered during several inflammatory disorders, with strong implications in disease treatment and prevention. Indeed, targeting of the long isoform of TSLP at the C-terminal portion, which is common to both isoforms, might lead to unwanted side effects caused by neutralization of the homeostatic short isoform.

Small extracellular vesicles (sEVs) present fairly distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid‐packing defects and a relative abundance in lipids such as phosphatidylserine and ceramide. sEV membrane could be then considered as a “universal” marker, alternative or complementary to traditional, characteristic, surface‐associated proteins. Here, we introduce the use of membrane‐sensing peptides as new, highly efficient ligands to directly integrate sEV capturing and analysis on a microarray platform. Samples were analysed by label‐free, single‐particle counting and sizing, and by fluorescence co‐localisation immune staining with fluorescent anti‐CD9/anti‐CD63/anti‐CD81 antibodies. Peptides performed as selective yet general sEV baits and showed a binding capacity higher than anti‐tetraspanins antibodies. Insights into surface chemistry for optimal peptide performances are also discussed, as capturing efficiency is strictly bound to probes surface orientation effects. We anticipate that this new class of ligands, also due to the versatility and limited costs of synthetic peptides, may greatly enrich the molecular toolbox for EV analysis.

Objective: Patient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies. Design: We undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions. Results: Several unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8⁺ and CD4⁺ T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8⁺ T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo. Conclusions: These results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.