Tumor onset is accelerated in TRAMP mice lacking iNKT cells.

<p>Age- and sex-matched male TRAMP (white bars; n = 97), TRAMPJα18^−/− (black bars; n = 51) and WT mice (gray bars; n = 97) were killed at the indicated week after birth and the anatomy and histology of the UGA was analyzed as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008646#s2" target="_blank">Material and Methods</a> section. Animals affected by neuroendocrine tumors were excluded. A, UGA weight (g), expressed as average±SEM, of TRAMP, TRAMPJα18^−/− and WT mice killed at weeks 6–8 (n = 5, 8 and 19, respectively), 12–16 (n = 16, 17 and 26), 17–24 (n = 30, 15 and 26) and 25–28 (n = 46, 11 and 26), respectively. B, ratio of the weight of UGA and body weight subtracted of the UGA in the groups of animals reported above. C, Disease score, expressed as average±SEM, of the groups of animals reported above. Statistical analysis of collected data was performed using the Student's <i>t</i>-test (A and B) and the Mann-Whitney test (C); ***p<0.001, **0.001</p

Survival of TRAMP mice lacking iNKT cells is reduced.

<p>Kaplan-Maier plot reporting the survival curves of groups of male TRAMP (black squares; n = 21) and TRAMPJα18^−/− mice (black circles; n = 21). Animals were examined twice a week and euthanized when signs of bulky prostate tumor and/or distress were manifest. At necropsy, the anatomy and histology of the UGA was analyzed as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008646#s2" target="_blank">Material and Methods</a> section. Animals were attributed a disease score≥4. Statistical comparison (Log-Rank test) between the survival curves: p<0.0001.</p

αGal-Cer-mediated release of iNKT-associated cytokines.

<p>αGalCer (2 µg/mouse) was administered i.v. to TRAMP (white circles) and WT (black circles) littermates, and blood samples were collected from the tail vein 2, 6 and 24 hours later, and the serum content of IL-4 and IFN-γ was determined by standard ELISA. Values are reported as concentration of the cytokine (pg/ml) in the sera of each animal (3–4/experimental group) analyzed at the indicated time points. Reported data are representative of at least two independent experiments.</p

Presentation_1_Circulating CD81-expressing extracellular vesicles as biomarkers of response for immune-checkpoint inhibitors in advanced NSCLC.pptx

PD-L1 in tumor cells is the only used biomarker for anti PD1/PD-L1 immune-checkpoints inhibitors (ICI) in Non Small Cell Lung Cancer (NSCLC) patients. However, this parameter is inaccurate to predict response, especially in patients with low tumor PD-L1. Here, we evaluated circulating EVs as possible biomarkers for ICI in advanced NSCLC patients with low tumoral PD-L1. EVs were isolated from plasma of 64 PD-L1 low, ICI-treated NSCLC patients, classified either as responders (R; complete or partial response by RECIST 1.1) or non-responders (NR). EVs were characterized following MISEV guidelines and by flow cytometry. T cells from healthy donors were triggered in vitro using patients’ EVs. Unsupervised statistical approach was applied to correlate EVs’ and patients’ features to clinical response. R-EVs showed higher levels of tetraspanins (CD9, CD81, CD63) than NR-EVs, significantly associated to better overall response rate (ORR). In multivariable analysis CD81-EVs correlated with ORR. Unsupervised analysis revealed a cluster of variables on EVs, including tetraspanins, significantly associated with ORR and improved survival. R-EVs expressed more costimulatory molecules than NR-EVs although both increased T cell proliferation and partially, activation. Tetraspanins levels on EVs could represent promising biomarkers for ICI response in NSCLC.</p

Table_1_Circulating CD81-expressing extracellular vesicles as biomarkers of response for immune-checkpoint inhibitors in advanced NSCLC.docx

PD-L1 in tumor cells is the only used biomarker for anti PD1/PD-L1 immune-checkpoints inhibitors (ICI) in Non Small Cell Lung Cancer (NSCLC) patients. However, this parameter is inaccurate to predict response, especially in patients with low tumor PD-L1. Here, we evaluated circulating EVs as possible biomarkers for ICI in advanced NSCLC patients with low tumoral PD-L1. EVs were isolated from plasma of 64 PD-L1 low, ICI-treated NSCLC patients, classified either as responders (R; complete or partial response by RECIST 1.1) or non-responders (NR). EVs were characterized following MISEV guidelines and by flow cytometry. T cells from healthy donors were triggered in vitro using patients’ EVs. Unsupervised statistical approach was applied to correlate EVs’ and patients’ features to clinical response. R-EVs showed higher levels of tetraspanins (CD9, CD81, CD63) than NR-EVs, significantly associated to better overall response rate (ORR). In multivariable analysis CD81-EVs correlated with ORR. Unsupervised analysis revealed a cluster of variables on EVs, including tetraspanins, significantly associated with ORR and improved survival. R-EVs expressed more costimulatory molecules than NR-EVs although both increased T cell proliferation and partially, activation. Tetraspanins levels on EVs could represent promising biomarkers for ICI response in NSCLC.</p