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Purpose: The success of immune checkpoint inhibitors strengthens the notion that tumor growth and regression are immune regulated. To determine whether distinct tissue immune microenvironments differentially affect clinical outcome in nonsmall cell lung cancer (NSCLC), an extended analysis of PD-L1 and tumor-infiltrating lymphocytes (TIL) was performed.Experimental Design: Samples from resected adenocarcinoma (ADC 42), squamous cell carcinoma (SCC 58), and 26 advanced diseases (13 ADC and 13 SCC) treated with nivolumab were analyzed. PD-L1 expression and the incidence of CD3, CD8, CD4, PD-1, CD57, FOXP3, CD25, and Granzyme B TILs were immunohistochemically assessed.Results: PD-L1 levels inversely correlated with N involvement, although they did not show a statistically significant prognostic value in resected patients. The incidence and phenotype of TILs differed in SCC versus ADC, in which EGFR and KRAS mutations conditioned a different frequency and tissue localization of lymphocytes. NSCLC resected patients with high CD8 pos lymphocytes lacking PD-1 inhibitory receptor had a longer overall survival (OS: HR ¼ 2.268; 95% CI, 1.056-4.871, P ¼ 0.03). PD-1-to-CD8 ratio resulted in a prognostic factor both on univariate (HR ¼ 1.952; 95% CI, 1.34-3.12, P ¼ 0.001) and multivariate (HR ¼ 1.943; 95% CI, 1.38-2.86, P ¼ 0.009) analysis. Moreover, low PD-1 incidence among CD8 pos cells was a distinctive feature of nivolumab-treated patients, showing clinical benefit with a prolonged progressionfree survival (PFS: HR ¼ 4.51; 95% CI, 1.45-13.94, P ¼ 0.004).Conclusions: In the presence of intrinsic variability in PD-L1 expression, the reservoir of PD-1-negative effector T lymphocytes provides an immune-privileged microenvironment with a positive impact on survival of patients with resected disease and response to immunotherapy in advanced NSCLC.

Ribosome-inactivating proteins (RIPs) remove a specific adenine from 28S rRNA leading to inactivation of ribosomes and arrest of translation. Great interest as to a possible second physiological substrate for RIPs came from the observation that in vitro RIPs remove adenine from DNA. This paper addresses the problem of nuclear lesions induced by RIPs in human endothelial cells susceptible to the bacterial RIP Shiga toxin 1 and the plant RIP ricin. With both toxins, nuclear DNA damage as evaluated by two independent techniques (alkaline-halo assay and alkaline filter elution) appears early, concomitant with (ricin) or after (Shiga toxin 1) the inhibition of protein synthesis. At this time, the annexin V binding assay, caspase 3 activity, the formation of typical < or = 50 Kb DNA fragments, and changes in morphology associated with apoptosis were negative. Furthermore, a block of translation comparable to that induced by RIPs, but obtained with cycloheximide, did not induce nuclear damage. Such damage is consistent with the enzymatic activity (removal of adenine) of RIPs acting in vitro on RNA-free chromatin and DNA. The results unequivocally indicate that RIPs can damage nuclear DNA in whole cells by means that are not secondary to ribosome inactivation or apoptosis.

Naive T cells respond to T cell receptor (TCR) activation by leaving quiescence, remodeling metabolism, initiating expansion, and differentiating toward effector T cells. The molecular mechanisms coordinating the naive to effector transition are central to the functioning of the immune system, but remain elusive. Here, we discover that T cells fulfill this transitional process through translational control. Naive cells accumulate untranslated mRNAs encoding for glycolysis and fatty acid synthesis factors and possess a translational machinery poised for immediate protein synthesis. Upon TCR engagement, activation of the translational machinery leads to synthesis of GLUT1 protein to drive glucose entry. Subsequently, translation of ACC1 mRNA completes metabolic reprogramming toward an effector phenotype. Notably, inhibition of the eIF4F complex abrogates lymphocyte metabolic activation and differentiation, suggesting ACC1 to be a key regulatory node. Thus, our results demonstrate that translation is a direct mediator of T cell metabolism and indicate translation factors as targets for novel immunotherapeutic approaches.

Malignant pleural mesothelioma (MPM) is a progressive malignancy associated to the exposure of asbestos fibers. The most frequently inactivated tumor suppressor gene in MPM is CDKN2A/ARF, encoding for the cell cycle inhibitors p16INK4a and p14ARF, deleted in about 70% of MPM cases. Considering the high frequency of alterations of this gene, we tested in MPM cells the efficacy of palbociclib (PD-0332991), a highly selective inhibitor of cyclin-dependent kinase (CDK) 4/6. The analyses were performed on a panel of MPM cell lines and on two primary culture cells from pleural effusion of patients with MPM. All the MPM cell lines, as well as the primary cultures, were sensitive to palbociclib with a significant blockade in G0/G1 phase of the cell cycle and with the acquisition of a senescent phenotype. Palbociclib reduced the phosphorylation levels of CDK6 and Rb, the expression of myc with a concomitant increased phosphorylation of AKT. Based on these results, we tested the efficacy of the combination of palbociclib with the PI3K inhibitors NVP-BEZ235 or NVP-BYL719. After palbociclib treatment, the sequential association with PI3K inhibitors synergistically hampered cell proliferation and strongly increased the percentage of senescent cells. In addition, AKT activation was repressed while p53 and p21 were up-regulated. Interestingly, two cycles of sequential drug administration produced irreversible growth arrest and senescent phenotype that were maintained even after drug withdrawal. These findings suggest that the sequential association of palbociclib with PI3K inhibitors may represent a valuable therapeutic option for the treatment of MPM.