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La Lombardia è la regione italiana con il maggior numero di over 65enni: oltre 2 milioni, pari al 22,9% della popolazione (Istat, 2020). Molti soffrono di una o più patologie croniche, incidendo per il 70% sulla spesa sanitaria regionale (Dgr 4662/2015). Le evidenze di letteratu-ra indicano la sedentarietà tra le cause che concorrono allo sviluppo di patologie croniche e sottolineano la necessità di politiche e programmi per promuovere l'attività fisica per favorire l'invecchiamento attivo e in buona salute (Active and Healthy Ageing). In tal senso, dal 2009 Regione Lombardia ha individuato i Gruppi di Cammino come programma principe per pro-muovere e mantenere la salute della popolazione over 65, inserendolo nei propri Piani Regio-nali Prevenzione. Il programma consiste in un'attività organizzata dove un gruppo di persone si ritrova per camminare insieme, almeno due volte a settimana, seguendo un percorso urbano o extra-urbano, sotto la guida di un conduttore (Walking leader). Rappresentano una strategia capace di promuovere il movimento di adulti e anziani in sicurezza, la conoscenza di temi di salute, la partecipazione dei cittadini e le loro abilità sociali, rispondendo ai principi chiave delle politiche di promozione della salute e di Active Ageing (Oms, 2002). L'articolo, attraverso la descrizione dei differenti passaggi adottati per l'implementazione del programma secondo la lente dell'Interactive System Framework, darà evidenza di come il programma Gruppi di Cammino sia una strategia che ad oggi promuove il movimento di oltre 18600 over 65.

Supplementary figure legends

Mechanism-based strategies to overcome resistance to PD-1 blockade therapy are urgently needed. We developed genetic acquired resistant models of JAK1, JAK2, and B2M loss-of-function mutations by gene knockout in human and murine cell lines. Human melanoma cell lines with JAK1/2 knockout became insensitive to IFN-induced antitumor effects, while B2M knockout was no longer recognized by antigen-specific T cells and hence was resistant to cytotoxicity. All of these mutations led to resistance to anti–PD-1 therapy in vivo. JAK1/2-knockout resistance could be overcome with the activation of innate and adaptive immunity by intratumoral Toll-like receptor 9 agonist administration together with anti–PD-1, mediated by natural killer (NK) and CD8 T cells. B2M-knockout resistance could be overcome by NK-cell and CD4 T-cell activation using the CD122 preferential IL2 agonist bempegaldesleukin. Therefore, mechanistically designed combination therapies can overcome genetic resistance to PD-1 blockade therapy.Significance:The activation of IFN signaling through pattern recognition receptors and the stimulation of NK cells overcome genetic mechanisms of resistance to PD-1 blockade therapy mediated through deficient IFN receptor and antigen presentation pathways. These approaches are being tested in the clinic to improve the antitumor activity of PD-1 blockade therapy.This article is highlighted in the In This Issue feature, p. 1079

IGV plot of RNA-Seq from YUMM1.1, YUMM1.7 and YUMM2.1; western blot analysis of cytoplasmic and nuclear beta-catenin in YUMM1.7 and YUMM2.1 cell lines with or without exposure to 10 uM 4HT for 48 hours; top-flash activity of total beta-catenin in YUMM1.7 and YUMM2.1 with or without exposure to 10uM 4HT for 48 hours; representative immunofluorescence of beta-catenin stained non-treated tumors.

Supplementary Figure 1. Exome sequencing and copy number changes in M431 and corresponding whole-tumor biopsy. Supplementary Figure 2. Mutations in antigen presentation machinery from anti-PD1 treated melanoma cohort. Supplementary Figure 3. Immunohistochemistry (IHC) analysis of biopsies from melanoma metastases with a JAK1 loss of function mutation. Supplementary Figure 4. Selection of PD-L1 flow cytometry antibody and impact of phosphatase inhibitor as well as temperature on measuring surface PD-L1 expression and pAKT. Supplementary Figure 5. Flow cytometry gating strategy and dose response curve of interferon alpha, beta and gamma to determine the optimal concentrations. Supplementary Figure 6. Time course of PD-L1 surface expression upon interferon alpha, beta and gamma treatment for selected cell lines to determine the optimal time point for the screening. Supplementary Figure 7. Time course of PD-L1 expression upon interferon alpha, beta or gamma treatment for the cell lines with poor or no up-regulation upon 18 hours exposure. Supplementary Figure 8. Interferon signaling pathway in good and poorly responding cell lines. Supplementary Figure 9. PD-L1 expression upon interferon alpha and beta exposure. Supplementary Figure 10. Predicted functional consequences of M368 JAK2 D313 splice site mutation. Supplementary Figure 11. Immunohistochemistry (IHC) analysis of biopsies from melanoma metastases with a JAK1 loss of function mutation. Supplementary Figure 12. JAK1 wild-type lentiviral vector transduction. Supplementary Figure 13. Mutations in antigen presentation machinery from anti-PD1 treated colorectal cohort. Supplementary Figure 14. DNA damage repair gene mutations in endometrial cancer cell lines with JAK1/2 mutations. Supplementary Figure 15. Frequency of JAK1 and JAK2 alterations and their association with overall survival in additional TCGA datasets. Supplementary Figure 16. Functional effect of genetic loss of reactive PD-L1 on responses to PD-1 blockade. Supplementary Database 1. Whole exome sequencing of 23 baseline biopsies from patients analyzed in Fig. 1, and whole exome sequencing of the M431 cell line (Accession code: SRP067938).

IGV plot exome sequencing from YUMM1.1, YUMM1.7 and YUMM2.1, tumor growth curve of YUMM1.7 and B16 with 4 mice in each group, analysis of the non-synonymous mutational load compared to a strain-matched normal with known dbSNP variants excluded.

Gating strategy of CD11c+B220-, CD11c+B220+, CD11c+B220-CD8+ and CD11c+B220-CD103+ cells; gating strategy of CD11b+MHC-IIhigh DCs in CD11c+ cells; gating strategy of CD11b+F4/80+TAMs, CD11b+F4/80+MHC-IIlow TAMs and CD11b+F4/80+MHC-IIhigh TAMs; gating strategy of MO-MDSC (CD11b+Ly6ChighLy6Glow) and PMN-MDSC (CD11b+Ly6ClowLy6Ghigh); gating strategy of Tregs (CD4+CD25+FoxP3+); corresponding normalized enrichment scores (NES), P values and false discovery rates (FDR) of the GSEA plots for YUMM2.1 versus YUMM1.1 enriched pathways involved in immune response, cytokine production and inflammatory response.

Quantification of CD3+CD8+ (CD8 T-cells) and (B) CD3+CD4+ (CD4 T-cells) in both tumors and spleens in YUMM2.1; gating strategy after exclusion of dead cells of CD3+CD8+ and CD3+CD4+ cells after gating for CD3+ cells; quantification of CD3+CD8+ (CD8 T-cells) in MC38 and YUMM2.1 spleens; CD3+CD4+ (CD4 T-cells) in MC38 and YUMM 2.1 spleens CD8 T-cells in both tumors and spleens in YUMM1.1.

The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues, and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor–host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN−/−). Anti–PD-1 or anti–PD-L1 therapy engendered strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. PD-L1 expression did not differ between the three models at baseline or upon interferon stimulation. Whereas mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T cells, as well as CD28 and CD80/86 costimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor-associated macrophages in the tumors after PD-1 blockade. Compared with YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in expression of chemokine-trafficking genes that are related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells and costimulation that is mediated by dendritic cells and macrophages. Cancer Immunol Res; 4(10); 845–57. ©2016 AACR.

Supplementary Figures

Supplemental Figure 1: Hypothetical model of peripheral blood TCR-transgenic cell repopulation. Supplemental Figure 2: GLP team organizational chart. Supplemental Figure 3. Bone marrow transplant (BMT) optimization studies in HLA-A2/Kb transgenic mice. Supplemental Figure 4. Body weight and hematology assessment at day 5 and 3 months after BMT. Supplemental Figure 5. Spleen and bone marrow cellularity at day 5 and 3 months after BMT. Supplemental Figure 6. Serum chemistry at 3 months after BMT. Supplemental Figure 7. Flow cytometry gating strategy for bone marrow and splenocytes phenotype characterization. Supplemental Figure 8. Survival and hematology 3 months after BMT with Lin- cells transduced with LV-empty, LV-NY-ESO-1 TCR or LV-NY-ESO-1 TCR/sr39TK. Supplemental Figure 9. Differentiation of NYESO TCR/sr39TK-engineered T cells in ATOs (artificial thymic organoids). Supplemental Table 1. Certificate of Analysis of the GMP-comparable LV-NY-ESO-1 TCR/sr39TK (RRL-MSCV-optNYESO-optsr39TK-WPRE, production volume: 20L). Supplemental Table 2. Certificate of Analysis of the Clinical Grade Lentivirus LV-NY-ESO- 1 TCR/sr39TK (RRL-MSCV-optNYESO-optsr39TK-WPRE, production volume 60L). Supplemental Table 3. Certificate of Analysis of the clinical grade RV-NY-ESO-1 TCR (MSGV1-A2aB-1G4A-LY3H10) (Production volume 18L)*. Supplemental Table 4. Certificate of Analysis of the GMP comparable RV-NY-ESO-1 TCR (MSGV1-A2ab-1G4A-Ly3H10, Production volume: 3L). Supplemental Table 5. Co-Administration of NY-ESO-1 TCR Genetically Modified T cells and Hematopoietic Stem Cells (HSCs) in HLA-A2.1/Kb mice. GLP studies cohort distribution. Supplemental Table 6. Cell manufacturing acceptance criteria. Supplemental Table 7. List of protocol-specific organs. Supplemental Table 8. Manufacturing validation runs. Supplemental Table 9. Comparison between fresh and cryopreserved product at 1, 30, 90 and 180# days.

Purpose:To improve persistence of adoptively transferred T-cell receptor (TCR)–engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application.Experimental Design:HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)–compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)–compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use.Results:TCR genetically modified and ex vivo–cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality.Conclusions:Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.

We developed a new Italian short version of the Geriatric Anxiety Scale (GAS-12) and evaluated its psychometric properties. The GAS-12 specifically screens for anxiety symptoms in the Italian older adult population by identifying items that best discriminate anxiety in this population.In Study 1, we administered the full-length Italian translation of the GAS to 517 older adults and used item response theory to identify the most discriminating items and to develop the short form used in Study 2. In Study 2, we evaluated the functioning of the new short form of the questionnaire in a new sample of 427 older adults using Confirmatory factor analysis.Analyses indicated 12 items that discriminated well between anxious and non-anxious participants and distributed along the latent continuum of each trait. The GAS-12 fits a three-factor structure. There was also evidence for convergent and divergent validity.The Italian GAS-12 appears to be a useful instrument for the quantitative screening of anxiety in Italian older adults.Anxiety imposes significant impairment thus making imperative the screening and assessment of anxiety symptoms. The GAS-12 is particularly indicated with limited time and many scales in a clinical assessment or research protocols.

L'empowerment di comunità è una strategia centrale per la promozione della salute. Obiet-tivo del case-study è analizzare il ruolo delle "Comunità Competenti" nei programmi lombardi di prevenzione e contrasto al Gioco d'Azzardo Patologico. Identificando cinque principi chiave emersi dai documenti "Salute in tutte le politiche" e "Tutti per la salute" si analizzerà la concretizzazione delle politiche lombarde in tema GAP, declinate localmente dalle Agenzie di Tutela della Salute (ATS). Sarà evidenziato come l'azione di governance e il coinvolgimento intersettoriale, la condi-visione di buone pratiche, la co-costruzione di interventi incrementino conoscenze, percezione di controllo e motivazioni delle comunità, estendendo il loro impatto anche ad altri determinanti di salute.

Italy faced the covid-19 emergency through the so-called “mitigation” approach (Giarelli & Vicarelli, 2020), having a significant impact on health conditions and people's behaviours, like gambling. Gambling is a growing phenomenon affecting about 36.4% of the Italian population with implications on social relations, economy and public health. Specifically, during the lockdown period, the access to Italian on-site game rooms was reduced (Prime Ministerial Decree of 8/3/20) by limiting game opportunities, except for online ones. This fact led to a change in gambling, highlighting the importance of both online gambling and the development of public prevention measures. The paper aims to study how environmental variables, such as Covid-19 containment measures, could affect gambling behaviour and whether this may have an impact on public policies. The study was carried out through a comparative analysis of scientific articles, regulations and statistical data about how gambling change during the Covid-19 pandemic. Unlike the international context, there are not neither scientific articles nor official data about the development of gambling behaviour during the lockdown in Italy yet, except for a research conducted by CNR’s Institute of Clinical Physiology. Findings show just a little portion of onsite gamblers moved to online gambling offer. However, unofficial data about gambling consumption show opposite results, i.e. it occurred a significant onsite gambling reduction in favour of an increase of online activity. Furthermore, since June data show a growth in gambling behaviour, thus, together with online values, gambling consumption does not seem to be reduced in the first quarter of 2020. In conclusion, limited official data show gambling is a complex and widespread phenomenon embedded in Italy, which hasn’t suffered a significant decrease despite all the changes occurred. Moreover, the pandemic highlighted the importance of online gambling, which needs to be more considered from public health policies.

The biopsychosocial paradigm is a model of care that has been proposed in order to improve the effectiveness of health care by promoting collaboration between different professions and disciplines. However, its application still faces several issues. A quantitative-qualitative survey was conducted on a sample of general practitioners (GPs) from Milan, Italy, to investigate their attitudes and beliefs regarding the role of the psychologist, the approach adopted to manage psychological diseases, and their experiences of collaboration with psychologists. The results show a partial view of the psychologist’s profession that limits the potential of integration between medicine and psychology in primary care. GPs recognized that many patients (66%) would often benefit from psychological intervention, but only in a few cases (9%) were these patients regularly referred to a psychologist. Furthermore, the referral represents an almost exclusive form of collaboration present in the opinions of GPs. Only 8% of GPs would consider the joint and integrated work of the psychologist and doctor useful within the primary health care setting. This vision of the role of psychologists among GPs represents a constraint in implementing a comprehensive primary health care approach, as advocated by the World Health Organization.

Defects in tumor-intrinsic interferon (IFN) signaling result in failure of immune checkpoint blockade (ICB) against cancer, but these tumors may still maintain sensitivity to T cell–based adoptive cell therapy (ACT). We generated models of IFN signaling defects in B16 murine melanoma observed in patients with acquired resistance to ICB. Tumors lacking Jak1 or Jak2 did not respond to ICB, whereas ACT was effective against Jak2(KO) tumors, but not Jak1(KO) tumors, where both type I and II tumor IFN signaling were defective. This was a direct result of low baseline class I major histocompatibility complex (MHC I) expression in B16 and the dependency of MHC I expression on either type I or type II IFN signaling. We used genetic and pharmacologic approaches to uncouple this dependency and restore MHC I expression. Through independent mechanisms, overexpression of NLRC5 (nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain containing 5) and intratumoral delivery of BO-112, a potent nanoplexed version of polyinosinic:polycytidylic acid (poly I:C), each restored the efficacy of ACT against B16-Jak1(KO) tumors. BO-112 activated double-stranded RNA (dsRNA) sensing (via protein kinase R and Toll-like receptor 3) and induced MHC I expression via nuclear factor κB, independent of both IFN signaling and NLRC5. In summary, we demonstrated that in the absence of tumor IFN signaling, MHC I expression is essential and sufficient for the efficacy of ACT. For tumors lacking MHC I expression due to deficient IFN signaling, activation of dsRNA sensors by BO-112 affords an alternative approach to restore the efficacy of ACT.

Mechanism-based strategies to overcome resistance to PD-1 blockade therapy are urgently needed. We developed genetic acquired resistant models of JAK1, JAK2, and B2M loss-of-function mutations by gene knockout in human and murine cell lines. Human melanoma cell lines with JAK1/2 knockout became insensitive to IFN-induced antitumor effects, while B2M knockout was no longer recognized by antigen-specific T cells and hence was resistant to cytotoxicity. All of these mutations led to resistance to anti¿PD-1 therapy in vivo. JAK1/2-knockout resistance could be overcome with the activation of innate and adaptive immunity by intratumoral Toll-like receptor 9 agonist administration together with anti¿PD-1, mediated by natural killer (NK) and CD8 T cells. B2M-knockout resistance could be overcome by NK-cell and CD4 T-cell activation using the CD122 preferential IL2 agonist bempegaldesleukin. Therefore, mechanistically designed combination therapies can overcome genetic resistance to PD-1 blockade therapy. Significance: The activation of IFN signaling through pattern recognition receptors and the stimulation of NK cells overcome genetic mechanisms of resistance to PD-1 blockade therapy mediated through deficient IFN receptor and antigen presentation pathways. These approaches are being tested in the clinic to improve the antitumor activity of PD-1 blockade therapy., This study was funded in part by the Parker Institute for Cancer Immunotherapy, NIH grants R35 CA197633 and P01 CA244118, the Ressler Family Fund, and support from Ken and Donna Schultz (all to A. Ribas). D.Y. Torrejon was supported by a Young Investigator Award from ASCO, a grant from the Spanish Society of Medical Oncology for Translational Research in Reference Centers, and the V Foundation-Gil Nickel Family Endowed Fellowship in Melanoma Research. It has been developed within the framework of a medical doctorate at the Autonomous University of Barcelona. G. Abril-Rodriguez was supported by the Isabel & Harvey Kibel Fellowship Award and the Alan Ghitis Fellowship Award for Melanoma Research. J. Tsoi and K.M. Campbell were supported by the NIH Ruth L. Kirschstein Institutional National Research Service Award #T32-CA009120 and the UCLA Tumor Immunology Training Grant (T32CA009120). G. Parisi was supported by the V Foundation-Gil Nickel Family Endowed Fellowship in Melanoma Research. J.M. Zaretsky was in the UCLA Medical Scientist Training Program supported by NIH training grant GM08042. S. Hu-Lieskovan was supported by a Conquer Cancer Foundation ASCO Career Development Award, a UCLA KL2 Translational Research Award, and a Melanoma Research Alliance Young Investigator Award. Flow and mass cytometry were performed in the UCLA Jonsson Comprehensive Cancer Center (JCCC) and Center for AIDS Research Flow Cytometry Core Facility that is supported by NIH awards P30 CA016042 and 5P30 AI028697, and by the JCCC, the UCLA AIDS Institute, and the David Geffen School of Medicine at UCLA. The purchase of the Helios mass cytometer that was used in this work was supported, in part, by funds provided by the James B. Pendleton Charitable Trust. We want to thank Dr. Cristiana Guiducci from Dynavax and Drs. Deborah Charych and Willem Overwijk from Nektar Therapeutics for helpful guidance in the performance of in vivo studies with SD-101 and bempegaldesleukin, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Interleukin-2 (IL-2) is a component of most protocols of adoptive cell transfer (ACT) therapy for cancer, but is limited by short exposure and high toxicities. NKTR-214 is a kinetically-engineered IL-2 receptor βγ (IL-2Rβγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rβγ. We report that ACT supported by NKTR-214 increases the proliferation, homing and persistence of anti-tumor T cells compared to ACT with IL-2, resulting in superior antitumor activity in a B16-F10 murine melanoma model. The use of NKTR-214 increases the number of polyfunctional T cells in murine spleens and tumors compared to IL-2, and enhances the polyfunctionality of T and NK cells in the peripheral blood of patients receiving NKTR-214 in a phase 1 trial. In conclusion, NKTR-214 may have the potential to improve the antitumor activity of ACT in humans through increased in vivo expansion and polyfunctionality of the adoptively transferred T cells., Adoptive cell transfer (ACT) of T cells for tumor treatment often requires IL-2 administration. Here, the authors show that a modified IL-2 cytokine (NKTR-214) can outperform IL-2 in a melanoma mouse model.

Production of high-quality sparkling wines relies on an optimal balance between sugar and acidity in the fruit at harvest. Warming trends favor a too-fast sugar increase at the expense of adequate acidity. The removal of photosynthetically active leaves from the distal part of the canopy around veraison has been proposed as a simple technique to delay sugar accumulation. Over three seasons (2015 to 2017), we compared the effects of mechanical mid-shoot leaf removal in the white variety Ortrugo, either performed before veraison (PRE-LR) or at a total soluble solids (TSS) concentration of ~12 Brix (POST-LR), with non-defoliated vines (C). PRE-LR shifted the sugar increase in all the three seasons, but the effects held until harvest only in 2015 and 2016 (−1.1 Brix and −1.4 Brix, respectively, if compared to C). Acidity was unaffected. POST-LR was less effective in changing the composition of grapes during ripening. Both leaf removal treatments reduced berry size (−5%) and yield (−19% PRE-LR and −14% POST-LR, in comparison with C). Finally, the treatments changed the berry morphology and the proportion between its components, with some peculiarities due to the seasons, but the relative skin mass generally increased. Overall, the efficacy of the technique was below expectations, primarily because the delay in general ripening expected from removing a significant portion of the mature and functioning leaves was offset by a decrease in malic acid due to excess heat summation for this variety.

There is a critical need for more effective therapy for acute myelogenous leukemia (AML). Although many patients achieve remission, most relapse with poor outcomes. Even after allogeneic Stem Cell Transplantation (SCT), 30-50% of patients relapse due to the persistence of residual disease. To address the poor immunogenicity of AML cells and the diminished immune responsiveness of patients, our candidate autologous AML vaccine is lentivirally engineered, in each patient's leukemic cells, to express CD80, IL-15, and IL-15 Receptor alpha (IL-15Rα). In prior studies in a syngeneic 32Dp210 murine AML model, CD80-mediated co-stimulation of T-cells combined with immune activation by the IL-15/IL-15Rα heterodimer showed unprecedented synergy in induction of anti-leukemic cytolytic activity (Shi, Y. et al, 2018). This was observed in both ex vivo co-culture and in vivo where vaccinated leukemic mice had >80% cure rates. No local skin, organ, or systemic toxicity was observed, nor was there evidence of systemic cytokine release of IL-6 or TNFα after SC or IV injection of up to 10 8 transduced irradiated AML cells. We confirmed the feasibility of producing patient-derived AML vaccines by transduction of 16 independent AML samples with a tri-cistronic lentiviral vector (TLV) that contains human CD80, IL-15 and IL-15Rα. Transduction levels were 11-71% of cells (median 38.6%). To define the minimum transduction level required for PBMC activation and to assess synergy of co-expressed human CD80, IL-15, and IL-15Rα, allogenic U937 leukemia cells were initially used as stimulators. Transduced U937 (U937-TLV) had high-level surface expression of CD80 and IL-15, secreted IL-15 (7 ng/ml/24 hours from 2 x 10 6 cells/ml) and activated CD3+ T-cells from an AML patient (Fig.1). Mixtures of irradiated U937-TLV with non-transduced U937 were created at fixed ratios (100%, 80%, 40%, 20%, 10%, 5%, 0%) for overnight co-culture with patient PBMC. At 24 hours, the T-cells were analyzed for activation by measurement of the frequency of CD69+ CD4 or CD8 T cells (Fig. 1), normalized to expression of unstimulated PBMC (0%) and the percentage of maximal CD69 expression with 100% U937-TLV (100%). Background levels of activation due to the presence of allogenic U937 were negligible. Co-culture with as little as 10% transduced U937-TLV reliably activated patient T-cells. To assess the synergy of CD80, IL-15 and IL-15Rα expression, parallel experiments were performed with PBMC co-cultured in IL-15 containing supernatants from U937-TLV cells (Fig. 1). The frequencies of activated T-cells were significantly higher after co-culture with U937/U937-TLV cells than after stimulation with IL-15-containing supernatants from similar ratios of U937/U937-TLV, confirming the synergy of CD80 and IL-15/IL-15Rα in the transduced cells. To better, model the clinical setting, we assessed induction of immune responses of patient T cells to autologous transduced AML. PBMC were stimulated with transduced or non-transduced autologous AML cells vs stimulation with allogeneic U937-TLV, or with anti-CD3/CD28 beads to define maximal stimulation. Negative controls included culture of PBMC alone. All patients had T-cell activation, as measured by induction of CD69, HLA-DR and CD95 (Fas) expression, although there was heterogeneity in the nature of responses, e.g., disparate induction of the markers in individual patients (Fig. 2A and B). Induction of cytotoxic effector pathways was confirmed by detection of CD178 (FasL) and perforin expression (Figure 2C and D). Overall, all patients' PBMC had the capacity to mount T-cell responses of similar magnitude to both allogeneic U937-TLV and autologous vaccine. These studies establish that autologous AML cells transduced with CD80, IL-15 and IL-15Rα can elicit specific anti-leukemic T-cell responses, even in the face of prior lymphodepleting chemotherapy. A strength of this autologous vaccine strategy is that it is agnostic to which AML proteins are immunogenic for each patient. Although uniformly detected, there was heterogeneity in the induction of activation markers and effector pathways, which may reflect host and/or disease-related differences. The mechanisms underlying differences in the nature of responses in patients will be important to understand and will provide the basis for future immune correlative studies for our Phase 1 vaccine trial in transplant ineligible AML patients. Figure 1 Figure 1. Disclosures Kohn: Lyrik Therapeutics: Membership on an entity's Board of Directors or advisory committees; MyoGene Bio: Membership on an entity's Board of Directors or advisory committees; ImmunoVec: Membership on an entity's Board of Directors or advisory committees; Pluto Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; UC Regents: Patents & Royalties; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Sangamo Biosciences: Membership on an entity's Board of Directors or advisory committees.

Purpose: To improve persistence of adoptively transferred T-cell receptor (TCR)–engineered T cells and durable clinical responses, we designed a clinical trial to transplant genetically-modified hematopoietic stem cells (HSCs) together with adoptive cell transfer of T cells both engineered to express an NY-ESO-1 TCR. Here, we report the preclinical studies performed to enable an investigational new drug (IND) application. Experimental Design: HSCs transduced with a lentiviral vector expressing NY-ESO-1 TCR and the PET reporter/suicide gene HSV1-sr39TK and T cells transduced with a retroviral vector expressing NY-ESO-1 TCR were coadministered to myelodepleted HLA-A2/Kb mice within a formal Good Laboratory Practice (GLP)–compliant study to demonstrate safety, persistence, and HSC differentiation into all blood lineages. Non-GLP experiments included assessment of transgene immunogenicity and in vitro viral insertion safety studies. Furthermore, Good Manufacturing Practice (GMP)–compliant cell production qualification runs were performed to establish the manufacturing protocols for clinical use. Results: TCR genetically modified and ex vivo–cultured HSCs differentiated into all blood subsets in vivo after HSC transplantation, and coadministration of TCR-transduced T cells did not result in increased toxicity. The expression of NY-ESO-1 TCR and sr39TK transgenes did not have a detrimental effect on gene-modified HSC's differentiation to all blood cell lineages. There was no evidence of genotoxicity induced by the lentiviral vector. GMP batches of clinical-grade transgenic cells produced during qualification runs had adequate stability and functionality. Conclusions: Coadministration of HSCs and T cells expressing an NY-ESO-1 TCR is safe in preclinical models. The results presented in this article led to the FDA approval of IND 17471.

Engineering cytokine-receptor pairs Interleukin-2 (IL-2) is an important cytokine that helps T cells destroy tumors and virus-infected cells. IL-2 has great therapeutic promise but is limited by toxic side effects and its capacity to both activate and repress immune responses. Sockolosky et al. set out to improve IL-2–based immunotherapy by engineering synthetic IL-2–receptor pairs (i.e., IL-2 and its receptor, IL-2R) (see the Perspective by Mackall). Engineered complexes transmitted IL-2 signals but only interacted with each other and not with endogenous IL-2/IL-2R. Treatment of mice with IL-2 improved the ability of engineered T cells to reject tumors with no obvious side effects. This type of approach may provide a way to mitigate toxicities associated with some cytokine-based immunotherapies. Science , this issue p. 1037 ; see also p. 990

SUMMARY PD-L1 and PD-L2 are ligands for the PD-1 immune inhibiting checkpoint that can be induced in tumors by interferon exposure, leading to immune evasion. This process is important for immunotherapy based on PD-1 blockade. We examined the specific molecules involved in interferon-induced signaling that regulates PD-L1 and PD-L2 expression in melanoma cells. These studies revealed that the interferon-gamma-JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis primarily regulates PD-L1 expression, with IRF1 binding to its promoter. PD-L2 responded equally to interferon beta and gamma and is regulated through both IRF1 and STAT3, which bind to the PD-L2 promoter. Analysis of biopsy specimens from patients with melanoma confirmed interferon signature enrichment and upregulation of gene targets for STAT1/STAT2/STAT3 and IRF1 in anti-PD-1-responding tumors. Therefore, these studies map the signaling pathway of interferon-gamma-inducible PD-1 ligand expression., In Brief Garcia-Diaz et al. performed a small hairpin RNA screen and genetic and functional studies to map the signaling pathways that result in reactive PD-L1 and PD-L2 on melanoma cells upon interferon gamma exposure. The authors highlight the importance of the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 axis for clinical responses to PD-1 blockade therapy.

Therapeutic resistance is a major obstacle to achieving durable clinical responses with targeted therapies, highlighting a need to elucidate the underlying mechanisms responsible for resistance and identify strategies to overcome this challenge. An emerging body of data implicates the tyrosine kinase MET in mediating resistance to BRAF inhibitors in BRAFV600E mutant melanoma. In this study we observed a dominant role for the HGF/MET axis in mediating resistance to BRAF and MEK inhibitors in models of BRAFV600E and NRAS mutant melanoma. In addition, we showed that MAPK pathway inhibition induced rapid increases in MET and GAB1 levels, providing novel mechanistic insight into how BRAFV600E mutant melanoma is primed for HGF-mediated rescue. We also determined that tumor-derived HGF, not systemic HGF, may be required to convey resistance to BRAF inhibition in vivo and that resistance could be reversed following treatment with AMG 337, a selective MET inhibitor. In summary, these findings support the clinical evaluation of MET-directed targeted therapy to circumvent resistance to BRAF and MEK inhibitors in BRAFV600E mutant melanoma. In addition, the induction of MET following treatment with BRAF and MEK inhibitors has the potential to serve as a predictive biomarker for identifying patients best suited for MET inhibitor combination therapy.

Loss-of-function mutations in JAK1/2 can lead to acquired resistance to anti-programmed death protein 1 (PD-1) therapy. We reasoned that they may also be involved in primary resistance to anti–PD-1 therapy. JAK1/2-inactivating mutations were noted in tumor biopsies of 1 of 23 patients with melanoma and in 1 of 16 patients with mismatch repair–deficient colon cancer treated with PD-1 blockade. Both cases had a high mutational load but did not respond to anti–PD-1 therapy. Two out of 48 human melanoma cell lines had JAK1/2 mutations, which led to a lack of PD-L1 expression upon interferon gamma exposure mediated by an inability to signal through the interferon gamma receptor pathway. JAK1/2 loss-of-function alterations in The Cancer Genome Atlas confer adverse outcomes in patients. We propose that JAK1/2 loss-of-function mutations are a genetic mechanism of lack of reactive PD-L1 expression and response to interferon gamma, leading to primary resistance to PD-1 blockade therapy. Significance: A key functional result from somatic JAK1/2 mutations in a cancer cell is the inability to respond to interferon gamma by expressing PD-L1 and many other interferon-stimulated genes. These mutations result in a genetic mechanism for the absence of reactive PD-L1 expression, and patients harboring such tumors would be unlikely to respond to PD-1 blockade therapy. Cancer Discov; 7(2); 188–201. ©2016 AACR. See related commentary by Marabelle et al., p. 128. This article is highlighted in the In This Issue feature, p. 115

Background and Aims The increasing interest by the wine market in sparkling white wines challenges how the desired grape composition can be achieved under the pressure of global warming. The aim of the present study was to assess the viability of summer pruning as a tool to pilot ripening towards desired compositional patterns. Methods and Results Ortrugo was subjected to basal leaf removal applied either at pre-flowering (ELR) or in lag-phase (LLR) and to bunch thinning (BT; removal of 50% of crop at lag-phase) in comparison with untreated control (C). Treatments induced large variation in bunch mass (30% less in ELR vs BT), yield per vine (37 and 21% less in BT and ELR vs C) and total soluble solids at harvest (BT scored 2.9°Brix higher than that of C). Conversely, given the same harvest date, all practices failed to maintain titratable acidity (TA) at the threshold of 6.5 g/L. Conclusions The data suggest that crop regulation achieved in the high yielding Ortrugo, through either BT or ELR, increases sugar accumulation rate so that concurrent optimal TA level (≅6.5 g/L) can be easily achieved by slightly anticipating harvest date. Under conditions of thermal or light stress, or weather conducive to bunch rot, preference should be given to ELR. Significance of the Study Suitable summer pruning techniques might be used as flexible and powerful tools to direct ripening towards the desired crop composition.

2584 Background: Mechanism-based strategies to overcome resistance to anti-PD1 therapy are urgently needed. Using CRISPR/Cas9 genome editing tools, we developed acquired resistant models through JAK1/2 and B2M loss of function (LoF) mutations in human melanoma cell lines and in the murine MC38 colon carcinoma, known for high mutational load and good response to anti-PD-1. We hypothesized that the downstream activation of the IFN-receptor pathway or the activation of natural killer (NK) cells would overcome this resistance. Methods: We studied signaling changes in four human cell lines (parental and LoFs) exposed to IFN-gamma using RNAseq. In addition, we analyzed the in-vivo antitumor activity in MC38 variants with anti-PD1 and characterized the tumor microenvironment using mass cytometry (CyTOF). Finally, we tested strategies to overcome resistance mechanisms with SD-101 (TLR-9 agonist) and bempegaldesleukin (NKTR-214, CD-122 biased agonist) with the extent of CD8 and NK1.1 depletion. Results: RNAseq differential gene expression analysis showed that the IFN-gamma induced increased expression of antigen presenting machinery, IFN-gamma signaling and chemokines (CXCL9/10) was lost in JAK1/2-LoF human melanoma cell lines. The significant antitumor activity of anti-PD-1 against MC38 parental cell line was lost in JAK1/2 and B2M LoF sublines, and CyTOF analysis revealed that anti-PD-1 therapy was unable to increase tumor CD8+ T-effectors in these LoF tumors. The intratumoral administration of SD-101 (50 μg/injection q4dx3wks) was able to overcome local resistance even in non-injected sites in JAK1/2 and IFNAR-type-I LoF tumors, and systemic administration of bempegaldesleukin (0.8 mg/kg, q9dx2, i.v.) was able to overcome resistance in B2M LoF with significantly increased survival (Table). Depletion studies showed complete abrogation of anti-tumor response with anti-NK1.1 in JAK1 LoF and B2M LoF, and partial abrogation with anti-NK1.1 or anti-CD8 in JAK2 LoF tumors. Conclusions: Even in the extreme setting of genetic resistance to PD-1 blockade by JAK1/2 LoF, resistance can be overcome by SD-101, a TLR9 agonist, while resistance of B2M LoF can be overcome by bempegaldesleukin (NKTR-214), a CD-122 biased agonist. Our findings support the testing of these rational mechanistic strategies in patients with a-PD1 resistance. [Table: see text]

The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues, and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor–host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN−/−). Anti–PD-1 or anti–PD-L1 therapy engendered strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. PD-L1 expression did not differ between the three models at baseline or upon interferon stimulation. Whereas mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T cells, as well as CD28 and CD80/86 costimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor-associated macrophages in the tumors after PD-1 blockade. Compared with YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in expression of chemokine-trafficking genes that are related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells and costimulation that is mediated by dendritic cells and macrophages. Cancer Immunol Res; 4(10); 845–57. ©2016 AACR.

Viticulture is in great need of new rootstocks sharing features of scion growth control and tolerance to major biotic and abiotic stress factors. A two year study was carried out in pots to assess performances of the two new M1 and M3 rootstocks vs. those of the commercial rootstocks 1103P and 101-14. Potted vines of M1, M3, 1103P and 101-14 rootstocks were grown in a calcareous and non calcareous soil and for two consecutive seasons subjected to three N supply levels at 0, 2 and 4 g of N per pot. Vegetative growth, leaf gas exchange, leaf greenness index (GI) and leaf blade nutrition were assessed. M1 and 1103P were the least vigorous genotypes in terms of total pruning weight; M1 also manifested a stronger apical dominance. Both M rootstocks and 101-14 showed increased leaf WUE at both N supply levels which was due to ability to maintain, at increasing N supply, similar leaf assimilation rates while significantly reducing leaf transpiration. Common tendency of any rootstocks was that increasing N supply corresponded to lowered leaf concentration of K, P, Mg and B. M1 was able to combine a series of desirable features including lower vigor, strong apical dominance, higher WUE at increasing N supply and quite well balanced leaf nutritional pattern. In the present trial M3 did not have the expected devigorating effect.