Development and Validation of a Short Form of the Geriatric Anxiety Scale (GAS-12) among Italian Older Adults.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The Italian GAS-12 appears to be a useful instrument for the quantitative screening of anxiety in Italian older adults. CLINICAL IMPLICATIONS: 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.

Immunomodulatory activity of SGI-110, a 5-aza-2'-deoxycytidine-containing demethylating dinucleotide.

PURPOSE: Pharmacologic DNA hypomethylation holds strong promises in cancer immunotherapy due to its immunomodulatory activity on neoplastic cells. Searching for more efficient DNA hypomethylating agents to be utilized to design novel immunotherapeutic strategies in cancer, we investigated the immunomodulatory properties of the new DNA hypomethylating agent SGI-110, that is resistant to in vivo inactivation by cytidine deaminase. EXPERIMENTAL DESIGN: Cutaneous melanoma, mesothelioma, renal cell carcinoma, and sarcoma cells were treated in vitro with SGI-110. RT-PCR, quantitative RT-PCR, quantitative methylation-specific PCR, and flow cytometric analyses were performed to investigate changes induced by SGI-110 in the constitutive immune profile of cancer cells. The recognition by gp100-specific CTL of gp100-positive melanoma cells, treated or not with SGI-110, was tested by LDH release assays. RESULTS: SGI-110 induced/up-regulated the expression of investigated cancer/testis antigens (CTA) (i.e., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A10, GAGE 1-2, GAGE 1-6, NY-ESO-1, and SSX 1-5) in all cancer cell lines studied, both at mRNA and at protein levels. Quantitative methylation-specific PCR analyses identified a hypomethylation of MAGE-A1 and NY-ESO-1 promoters in SGI-110-treated neoplastic cells, demonstrating a direct role of pharmacologic DNA demethylation in CTA induction. SGI-110 also up-regulated the expression of HLA class I antigens and of ICAM-1, resulting in an improved recognition of cancer cells by gp100-specific CTL. CONCLUSIONS: Our findings show that SGI-110 is a highly attractive therapeutic agent to comprehensively increase immunogenicity and immune recognition of neoplastic cells, and provide the scientific rationale for its clinical development to design novel chemo-immunotherapeutic approaches in cancer patients.

Whole genome methylation profiles as independent markers of survival in stage IIIC melanoma patients.

BACKGROUND: The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis. This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM. METHODS: Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients. Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles. Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm. The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses. RESULTS: Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients. Patients with a "favorable" methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis. Median OS of stage IIIC patients with a "favorable" vs. "unfavorable" methylation profile were 31.5 and 10.4 months, respectively. The 5 year OS for stage IIIC patients with a "favorable" methylation profile was 41.2% as compared to 0% for patients with an "unfavorable" methylation profile. Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for "unfavorable" methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045). A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified. CONCLUSIONS: A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients. Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

Methylation levels of the "long interspersed nucleotide element-1" repetitive sequences predict survival of melanoma patients.

BACKGROUND: The prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established. Genome-wide alterations in DNA methylation are a common event in cancer. This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients. METHODS: Overall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients. The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis. RESULTS: Hypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association. Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis. Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively. The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences. Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01). CONCLUSION: LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC. Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients.

Analysis of General Practitioners' Attitudes and Beliefs about Psychological Intervention and the Medicine-Psychology Relationship in Primary Care: Toward a New Comprehensive Approach to Primary Health Care.

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.

Epigenetics of human cutaneous melanoma: setting the stage for new therapeutic strategies.

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.

IL-32γ potentiates tumor immunity in melanoma.

Myeloid cells orchestrate the antitumor immune response and influence the efficacy of immune checkpoint blockade (ICB) therapies. We and others have previously shown that IL-32 mediates DC differentiation and macrophage activation. Here, we demonstrate that IL-32 expression in human melanoma positively correlates with overall survival, response to ICB, and an immune-inflamed tumor microenvironment (TME) enriched in mature DC, M1 macrophages, and CD8+ T cells. Treatment of B16F10 murine melanomas with IL-32 increased the frequencies of activated, tumor-specific CD8+ T cells, leading to the induction of systemic tumor immunity. Our mechanistic in vivo studies revealed a potentially novel role of IL-32 in activating intratumoral DC and macrophages to act in concert to prime CD8+ T cells and recruit them into the TME through CCL5. Thereby, IL-32 treatment reduced tumor growth and rendered ICB-resistant B16F10 tumors responsive to anti-PD-1 therapy without toxicity. Furthermore, increased baseline IL-32 gene expression was associated with response to nivolumab and pembrolizumab in 2 independent cohorts of patients with melanoma, implying that IL-32 is a predictive biomarker for anti-PD-1 therapy. Collectively, this study suggests IL-32 as a potent adjuvant in immunotherapy to enhance the efficacy of ICB in patients with non-T cell-inflamed TME.

Persistence of adoptively transferred T cells with a kinetically engineered IL-2 receptor agonist.

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 ßγ (IL-2Rßγ)-biased agonist consisting of IL-2 conjugated to multiple releasable polyethylene glycol chains resulting in sustained signaling through IL-2Rßγ. 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.

Uncoupling interferon signaling and antigen presentation to overcome immunotherapy resistance due to JAK1 loss in melanoma.

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.

Overcoming Genetically Based Resistance Mechanisms to PD-1 Blockade.

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.

Hyperhomocysteinemia in patients with epilepsy: does it play a role in the pathogenesis of brain atrophy? A preliminary report.

PURPOSE: Brain atrophy (BA) is observed in 20-50% of patients with epilepsy. Hyper-total-homocysteinemia (hyper-tHcy), which occurs in 10-40% of patients, is considered to be a risk factor for cardiovascular diseases and BA. The present study was aimed at investigating the possible association of hyper-tHcy with BA in a population of patients with epilepsy. METHODS: Fifty-eight patients (33 M/25 F, 43.5 +/- 13.1 years of age) chronically treated with antiepileptic drugs (AEDs) and 60 controls matched for age and sex were enrolled. All participants underwent determination of plasma tHcy, folate, vitamin B(12), and C677T methylene-tetrahydrofolate-reductase (MTHFR) polymorphism genotyping, and brain magnetic resonance imaging (MRI). RESULTS: Patients exhibited significantly higher tHcy and lower folate levels than controls; hyper-tHcy was significantly associated with the variables group (patients vs. controls), MTHFR genotype, and their interaction terms. BA was observed in 30.1% of patients and was significantly associated with hyper-tHcy (beta = 0.45, p = 0.003) and polytherapy (beta = 0.31, p < 0.001). DISCUSSION: Our investigation suggests that hyper-tHcy plays a role in the development of BA in patients with epilepsy. Although the real origin of this phenomenon is not yet fully elucidated, experimental data support the hypothesis of a link of the neuronal Hcy-mediated damage with oxidative stress and excitotoxicity.

IND-Enabling Studies for a Clinical Trial to Genetically Program a Persistent Cancer-Targeted Immune System.

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.

NF-kappaB activation is associated with homocysteine-induced injury in Neuro2a cells.

BACKGROUND: Perinatal exposure to hyperhomocysteinemia might disturb neurogenesis during brain development and growth. Also, high levels of homocysteine trigger neurodegeneration in several experimental models. However, the putative mechanisms of homocysteine-induced toxicity in the developing nervous system have poorly been elucidated. This study was aimed to investigate homocysteine effects in undifferentiated neuroblastoma cells, Neuro2a. RESULTS: A 4 h exposure to homocysteine in a concentration range of 10-100 microM did not affect cell viability and ROS production in Neuro2a cell cultures. Instead, ROS levels were increased by two-three folds in cells treated with 250 microM and 500 microM homocysteine, respectively, in comparison with control cells. Also, the highest homocysteine dose significantly reduced the viable cell number by 40%. Notably, the treatment with homocysteine (250 microM-500 microM) in the presence of antioxidants, such as N-acetylcysteine and IRFI 016, a synthetic alpha-tocopherol analogue, recovered cell viability and significantly reduced homocysteine-evoked increases in ROS production. Moreover, antioxidants, particularly IRFI 016, were able to counteract NF-kappaB activation induced by 250 microM homocysteine. Cell treatment with 250 microM homocysteine also triggered the onset of apoptosis, as demonstrated by the increased expression of early apoptotic markers such as Bax, caspase-3 and p53. In contrast, Bcl2 expression was not affected by homocysteine exposure. Interestingly, the specific inhibition of NF-kappaB nuclear translocation by the synthetic peptide SN50 was able to almost completely suppress the homocysteine-evoked rises in pro-apoptotic protein expression as well as in caspase-3 activity. Further, also IRFI 016 and N-acetylcysteine were able to significantly reduce caspase-3 activation induced by homocysteine treatment. CONCLUSION: These observations suggest an involvement of redox state alterations and activated NF-kappaB in apoptosis onset triggered by homocysteine in neuroblastoma cells Neuro2a. However, further investigations are needed to characterize molecular events involved in the NF-kappaB activation induced by homocysteine.

Transglutaminase 2 and NF-kappaB interplay during NGF-induced differentiation of neuroblastoma cells.

NGF treatment of neuroblastoma cells stimulates outgrowth of neurite processes associated with the expression of TrkA receptor and several differentiation markers. In this study, a 6 DIV exposure to NGF (50 ng/ml) increased immunostaining for alpha-tubulin, and expression of both alpha-tubulin and protein kinase C in the neuroblastoma cell line Neuro2a. Further, up-regulation of transglutaminase 1 and transglutaminase 2 expression, and reduction of transglutaminase 3 levels, were also observed in NGF-treated cells in comparison to untreated cells. Moreover, when Neuro2a cells were treated with the specific NF-kappaB inhibitor SN-50, the strong reduction of NF-kappaB activation was concomitant with a significant decrease of transglutaminase 2 expression, suggesting that NGF-evoked transglutaminase 2 induction could be related to NF-kappaB activation. To characterize the possible transglutaminase 2/NF-kappaB interplay, NGF treatment was carried out in Neuro2a cells which already over-expressed transglutaminase 2 after retinoic acid treatment. An additive effect of NGF was observed on the retinoic acid-induced transglutaminase 2 expression and enzyme activity, and NF-kappaB activation. However, a cystamine-mediated significant inhibition of transglutaminase activity (70%) was accompanied by a drastically reduced NF-kappaB activation only in cells exposed to NGF following retinoic acid treatment. We hypothesize that NF-kappaB activation was dependent on the transamidating activity related to high levels of TG2, and NGF enhanced NF-kappaB activation by a different, synergistically acting, pathway. These data suggest that the combined use of NGF and retinoic acid, or mimicking drugs, may provide the basics for the development of novel strategies in the therapeutic management of neuroblastomas.

Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes.

Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rß into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4+ and CD8+ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

Effects of nickel on human and fish red blood cells.

The objective of this study was to assess the effects of nickel chloride on human and rainbow trout erythrocytes in vitro. The cells were incubated with 0, 0.5 and 1 mM nickel chloride for 1 h at pH 7.40 and 25 degrees C, then K(+) efflux, SO (4) (2-) uptake and GSH and GSSG concentrations were measured. In both kind of cells, "high concentration" nickel treatment increased KCl efflux with respect to the control. The SO (4) (2-) uptake was not significantly different at "low nickel concentration" but was lower in erythrocytes treated with 1 mM nickel chloride; the rate constant of SO (4) (2-) uptake decreased by 35% in human erythrocytes and by 44% in fish erythrocytes. Nickel chloride also acts on cellular metabolism and in particular on erythrocyte glutathione peroxidase with consequent increase in oxidative stress; the data show a significant decrease in intracellular GSH in both human (25%) and fish erythrocytes (18%) after treatment with nickel chloride, with concomitantly high GSSG concentrations and lower GSH/GSSG ratios.

MAPK pathway inhibition induces MET and GAB1 levels, priming BRAF mutant melanoma for rescue by hepatocyte growth factor.

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.

Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression.

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.