SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion.

Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 limits endogenous DNA damage, thereby suppressing cGAS-STING-dependent signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a PD-L1 transcriptional regulatory element, thereby promoting PD-L1 expression in cancer cells. SMARCAL1 loss hinders the ability of tumor cells to induce PD-L1 in response to genomic instability, enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a promising target for cancer immunotherapy.

Cytoreductive Nephrectomy Following Immune Checkpoint Inhibitor Therapy Is Safe and Facilitates Treatment-free Intervals.

Patients with metastatic renal cell cancer (mRCC) who respond to upfront immune checkpoint inhibitor (ICI) combination therapies may be treated with cytoreductive nephrectomy (CN) to remove radiographically viable primary tumors. Early data for post-ICI CN suggested that ICI therapies induce desmoplastic reactions in some patients, increasing the risk of surgical complications and perioperative mortality. We evaluated perioperative outcomes for 75 consecutive patients treated with post-ICI CN at four institutions from 2017 to 2022. Our cohort of 75 patients had minimal or no residual metastatic disease but radiographically enhancing primary tumors after ICI and were treated with CN. Intraoperative complications were identified in 3/75 patients (4%) and 90-d postoperative complications in 19/75 (25%), including two patients (3%) with high-grade (Clavien ≥III) complications. One patient was readmitted within 30 d. No patients died within 90 d after surgery. Viable tumor was present in all but one specimen. Approximately half of the patients (36/75, 48%) remained off systemic therapy at last follow-up. These data suggest that CN following ICI therapy is safe and associated with low rates of major postoperative complications in appropriately selected patients at experienced centers. Post-ICI CN may facilitate observation without additional systemic therapy in patients without significant residual metastatic disease. Patient summary: Current first-line treatment for patients with kidney cancer that has spread to other sites (metastatic cancer) is immunotherapy. For cases in which metastatic sites respond to this therapy but primary tumor is still detected in the kidney, surgical treatment of the tumor is feasible and has a low rate of complications, and may delay the need for further chemotherapy.

Mycobacterium Bovis Causing Mycotic Aneurysm Secondary to Intravesical Treatment with Bacillus Calmette-Guérin: A Case Report.

BACKGROUND: Bacillus Calmette-Guerin (BCG) is a live, attenuated strain of Mycobacterium bovis that is used in the treatment of non-muscle invasive bladder cancer (NMIBC). Vascular complications, including mycotic aneurysms, after BCG therapy are exceedingly rare. In this patient population, the diagnosis of mycotic aneurysms can be delayed or missed due to their non-specific clinical and radiologic presentation. Literature review reveals management of mycotic aneurysms attributable to BCG therapy is widely varied.2,5-8,12,15 CASE REPORT: We report a patient who presented with mycotic aneurysm formation secondary to BCG treatment for bladder cancer that was repaired with in-line reconstruction utilizing cryoartery and buttressed with omental flap. We suggest this as an alternative treatment to in-line prosthetic graft or extra-anatomic reconstruction.

Blocking IL1 Beta Promotes Tumor Regression and Remodeling of the Myeloid Compartment in a Renal Cell Carcinoma Model: Multidimensional Analyses.

PURPOSE: Intratumoral immunosuppression mediated by myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) represents a potential mechanism of immune checkpoint inhibitor (ICI) resistance in solid tumors. By promoting TAM and MDSC infiltration, IL1ß may drive adaptive and innate immune resistance in renal cell carcinoma (RCC) and in other tumor types. EXPERIMENTAL DESIGN: Using the RENCA model of RCC, we evaluated clinically relevant combinations of anti-IL1ß plus either anti-PD-1 or the multitargeted tyrosine kinase inhibitor (TKI), cabozantinib. We performed comprehensive immune profiling of established RENCA tumors via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA sequencing (RNA-seq). Similar analyses were extended to the MC38 tumor model. RESULTS: Analyses via multiparameter flow cytometry, tumor cytokine profiling, and single-cell RNA-seq showed that anti-IL1ß reduces infiltration of polymorphonuclear MDSCs and TAMs. Combination treatment with anti-IL1ß plus anti-PD-1 or cabozantinib showed increased antitumor activity that was associated with decreases in immunosuppressive MDSCs and increases in M1-like TAMs. CONCLUSIONS: Single-cell RNA-seq analyses show that IL1ß blockade and ICI or TKI remodel the myeloid compartment through nonredundant, relatively T-cell-independent mechanisms. IL1ß is an upstream mediator of adaptive myeloid resistance and represents a potential target for kidney cancer immunotherapy.

A conserved intratumoral regulatory T cell signature identifies 4-1BB as a pan-cancer target.

Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB-expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB-expressing Tregs represents a strategy with potential activity across cancer types.

Prevalence of astrovirus, adenovirus, and sapovirus infections among Iranian children with acute gastroenteritis.

AIM: This study aimed to determine the prevalence of Human Astroviruses (HAstVs), enteric Adenoviruses (HAdVs), and Sapoviruses (SaVs) in acute diarrhea patients, as well as their relation to age, sex, and season. BACKGROUND: Acute gastroenteritis is one of the most common diseases affecting children <5 years old and viral agents with approximately >75% are the major causative agent of acute infectious diarrhea. After Rotavirus and Norovirus, the greater viral agents of acute gastroenteritis include HAstVs, HAdVs, and SaVs. To the best of our knowledge, there are sparse studies in Iran detecting at least three enteric viruses as causative agents of diarrhea simultaneously. METHODS: The sample was collected from children referring to pediatric medical centers in Tehran, Iran; they were tested for Astrovirus, enteric Adenovirus, and Sapovirus by conventional PCR method. The association of incidence of viral enteric agents was evaluated with age, sex and seasonal pattern in children <5 years old. RESULTS: The positive case number among acute gastroenteritis patients was 17/120 (14.1%). Patients ranged in age within 1-60 months, but 52.9% were aged ≤ 12 months. Males comprised the majority (70.6), and the male: female ratio was 2.4. HAstV was the most frequently detected virus (6.7%), while SaVs were detected only in 2.5% of cases. Mixed infections were not detected in these samples. The highest rate of HAstV was identified in winter (66.7%), HAdV in fall (66.7%), and SaV in winter (33.3%). CONCLUSION: These findings underscore the importance of monitoring the epidemiology of HAstV, HAdV, and SaV as causative agents of viral diarrhea infections.

Immunogenicity of prostate cancer is augmented by BET bromodomain inhibition.

BACKGROUND: Prostate cancer responds poorly to current immunotherapies. Epigenetic therapies such as BET Bromodomain inhibition can change the transcriptome of tumor cells, possibly making them more immunogenic and thus susceptible to immune targeting. METHODS: We characterized the effects of BET bromodomain inhibition using JQ1 on PD-L1 and HLA-ABC expression in two human prostate cell lines, DU145 and PC3. RNA-Seq was performed to assess changes on a genome-wide level. A cytotoxic T cell killing assay was performed in MC38-OVA cells treated with JQ1 to demonstrate increased immunogenicity. In vivo experiments in the Myc-Cap model were conducted to show the effects of JQ1 administration in concert with anti-CTLA-4 checkpoint blockade. RESULTS: Here, we show that targeting BET bromodomains using the small molecule inhibitor JQ1 decreased PD-L1 expression and mitigated tumor progression in prostate cancer models. Mechanistically, BET bromodomain inhibition increased MHC I expression and increased the immunogenicity of tumor cells. Transcriptional profiling showed that BET bromodomain inhibition regulates distinct networks of antigen processing and immune checkpoint molecules. In murine models, treatment with JQ1 was additive with anti-CTLA-4 immunotherapy, resulting in an increased CD8/Treg ratio. CONCLUSIONS: BET Bromodomain inhibition can mediate changes in expression at a genome wide level in prostate cancer cells, resulting in an increased susceptibility to CD8 T cell targeting. These data suggest that combining BET bromodomain inhibition with immune checkpoint blockade may have clinical activity in prostate cancer patients.

Anticancer potential of rosmarinic acid and its improved production through biotechnological interventions and functional genomics.

Rosmarinic acid (RA) is a highly valued natural phenolic compound that is very commonly found in plants of the families Lamiaceae and Boraginaceae, including Coleus blumei, Heliotropium foertherianum, Rosmarinus officinalis, Perilla frutescens, and Salvia officinalis. RA is also found in other members of higher plant families and in some fern and horned liverwort species. The biosynthesis of RA is catalyzed by the enzymes phenylalanine ammonia lyase and cytochrome P450-dependent hydroxylase using the amino acids tyrosine and phenylalanine. Chemically, RA can be produced via methods involving the esterification of 3,4-dihydroxyphenyllactic acid and caffeic acid. Some of the derivatives of RA include melitric acid, salvianolic acid, lithospermic acid, and yunnaneic acid. In plants, RA is known to have growth-promoting and defensive roles. Studies have elucidated the varied pharmacological potential of RA and its derived molecules, including anticancer, antiangiogenic, anti-inflammatory, antioxidant, and antimicrobial activities. The demand for RA is therefore, very high in the pharmaceutical industry, but this demand cannot be met by plants alone because RA content in plant organs is very low. Further, many plants that synthesize RA are under threat and near extinction owing to biodiversity loss caused by unscientific harvesting, over-collection, environmental changes, and other inherent features. Moreover, the chemical synthesis of RA is complicated and expensive. Alternative approaches using biotechnological methodologies could overcome these problems. This review provides the state of the art information on the chemistry, sources, and biosynthetic pathways of RA, as well as its anticancer properties against different cancer types. Biotechnological methods are also discussed for producing RA using plant cell, tissue, and organ cultures and hairy-root cultures using flasks and bioreactors. The recent developments and applications of the functional genomics approach and heterologous production of RA in microbes are also highlighted. This chapter will be of benefit to readers aiming to design studies on RA and its applicability as an anticancer agent.

Elective Nodal Irradiation Attenuates the Combinatorial Efficacy of Stereotactic Radiation Therapy and Immunotherapy.

Purpose: In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB).Experimental Design: We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN.Results: Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8+ effector T cells as well as FoxP3+ regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8+ T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1.Conclusions: Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8+ T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy. Clin Cancer Res; 24(20); 5058-71. ©2018 AACR.

Phytochemical constituents, antioxidant activity, and antiproliferative properties of black, red, and brown rice bran.

BACKGROUND: In the recent years, the health benefits of the pigmented rice varieties have reported due to the presence of bioactive compounds. In this study, the phytochemical constituents (total phenolic, flavonoid and anthocyanin content) and individual phenolics and flavonoids of the extracts of sixteen genotypes of pigmented rice bran were evaluated using spectrophotometric and ultra-high performance liquid chromatography method. Antioxidative properties of the free and bound fractions were evaluated using nitric oxide and 1,1-diphenyl-2-picrylhydrazyl scavenging assays. Extracts were evaluated for antiproliferative activity against breast cancer cell lines (MCF-7 and MDA-MB-231) using the MTT assay. RESULTS: Signifficant diferences were observed in the concentrations of phytochemicals and biological activities among different pigmented rice brans. The highest phytochemical content was observed in black rice bran followed by red and brown rice bran. The concentration of free individual flavonoids and phenolic compounds were significantly higher than those of bound compounds except those of ferulic acid and p-coumaric acid. Highest antioxidant activities were observed in black rice bran, followed by red and brown rice bran extracts. Extracts of black rice bran exhibited potent antiproliferative activity, with half maximal inhibitory concentrations (IC50) of 148.6 and 119.2 mg/mL against MCF-7 and MDA-MB-231 cell lines, respectively, compared to the activity of the extracts of red rice bran (175.0 and 151.0 mg/mL, respectively) and brown rice bran (382.3 and 346.1 mg/mL, respectively). CONCLUSIONS: Black rice bran contains high levels of phytochemicals, and thus has potent pharmaceutical activity. This highlights opportunities for researcher to breed new genotypes of rice with higher nutritional values, which the food industry can use to develop new products that will compete in expanding functional food markets.

TNFα and Radioresistant Stromal Cells Are Essential for Therapeutic Efficacy of Cyclic Dinucleotide STING Agonists in Nonimmunogenic Tumors.

The cGAS-STING cytosolic DNA sensing pathway may play an integral role in the initiation of antitumor immune responses. Studies evaluating the immunogenicity of various cyclic dinucleotide (CDN) STING agonists administered by intratumoral (i.t.) injection showed potent induction of inflammation, tumor necrosis, and, in some cases, durable tumor-specific adaptive immunity. However, the specific immune mechanisms underlying these responses remain incompletely defined. The majority of these studies have focused on the effect of CDNs on immune cells but have not conclusively interrogated the role of stromal cells in the acute rejection of the CDN-injected tumor. Here, we revealed a mechanism of STING agonist-mediated tumor response that relied on both stromal and immune cells to achieve tumor regression and clearance. Using knockout and bone marrow chimeric mice, we showed that although bone marrow-derived TNFα was necessary for CDN-induced necrosis, STING signaling in radioresistant stromal cells was also essential for CDN-mediated tumor rejection. These results provide evidence for crosstalk between stromal and hematopoietic cells during CDN-mediated tumor collapse after i.t. administration. These mechanistic insights may prove critical in the clinical development of STING agonists. Cancer Immunol Res; 6(4); 422-33. ©2018 AACR.

Combining intratumoral Treg depletion with androgen deprivation therapy (ADT): preclinical activity in the Myc-CaP model.

BACKGROUND: Immune checkpoint blockade has shown promising antitumor activity against a variety of tumor types. However, responses in castration-resistant prostate cancer remain relatively rare-potentially due to low baseline levels of infiltration. Using an immunocompetent cMyc-driven model (Myc-CaP), we sought to understand the immune infiltrate induced by androgen deprivation therapy (ADT) and to leverage that infiltration toward therapeutic benefit. METHODS: Using flow cytometry, qPCR and IHC, we quantified ADT-induced immune infiltration in terms of cell type and function. Preclinical treatment studies tested the combinatorial effects of ADT and immune checkpoint blockade using tumor outgrowth and overall survival as end points. RESULTS: ADT induces a complex pro-inflammatory infiltrate. This pro-inflammatory infiltrate was apparent in the early postcastration period but diminished as castration resistance emerged. Combining ADT with tumor-infiltrating regulatory T cell (Treg) depletion using a depleting anti-CTLA-4 antibody significantly delayed the development of castration resistance and prolonged survival of a fraction of tumor-bearing mice. Immunotherapy as a monotherapy failed to provide a survival benefit and was ineffective if not administered in the peri-castration period. CONCLUSIONS: The immune infiltrate induced by ADT is diverse and varies over time. Therapeutic strategies focusing on depleting Tregs in the peri-castration period are of particular interest.

Stereotactic Radiotherapy Increases Functionally Suppressive Regulatory T Cells in the Tumor Microenvironment.

Radiotherapy (RT) enhances innate and adaptive antitumor immunity; however, the effects of radiation on suppressive immune cells, such as regulatory T cells (Treg), in the tumor microenvironment (TME) are not fully elucidated. Although previous reports suggest an increased Treg infiltration after radiation, whether these Tregs are functionally suppressive remains undetermined. To test the hypothesis that RT enhances the suppressive function of Treg in the TME, we selectively irradiated implanted tumors using the small animal radiation research platform (SARRP), which models stereotactic radiotherapy in human patients. We then analyzed tumor-infiltrating lymphocytes (TIL) with flow-cytometry and functional assays. Our data showed that RT significantly increased tumor-infiltrating Tregs (TIL-Treg), which had higher expression of CTLA-4, 4-1BB, and Helios compared with Tregs in nonirradiated tumors. This observation held true across several tumor models (B16/F10, RENCA, and MC38). We found that TIL-Tregs from irradiated tumors had equal or improved suppressive capacity compared with nonirradiated tumors. Our data also indicated that after RT, Tregs proliferated more robustly than other T-cell subsets in the TME. In addition, after RT, expansion of Tregs occurred when T-cell migration was inhibited using Fingolimod, suggesting that the increased Treg frequency was likely due to preferential proliferation of intratumoral Treg after radiation. Our data also suggested that Treg expansion after irradiation was independent of TGFß and IL33 signaling. These data demonstrate that RT increased phenotypically and functionally suppressive Tregs in the TME. Our results suggest that RT might be combined effectively with Treg-targeting agents to maximize antitumor efficacy. Cancer Immunol Res; 5(11); 992-1004. ©2017 AACR.

Optimization of microwave-assisted extraction of zerumbone from Zingiber zerumbet L. rhizome and evaluation of antiproliferative activity of optimized extracts.

BACKGROUND: The extraction of bioactive compounds from herbal materials requires optimization in order to recover the highest active dose. Response surface methodology was used to optimize variables affecting the microwave extraction of zerumbone from Zingiber zerumbet using the Box-Behnken design. The influence of variables, such as ethanol concentration (X1), microwave power (X2), irradiation time (X3), and liquid-to-solid ratio (X4), on the extraction of zerumbone was modeled using a second-order regression equation. The antiproliferative activity of optimized and non-optimized extracts was evaluated against the HeLa cancer cell line using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS: Two linear parameters, X1 and X4, and their quadratic parameters were highly significant at the P < 0.01 level. Two interaction parameters, X1X4 and X2X3 were significant, whereas interactions of X1X2, X1X3, X2X4 and X3X4 were insignificant (P > 0.05). The optimum microwave extraction conditions were as follows: ethanol concentration, 44%; microwave power, 518 W; irradiation time, 38.5 s; and liquid-to-solid ratio, 38 mL/g. Under these conditions, the maximum zerumbone yield was 5.88 mg/g DM, which was similar to the predicted value (5.946 mg/g DM). Optimized and non-optimized Z. zerumbet rhizome extracts exhibited significant antiproliferative activity against HeLa cancer cells, with half-maximal inhibitory concentration (IC50) values of 4.3 and 7.8 µg/mL, respectively, compared with 1.68 µg/mL for the anticancer drug cisplatin. When the extract concentration increased from 4.3 to 16.0 µg/mL, the inhibition of cancer cell growth increased from 50.0 to 79.5%. CONCLUSIONS: In this study, the optimized microwave protocol developed for extracting zerumbone from Z. zerumbet was faster and consumed less solvent than previous methods, while improving and enhancing the antiproliferative activity.

Erratum to: Agonist anti-GITR monoclonal antibody and stereotactic radiation induce immune-mediated survival advantage in murine intracranial glioma.

[This corrects the article DOI: 10.1186/s40425-016-0132-2.].

Changes in antioxidant and antibacterial activities as well as phytochemical constituents associated with ginger storage and polyphenol oxidase activity.

BACKGROUND: Herbal materials should be stored at optimal conditions in order to retain their nutritional quality. Proper storage has a significant impact on the quality of the herbs and spices. METHODS: The effects of storage temperature (5 and 15 °C) and time (4 and 8 months) on the phytochemical constituents associated with the antioxidant and antibacterial activities of ginger varieties (Halia bentong and Halia bara) were evaluated to determine the optimal storage conditions for ginger rhizomes. Total flavonoid content (TFC) and Total phenolic content (TPC) were measured using the spectrophotometric method. Individual phenolic acids and flavonoids, 6-gingerol and 6-shogaol were identified by ultra-high performance liquid chromatography. Ferric reducing antioxidant potential (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used for evaluation of antioxidant activities. An antibacterial property of ginger varieties was evaluated using well diffusion method. RESULTS: Dry matter, TPC, TFC and individual phenolics and flavonoids content, 6-gingerol and 6-shogaol content noticeably decreased at 5 and 15 °C during the storage times from 4 to 8 months. Highest content of flavonoids, phenolic acids, 6-gingerol, and 6-shogaol was observed in fresh samples followed by rhizomes stored at 5 °C for 4 months. Storage at 15 °C for 4 months reduced the phytochemical content significantly. Cinnamic acid and tannic acid were not detected in those variety stored at 15 °C for 4 and 8 months. Polyphenol oxidase (PPO) activity was associated significantly with storage time and temperature. Highest and lowest PPO activity was observed in stored and fresh rhizomes respectively. Antioxidant and antimicrobial activities gradually declined with the increase of storage temperature (from 5 to 15 °C) and duration (from 4 to 8 months) in both the varieties. Freshly harvested Halia bara variety had higher antioxidant and antibacterial activity compared to the Halia bentong variety. CONCLUSIONS: Halia bara exhibited valuable phytochemical content and antioxidant and antibacterial activities at higher levels compared to that exhibited by Halia bentong rhizomes. In general, storage of Malaysian ginger varieties at temperature of 5 °C is recommended and the storage time should be not more than 4 months. This storage condition will provide greater stability to the concentration of the phytochemical constituents more similar to the fresh material.

TGFß1-Mediated SMAD3 Enhances PD-1 Expression on Antigen-Specific T Cells in Cancer.

Programmed death-1 (PD-1) is a coinhibitory receptor that downregulates the activity of tumor-infiltrating lymphocytes (TIL) in cancer and of virus-specific T cells in chronic infection. The molecular mechanisms driving high PD-1 expression on TILs have not been fully investigated. We demonstrate that TGFß1 enhances antigen-induced PD-1 expression through SMAD3-dependent, SMAD2-independent transcriptional activation in T cells in vitro and in TILs in vivo The PD-1hi subset seen in CD8+ TILs is absent in Smad3-deficient tumor-specific CD8+ TILs, resulting in enhanced cytokine production by TILs and in draining lymph nodes and antitumor activity. In addition to TGFß1's previously known effects on T-cell function, our findings suggest that TGFß1 mediates T-cell suppression via PD-1 upregulation in the tumor microenvironment (TME). They highlight bidirectional cross-talk between effector TILs and TGFß-producing cells that upregulates multiple components of the PD-1 signaling pathway to inhibit antitumor immunity. SIGNIFICANCE: Engagement of the coinhibitory receptor PD-1 or its ligand, PD-L1, dramatically inhibits the antitumor function of TILs within the TME. Our findings represent a novel immunosuppressive function of TGFß and demonstrate that TGFß1 allows tumors to evade host immune responses in part through enhanced SMAD3-mediated PD-1 expression on TILs. Cancer Discov; 6(12); 1366-81. ©2016 AACRThis article is highlighted in the In This Issue feature, p. 1293.

Improvement in Flavonoids and Phenolic Acids Production and Pharmaceutical Quality of Sweet Basil (Ocimum basilicum L.) by Ultraviolet-B Irradiation.

Sweet basil (Ocimum basilicum Linnaeus) is aromatic herb that has been utilized in traditional medicine. To improve the phytochemical constituents and pharmaceutical quality of sweet basil leaves, ultraviolet (UV)-B irradiation at different intensities (2.30, 3.60, and 4.80 W/m²) and durations (4, 6, 8, and 10-h) was applied at the post-harvest stage. Total flavonoid content (TFC) and total phenolic content (TPC) were measured using spectrophotometric method, and individual flavonoids and phenolic acids were identified using ultra-high performance liquid chromatography. As a key enzyme for the metabolism of flavonoids, chalcone synthase (CHS) activity, was measured using a CHS assay. Antioxidant activity and antiproliferative activity of extracts against a breast cancer cell line (MCF-7) were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, respectively. UV-B irradiation at an intensity of 3.60 W/m² increased TFC approximately 0.85-fold and also increased quercetin (0.41-fold), catechin (0.85-fold), kaempferol (0.65-fold) rutin (0.68-fold) and luteolin (1.00-fold) content. The highest TPC and individual phenolic acid (gallic acid, cinnamic acid and ferulic acid) was observed in the 3.60 W/m² of UV-B treatment. Cinnamic acid and luteolin were not detected in the control plants, production being induced by UV-B irradiation. Production of these secondary metabolites was also significantly influenced by the duration of UV-B irradiation. Irradiation for 8-h led to higher TFC, TPC and individual flavonoids and phenolic acids than for the other durations (4, 8, and 10-h) except for cinnamic acid, which was detected at higher concentration when irradiated for 6-h. Irradiation for 10-h significantly decreased the secondary metabolite production in sweet basil leaves. CHS activity was induced by UV-B irradiation and highest activity was observed at 3.60 W/m² of UV-B irradiation. UV-B treated leaves presented the highest DPPH activity and antiproliferative activity with a half-maximal inhibitory concentration (IC50) value of 56.0 and 40.8 µg/mL, respectively, over that of the control plants (78.0 and 58.2 µg/mL, respectively). These observations suggest that post-harvest irradiation with UV-B can be considered a promising technique to improve the healthy-nutritional and pharmaceutical properties of sweet basil leaves.

Variation of the Phytochemical Constituents and Antioxidant Activities of Zingiber officinale var. rubrum Theilade Associated with Different Drying Methods and Polyphenol Oxidase Activity.

The effects of different drying methods (freeze drying, vacuum oven drying, and shade drying) on the phytochemical constituents associated with the antioxidant activities of Z. officinale var. rubrum Theilade were evaluated to determine the optimal drying process for these rhizomes. Total flavonoid content (TFC), total phenolic content (TPC), and polyphenol oxidase (PPO) activity were measured using the spectrophotometric method. Individual phenolic acids and flavonoids, 6- and 8-gingerol and shogaol were identified by ultra-high performance liquid chromatography method. Ferric reducing antioxidant potential (FRAP) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays were used for the evaluation of antioxidant activities. The highest reduction in moisture content was observed after freeze drying (82.97%), followed by vacuum oven drying (80.43%) and shade drying (72.65%). The highest TPC, TFC, and 6- and 8-shogaol contents were observed in samples dried by the vacuum oven drying method compared to other drying methods. The highest content of 6- and 8-gingerol was observed after freeze drying, followed by vacuum oven drying and shade drying methods. Fresh samples had the highest PPO activity and lowest content of flavonoid and phenolic acid compounds compared to dried samples. Rhizomes dried by the vacuum oven drying method represent the highest DPPH (52.9%) and FRAP activities (566.5 µM of Fe (II)/g DM), followed by freeze drying (48.3% and 527.1 µM of Fe (II)/g DM, respectively) and shade drying methods (37.64% and 471.8 µM of Fe (II)/g DM, respectively) with IC50 values of 27.2, 29.1, and 34.8 µg/mL, respectively. Negative and significant correlations were observed between PPO and antioxidant activity of rhizomes. Vacuum oven dried rhizomes can be utilized as an ingredient for the development of value-added food products as they contain high contents of phytochemicals with valuable antioxidant potential.

Agonist anti-GITR monoclonal antibody and stereotactic radiation induce immune-mediated survival advantage in murine intracranial glioma.

BACKGROUND: Glioblastoma (GBM) is a poorly immunogenic neoplasm treated with focused radiation. Immunotherapy has demonstrated synergistic survival effects with stereotactic radiosurgery (SRS) in murine GBM. GITR is a co-stimulatory molecule expressed constitutively on regulatory T-cells and by effector T-cells upon activation. We tested the hypothesis that anti-GITR monoclonal antibody (mAb) and SRS together would confer an immune-mediated survival benefit in glioma using the orthotopic GL261 glioma model. METHODS: Mice received SRS and anti-GITR 10 days after implantation. The anti-GITR mAbs tested were formatted as mouse IgG1 D265A (anti-GITR (1)) and IgG2a (anti-GITR (2a)) isotypes. Mice were randomized to four treatment groups: (1) control; (2) SRS; (3) anti-GITR; (4) anti-GITR/SRS. SRS was delivered to the tumor in one fraction, and mice were treated with mAb thrice. Mice were euthanized on day 21 to analyze the immunologic profile of tumor, spleen, and tumor draining lymph nodes. RESULTS: Anti-GITR (1)/SRS significantly improved survival over either treatment alone (p < .0001) with a cure rate of 24 % versus 0 % in a T-lymphocyte-dependent manner. There was elevated intratumoral CD4+ effector cell infiltration relative to Treg infiltration in mice treated with anti-GITR (1)/SRS, as well as significantly elevated IFNγ and IL-2 production by CD4+ T-cells and elevated IFNγ and TNFα production by CD8+ T-cells. There was increased mRNA expression of M1 markers and decreased expression of M2 markers in tumor infiltrating mononuclear cells. The anti-GITR (2a)/SRS combination did not improve survival, induce tumor regression, or result in Treg depletion. CONCLUSIONS: These findings provide preclinical evidence for the use of anti-GITR (1) non-depleting antibodies in combination with SRS in GBM.