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Hybrid thin films with a high loading and homogeneous dispersion of functional nanoparticles (and/or molecules) find applications in (bio)-sensors and electronic devices. The fabrication of such hybrid thin films, however, suffers from the complex and diverse surface and physicochemical properties of individual nanoparticles. To address this challenge, a facile and general strategy toward compartmentalized thin films through the interfacial cross-linking of viral protein cages is reported. Employing these protein cages, gold nanoparticles, as well as enzyme horseradish peroxidase, are encapsulated into virus-like particles and then cross-linked into thin films with a thickness varying from monolayer to submicron dimensions. These compartmentalized thin films not only ensure that the cargo is homogeneously dispersed, but also display good catalytic activity. This strategy is, in principle, applicable for a wide range of (bio)-organic nanocontainers, enabling the versatile fabrication of 2D thin films with extensive application prospects.

Systemic lupus erythematosus (SLE) is an autoimmune disease caused by an immune response to many self-antigens, including DNA, histones, and RNA-binding proteins. One of the most physiologically relevant mouse models is the NZB/NZW F1 hybrid mouse, which develops SLE at high penetrance, in contrast to either parental strain. However, the critical genetic contributions of each parent, which must somehow interact to cause SLE, have remained unresolved. We have determined that NZB;NZW embryonic chimeras develop an accelerated form of autoimmunity characterized by the presence of auto-antibodies 6 months earlier, and at titers 10-fold greater, than the NZB/NZW F1 hybrids. The chimeras also developed glomerulonephritis, severe lymphadenopathy, and splenomegaly. Within each chimera, expansion of activated T cells of NZB/BINJ origin and a predominance of B cells of NZW/LacJ origin was observed with respect to the relative proportions of myeloid cells. Furthermore, the expansion of NZB/BINJ T cells correlates with autoantibody production. This SLE phenotype was specific for the NZB/BINJ;NZW/LacJ combination, and occurs despite the fact that neither parental strain develops SLE-like autoimmunity in a non-chimeric environment. Understanding the pathogenesis of SLE in the NZB;NZW embryonic chimeras may elucidate causes of autoimmunity by identifying the intercellular transactions between these two strains that lead to disease.

Cytotoxic T cells (CTLs) are an important component of adaptive immunity. The study of antigen-specific CTLs in vivo is desirable yet difficult. Identification of the class I-restricted peptide used by CTLs for target recognition is often required for detailed studies, but is generally not known for most antigens. Toxoplasma gondii is a medically important, obligate intracellular parasite and is often used as a model for studies of parasite immunology. No class I-restricted peptides for CTLs are known. We show here a new and convenient method to detect T. gondii-specific CTLs in vivo. We engineered T. gondii tachyzoites to express the model antigen ovalbumin, for which many useful reagents and transgenic mice are available. Using ovalbumin-transgenic T. gondii tachyzoites, antigen-specific CTLs were detected in vivo, and at much earlier time points post-infection than previously reported. This new method has several additional advantages over current methods to detect T. gondii-specific CTLs.

CD73, originally defined as a lymphocyte differentiation antigen, is thought to function as a cosignaling molecule on T lymphocytes and an adhesion molecule that is required for lymphocyte binding to endothelium. We show here that CD73 is widely expressed on many tumor cell lines and is upregulated in cancerous tissues. Because the ecto-5′-nucleotidase activity of CD73 catalyzes AMP breakdown to immunosuppressive adenosine, we hypothesized that CD73-generated adenosine prevents tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that combining tumor CD73 knockdown and tumor-specific T-cell transfer cured all tumor-bearing mice. In striking contrast, there was no therapeutic benefit of adoptive T-cell immunotherapy in mice bearing tumors without CD73 knockdown. Moreover, blockade of the A2A adenosine receptor with a selective antagonist also augmented the efficacy of adoptive T-cell therapy. These findings identify a potential mechanism for CD73-mediated tumor immune evasion and point to a novel cancer immunotherapy strategy by targeting the enzymatic activity of tumor CD73. Cancer Res; 70(6); 2245–55

Summary The mammalian (mechanistic) target of rapamycin (mTOR) regulates critical immune processes that remain incompletely defined. Interest in mTOR inhibitor drugs is heightened by recent demonstrations that the mTOR inhibitor rapamycin extends lifespan and healthspan in mice. Rapamycin or related analogues (rapalogues) also mitigate age‐related debilities including increasing antigen‐specific immunity, improving vaccine responses in elderly humans, and treating cancers and autoimmunity, suggesting important new clinical applications. Nonetheless, immune toxicity concerns for long‐term mTOR inhibition, particularly immunosuppression, persist. Although mTOR is pivotal to fundamental, important immune pathways, little is reported on immune effects of mTOR inhibition in lifespan or healthspan extension, or with chronic mTOR inhibitor use. We comprehensively analyzed immune effects of rapamycin as used in lifespan extension studies. Gene expression profiling found many and novel changes in genes affecting differentiation, function, homeostasis, exhaustion, cell death, and inflammation in distinct T‐ and B‐lymphocyte and myeloid cell subpopulations. Immune functions relevant to aging and inflammation, and to cancer and infections, and innate lymphoid cell effects were validated in vitro and in vivo. Rapamycin markedly prolonged lifespan and healthspan in cancer‐ and infection‐prone mice supporting disease mitigation as a mechanism for mTOR suppression‐mediated longevity extension. It modestly altered gut metagenomes, and some metagenomic effects were linked to immune outcomes. Our data show novel mTOR inhibitor immune effects meriting further studies in relation to longevity and healthspan extension.

The majority of children with Dravet syndrome (DS) are caused by de novo SCN1A mutations. To investigate the origin of the mutations, we developed and applied a new method that combined deep amplicon resequencing with a Bayesian model to detect and quantify allelic fractions with improved sensitivity. Of 174 SCN1A mutations in DS probands which were considered “de novo” by Sanger sequencing, we identified 15 cases (8.6%) of parental mosaicism. We identified another five cases of parental mosaicism that were also detectable by Sanger sequencing. Fraction of mutant alleles in the 20 cases of parental mosaicism ranged from 1.1% to 32.6%. Thirteen (65% of 20) mutations originated paternally and seven (35% of 20) maternally. Twelve (60% of 20) mosaic parents did not have any epileptic symptoms. Their mutant allelic fractions were significantly lower than those in mosaic parents with epileptic symptoms (P = 0.016). We identified mosaicism with varied allelic fractions in blood, saliva, urine, hair follicle, oral epithelium, and semen, demonstrating that postzygotic mutations could affect multiple somatic cells as well as germ cells. Our results suggest that more sensitive tools for detecting low‐level mosaicism in parents of families with seemingly “de novo” mutations will allow for better informed genetic counseling.

Etomidate is a unique drug used for induction of general anesthesia and sedation, and is usually used through intravenous injection clinically. Before targeting to the receptor, etomidate binds proteins in blood when it comes into veins. Thus to study the interaction of etomidate and serum albumin would be of great toxicological and pharmacological importance. In this study, the interaction between etomidate and human serum albumin (HSA) was studied using fluorescence spectroscopy, UV–vis absorption spectroscopy, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD) spectroscopy, site maker displacement and molecular modeling methods. Investigations of the binding constant (K = 3.55 × 105 M−1, 295 K), the number of binding sites (n = 1.16), thermodynamic parameters (ΔG = 3.13 × 104 J·mol−1, ΔS = 364 J·mol−1·K−1 and ΔH = −6.85 × 105 J·mol−1) for the reaction and changes to the binding sites and conformation in HSA in response to etomidate were presented. Results show that etomidate can bind HSA tightly through electrostatic forces, and the protein skeleton conformation and secondary structure changes thereby. This is the first spectroscopic report for etomidate–HSA interactions which illustrates the complex nature of this subject.

To understand how chloroquine (CQ) enhances transgene expression in polycation-based, nonviral gene delivery systems, a number of CQ analogues with variations in the aliphatic amino side chain or in the aromatic ring are synthesized and investigated. Our studies indicate that the aliphatic amino moiety of CQ is essential to provide increased gene expression. Further, the enhancements are more dramatically affected by changes to the aromatic ring and are positively correlated to the strength of intercalation between DNA and the CQ analogues. Quinacrine (QC), a CQ analogue with a fused acridinyl structure that can strongly intercalate DNA, enhances transfection similarly to CQ at a concentration 10 times lower, while N(4)-(4-pyridinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CP), a CQ analogue that has a weakly intercalating pyridinyl ring, shows no effect on gene expression. Subtle change on the 7-substituent of the chloroquine aromatic structure can also greatly affect the ability of the CQ analogues to enhance transgene expression. Transfection in the presence of N(4)-(7-trifluoromethyl-4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamin e (CQ7a) shows expression efficiency 10 times higher than in the presence of CQ at same concentration, while transfection in the presence of N(4)-(4-quinolinyl)-N(1),N(1)-diethyl-1,4-pentanediamine (CQ7b) does not reveal any enhancing effects on expression. Through a number of comparative studies with CQ and its analogues, we conclude that there are at least three mechanistic features of CQ that lead to the enhancement in gene expression: (i) pH buffering in endocytic vesicles, (ii) displacement of polycations from the nucleic acids in polyplexes, and (iii) alteration of the biophysical properties of the released nucleic acid.

The IL-2 receptor (IL-2R) is an attractive target for cancer immunotherapy as it controls both immune-suppressive regulatory T cells (Tregs) and anti-tumor T cells. We tested depleting Tregs as immunotherapy using anti-CD25 (high-affinity IL-2R subunit) antibodies (αCD25) in ID8agg mouse ovarian cancer (OC). αCD25 reduced ascites and Treg numbers but failed to reduce tumor burden, possibly because it depleted newly activated anti-tumor T cells in tumor-draining lymph nodes. Thus, αCD25 could be novel malignant ascites palliation, but has limited stand-alone efficacy. We then tested IL-2/anti-IL-2 complexes (IL-2c) that selectively stimulate medium-affinity (CD122/CD132) IL-2R thought to expand anti-tumor T cells preferentially, but with little Treg effects. In contrast to several single agents we tested that failed to treat ID8agg (e.g., αCD25, αPD-L1, IL-2 fusion toxin denileukin diftitox), IL-2c alone durably reduced ID8agg tumor burden despite lowering the tumor microenvironmental CD8+/Treg ratio. Thus, we hypothesized that IL-2c improved CD8+ function, reduced Treg function, or both. IL-2c increased polyfunctional IFN-γ+TNF-α+ anti-tumor T cells as expected, an effect that persists weeks after drug clearance. IL-2c also increased anti-tumor T cell CD25 expression that increased IL-2 sensitivity and STAT5 phosphorylation, a likely mechanism for increased polyfunctionality. Unexpectedly, IL-2c reduced the Treg functional mediators CD25, TIGIT and granzyme B, and reduced Treg suppressive function. Thus, favorable Treg modifications are a novel IL-2c mechanism of action. Adding αCD25 to IL-2c to deplete Tregs further unexpectedly worsened IL-2c efficacy in ID8agg and reduced effector memory T cells and polyfunctional T cells in the tumor microenvironment, suggesting a previously unappreciated role for CD25 in IL-2c therapy. Similar data were seen in B16 melanoma, suggesting αCD25 reduction of IL-2c efficacy is not tumor or compartment-specific (ID8agg is peritoneal and B16 is subcutaneous). αPD-L1, an ineffective monotherapy in ID8agg, combined with IL-2c to promote complete responses, suggesting potential for potent, novel combinatorial approaches. Our data suggest that antagonizing high affinity IL-2R (such as to deplete Tregs with αCD25) has limited cancer immunotherapy utility without more specific Treg targeting. In contrast, stimulating medium-affinity IL-2R with CD122-selective IL-2c has great translational promise by simultaneously improving beneficial anti-tumor T cells and reducing detrimental Treg function. Citation Format: Justin M. Drerup, Sri Lakshmi Pandeswara, Aijie Liu, Curtis A. Clark, Alvaro S. Padron, Wanjiao Chen, Vincent Hurez, Tyler J. Curiel. CD122-selective IL-2/anti-IL-2 complexes reduce regulatory T cell function and promote CD8+ T cell polyfunctionality for durable ovarian cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1608. doi:10.1158/1538-7445.AM2017-1608

Background Most cancer patients are aged but most preclinical studies are in young hosts. We tested age effects on immunotherapy to further our understanding and identify strategies. Methods Young (3 mos) and aged (~26 mos) BL6 mice were injected subcutaneously with syngeneic B16 melanoma cells. 7 days later, mice got 2 μg denileukin diftitox (DD), an FDA-approved IL-2/diphtheria toxin or 200 μg αCD25 (both to deplete Tregs) ± 50 μg αCTLA-4 or 100 μg αB7-H1. 5 days later mice got a 2nd treatment. Results αCTLA-4 depleted intra-tumor Tregs in young but not aged males, a major age effect. In young BL6 males or females αCTLA-4 + αB7-H1 rejected B16 tumors better than either single agent. In aged males, αCTLA4 + αB7-H1 treated B16, but surprisingly accelerated tumors in aged females. Individual agents were effective in young hosts but had negligible effects in the aged. αCTLA-4 + either DD or αCD25 to deplete Tregs significantly reduced tumor growth in young mice but DD was better than αCD25. Neither combo treated B16 in aged mice. This DD dose did not deplete Tregs in aged mice as well as young, but higher DD doses were equally effective. In support, in Foxp3DTR mice, in which we deplete ~100% Treg (versus 20–50% with DD or αCD25), aged mice potently rejected B16. Conclusions Cancer immunotherapy studies in young give an incomplete understanding of clinical effects and mechanisms in the aged most at risk for cancer. We identify major Treg effects and differential Treg depletion efficacy of αCTLA4 and DD contributing to treatment failures in the aged and means to mitigate effects. With appropriate treatments, even very old mice can mount effective anti-tumor immunity. Novel sex differences in aged are unlikely estrogen-related and require further study.

Age is the greatest cancer risk factor, but contributions of aging to tumor intrinsic properties and immunotherapy responses are poorly understood as most preclinical studies are in young hosts. We assessed age effects on tumor properties, progression, immunity and response to immunotherapies in an aggressive ID8 ovarian cancer line (ID8agg) that is poorly immunogenic and fatal in ~5 weeks. Young (3 mos) and aged (24 mos) BL6 females were injected intraperitoneally with 4×106 syngeneic ID8agg cells. In young mice, ID8agg ascites had more regulatory T cells (Tregs) and myeloid suppressor cells (MDSCs). Young mice gained 30% weight from ascites ~30 days after ID8agg challenge and formed 1–2 large, discrete peritoneal tumor masses. Aged mice tended to lose weight although ascites volumes were comparable to young, suggesting cachexia, and formed multiple small peritoneal nodules. Aged ascites had more tumor cells, Tregs, monocytic MDSCs and PD-1+ T cells versus young. Strikingly, tumor cells and MDSCs in aged ascites upregulated the immune checkpoint molecule B7-H1 greater than in young ascites. The combination of depleting Tregs and MDSCs with denileukin diftitox (DD), an FDA-approved IL-2/diphtheria toxin + αGr-1 temporarily slowed tumor progression in aged mice with increased IFNγ+ CD8 T cells, consistent with our prior B16 data. Treg depletion with either DD or αCD25 alone, or plus αCTLA4 did not significantly prevent ID8agg progression in young or aged mice. In young hosts, αB7-H1 slowed tumor growth and increased survival, with both clinical outcomes potentiated by adding αCTLA4. αCTLA4 + αB7-H1 effects in aged hosts are under study. Thus, we identified major age effects on tumor progression, and immune and immunotherapy responses.

Polyaniline-copper (PANI-Cu) and polyaniline-silver (PANI-Ag) composites were prepared by electrical explosion of wire (EEW) in presence and the absence of polyacrylic acid (PAA) surfactant. Structure characteristics of the composites were studied by field emission scanning electron microscope (FE-SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), UV-vis spectroscopy and electrical measurement. The conductivity of polyaniline-metal increased several magnitude compared to pure PANI. The characteristics of composites changed significantly when PAA was used. The stability of composite colloids increased; the surface of composite was smoother and the conductivity decreased. PANI-Ag composite have higher conductivity than PANI-Cu.

In this work, polyaniline - copper (PANI-Cu) and polyaniline/polyacrylic acid - copper (PANI/PAA-Cu) composites were synthesized by electrical explosion of wire (EEW) in the liquid. The PANI and PAA polymers in ethanol were stirred for 24 hrs. and sonicated for 2 hrs. The polymer solutions used as a liquid for explosion process to fabricate the Cu nanoparticle. X-ray diffraction (XRD) results of the composites showed that the Cu was polycrystal. Field emission scanning electron microscope (FE-SEM) was used to observe the microstructure of the composites. The PANI particles were in round shape and covered on the as-prepared Cu nanoparticles. The Cu nanoparticles were spherical and dispersed well in the polymer matrix. Crystalline size of the Cu nanoparticles was about 25nm. The obtained composites were extensively characterized by Fourier Transform Infrared (FTIR) spectroscopy, and electrical measurements.

Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, prevents distinct cancers in mice and prolongs life, making it a candidate cancer prevention (and life extension) agent. We hypothesized that eRapa cancer prevention includes immune effects, as mTOR is pivotal in immunity. We tested immune effects in carcinogen (DMBA) and inflammation (TPA)-induced dermal carcinogenesis with eRapa daily at 14 ppm, which protected 100% from skin cancer in wild type (WT) mice. Tumor mTORC1 was not suppressed, supporting eRapa immune effects. γδ T cells and IFN-γ are protective in this model. In δ TCR KO (no γδ T cells) and IFN-γ KO mice, eRapa cancer protection was lost, confirming immune mechanisms. IFN-γ KO mice had significantly reduced CD69+Lamp1+GzB+CXCR3+ epidermal γδ T cells and CXCL10, suggesting IFN-γ is required for epidermal γδ T cell migration (via CXCR3/CXCL10), activation and cytolysis. Bone marrow (BM) chimeras showed eRapa skin cancer protection and maximum γδ T cell epidermal infiltration requires IFN-γ signals in BM (e.g., γδ T cells) and non-BM (e.g., epidermal) cells. Intratumor injections of WT but not Prf1 (perforin) KO γδ T cells regressed DMBA/TPA tumors in δ TCR KO mice on eRapa but not control, showing eRapa boosts anti-tumor γδ T cell cytotoxicity. Our data confirm eRapa cancer prevention through immune mechanisms, challenging the paradigm of mTOR effects solely on cancer cells for prevention, and suggesting novel immune-mediated life extension mechanisms.

mTOR regulates many important immune processes. Interest in immune effects of mTOR inhibitors is heightened by recent findings of ability to boost specific immunity and vaccine response in elderly humans, treat cancers and autoimmunity, and extend life and healthspan in mice, suggesting important new clinical applications. However, immune toxicity concerns for long-term mTOR inhibition, particularly immunosuppression, persist. We comprehensively analyzed immune effects of long-term rapamycin in mice. Gene expression profiling of purified T and B cells (CD4+PD1+, CD4+PD1-, CD8+PD1+, CD8+PD1-, B220+), CD11b+CD11c- myeloid cells and CD11c+ dendritic cells showed numerous, novel changes in genes affecting differentiation, function, homeostasis, migration, exhaustion, cell death and inflammation. Novel effects on Th9, Th22 and Tfh differentiation were seen as was improved function of exhausted PD1+ T cells (supported by human clinical data) and increased numbers and function of innate lymphoid cells. Immune functions relevant to cancer, infections, aging and inflammation, and innate lymphoid cell effects were validated in vitro and in vivo. eRapa prolonged life in RAG KO mice (without T or B cells) >12x and in IFN-γ KO mice >3x over wild type mice, supporting mTOR suppression immune effects mitigating disease as a mechanism for longevity extension. Metagenomic effects were linked to immune outcomes. Our data demonstrate novel mTOR immune effects meriting further investigations.

mTOR signals through 2 complexes: mTORC1 and mTORC2. Approved cancer mTOR inhibitor drugs inhibit mTORC1 and are poorly effective against carcinomas and B cell lymphomas. There is thought that dual mTORC1/2 inhibitors could have better clinical efficacy. As mTOR regulates important T cell functions, we tested mTOR inhibitors in mouse EL4 T cell lymphoma. The mTORC1 inhibitor rapamycin potently suppressed EL4 proliferation in vitro, and significantly reduced EL4 tumor growth in vivo. AZD8055, a dual mTORC1/2 inhibitor, also inhibited EL4 cell proliferation in vitro, decreased in vivo tumor burden and improved survival in EL4 challenged mice, but no better than rapamycin. EL4 tumor growth was slower in females versus males, suggesting hormonal effects. As estrogen receptor (ER)β agonists reportedly can treat lymphomas we tested them. We found that ERβ agonists altered EL4 mTOR signals and a novel ERβ/mTOR physical interaction (mTORC2>>mTORC1) in EL4 by GST pull-down. The ERβ agonist S-equol synergized with AZD8055 to inhibit EL4 cell proliferation in vitro but did not alter EL4 tumor growth in vivo nor boost AZD8055-mediated EL4 growth suppression or survival in vivo. EL4 tumors from AZD8055 treated mice had reduced viability and proliferation (Ki67) without affecting CD3+ T cell Ki67. AZD8055 treated ID8 ovarian cancer in vivo but rapamycin did not, suggesting T cell lymphomas could respond to mTOR inhibitors better than carcinomas. ERβ effects also deserve more study.

Immunotherapy shows promise in carcinoma treatments. As less is known about effects on lymphomas, we tested EL4 T cell lymphoma. We found that EL4 cells were CD25-CTLA4-B7H1loPD1hi by flow cytometry. Immune checkpoint blockade with αCTLA4, αB7H1, or αPD1 antibodies had no effect on tumor growth in EL4-challenged mice. αCD25 worked as prophylaxis (before EL4 challenge) but not therapy, despite depleting regulatory T cells (Tregs). In Foxp3DTR mice, specific Treg depletion reduced EL4 tumor growth, suggesting Treg depletion as a good treatment strategy. Denileukin diftitox (DD) is a relatively-specific Treg depletion drug with clinical benefits in mouse and human cancer. DD (5 μg/4 days starting 4 days after EL4 challenge) significantly delayed EL4 tumor growth and increased survival (P=.002) in WT mice. DD protection was lost in βδ TCR KO mice lacking all T cells, suggesting a T cell dependent treatment mechanism. In EL4-challenged WT, DD depleted Tregs > αCD25 but significantly increased ICOS by 2-fold on remaining Tregs > than on CD4+ or CD8+ T cells in tumor draining lymph nodes. Nearly all Tregs within tumors were ICOS+. ICOS+ Tregs were mostly CD44+ with hi Ki-67, suggesting activation that reduced DD treatment effects. In support, αICOS (100 μg/4 days starting 1 day after DD) enhanced DD-mediated EL4 tumor growth inhibition, but did not improve overall survival. DD-mediated Treg depletion plus αICOS and/or other adjuncts could lead to effective lymphoma immunotherapy.

In ovarian cancer (OC) regulatory T cells (Tregs) block anticancer immunity, suggesting Treg depletion could be beneficial. In our phase 2 denileukin diftitox (DDT) trial we found no clinical efficacy in 19 OC patients despite Treg depletion. αCD25 depletes Tregs but reportedly only works as cancer prophylaxis. We found that αCD25 did not treat subcutaneous B16 mouse melanoma whereas DDT did, and both reduced Tregs equally. We further show that in intraperitoneal ID8 challenge, αCD25 depletes Tregs in ascites and strikingly extends survival better than DDT, the first demonstration that αCD25 is effective cancer treatment (vs prophylaxis). Treg suppression of T cell proliferation in vitro from ID8-bearing mice was equal after DDT or αCD25. DDT and αCD25 could thus target or modulate Treg subsets or other immune cells differentially, as neither is Treg-specific. Despite reduced clinical efficacy, DDT increased beneficial IFN-γ+CD4+ and ID8-specific CD8+ T cells and NK cells in ascites better than αCD25, but also increased pro-tumorigenic IL-17+ and TNF-α+ CD4+ T cells, whereas αCD25 did not. αCD25 increased numbers of ascites GzB+CD107a+CD8+ T cells whereas DDT did not, suggesting increased cytotoxicity that could explain better clinical efficacy. These observations show that Treg depletion agents available for clinical use have distinct effects on antitumor immunity, help define clinical indications for αCD25 versus DDT and identify responsive cancers or anatomic compartments.

Background Cancer prevention is a cost-effective alternative to treatment. Oral rapamycin (eRapa) prevents distinct cancers and extends life in mice [1] , making it a candidate broad-spectrum cancer prevention agent. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), has significant immune effects that are unstudied in cancer. We hypothesize that eRapa prevents cancer through improved cancer immune surveillance, which we tested in a carcinogen-induced, inflammation-driven skin cancer model. Methods eRapa was used at 14 ppm microencapsulated rapamycin. Skin cancer. Wild-type C57BL/6 (WT) or syngeneic β δ knockout (KO) and interferon (IFN)- γ KO mice (lacking all T cells or IFN- γ , respectively) got eRapa or control for 1 month, followed once with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), then twice weekly applications with the inflammatory agent 12-O-tetradecanoylphorbol (TPA) for 24 weeks. Flow cytometry assessed cells and IFN- γ . Statistics. Tumors (unpaired t test and 2-way ANOVA) and malignant degeneration (Fischer exact test). Results eRapa reduced benign ( p = .004) and malignant ( p = .03) tumors in WT mice. T cells and IFN- γ mediate cancer immune surveillance so their effects on eRapa cancer prevention were studied. eRapa reduced skin tumors in β δ KO mice lacking all T cells ( p = .04), but not in IFN- γ KO mice ( p = .13), consistent with loss of beneficial eRapa-induced, non-T cell IFN- γ . In support, WT or IFN- γ KO T cell transfer into IFN- γ KO mice did not alter eRapa cancer prevention in this model. In WT mice on DMBA/TPA, eRapa increased IFN- γ producing dermal natural killer (NK) cells ( p γ KO mice, suggesting that eRapa-mediated IFN- γ facilitated local NK cell accumulation. Conclusions eRapa prevents carcinogen and inflammation-induced skin cancer, which involves a non-T cell that could be an NK cell, and is IFN- γ dependent. We also show that eRapa prevents cancer and extends life in distinct mouse cancer models, which paves the way for use of mTOR inhibitors as broad spectrum human cancer prevention agents.

Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with beta-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 microg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.

Tregs hinder anti-tumor immunity, and depleting them treats cancers effectively in mice. By contrast, Treg depletion in human trials has limited efficacy. In a phase I trial of advanced stage carcinomas (breast, lung, melanoma, ovarian, bladder), we showed that the IL-2/diphtheria fusion toxin denileukin diftitox (DT) significantly depleted functional CD4+CD25hiFoxp3+ Tregs in blood with improved T cell function (increased Ki-67 and interferon-γ) with minimal effects on other blood mononuclear cells. One patient with stage IV ovarian carcinoma experienced significant reduction of metastatic tumor burden with denileukin diftitox at 12 μg/kg. Additional work showed that Treg depletion effects could last up to 4 weeks, and that weekly DT at 12 μg/kg eventually depleted anti-tumor and tumor-specific CD8+ T cells. Based on these data, we conducted a phase II clinical trial of epithelial ovarian carcinomas FIGO stage III-IV failing standard treatments, using DT as a single agent at 12 μg/kg by intravenous infusion every 3-4 weeks. In this trial, DT significantly depleted blood and tumor microenvironmental Tregs with only grade I-II toxicities, but with minimal clinical efficacy in 28 consecutive patients. This trial was halted for futility according to the Simon 2-stage design. Interferon-α alone does not treat ovarian cancer, but we now show that it significantly improves clinical and immune DT-mediated Treg depletion efficacy in human ovarian cancer. In the ID8 mouse ovarian carcinoma model, DT modestly increased survival and anti-tumor immunity. Interferon-α alone did not affect Treg numbers or function, but enhanced CD8+ T cell anti-tumor immunity. Interferon-α plus DT increased mouse survival significantly over either individual drug. In type I IFNR-/- mice unable to mediate interferon-α signals, interferon-α directly increased adoptively transferred IFNR+CD8+ T cell function independent of CD4+ T cell help. When combined with DT, IFN-α reduced Treg function without further numerical Treg reduction by indirect effects on tumor microenvironmental dendritic cells. In vitro studies identified IFN-α-driven dendritic cell IL-6 as a mechanism for reducing Treg function. When three ovarian cancer patients failed DT alone in the phase II trial, two had clinical and immune benefit by adding pegylated interferon-α2a in a separate trial, with acceptable toxicities. This trial was halted due to a lack of further DT. These studies demonstrate that DT depletes Tregs in distinct human carcinomas but is unlikely to be clinically effective as a single agent. We identified novel IFN-α mechanisms that improve Treg depletion effects using FDA-approved agents that can be rapidly translated. More selective Treg depletion agents and rationale combinations with other agents could improve clinical efficacy further. Citation Format: Suzanne Thibodeaux, Vincent Hurez, Shawna Wall, Srilakshmi Pandeswara, Benjamin Daniel, Aijie Liu, Lishi Sun, Leslie Wood, Weiping Zou, Tyler Curiel. Interferon-α enhances clinical benefits of regulatory T cell depletion in ovarian cancer through direct T cell effects and by inducing bystander IL-6. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-259. doi:10.1158/1538-7445.AM2014-LB-259

eRapa extends life in mice and cancer prevention could be a mechanism. Rapamycin inhibits mTOR, which has significant immune effects that are surprisingly unstudied in cancer therapy or prevention. We hypothesize that cancer prevention by eRapa is mediated by improved cancer immune surveillance, which we tested in the well-established DMBA/TPA carcinogen-induced skin cancer model. Mice got eRapa or control chow, and skin tumors were induced with DMBA/TPA over 24 weeks. eRapa reduced benign (p=.001) and malignant (p=.05) tumors in WT mice. T cells and IFN-γ mediate cancer immune surveillance. eRapa reduced skin tumors in βδ KO mice lacking all T cells (p=.04), but not in IFN-γ KO mice (p=.13), consistent with loss of beneficial eRapa-induced, non-T cell IFN-γ. In support, WT or IFN-γ KO T cell transfer into IFN-γ KO mice did not alter eRapa cancer prevention in DMBA/TPA. In WT mice on DMBA/TPA, eRapa increased IFN-γ-producing natural killer cells (p=.01) that could mediate skin cancer immune surveillance, and decreased CD34+CD49fint skin cancer stem cells (p=.01) and CXCR3+ T cells (p

Tregs blunt anti-tumor immunity, and their depletion effectively treats tumor in mouse cancer models. However, Treg depletion in human trials is only partially effective. In our phase II ovarian cancer trial, denileukin diftitox (DT) effected significant Treg depletion in blood and the tumor microenvironment but did not produce significant clinical efficacy. Interferon-α is not an effective ovarian cancer treatment, but we show here that it significantly augments immune and clinical DT-mediated Treg depletion efficacy in human ovarian cancer. In a mouse ovarian cancer model, DT modestly improved anti-tumor immunity and survival. Interferon-α alone did not alter Treg numbers or function, but boosted CD8+ T cell anti-tumor immunity. Interferon-α plus DT significantly prolonged mouse survival over either individual agent. Using type I IFNR-/- mice that cannot mediate interferon-α signals, we showed that interferon-α directly boosted CD8+ T cell function independent of CD4+ T cell help, and in combination with DT reduced Treg function (without further numerical reduction) through indirect effects on tumor microenvironmental dendritic cells. When three ovarian cancer patients failed DT alone, two experienced immunologic and clinical benefit by adding pegylated interferon-α2a, with manageable toxicities. These studies demonstrate novel immune and clinical interferon-α anti-cancer benefits that augment Treg depletion using FDA-approved agents for rapid clinical translation.

Chloroquine has been widely used as a reagent to enhance transfection in many non-viral gene delivery systems. The mechanism of its action is not well understood and has not been extensively investigated. We synthesized a number of CQ analogues with variations either on the alkyl amino side chain or on the aromatic ring, and investigated their structure-function correlations as transgene expression enhancers. We found that the aliphatic amino moiety is essential to CQ's role as a gene expression enhancer, as its removal eliminates any enhancement. Further, the enhancement is dramatically affected by changes to the aromatic ring, and is positively correlated to interactions between DNA and CQ analogues. Quinacrine, a CQ analogue with a tricyclic aromatic acridinyl structure that can strongly intercalate DNA, can enhance transfection similarly to CQ at a concentration ten times lower. In contrast, N4-(7-Chloro-4-pyridinyl)-N1,N1-diethyl-1,4-pentanediamine, a CQ analogue that has a weak intercalating pyridinyl ring, has no effect on gene expression. Subtle change of the 7-substituent of the CQ aromatic structure can also greatly affect its ability to enhance transgene expression. Transfection in the presence of N4-(7-trifluoromethyl-4-quinolinyl)-N1,N1-diethyl-1,4-pentanediamine (TFCQ), a 7- trifluoromethyl CQ analogue, shows expression at least one order of magnitude higher than transfection in the presence of CQ at the same concentration, while transfection in the presence of N4-(4-quinolinyl)-N1,N1-diethyl-1,4-pentanediamine, a 7-hydro CQ analogue, shows no enhancement in expression. CQ and TFCQ demonstrate similar activity in buffering the pH experienced by polyplexes within the endocytic pathway, but TFCQ is more potent in enhancing gene transfer. TFCQ has higher binding affinity to DNA than CQ as well as a greater tendency to competitively displace the polycation from polyplexes at an achievable intracellular concentration. These findings suggest that in addition to pH-buffering activity, CQ's competitive displacement of polycations and its interaction with nucleic acids are important to its enhancement of gene expression in non-viral nucleic acid delivery systems. Through a number of comparative studies on CQ and its analogues, we elucidated for the first time the important structural features of CQ that confer its capability to accumulate inside cells, buffer intracellular pH, and competitively displace polycations and thus unpackage DNA.

A widely applicable cancer prevention strategy would be a cost-effective approach to lessen the enormous burden of cancer. Enterically-released microencapsulated rapamycin (eRapa) extends lifespan in mice and delays or prevents cancer development, suggesting cancer prevention as a longevity mechanism. Rapamycin acts through inhibition of mechanistic target of rapamycin (mTOR), which has significant immune effects that are surprisingly little studied in cancer treatment or prevention. In naive mice, chronic eRapa significantly reduced Th1 (CXCR3 + ) cell numbers ( p = .03) and T cell interferon (IFN)- γ ( p = .04), increased Th17 (CCR6 + ) cell numbers ( p = .04) and T cell IL-17 ( p = .03), increased T cell IL-22 ( p = .02), and reduced immune suppressing regulatory T cells ( p βδ −/− and IFN- γ −/− mice (lacking all T cells or IFN- γ , respectively) were given eRapa or control, and skin tumors were induced with the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) followed by the inflammatory promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). eRapa reduced papilloma size in WT BL6 mice ( p = .02) versus controls, the first demonstration that oral rapamycin prevents inflammation-mediated neoplasia. eRapa also significantly reduced papillomas in βδ −/− mice ( p = .04) and increased papillomas in IFN- γ −/− mice ( p = .05). These data are consistent with the concept that eRapa protects from carcinogen-induced dermal carcinogenesis through a non-T cell, IFN- γ -dependent mechanism, which we suspect includes natural killer cells. We have also shown that eRapa prevents cancer in a variety of mouse cancer models, in which we are further studying immune mechanisms. Immune contributions from mTOR inhibitors in cancer prevention (and treatment) require more attention.

Age is the biggest risk factor for cancer but most studies are in young hosts. We mitigated age-specific tumor-related immune dysfunction to develop immunotherapy for B16 melanoma effective in aged hosts. Young and aged CD4+CD25hi regulatory T cells (Tregs) had similar in vitro function and in vivo tumor-associated increases, but denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. Aged mice had more basal and tumor-related myeloid derived suppressor cells (MDSC). Depleting MDSC with anti-Gr-1 Ab improved tumor-specific immunity and clinical response better in aged versus young mice. Treg depletion significantly increased MDSC in aged but not young mice, suggesting age-related Treg control of MDSC. In vitro, Treg from aged tumor bearing mice suppressed GM-CSF-induced bone marrow MDSC differentiation. Treg depletion + anti-Gr-1 Ab was more efficacious than anti-Gr-1 alone in aged B16-bearing mice but was no better than Treg depletion alone in young mice. In MC-38 colon cancer, DT and anti-Gr-1 Ab effects were similar in young and aged hosts. Thus aged anti-tumor immune effector cells are competent to combat tumor when tumor-associated immune dysfunction is appropriately mitigated, and age-related immune dysfunction is tumor-dependent. Tailoring immunotherapy to counter age-related, tumor-associated immune dysfunctions remarkably improves cancer immunotherapy for aged patients with specific tumors.

During tumor progression, tumor cells foster a tolerant microenvironment and the activation of many immunosuppressive mechanisms that act in concert to counteract effective immune responses. Therefore, to accomplish cancer immune eradication, tumor-induced immunosuppression must be overcome. We demonstrate that CD73 is widely expressed on many tumor cell lines and upregulated in cancerous tissues with active enzymatic activity. Notably, CD73 expression in malignant cancer patients is closely associated with poor immune status of tumor infiltrating effector T cells. Because CD73 catalyzes AMP breakdown to immunosuppressive adenosine, we propose that CD73-generated adenosine prevents tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that inhibition of CD73 by selective inhibitor APCP slowed tumor growth and increased mice survival, and more importantly, augmented CTL therapy of cancer. Using genetic ablation of CD73 on tumor cells by siRNA and CD73 deficient mice, we found that both tumor and host CD73 synergistically controlled tumor progression by impairing antitumor T cell immunity. Thus, counteracting adenosinergic effects of CD73 in the tumor microenvironment represents a novel and feasible strategy of cancer treatment and is complementary to other approaches directed at improving the immune function of antitumor T cells such as adoptive T cell therapy or dendritic cell vaccines.

Interleukin (IL)-12 is a proinflammatory cytokine generally regarded as a master regulator of type 1 T cell mediated immunity against intracellular pathogens, including T. gondii and cancers. A critical measure of IL-12 activity is its ability to induce IFN-γ production by NK and T cells. Using a transgenic T. gondii expressing ovalbumin (OVA), we show here that by flow cytometry analysis T. gondii-OVA infected BL6 IL-12-/- mice generate OVA-pentamer+CD8+ T cells similar to WT and surprisingly, T. gondii-infected IL-12-/- mice produce more IFN-γ +CD8+ T cells than T. gondii-infected WT mice (avg 9.6% in IL-12-/- vs 3.7% in WT). We confirmed that function of OVA-specific CD8+ T cells in IL-12-/- was comparable to BL6 WT using an in vivo OVA+ target lysis assay. As expected, IFN-γ +CD4+ T cells decreased from an avg 29% to 9% in WT mice compared to IL-12-/- mice respectively when infected with T. gondii. These results demonstrate a novel IL-12-independent pathway to CD8+ CTL that may be less dependent of CD4+ T cell help than previously thought.

CD73, originally defined as a lymphocyte differentiation antigen, is thought to function as a costimulatory molecule on T lymphocytes and an adhesion molecule that is important for lymphocyte binding to endothelium. We show here that CD73 is widely expressed on many tumor cell lines and upregulated in cancerous tissues. Because CD73 ecto-5'-nucleotidase catalyzes AMP breakdown to immunosuppressive adenosine, we propose that CD73-generated adenosine may prevent tumor destruction by inhibiting antitumor immunity. We confirmed this hypothesis by showing that genetic ablation of CD73 on ID8 ovarian tumor cells by siRNA reduced its enzymatic activity and increased survival and decreased tumor burden in immunocompetent but not immunodificient Rag1-/- mice. CD73 knockdown improved tumor antigen-specific T cell proliferation and CTL in vivo killing function and decreased apoptosis of tumor activated microenvironmental T cells. Notably, there was no therapeutic benefit of adoptive T-cell immunotherapy in mice bearing tumors without CD73 knockdown. By contrast, combining tumor CD73 knockdown and T cell transfer cured all mice. Moreover, blockade of A2A adenosine receptor with its antagonist SCH58261 also augmented the efficacy of T cell therapy. These findings identify a potential mechanism for CD73-mediated tumor immune evasion and point to a novel cancer immunotherapy strategy by targeting tumor CD73.