Identification and Validation of Nutrient State-Dependent Serum Protein Mediators of Human CD4+ T Cell Responsiveness.

Intermittent fasting and fasting mimetic diets ameliorate inflammation. Similarly, serum extracted from fasted healthy and asthmatic subjects' blunt inflammation in vitro, implicating serum components in this immunomodulation. To identify the proteins orchestrating these effects, SOMAScan technology was employed to evaluate serum protein levels in healthy subjects following an overnight, 24-h fast and 3 h after refeeding. Partial least square discriminant analysis identified several serum proteins as potential candidates to confer feeding status immunomodulation. The characterization of recombinant IGFBP1 (elevated following 24 h of fasting) and PYY (elevated following refeeding) in primary human CD4+ T cells found that they blunted and induced immune activation, respectively. Furthermore, integrated univariate serum protein analysis compared to RNA-seq analysis from peripheral blood mononuclear cells identified the induction of IL1RL1 and MFGE8 levels in refeeding compared to the 24-h fasting in the same study. Subsequent quantitation of these candidate proteins in lean versus obese individuals identified an inverse regulation of serum levels in the fasted subjects compared to the obese subjects. In parallel, IL1RL1 and MFGE8 supplementation promoted increased CD4+ T responsiveness to T cell receptor activation. Together, these data show that caloric load-linked conditions evoke serological protein changes, which in turn confer biological effects on circulating CD4+ T cell immune responsiveness.

Fasting-induced FOXO4 blunts human CD4+ T helper cell responsiveness.

Intermittent fasting blunts inflammation in asthma1 and rheumatoid arthritis2, suggesting that fasting may be exploited as an immune-modulatory intervention. However, the mechanisms underpinning the anti-inflammatory effects of fasting are poorly characterized3-5. Here, we show that fasting in humans is sufficient to blunt CD4+ T helper cell responsiveness. RNA sequencing and flow cytometry immunophenotyping of peripheral blood mononuclear cells from volunteers subjected to overnight or 24-h fasting and 3 h of refeeding suggest that fasting blunts CD4+ T helper cell activation and differentiation. Transcriptomic analysis reveals that longer fasting has a more robust effect on CD4+ T-cell biology. Through bioinformatics analyses, we identify the transcription factor FOXO4 and its canonical target FK506-binding protein 5 (FKBP5) as a potential fasting-responsive regulatory axis. Genetic gain- or loss-of-function of FOXO4 and FKBP5 is sufficient to modulate TH1 and TH17 cytokine production. Moreover, we find that fasting-induced or genetic overexpression of FOXO4 and FKBP5 is sufficient to downregulate mammalian target of rapamycin complex 1 signalling and suppress signal transducer and activator of transcription 1/3 activation. Our results identify FOXO4-FKBP5 as a new fasting-induced, signal transducer and activator of transcription-mediated regulatory pathway to blunt human CD4+ T helper cell responsiveness.

Web Tool for Navigating and Plotting SomaLogic ADAT Files.

SOMAscan™ is a complex proteomic platform created by SomaLogic. Experimental data resulting from the assay is provided by SomaLogic in a proprietary text-based format called ADAT. This manuscript describes a user-friendly point and click open source, platform-independent software tool designed to be used for navigating and plotting data from an ADAT file. This tool was used either alone or in conjunction with other tools as a first pass analysis of the data on several different on-going research projects. We have seen a need from our experience for a web interface to the ADAT file so that users can navigate, generate plots, perform QC and conduct statistical analysis on their own data in a point and click manner. After several rounds of interacting with biologists and their requirements with respect to data analysis, we present an online interactive Shiny Web Tool for Navigating and Plotting data contained within the ADAT file. Extensive video tutorials, example data, the tool and the source code are available online.

An Immune-Inflammation Gene Expression Signature in Prostate Tumors of Smokers.

Smokers develop metastatic prostate cancer more frequently than nonsmokers, suggesting that a tobacco-derived factor is driving metastatic progression. To identify smoking-induced alterations in human prostate cancer, we analyzed gene and protein expression patterns in tumors collected from current, past, and never smokers. By this route, we elucidated a distinct pattern of molecular alterations characterized by an immune and inflammation signature in tumors from current smokers that were either attenuated or absent in past and never smokers. Specifically, this signature included elevated immunoglobulin expression by tumor-infiltrating B cells, NF-κB activation, and increased chemokine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects of nicotine in human prostate cancer cells and prostate cancer-prone TRAMP mice. These investigations showed that nicotine increased glutamine consumption and invasiveness of cancer cells in vitro and accelerated metastatic progression in tumor-bearing TRAMP mice. Overall, our findings suggest that nicotine is sufficient to induce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel candidate driver underlying metastatic prostate cancer in current smokers.

T cell avidity and tumor immunity: problems and solutions.

A potent T cell response is an important component of durable anti-tumor immunity. The quality of the T cell response can, in-part, be measured by the avidity of the T cell for its tumor antigen-expressing target. While convention suggests that raising the avidity of the responding T cells may make for a more potent anti-tumor immune response, the threshold for effective tumor immunity remains unclear, as do some of the adverse effects of an inappropriately high avidity response. In this review, we discuss the relationship between T cell avidity and anti-tumor immunity, considering both experimental model systems as well as human clinical trials.

DEXterity of tolerogenic APCs.

A promising therapeutic approach for inducing tolerance in autoreactive T cells is the use of APCs such as DCs and macrophages. In this issue of the European Journal of Immunology, Zheng et al. [Eur. J. Immunol. 2013. 43: 219-227] study the concept of "tolerogenic adjuvants" to induce tolerance via vaccination. These authors have previously identified dexamethasone (Dex) as an effective "tolerogenic adjuvant" and, in this study, they have identified a population of peripheral macrophages that is enriched by Dex treatment and that mediates Dex's tolerogenic effect. In addition to performing a phenotypic characterization of this population, the authors noted an increase in serum levels of IL-10 and Treg cells after Dex treatment of mice. As discussed in this Commentary, by employing Dex as a tolerogenic adjuvant in the presence of relevant peptides, we may have a means of restoring specific immune tolerance in cases of autoimmune disease and allergy.

Analysis of PSPHL as a Candidate Gene Influencing the Racial Disparity in Endometrial Cancer.

Endometrial cancer is the most commonly diagnosed gynecologic malignancy in the United States. A well recognized disparity by race in both incidence and survival outcome exists for this cancer. Specifically Caucasians are about two times more likely to develop endometrial cancer than are African-Americans. However, African-American women are more likely to die from this disease than are Caucasians. The basis for this disparity remains unknown. Previous studies have identified differences in the types and frequencies of gene mutations among endometrial cancers from Caucasians and African-Americans suggesting that the tumors from these two groups might have differing underlying genetic defects. We performed a gene expression microarray study in an effort to identify differentially expressed transcripts between African-American and Caucasian women's endometrial cancers. Our gene expression screen identified a list of potential biomarkers that are differentially expressed between these two groups of cancers. Of these we identified a poorly characterized transcript with a region of homology to phospho serine phosphatase (PSPH) and designated phospho serine phosphatase like (PSPHL) as the most differentially over-expressed gene in cancers from African-Americans. We further clarified the nature of expressed transcripts. Northern blot analysis confirmed the message was limited to a transcript of under 1 kB. Sequence analysis of transcripts confirmed two alternate open reading frame (ORF) isoforms due to alternative splicing events. Splice specific primer sets confirmed both isoforms were differentially expressed in tissues from Caucasians and African-Americans. We further examined the expression in other tissues from women to include normal endometrium, normal and malignant ovary. In all cases PSPHL expression was more often present in tissues from African-Americans than Caucasians. Our data confirm the African-American based expression of the PSPHL transcript in endometrial cancer and also identify its expression in other tissues from African-Americans including ovary and ovarian cancer. PSPHL represents a candidate gene that might influence the observed racial disparity in endometrial and other cancers.

FOXO3 programs tumor-associated DCs to become tolerogenic in human and murine prostate cancer.

The limited success of cancer immunotherapy is often attributed to the loss of antigen-specific T cell function in situ. However, the mechanism for this loss of function is unknown. In this study, we describe a population of tumor-associated DCs (TADCs) in both human and mouse prostate cancer that tolerizes and induces suppressive activity in tumor-specific T cells. In tumors from human prostate cancer patients and transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, TADCs expressed elevated levels of FOXO3 and Foxo3, respectively, which correlated with expression of suppressive genes that negatively regulate T cell function. Silencing FOXO3 and Foxo3 with siRNAs abrogated the ability of human and mouse TADCs, respectively, to tolerize and induce suppressive activity by T cells. Silencing Foxo3 in mouse TADCs was also associated with diminished expression of tolerogenic mediators, such as indoleamine-2,3-dioxygenase, arginase, and TGF-ß, and upregulated expression of costimulatory molecules and proinflammatory cytokines. Importantly, transfer of tumor-specific CD4+ Th cells into TRAMP mice abrogated TADC tolerogenicity, which was associated with reduced Foxo3 expression. These findings demonstrate that FOXO3 may play a critical role in mediating TADC-induced immune suppression. Moreover, our results identify what we believe to be a novel target for preventing CTL tolerance and enhancing immune responses to cancer by modulating the immunosuppressive activity of TADCs found in the tumor microenvironment.

Non-clinical efficacy and safety of HyVac4:IC31 vaccine administered in a BCG prime-boost regimen.

Despite the extensive success with the introduction of M. bovis Bacille Calmette-Guérin (BCG), tuberculosis (TB) remains a major global epidemic infecting between 8 and 9 million people annually with an estimated 1.7 million deaths each year. However, because of its demonstrated effectiveness against some of the most severe forms of childhood TB, it is now realized that BCG vaccination of newborns is unlikely to be replaced. Therefore, BCG or an improved BCG will continue to be used as a prime TB vaccine and there is a need to develop effective boost vaccines that would enhance and prolong the protective immunity induced by BCG prime immunization. We report on a heterologous booster approach using two highly immunogenic TB antigens comprising Ag85B and TB10.4 (HyVac4) delivered as a fusion molecule and formulated in the proprietary adjuvant IC31. This vaccine was found to be immunogenic and demonstrated greater protection in the more stringent guinea pig model of pulmonary tuberculosis than BCG alone when used in a prime/boost regimen. Significant difference in lung involvement was observed for all animals in the HyVac4 boosted group compared to BCG alone regardless of time to death or sacrifice. A vaccine toxicology study of the HyVac4:IC31 regimen was performed and it was judged safe to advance the vaccine into clinical trials. Therefore, all non-clinical data supports the suitability of HyVac4 as a safe, immunogenic, and effective vaccination in a prime-boost regimen with BCG.

Cutting Edge: Tumor-specific CD8+ T cells infiltrating prostatic tumors are induced to become suppressor cells.

We previously reported that naive, tumor-specific CD8(+) (TcR-I) T cells transferred into prostate tumor-bearing mice traffic to the prostate where they become tolerized. We now report that TcR-I cells suppress the proliferation of naive T cells. This suppression is mediated at least in part by secreted factors, and the suppressive activity can be blocked by Abs directed against TGF-beta. We further report that TcR-I cells must infiltrate the prostate to acquire suppressive activity. Delivery of tumor-specific CD4(+) T cells prevents the conversion of TcR-I cells into suppressor cells. Taken together, our findings may have critical implications for sustaining T cell responsiveness during immunotherapy, as the development of suppressor cells in the tumor microenvironment may eliminate the potency of T cells primed in the periphery or delivered during adoptive immunotherapy.

Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG(1331)) expresses a mutant form of perfringolysin O (PfoA(G137Q)), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA(G137Q) gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q) under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG(1331) DeltaureC::OmegapfoA(G137Q)) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains.

Tumor-derived p53 mutants induce NF-kappaB2 gene expression.

Overexpression of mutant p53 is a common theme in tumors, suggesting a selective pressure for p53 mutation in cancer development and progression. To determine how mutant p53 expression may lead to survival advantage in human cancer cells, we generated stable cell lines expressing p53 mutants p53-R175H, -R273H, and -D281G by use of p53-null human H1299 (lung carcinoma) cells. Compared to vector-transfected cells, H1299 cells expressing mutant p53 showed a survival advantage when treated with etoposide, a common chemotherapeutic agent; however, cells expressing the transactivation-deficient triple mutant p53-D281G (L22Q/W23S) had significantly lower resistance to etoposide. Gene expression profiling of cells expressing transcriptionally active mutant p53 proteins revealed the striking pattern that all three p53 mutants induced expression of approximately 100 genes involved in cell growth, survival, and adhesion. The gene NF-kappaB2 is a prominent member of this group, whose overexpression in H1299 cells also leads to chemoresistance. Treatment of H1299 cells expressing p53-R175H with small interfering RNA specific for NF-kappaB2 made these cells more sensitive to etoposide. We have also observed activation of the NF-kappaB2 pathway in mutant p53-expressing cells. Thus, one possible pathway through which mutants of p53 may induce loss of drug sensitivity is via the NF-kappaB2 pathway.

Modulation of gene expression by tumor-derived p53 mutants.

p53 mutants with a single amino acid substitution are overexpressed in a majority of human cancers containing a p53 mutation. Overexpression of the mutant protein suggests that there is a selection pressure on the cell indicative of an active functional role for mutant p53. Indeed, H1299 cells expressing mutant p53-R175H, p53-R273H or p53-D281G grow at a faster rate compared with a control cell line. Using p53-specific small interfering RNA, we show that the growth rate of mutant p53-expressing cells decreases as mutant p53 level decreases, demonstrating that the increased cellular growth is dependent on p53 expression. Increased growth rate is not observed for H1299 cell clones expressing mutant p53-D281G (L22Q/W23S), which has been shown to be defective in transactivation in transient transcriptional assays. This shows that the increased growth rate imparted by mutant p53 in H1299 cells requires the transactivation function of mutant p53. By performing microarray hybridization analyses, we show that constitutive expression of three common p53 mutants (p53-R175H, p53-R273H, and p53-D281G) in H1299 human lung carcinoma cells evokes regulation of a common set of genes, a significant number of which are involved in cell growth regulation. Predictably, H1299 cells expressing p53-D281G (L22Q/W23S) are defective in up-regulating a number of these genes. The differences in expression profiles induced by individual p53 mutants in the cells may be representative of the p53 mutants and how they can affect gene expression resulting in the observed "gain of function" phenotypes (i.e., increased growth rate, decreased sensitivity to chemotherapeutic agents, and so forth).

Tumor-derived p53 mutants induce oncogenesis by transactivating growth-promoting genes.

We have studied the mechanism of mutant p53-mediated oncogenesis using several tumor-derived mutants. Using a colony formation assay, we found that the majority of the mutants increased the number of colonies formed compared to the vector. Expression of tumor-derived p53 mutants increases the rate of cell growth, suggesting that the p53 mutants have 'gain of function' properties. We have studied the gene expression profile of cells expressing tumor-derived p53-D281G to identify genes transactivated by mutant p53. We report the transactivation of two genes, asparagine synthetase and human telomerase reverse transcriptase. Quantitative real-time PCR confirms this upregulation. Transient transfection promoter assays verify that tumor-derived p53 mutants transactivate these promoters significantly. An electrophoretic mobility shift assay shows that tumor-derived p53-mutants cannot bind to the wild-type p53 consensus sequence. The results presented here provide some evidence of a possible mechanism for mutant p53-mediated transactivation.

Real-time polymerase chain reaction quantitation of relative expression of genes modulated by p53 using SYBR Green I.

Real-time quantitative polymerase chain reaction (QPCR) using the Roche LightCycler was used to verify the expression of asparagine synthetase (ASNS) identified by microarray analysis as a target of p53 transrepression and mutant p53 transactivation. A p53-null cell line derived from lung carcinoma, H1299, was infected with recombinant adenovirus expressing wild-type (WT) p53, mutant p53-D281G, or beta-galactosidase as a control. After 24 h of infection, RNA was harvested and used for microarray analysis. ASNS was one of several genes whose expression was down-regulated by WT p53 and up-regulated in the presence of mutant p53. Expression levels of ASNS were measured relative to an exogenously applied quality-control nucleic acid template. Real-time PCR product accumulation was monitored using the intercalating dye, SYBR Green I, which exhibits a higher fluorescence upon binding of double-stranded DNA. Relative gene expression was calculated using conditions at the early stages of PCR, when amplification was logarithmic and, thus, could be correlated to initial copy number of gene transcripts. ASNS was found to be down-regulated in the presence of WT p53 and up-regulated by mutant p53.