Ena Wang

The gut microbiota influences both local and systemic inflammation. Inflammation contributes to development, progression, and treatment of cancer, but it remains unclear whether commensal bacteria affect inflammation in the sterile tumor microenvironment. Here, we show that disruption of the microbiota impairs the response of subcutaneous tumors to CpG-oligonucleotide immunotherapy and platinum chemotherapy. In antibiotics-treated or germ-free mice, tumor-infiltrating myeloid-derived cells responded poorly to therapy, resulting in lower cytokine production and tumor necrosis after CpG-oligonucleotide treatment and deficient production of reactive oxygen species and cytotoxicity after chemotherapy. Thus, optimal responses to cancer therapy require an intact commensal microbiota that mediates its effects by modulating myeloid-derived cell functions in the tumor microenvironment. These findings underscore the importance of the microbiota in the outcome of disease treatment.

Whether there exists a human memory T cell population with stem cell–like properties of self-renewal and multipotency is under active investigation. Here Gattinoni et al. characterize a subset of human T cells that phenotypically resemble naive T cells yet have properties associated with memory T cells. These T cells show enhanced ability to self renew and to give rise to differentiated memory cell subsets, suggesting a stem cell–like functionality. Immunological memory is thought to depend on a stem cell–like, self-renewing population of lymphocytes capable of differentiating into effector cells in response to antigen re-exposure. Here we describe a long-lived human memory T cell population that has an enhanced capacity for self-renewal and a multipotent ability to derive central memory, effector memory and effector T cells. These cells, specific to multiple viral and self-tumor antigens, were found within a CD45RO−, CCR7+, CD45RA+, CD62L+, CD27+, CD28+ and IL-7Rα+ T cell compartment characteristic of naive T cells. However, they expressed large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and showed numerous functional attributes distinctive of memory cells. Compared with known memory populations, these lymphocytes had increased proliferative capacity and more efficiently reconstituted immunodeficient hosts, and they mediated superior antitumor responses in a humanized mouse model. The identification of a human stem cell–like memory T cell population is of direct relevance to the design of vaccines and T cell therapies.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

A major goal of systems biology is the development of models that accurately predict responses to perturbation. Constructing such models requires the collection of dense measurements of system states, yet transformation of data into predictive constructs remains a challenge. To begin to model human immunity, we analyzed immune parameters in depth both at baseline and in response to influenza vaccination. Peripheral blood mononuclear cell transcriptomes, serum titers, cell subpopulation frequencies, and B cell responses were assessed in 63 individuals before and after vaccination and were used to develop a systematic framework to dissect inter- and intra-individual variation and build predictive models of postvaccination antibody responses. Strikingly, independent of age and pre-existing antibody titers, accurate models could be constructed using pre-perturbation cell populations alone, which were validated using independent baseline time points. Most of the parameters contributing to prediction delineated temporally stable baseline differences across individuals, raising the prospect of immune monitoring before intervention.

Signaling through the adaptor protein myeloid differentiation factor 88 (MyD88) promotes carcinogenesis in several cancer models. In contrast, MyD88 signaling has a protective role in the development of azoxymethane (AOM)/dextran sodium sulfate (DSS) colitis-associated cancer (CAC). The inability of Myd88−/− mice to heal ulcers generated upon injury creates an altered inflammatory environment that induces early alterations in expression of genes encoding proinflammatory factors, as well as pathways regulating cell proliferation, apoptosis, and DNA repair, resulting in a dramatic increase in adenoma formation and progression to infiltrating adenocarcinomas with frequent clonal mutations in the β-catenin gene. Others have reported that toll-like receptor (Tlr) 4–deficient mice have a similar susceptibility to colitis to Myd88-deficient mice but, unlike the latter, are resistant to CAC. We have observed that mice deficient for Tlr2 or Il1r do not show a differential susceptibility to colitis or CAC. However, upon AOM/DSS treatment Il18−/− and Il18r1−/− mice were more susceptible to colitis and polyp formation than wild-type mice, suggesting that the phenotype of Myd88−/− mice is, in part, a result of their inability to signal through the IL-18 receptor. This study revealed a previously unknown level of complexity surrounding MyD88 activities downstream of different receptors that impact tissue homeostasis and carcinogenesis.

Diverse autoimmune and allergic diseases are associated with polymorphisms in a locus encoding the transcription factor BACH2; here, BACH2 is shown to be a broad regulator of immune activation that stabilizes the differentiation of Treg cells by repressing commitment of CD4+ T cells to alternate cell fates. Polymorphisms within a locus encoding the transcription factor BACH2 are associated with a number of allergic and autoimmune diseases including asthma, multiple sclerosis, Crohn's disease, coeliac disease and type 1 diabetes. This paper identifies a mechanism by which BACH2 might contribute to autoimmunity. Roychoudhuri et al. show how BACH2 limits autoimmunity by repressing alternative cell fates through the stabilization of the differentiation of regulatory T cells. These findings suggest a role for BACH2 as a regulator of CD4+ T-cell differentiation, preventing inflammatory disease by controlling the balance between tolerance and immunity. Through their functional diversification, distinct lineages of CD4+ T cells can act to either drive or constrain immune-mediated pathology. Transcription factors are critical in the generation of cellular diversity, and negative regulators antagonistic to alternate fates often act in conjunction with positive regulators to stabilize lineage commitment1. Genetic polymorphisms within a single locus encoding the transcription factor BACH2 are associated with numerous autoimmune and allergic diseases including asthma2, Crohn’s disease3,4, coeliac disease5, vitiligo6, multiple sclerosis7 and type 1 diabetes8. Although these associations point to a shared mechanism underlying susceptibility to diverse immune-mediated diseases, a function for BACH2 in the maintenance of immune homeostasis has not been established. Here, by studying mice in which the Bach2 gene is disrupted, we define BACH2 as a broad regulator of immune activation that stabilizes immunoregulatory capacity while repressing the differentiation programs of multiple effector lineages in CD4+ T cells. BACH2 was required for efficient formation of regulatory (Treg) cells and consequently for suppression of lethal inflammation in a manner that was Treg-cell-dependent. Assessment of the genome-wide function of BACH2, however, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, BACH2 constrained full effector differentiation within TH1, TH2 and TH17 cell lineages. These findings identify BACH2 as a key regulator of CD4+ T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity.

The molecular drivers that determine histology in lung cancer are largely unknown. We investigated whether microRNA (miR) expression profiles can differentiate histologic subtypes and predict survival for non-small cell lung cancer.We analyzed miR expression in 165 adenocarcinoma and 125 squamous cell carcinoma (SQ) tissue samples from the Environment And Genetics in Lung cancer Etiology (EAGLE) study using a custom oligo array with 440 human mature antisense miRs. We compared miR expression profiles using t tests and F tests and accounted for multiple testing using global permutation tests. We assessed the association of miR expression with tobacco smoking using Spearman correlation coefficients and linear regression models, and with clinical outcome using log-rank tests, Cox proportional hazards, and survival risk prediction models, accounting for demographic and tumor characteristics.MiR expression profiles strongly differed between adenocarcinoma and SQ (P(global) < 0.0001), particularly in the early stages, and included miRs located on chromosome loci most often altered in lung cancer (e.g., 3p21-22). Most miRs, including all members of the let-7 family, were downregulated in SQ. Major findings were confirmed by quantitative real time-polymerase chain reaction (qRT-PCR) in EAGLE samples and in an independent set of lung cancer cases. In SQ, the low expression of miRs that are downregulated in the histology comparison was associated with 1.2- to 3.6-fold increased mortality risk. A five-miR signature significantly predicted survival for SQ.We identified a miR expression profile that strongly differentiated adenocarcinoma from SQ and had prognostic implications. These findings may lead to histology-based therapeutic approaches.

Cancers often arise as the end stage of inflammation in adults, but not in children. As such there is a complex interplay between host immune cells during neoplastic development, with both an ability to promote cancer and limit or eliminate it, most often complicit with the host. In humans, defining inflammation and the presence of inflammatory cells within or surrounding the tumor is a critical aspect of modern pathology. Groups defining staging for neoplasms are strongly encouraged to assess and incorporate measures of the presence of apoptosis, autophagy, and necrosis and also the nature and quality of the immune infiltrate. Both environmental and genetic factors enhance the risk of cigarette smoking, Helicobacter pylori, hepatitis B/C, human papilloma virus, solar irradiation, asbestos, pancreatitis, or other causes of chronic inflammation. Identifying suitable genetic polymorphisms in cytokines, cytokine receptors, and Toll-like receptors among other immune response genes is also seen as high value as genomic sequencing becomes less expensive. Animal models that incorporate and assess not only the genetic anlagen but also the inflammatory cells and the presence of microbial pathogens and damage-associated molecular pattern molecules are necessary. Identifying micro-RNAs involved in regulating the response to damage or injury are seen as highly promising. Although no therapeutic strategies to prevent or treat cancers based on insights into inflammatory pathways are currently approved for the common epithelial malignancies, there remains substantial interest in agents targeting COX2 or PPARgamma, ethyl pyruvate and steroids, and several novel agents on the horizon.

Solid tumors are complex masses with a local microenvironment, or stroma, that supports tumor growth and progression. Among the diverse tumor-supporting stromal cells is a heterogeneous population of myeloid-derived cells. These cells are alternatively activated and contribute to the immunosuppressive environment of the tumor; overcoming their immunosuppressive effects may improve the efficacy of cancer immunotherapies. We recently found that engineering tumor-specific CD8(+) T cells to secrete the inflammatory cytokine IL-12 improved their therapeutic efficacy in the B16 mouse model of established melanoma. Here, we report the mechanism underlying this finding. Surprisingly, direct binding of IL-12 to receptors on lymphocytes or NK cells was not required. Instead, IL-12 sensitized bone marrow-derived tumor stromal cells, including CD11b(+)F4/80(hi) macrophages, CD11b(+)MHCII(hi)CD11c(hi) dendritic cells, and CD11b(+)Gr-1(hi) myeloid-derived suppressor cells, causing them to enhance the effects of adoptively transferred CD8(+) T cells. This reprogramming of myeloid-derived cells occurred partly through IFN-γ. Surprisingly, direct presentation of antigen to the transferred CD8(+) T cells by tumor was not necessary; however, MHCI expression on host cells was essential for IL-12-mediated antitumor enhancements. These results are consistent with a model in which IL-12 enhances the ability of CD8(+) T cells to collapse large vascularized tumors by triggering programmatic changes in otherwise suppressive antigen-presenting cells within tumors and support the use of IL-12 as part of immunotherapy for the treatment of solid tumors.

Abstract Purpose: Cancer stem cells (CSC) have been isolated from human tumors, including glioblastoma multiforme (GBM). The aims of this study were the immunobiological characterization of GBM CSCs and the assessment of whether these cells represent suitable targets for immunotherapy. Experimental Design: GBM CSC lines and their fetal bovine serum (FBS)–cultured non-CSC pair lines were generated and examined by flow cytometry for expression of known tumor antigens, MHC-I and MHC-II molecules, antigen-processing machinery components, and NKG2D ligands. In addition, immunogenicity and immunosuppression of such cell lines for autologous or allogeneic T lymphocytes were tested by cytokine secretion (ELISPOT) or proliferation (carboxyfluorescein diacetate succinimidyl ester) assays, respectively. Results: Both GBM CSC and FBS lines were weakly positive and negative for MHC-I, MHC-II, and NKG2D ligand molecules, respectively. Antigen-processing machinery molecules were also defective in both cell types. Upregulation of most molecules was induced by IFNs or 5-Aza deoxycytidine, although more efficiently in FBS than in CSCs. Patient T-cell responses, mediated by both TH1 and the TH2 subsets, against autologous CSC could be induced in vitro. In addition, CSC but not their paired FBS tumor lines inhibited T-cell proliferation of healthy donors. Notably, a differential gene signature that was confirmed at the protein levels for some immunologic-related molecules was also found between CSC and FBS lines. Conclusions: These results indicate lower immunogenicity and higher suppressive activity of GBM CSC compared with FBS lines. The immunogenicity, however, could be rescued by immune modulation leading to anti-GBM T cell–mediated immune response. Clin Cancer Res; 16(3); 800–13

Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

Abstract Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TIL) results in complete regression of advanced cancer in some patients, but the efficacy of this potentially curative therapy may be limited by poor persistence of TIL after adoptive transfer. Pharmacologic inhibition of the serine/threonine kinase Akt has recently been shown to promote immunologic memory in virus-specific murine models, but whether this approach enhances features of memory (e.g., long-term persistence) in TIL that are characteristically exhausted and senescent is not established. Here, we show that pharmacologic inhibition of Akt enables expansion of TIL with the transcriptional, metabolic, and functional properties characteristic of memory T cells. Consequently, Akt inhibition results in enhanced persistence of TIL after adoptive transfer into an immunodeficient animal model and augments antitumor immunity of CD8 T cells in a mouse model of cell-based immunotherapy. Pharmacologic inhibition of Akt represents a novel immunometabolomic approach to enhance the persistence of antitumor T cells and improve the efficacy of cell-based immunotherapy for metastatic cancer. Cancer Res; 75(2); 296–305. ©2014 AACR.

Human malignant mesothelioma is an aggressive and highly lethal cancer that is believed to be caused by chronic exposure to asbestos and erionite. Prognosis for this cancer is generally poor because of late-stage diagnosis and resistance to current conventional therapies. The damage-associated molecular pattern protein HMGB1 has been implicated previously in transformation of mesothelial cells. Here we show that HMGB1 establishes an autocrine circuit in malignant mesothelioma cells that influences their proliferation and survival. Malignant mesothelioma cells strongly expressed HMGB1 and secreted it at high levels in vitro. Accordingly, HMGB1 levels in malignant mesothelioma patient sera were higher than that found in healthy individuals. The motility, survival, and anchorage-independent growth of HMGB1-secreting malignant mesothelioma cells was inhibited in vitro by treatment with monoclonal antibodies directed against HMGB1 or against the receptor for advanced glycation end products, a putative HMGB1 receptor. HMGB1 inhibition in vivo reduced the growth of malignant mesothelioma xenografts in severe-combined immunodeficient mice and extended host survival. Taken together, our findings indicate that malignant mesothelioma cells rely on HMGB1, and they offer a preclinical proof-of-principle that antibody-mediated ablation of HMBG1 is sufficient to elicit therapeutic activity, suggesting a novel therapeutic approach for malignant mesothelioma treatment.

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells.Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets.Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes.Using both mouse and human cells, we identified a T cell-T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells.This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT.The T memoryinduced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation.Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations.These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell-based immunotherapies.

Mounting evidence supports a role for the immune system in breast cancer outcomes. The ability to distinguish highly immunogenic tumors susceptible to anti-tumor immunity from weakly immunogenic or inherently immune-resistant tumors would guide development of therapeutic strategies in breast cancer. Genomic, transcriptomic and clinical data from The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) breast cancer cohorts were used to examine statistical associations between tumor mutational burden (TMB) and the survival of patients whose tumors were assigned to previously-described prognostic immune subclasses reflecting favorable, weak or poor immune-infiltrate dispositions (FID, WID or PID, respectively). Tumor immune subclasses were associated with survival in patients with high TMB (TMB-Hi, P < 0.001) but not in those with low TMB (TMB-Lo, P = 0.44). This statistical relationship was confirmed in the METABRIC cohort (TMB-Hi, P = 0.047; TMB-Lo, P = 0.39), and also found to hold true in the more-indolent Luminal A tumor subtype (TMB-Hi, P = 0.011; TMB-Lo, P = 0.91). In TMB-Hi tumors, the FID subclass was associated with prolonged survival independent of tumor stage, molecular subtype, age and treatment. Copy number analysis revealed the reproducible, preferential amplification of chromosome 1q immune-regulatory genes in the PID immune subclass. These findings demonstrate a previously unappreciated role for TMB as a determinant of immune-mediated survival of breast cancer patients and identify candidate immune-regulatory mechanisms associated with immunologically cold tumors. Immune subtyping of breast cancers may offer opportunities for therapeutic stratification.

The culmination of over a century's work to understand the role of the immune system in tumor control has led to the recent advances in cancer immunotherapies that have resulted in durable clinical responses in patients with a variety of malignancies. Cancer immunotherapies are rapidly changing traditional treatment paradigms and expanding the therapeutic landscape for cancer patients. However, despite the current success of these therapies, not all patients respond to immunotherapy and even those that do often experience toxicities. Thus, there is a growing need to identify predictive and prognostic biomarkers that enhance our understanding of the mechanisms underlying the complex interactions between the immune system and cancer. Therefore, the Society for Immunotherapy of Cancer (SITC) reconvened an Immune Biomarkers Task Force to review state of the art technologies, identify current hurdlers, and make recommendations for the field. As a product of this task force, Working Group 2 (WG2), consisting of international experts from academia and industry, assembled to identify and discuss promising technologies for biomarker discovery and validation. Thus, this WG2 consensus paper will focus on the current status of emerging biomarkers for immune checkpoint blockade therapy and discuss novel technologies as well as high dimensional data analysis platforms that will be pivotal for future biomarker research. In addition, this paper will include a brief overview of the current challenges with recommendations for future biomarker discovery.

Improving the functional avidity of effector T cells is critical in overcoming inhibitory factors within the tumor microenvironment and eliciting tumor regression. We have found that Cish, a member of the suppressor of cytokine signaling (SOCS) family, is induced by TCR stimulation in CD8(+) T cells and inhibits their functional avidity against tumors. Genetic deletion of Cish in CD8(+) T cells enhances their expansion, functional avidity, and cytokine polyfunctionality, resulting in pronounced and durable regression of established tumors. Although Cish is commonly thought to block STAT5 activation, we found that the primary molecular basis of Cish suppression is through inhibition of TCR signaling. Cish physically interacts with the TCR intermediate PLC-γ1, targeting it for proteasomal degradation after TCR stimulation. These findings establish a novel targetable interaction that regulates the functional avidity of tumor-specific CD8(+) T cells and can be manipulated to improve adoptive cancer immunotherapy.

Abstract The lack of multi-omics cancer datasets with extensive follow-up information hinders the identification of accurate biomarkers of clinical outcome. In this cohort study, we performed comprehensive genomic analyses on fresh-frozen samples from 348 patients affected by primary colon cancer, encompassing RNA, whole-exome, deep T cell receptor and 16S bacterial rRNA gene sequencing on tumor and matched healthy colon tissue, complemented with tumor whole-genome sequencing for further microbiome characterization. A type 1 helper T cell, cytotoxic, gene expression signature, called Immunologic Constant of Rejection, captured the presence of clonally expanded, tumor-enriched T cell clones and outperformed conventional prognostic molecular biomarkers, such as the consensus molecular subtype and the microsatellite instability classifications. Quantification of genetic immunoediting, defined as a lower number of neoantigens than expected, further refined its prognostic value. We identified a microbiome signature, driven by Ruminococcus bromii , associated with a favorable outcome. By combining microbiome signature and Immunologic Constant of Rejection, we developed and validated a composite score (mICRoScore), which identifies a group of patients with excellent survival probability. The publicly available multi-omics dataset provides a resource for better understanding colon cancer biology that could facilitate the discovery of personalized therapeutic approaches.

Abstract Although in vitro sensitization assays have shown increased melanoma Ag (MA)-specific CTL reactivity after vaccination with MA peptides, clinical responses have been uncommon. This paradox questions whether data obtained from the in vitro stimulation and expansion of T cells lead to an overestimation of the immune response to vaccines. Using HLA/peptide tetramer (tHLA), we enumerated MA-specific T cell precursor frequency (TCPF) directly in PBMC from 23 melanoma patients vaccinated with gp100:209–217(210M) (g209–2M) peptide. Vaccine-specific TCPF was higher in postvaccination PBMC from seven of seven patients treated with peptide alone and four of five patients treated with peptide plus IL-12 (range of postvaccination TCPF, 0.2–2.4% and 0.2–2.5%, respectively). The increased TCPF correlated with enhanced susceptibility to in vitro stimulation with the relevant epitope. Paradoxically, no increase in postvaccination TCPF was observed in most patients who had been concomitantly treated with IL-2 (1 of 11 patients; range of postvaccination TCPF, 0.02–1.0%), a combination associated with enhanced rates of tumor regression. The lack of increase in TCPF seen in these patients corresponded to inability to elicit expansion of vaccine-specific T cells in culture. This study shows that a peptide-based vaccine can effectively generate a quantifiable T cell-specific immune response in the PBMC of cancer patients, though such a response does not associate with a clinically evident regression of metastatic melanoma.

T-cell-based immunotherapies provide a promising means of cancer treatment although durable antitumor responses are infrequent. A potential reason for these shortcomings may lie in the observed lack of trafficking of specific T cells to tumor. Our increasing knowledge of the process of trafficking involving adhesion molecules and chemokines affords us the opportunity to intervene and correct deficiencies in this process. Chemokines can be expressed by a range of tumors and may serve as suitable targets for directing specific T cells toward tumor. We initially sought to identify which chemokines were produced by a range of human tumor cell lines, and which chemokines and chemokine receptors were expressed by cultured T cells. We identified two chemokines: Growth-Regulated Oncogene-alpha (Gro-alpha; CXCL1) and Regulated on Activation Normal T Cell-Expressed and Secreted (RANTES; CCL5), to be secreted by several human tumor cell lines. Expression was also detected in fine-needle aspirates of melanoma from patients. In addition, we determined the expression of several chemokine receptors on cultured human T cells including CCR1, CCR2, CCR4, CCR5, CXCR3, and CXCR4. Cultured, activated human T cells expressed the chemokines lymphotactin (XCL1), RANTES, macrophage inflammatory protein-1 alpha (MIP-1 alpha; CCL3) and MIP-1 beta (CCL4), but no appreciable Gro-alpha. In a strategy to direct T cells toward chemokines expressed by tumors we chose Gro-alpha as the target chemokine because it was produced by tumor and not by T cells themselves. However, T cells did not express the receptor for Gro-alpha, CXCR2, and therefore, T cells were transduced with a retroviral vector encoding CXCR2. Calcium ion mobilization, an important first step in chemokine receptor signaling, was subsequently demonstrated in transduced T cells in response to Gro-alpha. In addition, Gro-alpha was chemotactic for T cells expressing CXCR2 in vitro toward both recombinant protein and tumor-derived chemokine. Interestingly we demonstrate, for the first time, that Gro-alpha was able to induce interferon-gamma (IFN-gamma) secretion from transduced T cells, thereby extending our knowledge of other potential functions of CXCR2. This study demonstrates the feasibility of redirecting the migration properties of T cells toward chemokines secreted by tumors.

Over the last few decades a wealth of evidence has been gathered on the potential role that the immune system (IS) can play in the fight against cancer. Together with cell surface adhesion molecules, cytokines (CKs) mediate the activities of IS cells. Therefore, CK kinetics may represent a mirror of the immunologic phenomena occurring in the tumor microenvironment, where immune and malignant cells interact. Yet, CKs are currently used in a clinical setting to polarize the immune response against cancer. Despite the large amount of information available on IS physiology, little is known about the role of CKs in modulating the effectiveness of immunotherapy clinical trials aimed at the treatment of patients with cancer. This underscores our relative ignorance about the complex cascade of events that lead to tumor rejection. Here, we review the properties of some CKs believed to be particularly relevant to tumor immunology (i.e., interleukin [IL]-10, transforming growth factor-β, interferon-γ, IL-2, IL-4, and IL-12). We summarized the experience gained with these CKs in vitro, in animal models, and in human beings to illustrate the achievements, the controversies, and the challenges that characterize this fascinating field of oncology. In addition, we added a short section in which a broad view of CKs released in the tumor microenvironment is proposed to underline the variety of factors that contribute to the complexity of tumor-IS interactions.

Abstract Previously, we reported that a recombinant vaccinia virus (VACV) carrying a light-emitting fusion gene enters, replicates in, and reveals the locations of tumors in mice. A new recombinant VACV, GLV-1h68, as a simultaneous diagnostic and therapeutic agent, was constructed by inserting three expression cassettes (encoding Renilla luciferase–Aequorea green fluorescent protein fusion, β-galactosidase, and β-glucuronidase) into the F14.5L, J2R (encoding thymidine kinase) and A56R (encoding hemagglutinin) loci of the viral genome, respectively. I.v. injections of GLV-1h68 (1 × 107 plaque-forming unit per mouse) into nude mice with established (∼300–500 mm3) s.c. GI-101A human breast tumors were used to evaluate its toxicity, tumor targeting specificity, and oncolytic efficacy. GLV-1h68 showed an enhanced tumor targeting specificity and much reduced toxicity compared with its parental LIVP strains. The tumors colonized by GLV-1h68 exhibited growth, inhibition, and regression phases followed by tumor eradication within 130 days in 95% of the mice tested. Tumor regression in live animals was monitored in real time based on decreasing light emission, hence demonstrating the concept of a combined oncolytic virus–mediated tumor diagnosis and therapy system. Transcriptional profiling of regressing tumors based on a mouse-specific platform revealed gene expression signatures consistent with immune defense activation, inclusive of IFN-stimulated genes (STAT-1 and IRF-7), cytokines, chemokines, and innate immune effector function. These findings suggest that immune activation may combine with viral oncolysis to induce tumor eradication in this model, providing a novel perspective for the design of oncolytic viral therapies for human cancers. [Cancer Res 2007;67(20):10038–46]

Although the sequence of events leading to wound repair has been described at the cellular and, to a limited extent, at the protein level this process has yet to be fully elucidated. Genome wide transcriptional analysis tools promise to further define the global picture of this complex progression of events.This study was part of a placebo-controlled double-blind clinical trial in which basal cell carcinomas were treated topically with an immunomodifier--toll-like receptor 7 agonist: imiquimod. The fourteen patients with basal cell carcinoma in the placebo arm of the trial received placebo treatment consisting solely of vehicle cream. A skin punch biopsy was obtained immediately before treatment and at the end of the placebo treatment (after 2, 4 or 8 days). 17.5K cDNA microarrays were utilized to profile the biopsy material.Four gene signatures whose expression changed relative to baseline (before wound induction by the pre-treatment biopsy) were identified. The largest group was comprised predominantly of inflammatory genes whose expression was increased throughout the study. Two additional signatures were observed which included preferentially pro-inflammatory genes in the early post-treatment biopsies (2 days after pre-treatment biopsies) and repair and angiogenesis genes in the later (4 to 8 days) biopsies. The fourth and smallest set of genes was down-regulated throughout the study. Early in wound healing the expression of markers of both M1 and M2 macrophages were increased, but later M2 markers predominated.The initial response to a cutaneous wound induces powerful transcriptional activation of pro-inflammatory stimuli which may alert the host defense. Subsequently and in the absence of infection, inflammation subsides and it is replaced by angiogenesis and remodeling. Understanding this transition which may be driven by a change from a mixed macrophage population to predominantly M2 macrophages, may help the interpretation of the cellular and molecular events occurring in the microenvironment of serially biopsied tissues.

The understanding of tumor-host interactions remains elusive despite significant progress in the identification of tumor antigens (TAs) recognized by autologous T cells. In particular, most human tumors do not regress and continue to grow in spite of spontaneous or immunization-induced immune responses demonstrated in circulating lymphocytes. Indeed, systemic immune responses might insufficiently address the complexity of tumor-host interactions because of factors, such as (1) the lack of productive T-cell receptor (TCR) engagement with epitope owing to qualitative and/or quantitative defects in the generation and maintenance of the immune response, (2) insufficient costimulation provided by the host, (3) the lack of localization of the immune response in target tissues and (4) the complexity of tumor-host interactions within the tumor microenvironment caused by temporal changes in tumor phenotypes and an array of immune mediators expressed in the tumor microenvironment. Here, we will review current knowledge of the different 'levels' of immune response that might be necessary for immunotherapy to be effective in the treatment of cancer. Furthermore, we will discuss the information still required in order to understand the mechanism(s) governing tumor rejection by the immune system in response to TA-specific immunization.

Abstract Purpose: Tumor antigen–loaded dendritic cells (DC) are believed to activate antitumor immunity by stimulating T cells, and CTL-associated antigen 4 (CTLA4)–blocking antibodies should release a key negative regulatory pathway on T cells. The combination was tested in a phase I clinical trial in patients with advanced melanoma. Experimental Design: Autologous DC were pulsed with MART-126-35 peptide and administered with a dose escalation of the CTLA4-blocking antibody tremelimumab. Sixteen patients were accrued to five dose levels. Primary end points were safety and immune effects; clinical efficacy was a secondary end point. Results: Dose-limiting toxicities of grade 3 diarrhea and grade 2 hypophysitis developed in two of three patients receiving tremelimumab at 10 mg/kg monthly. Four patients had an objective tumor response, two partial responses and two complete responses, all melanoma free between 2 and 4 years after study initiation. There was no difference in immune monitoring results between patients with an objective tumor response and those without a response. Exploratory gene expression analysis suggested that immune-related gene signatures, in particular for B-cell function, may be important in predicting response. Conclusion: The combination of MART-1 peptide–pulsed DC and tremelimumab results in objective and durable tumor responses at the higher range of the expected response rate with either agent alone. (Clin Cancer Res 2009;15(19):6267–76)

Abstract The cloning of cancer Ags recognized by T cells has provided potentially new tools to enhance immunity against metastatic cancer. The biological monitoring of effective immunization has, however, remained a dilemma. We describe here a sensitive molecular quantitation methodology that allows analysis of in vivo immune response to vaccination. Metastatic melanoma patients were immunized with a synthetically modified peptide epitope (209-2M) from the melanoma self-Ag gp100. Using serial gene expression analysis, we report functional evidence of vaccine-induced CTL reactivity in fresh cells obtained directly from the peripheral blood of postimmunized patients. Further, we demonstrate in vivo localization of vaccine-induced immune response within the tumor microenvironment. The results of these molecular assays provide direct evidence that peptide immunization in humans can result in tumor-specific CTL that localize to metastatic sites.

The transcriptional repressor Blimp-1 promotes the differentiation of CD8(+) T cells into short-lived effector cells (SLECs) that express the lectin-like receptor KLRG-1, but how it operates remains poorly defined. Here we show that Blimp-1 bound to and repressed the promoter of the gene encoding the DNA-binding inhibitor Id3 in SLECs. Repression of Id3 by Blimp-1 was dispensable for SLEC development but limited the ability of SLECs to persist as memory cells. Enforced expression of Id3 was sufficient to restore SLEC survival and enhanced recall responses. Id3 function was mediated in part through inhibition of the transcriptional activity of E2A and induction of genes regulating genome stability. Our findings identify the Blimp-1-Id3-E2A axis as a key molecular switch that determines whether effector CD8(+) T cells are programmed to die or enter the memory pool.

The unique features of human embryonic stem (hES) cells make them the best candidate resource for both cell replacement therapy and development research. However, the molecular mechanisms responsible for the simultaneous maintenance of their self-renewal properties and undifferentiated state remain unclear. Non-coding microRNAs (miRNA) which regulate mRNA cleavage and inhibit encoded protein translation exhibit temporal or tissue-specific expression patterns and they play an important role in development timing.In this study, we analyzed miRNA and gene expression profiles among samples from 3 hES cell lines (H9, I6 and BG01v), differentiated embryoid bodies (EB) derived from H9 cells at different time points, and 5 adult cell types including Human Microvascular Endothelial Cells (HMVEC), Human Umbilical Vein Endothelial Cells (HUVEC), Umbilical Artery Smooth Muscle Cells (UASMC), Normal Human Astrocytes (NHA), and Lung Fibroblasts (LFB). This analysis rendered 104 miRNAs and 776 genes differentially expressed among the three cell types. Selected differentially expressed miRNAs and genes were further validated and confirmed by quantitative real-time-PCR (qRT-PCR). Especially, members of the miR-302 cluster on chromosome 4 and miR-520 cluster on chromosome 19 were highly expressed in undifferentiated hES cells. MiRNAs in these two clusters displayed similar expression levels. The members of these two clusters share a consensus 7-mer seed sequence and their targeted genes had overlapping functions. Among the targeted genes, genes with chromatin structure modification function are enriched suggesting a role in the maintenance of chromatin structure. We also found that the expression level of members of the two clusters, miR-520b and miR-302c, were negatively correlated with their targeted genes based on gene expression analysisWe identified the expression patterns of miRNAs and gene transcripts in the undifferentiation of human embryonic stem cells; among the miRNAs that are highly expressed in undifferentiated embryonic stem cells, the miR-520 cluster may be closely involved in hES cell function and its relevance to chromatin structure warrants further study.

Purpose High-dose interleukin-2 (IL-2) induces durable therapeutic responses in a small subset of patients with metastatic melanoma and renal cell carcinoma, but simple pretreatment predictors of response have not been identified. Patients and Methods To identify predictive biomarkers of clinical response, sera from patients treated with high-dose IL-2 were collected for analysis using a customized, multiplex antibody-targeted protein array platform that surveyed expression of soluble factors associated with tumor immunobiology. Soluble factors associated with clinical responses were analyzed using a multivariate permutation test, and survival outcomes were determined using Kaplan-Meier and log-rank tests. Results A training set from 10 patients identified 68 potentially relevant soluble factors that were then tested in an independent validation set of 49 patients. Class comparison revealed a cluster of 11 biomarkers that were associated with therapeutic outcome. Vascular endothelial growth factor (VEGF) and fibronectin were identified as independent predictors of response. In particular, high levels of these proteins were correlated with lack of clinical response and decreased overall survival. Conclusion Serum VEGF and fibronectin are easily measured pretreatment biomarkers that could serve to exclude patients unlikely to respond to IL-2 therapy.

The pathogenesis of nasopharyngeal carcinoma (NPC) is a complicated process involving genetic predisposition, Epstein-Bar Virus infection, and genetic alterations. Although some oncogenes and tumor suppressor genes have been previously reported in NPC, a complete understanding of the pathogenesis of NPC in the context of global gene expression, transcriptional pathways and biomarker assessment remains to be elucidated.Total RNA from 32 pathologically-confirmed cases of poorly-differentiated NPC was divided into pools inclusive of four consecutive specimens and each pool (T1 to T8) was co-hybridized with pooled RNA from 24 normal non-cancerous nasopharyngeal tissues (NP) to a human 8K cDNA array platform. The reliability of microarray data was validated for selected genes by semi-quantitative RT-PCR and immunohistochemistry.Stringent statistical filtering parameters identified 435 genes to be up-regulated and 257 genes to be down-regulated in NPC compared to NP. Seven up-regulated genes including CYC1, MIF, LAMB3, TUBB2, UBE2C and TRAP1 had been previously proposed as candidate common cancer biomarkers based on a previous extensive comparison among various cancers and normal tissues which did not, however, include NPC or NP. In addition, nine known oncogenes and tumor suppressor genes, MIF, BIRC5, PTTG1, ATM, FOXO1A, TGFBR2, PRKAR1A, KLF5 and PDCD4 were identified through the microarray literature-based annotation search engine MILANO, suggesting these genes may be specifically involved in the promotion of the malignant conversion of nasopharyngeal epithelium. Finally, we found that these differentially expressed genes were involved in apoptosis, MAPK, VEGF and B cell receptor signaling pathways and other functions associated with cell growth, signal transduction and immune system activation.This study identified potential candidate biomarkers, oncogenes/tumor suppressor genes involved in several pathways relevant to the oncogenesis of NPC. This information may facilitate the determination of diagnostic and therapeutic targets for NPC as well as provide insights about the molecular pathogenesis of NPC.

The clinical significance of tumor-infiltrating immune cells has been reported in a variety of human carcinomas including breast cancer. However, molecular signature of tumor-infiltrating immune cells and their prognostic value in breast cancer patients remain elusive. We hypothesized that a distinct network of immune function genes at the tumor site can predict a low risk versus high risk of distant relapse in breast cancer patients regardless of the status of ER, PR, or HER-2/neu in their tumors. We conducted retrospective studies in a diverse cohort of breast cancer patients with a 1–5 year tumor relapse versus those with up to 7 years relapse-free survival. The RNAs were extracted from the frozen tumor specimens at the time of diagnosis and subjected to microarray analysis and real-time RT-PCR. Paraffin-embedded tissues were also subjected to immunohistochemistry staining. We determined that a network of immune function genes involved in B cell development, interferon signaling associated with allograft rejection and autoimmune reaction, antigen presentation pathway, and cross talk between adaptive and innate immune responses were exclusively upregulated in patients with relapse-free survival. Among the 299 genes, five genes which included B cell response genes were found to predict with >85% accuracy relapse-free survival. Real-time RT-PCR confirmed the 5-gene prognostic signature that was distinct from an FDA-cleared 70-gene signature of MammaPrint panel and from the Oncotype DX recurrence score assay panel. These data suggest that neoadjuvant immunotherapy in patients with high risk of relapse may reduce tumor recurrence by inducing the immune function genes.

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.

The complexity underlying a pathologic process does not necessarily require a complex explanation. The biology determining allograft or cancer rejection, autoimmunity or tissue damage during pathogen infections is complex; however, common patterns are emerging that lead to a common final outcome. For instance, tissue destruction occurs with resolution of the pathogenic process (cancer, infection) or tissue damage and organ failure (autoimmunity, allograft rejection). Observations in humans based on transcriptional profiling converge into what we call an 'immunologic constant of rejection' that characterizes such occurrences. This constant includes the coordinate activation of interferon-stimulated genes (ISGs) and immune effector functions (IEFs). Understanding this final effector pathway may suggest novel strategies for the induction or inhibition of tissue-specific destruction with therapeutic intent in cancer and other immune pathologies.

Cancer immunotherapy is revolutionizing the clinical management of several tumors, but has demonstrated limited activity in breast cancer. The development of more effective treatments is hindered by incomplete knowledge of the genetic determinant of immune responsiveness. To fill this gap, we mined copy number alteration, somatic mutation, and expression data from The Cancer Genome Atlas (TCGA). By using RNA-sequencing data from 1,004 breast cancers, we defined distinct immune phenotypes characterized by progressive expression of transcripts previously associated with immune-mediated rejection. The T helper 1 (Th-1) phenotype (ICR4), which also displays upregulation of immune-regulatory transcripts such as PDL1, PD1, FOXP3, IDO1, and CTLA4, was associated with prolonged patients' survival. We validated these findings in an independent meta-cohort of 1,954 breast cancer gene expression data. Chromosome segment 4q21, which includes genes encoding for the Th-1 chemokines CXCL9-11, was significantly amplified only in the immune favorable phenotype (ICR4). The mutation and neoantigen load progressively decreased from ICR4 to ICR1 but could not fully explain immune phenotypic differences. Mutations of TP53 were enriched in the immune favorable phenotype (ICR4). Conversely, the presence of MAP3K1 and MAP2K4 mutations were tightly associated with an immune-unfavorable phenotype (ICR1). Using both the TCGA and the validation dataset, the degree of MAPK deregulation segregates breast tumors according to their immune disposition. These findings suggest that mutation-driven perturbations of MAPK pathways are linked to the negative regulation of intratumoral immune response in breast cancer. Modulations of MAPK pathways could be experimentally tested to enhance breast cancer immune sensitivity.

Tumors grow in the presence of antigen-specific T cells, suggesting the existence of intrinsic cancer cell escape mechanisms. We hypothesized that a histone deacetylase (HDAC) inhibitor could sensitize tumor cells to immunotherapy because this class of agents has been reported to increase tumor antigen expression and shift gene expression to a proapoptotic milieu in cancer cells. To test this question, we treated B16 murine melanoma with the combination of the HDAC inhibitor LAQ824 and the adoptive transfer of gp100 melanoma antigen-specific pmel-1 T cells. The combined therapy significantly improved antitumor activity through several mechanisms: (a) increase in MHC and tumor-associated antigen expression by tumor cells; (b) decrease in competing endogenous lymphocytes in recipient mice, resulting in a proliferative advantage for the adoptively transferred cells; and (c) improvement in the functional activity of the adoptively transferred lymphocytes. We confirmed the beneficial effects of this HDAC inhibitor as a sensitizer to immunotherapy in a different model of prophylactic prime-boost vaccination with the melanoma antigen tyrosinase-related protein 2, which also showed a significant improvement in antitumor activity against B16 melanoma. In conclusion, the HDAC inhibitor LAQ824 significantly enhances tumor immunotherapy through effects on target tumor cells as well as improving the antitumor activity of tumor antigen-specific lymphocytes.

Abstract Background: MicroRNAs (miR) are endogenous, noncoding RNAs involved in many cellular processes and have been associated with the development and progression of cancer. There are many different ways to evaluate miRs. Methods: We described some of the most commonly used and promising miR detection methods. Results: Each miR detection method has benefits and limitations. Microarray profiling and quantitative real-time reverse-transcription PCR are the two most common methods to evaluate miR expression. However, the results from microarray and quantitative real-time reverse-transcription PCR do not always agree. High-throughput, high-resolution next-generation sequencing of small RNAs may offer the opportunity to quickly and accurately discover new miRs and confirm the presence of known miRs in the near future. Conclusions: All of the current and new technologies have benefits and limitations to consider when designing miR studies. Results can vary across platforms, requiring careful and critical evaluation when interpreting findings. Impact: Although miR detection and expression analyses are rapidly improving, there are still many technical challenges to overcome. The old molecular epidemiology tenet of rigorous biomarker validation and confirmation in independent studies remains essential. Cancer Epidemiol Biomarkers Prev; 19(4); 907–11. ©2010 AACR.

Corticosteroids have been used for decades to modulate inflammation therapeutically, yet there is a paucity of data on their effects in humans. We examined the changes in cellular and molecular immune system parameters, or "immunome", in healthy humans after systemic corticosteroid administration. We used multiplexed techniques to query the immunome in 20 volunteers at baseline, and after intravenous hydrocortisone (HC) administered at moderate (250 mg) and low (50 mg) doses, to provide insight into how corticosteroids exert their effects. We performed comprehensive phenotyping of 120 lymphocyte subsets by high dimensional flow cytometry, and observed a decline in circulating specific B and T cell subsets, which reached their nadir 4-8 hours after administration of HC. However, B and T cells rebounded above baseline 24 hours after HC infusion, while NK cell numbers remained stable. Whole transcriptome profiling revealed down regulation of NF-κB signaling, apoptosis, and cell death signaling transcripts that preceded lymphocyte population changes, with activation of NK cell and glucocorticoid receptor signaling transcripts. Our study is the first to systematically characterize the effects of corticosteroids on the human immunome, and we demonstrate that HC exerts differential effects on B and T lymphocytes and natural killer cells in humans.

<h2>Summary</h2> Tumor progression is accompanied by an altered myelopoiesis causing the accumulation of immunosuppressive cells. Here, we showed that miR-142-3p downregulation promoted macrophage differentiation and determined the acquisition of their immunosuppressive function in tumor. Tumor-released cytokines signaling through gp130, the common subunit of the interleukin-6 cytokine receptor family, induced the LAP^∗ isoform of C/EBPβ transcription factor, promoting macrophage generation. miR-142-3p downregulated gp130 by canonical binding to its messenger RNA (mRNA) 3' UTR and repressed C/EBPβ LAP^∗ by noncanonical binding to its 5' mRNA coding sequence. Enforced miR expression impaired macrophage differentiation both in vitro and in vivo. Mice constitutively expressing miR-142-3p in the bone marrow showed a marked increase in survival following immunotherapy with tumor-specific T lymphocytes. By modulating a specific miR in bone marrow precursors, we thus demonstrated the feasibility of altering tumor-induced macrophage differentiation as a potent tool to improve the efficacy of cancer immunotherapy.

Whereas preclinical investigations and clinical studies have established that CD8+ T cells can profoundly affect cancer progression, the underlying mechanisms are still elusive. Challenging the prevalent view that the beneficial effect of CD8+ T cells in cancer is solely attributable to their cytotoxic activity, several reports have indicated that the ability of CD8+ T cells to promote tumor regression is dependent on their cytokine secretion profile and their ability to self-renew. Evidence has also shown that the tumor microenvironment can disarm CD8+ T cell immunity, leading to the emergence of dysfunctional CD8+ T cells. The existence of different types of CD8+ T cells in cancer calls for a more precise definition of the CD8+ T cell immune phenotypes in cancer and the abandonment of the generic terms “pro-tumor” and “antitumor.” Based on recent studies investigating the functions of CD8+ T cells in cancer, we here propose some guidelines to precisely define the functional states of CD8+ T cells in cancer.

&lt;b&gt;&lt;i&gt;Background:&lt;/i&gt;&lt;/b&gt; Vitamin D is a secosteroid, which was initially known for its skeletal role; however, in recent years, its functions in different organs have been increasingly recognized. In this review, we will provide an overview of vitamin D functions in the skin physiology with specific focus on its role in certain inflammatory skin conditions such as psoriasis and atopic dermatitis. &lt;b&gt;&lt;i&gt;Methods:&lt;/i&gt;&lt;/b&gt; A comprehensive literature search was carried out in PubMed and Google Scholar databases using keywords like “vitamin D,” “skin,” “atopic dermatitis,” and “psoriasis.” Only articles published in English and related to the study topic were included in this review. &lt;b&gt;&lt;i&gt;Results:&lt;/i&gt;&lt;/b&gt; Vitamin D is integrally connected to the skin for its synthesis, metabolism, and activity. It regulates many physiological processes in the skin ranging from cellular proliferation, differentiation, and apoptosis to barrier maintenance and immune functions. Vitamin D deficiency is associated with the risk of psoriasis and atopic dermatitis, and several clinical/observational studies have suggested the beneficial effect of vitamin D in the therapy of these 2 inflammatory skin disorders. &lt;b&gt;&lt;i&gt;Conclusions:&lt;/i&gt;&lt;/b&gt; Vitamin D exerts a pleiotropic effect in the skin and could be an important therapeutic option for psoriasis and atopic dermatitis.

Dendritic cells (DCs) are often produced by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) stimulation of monocytes. To improve the effectiveness of DC adoptive immune cancer therapy, many different agents have been used to mature DCs. We analyzed the kinetics of DC maturation by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) induction in order to characterize the usefulness of mature DCs (mDCs) for immune therapy and to identify biomarkers for assessing the quality of mDCs.Peripheral blood mononuclear cells were collected from 6 healthy subjects by apheresis, monocytes were isolated by elutriation, and immature DCs (iDCs) were produced by 3 days of culture with GM-CSF and IL-4. The iDCs were sampled after 4, 8 and 24 hours in culture with LPS and IFN-gamma and were then assessed by flow cytometry, ELISA, and global gene and microRNA (miRNA) expression analysis.After 24 hours of LPS and IFN-gamma stimulation, DC surface expression of CD80, CD83, CD86, and HLA Class II antigens were up-regulated. Th1 attractant genes such as CXCL9, CXCL10, CXCL11 and CCL5 were up-regulated during maturation but not Treg attractants such as CCL22 and CXCL12. The expression of classical mDC biomarker genes CD83, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1 and HES4 were up-regulated throughout maturation while MTIB, MTIE, MTIG, MTIH, GADD45A and LAMP3 were only up-regulated late in maturation. The expression of miR-155 was up-regulated 8-fold in mDCs.DCs, matured with LPS and IFN-gamma, were characterized by increased levels of Th1 attractants as opposed to Treg attractants and may be particularly effective for adoptive immune cancer therapy.

Because oncogene MET and EGF receptor (EGFR) inhibitors are in clinical development against several types of cancer, including glioblastoma, it is important to identify predictive markers that indicate patient subgroups suitable for such therapies. We investigated in vivo glioblastoma models characterized by hepatocyte growth factor (HGF) autocrine or paracrine activation, or by MET or EGFR amplification, for their susceptibility to MET inhibitors. HGF autocrine expression correlated with high phospho-MET levels in HGF autocrine cell lines, and these lines showed high sensitivity to MET inhibition in vivo. An HGF paracrine environment may enhance glioblastoma growth in vivo but did not indicate sensitivity to MET inhibition. EGFRvIII amplification predicted sensitivity to EGFR inhibition, but in the same tumor, increased copies of MET from gains of chromosome 7 did not result in increased MET activity and did not predict sensitivity to MET inhibitors. Thus, HGF autocrine glioblastoma bears an activated MET signaling pathway that may predict sensitivity to MET inhibitors. Moreover, serum HGF levels may serve as a biomarker for the presence of autocrine tumors and their responsiveness to MET therapeutics.

Low-dose interleukin-2 (IL-2) expands regulatory T cells (T_regs) and natural killer (NK) cells after stem cell transplantation (SCT) and may reduce graft-versus-host disease (GVHD). We hypothesized that ultra-low dose (ULD) IL-2 could serve as an immune-modulating agent for stem cell donors to prevent GVHD following SCT. However, the safety, dose level, and immune signatures of ULD IL-2 in immune-competent healthy subjects remain unknown. Here, we have characterized the phenotype and function of T_regs and NK cells as well as the gene expression and cytokine profiles of 21 healthy volunteers receiving 50,000 to 200,000 units/m²/day IL-2 for 5 days. ULD IL-2 was well tolerated and induced a significant increase in the frequency of T_regs with increased suppressive function. There was a marked expansion of CD56^bright NK cells with enhanced interferon-γ (IFN-γ) production. Serum cytokine profiling demonstrated increase of IFN-γ induced protein 10 (IP-10). Gene expression analysis revealed significant changes in a highly restricted set of genes, including <i>FOXP3</i>, <i>IL-2RA,</i> and <i>CISH.</i> This is the first study to evaluate global immune-modulating function of ULD IL-2 in healthy subjects and to support the future studies administrating ULD IL-2 to stem cell donors.

The immune system has a substantial effect on colorectal cancer (CRC) progression. Additionally, the response to immunotherapeutics and conventional treatment options (e.g., chemotherapy, radiotherapy and targeted therapies) is influenced by the immune system. The molecular characterization of colorectal cancer (CRC) has led to the identification of favorable and unfavorable immunological attributes linked to clinical outcome. With the definition of consensus molecular subtypes (CMSs) based on transcriptomic profiles, multiple characteristics have been proposed to be responsible for the development of the tumor immune microenvironment and corresponding mechanisms of immune escape. In this review, a detailed description of proposed immune phenotypes as well as their interaction with different therapeutic modalities will be provided. Finally, possible strategies to shift the CRC immune phenotype towards a reactive, anti-tumor orientation are proposed per CMS.

To better elucidate epigenetic mechanisms that correlate with the dynamic gene expression program observed upon T-cell differentiation, we investigated the genomic landscape of histone modifications in naive and memory CD8+ T cells. Using a ChIP-Seq approach coupled with global gene expression profiling, we generated genome-wide histone H3 lysine 4 (H3K4me3) and H3 lysine 27 (H3K27me3) trimethylation maps in naive, T memory stem cells, central memory cells, and effector memory cells in order to gain insight into how histone architecture is remodeled during T cell differentiation. We show that H3K4me3 histone modifications are associated with activation of genes, while H3K27me3 is negatively correlated with gene expression at canonical loci and enhancers associated with T-cell metabolism, effector function, and memory. Our results also reveal histone modifications and gene expression signatures that distinguish the recently identified T memory stem cells from other CD8+ T-cell subsets. Taken together, our results suggest that CD8+ lymphocytes undergo chromatin remodeling in a progressive fashion. These findings have major implications for our understanding of peripheral T-cell ontogeny and the formation of immunological memory.

Recent observations suggest that immune-mediated tissue destruction is dependent upon coordinate activation of immune genes expressed by cells of the innate and adaptive immune systems.Here, we performed a retrospective pilot study to investigate whether the coordinate expression of molecular signature mostly associated with NK cells could be used to segregate breast cancer patients into relapse and relapse-free outcomes.By analyzing primary breast cancer specimens derived from patients who experienced either 58-116 months (~5-9 years) relapse-free survival or developed tumor relapse within 9-76 months (~1-6 years) we found that the expression of molecules involved in activating signaling of NK cells and in NK cells: target interaction is increased in patients with favorable prognosis.The parameters identified in this study, together with the prognostic signature previously reported by our group, highlight the cooperation between the innate and adaptive immune components within the tumor microenvironment.

MiR-17-92 cluster and its paralogues have emerged as crucial regulators of many oncogenes and tumor suppressors. Transforming growth factor-β receptor II (TGFβR2), as an important tumor suppressor, is involved in various cancer types. However, it is in cancer that only two miRNAs of this cluster and its paralogues have been reported so far to regulate TGFβR2. MiR-93 is oncogenic, but its targetome in cancer has not been fully defined. The role of miR-93 in nasopharyngeal carcinoma (NPC) still remains largely unknown.We firstly evaluated the clinical signature of TGFβR2 down-regulation in clinical samples, and next used a miRNA expression profiling analysis followed by multi-validations, including Luciferase reporter assay, to identify miRNAs targeting TGFβR2 in NPC. In vitro and in vivo studies were performed to further investigate the effects of miRNA-mediated TGFβR2 down-regulation on NPC aggressiveness. Finally, mechanism studies were conducted to explore the associated pathway and genes influenced by this miRNA-mediated TGFβR2 down-regulation.TGFβR2 was down-regulated in more than 50% of NPC patients. It is an unfavorable prognosis factor contributing to clinical NPC aggressiveness. A cluster set of 4 TGFβR2-associated miRNAs was identified; they are all from miR-17-92 cluster and its paralogues, of which miR-93 was one of the most significant miRNAs, directly targeting TGFβR2, promoting cell proliferation, invasion and metastasis in vitro and in vivo. Moreover, miR-93 resulted in the attenuation of Smad-dependent TGF-β signaling and the activation of PI3K/Akt pathway by suppressing TGFβR2, further promoting NPC cell uncontrolled growth, invasion, metastasis and EMT-like process. Impressively, the knockdown of TGFβR2 by siRNA displayed a consentaneous phenocopy with the effect of miR-93 in NPC cells, supporting TGFβR2 is a major target of miR-93. Our findings were also substantiated by investigation of the clinical signatures of miR-93 and TGFβR2 in NPC.The present study reports an involvement of miR-93-mediated TGFβR2 down-regulation in NPC aggressiveness, thus giving extended insights into molecular mechanisms underlying cancer aggressiveness. Approaches aimed at blocking miR-93 may serve as a promising therapeutic strategy for treating NPC patients.

The abundance and functional orientation of tumor-infiltrating lymphocytes in breast cancer is associated with distant metastasis-free survival, yet how this association is influenced by tumor phenotypic heterogeneity is poorly understood. Here, a bioinformatics approach defined tumor biologic attributes that influence this association and delineated tumor subtypes that may differ in their ability to sustain durable antitumor immune responses. A large database of breast tumor expression profiles and associated clinical data was compiled, from which the ability of phenotypic markers to significantly influence the prognostic performance of a classification model that incorporates immune cell-specific gene signatures was ascertained. Markers of cell proliferation and intrinsic molecular subtype reproducibly distinguished two breast cancer subtypes that we refer to as immune benefit-enabled (IBE) and immune benefit-disabled (IBD). The IBE tumors, comprised mostly of highly proliferative tumors of the basal-like, HER2-enriched, and luminal B subtypes, could be stratified by the immune classifier into significantly different prognostic groups, while IBD tumors could not, indicating the potential for productive engagement of metastasis-protective immunity in IBE tumors, but not in IBD tumors. The prognostic stratification in IBE was independent of conventional variables. Gene network analysis predicted the activation of TNFα/IFNγ signaling pathways in IBE tumors and the activation of the transforming growth factor-β pathway in IBD tumors. This prediction supports a model in which breast tumors can be distinguished on the basis of their potential for metastasis-protective immune responsiveness. Whether IBE and IBD represent clinically relevant contexts for evaluating sensitivity to immunotherapeutic agents warrants further investigation. Cancer Immunol Res; 4(7); 600-10. ©2016 AACR.

The ability to identify key biomolecules and molecular changes associated with cancer malignancy and the capacity to monitor the therapeutic outcome against these targets is critically important for cancer treatment. Recent developments in molecular imaging based on magnetic resonance (MR) techniques have provided researchers and clinicians with new tools to improve most facets of cancer care. Molecular imaging is broadly described as imaging techniques used to detect molecular signature at the cellular and gene expression levels. This article reviews both established and emerging molecular MR techniques in oncology and discusses the potential of these techniques in improving the clinical cancer care. It also discusses how molecular MR, in conjunction with other structural and functional MR imaging techniques, paves the way for developing tailored treatment strategies to enhance cancer care.

An immune active cancer phenotype typified by a T helper 1 (Th-1) immune response has been associated with increased responsiveness to immunotherapy and favorable prognosis in some but not all cancer types. The reason of this differential prognostic connotation remains unknown.To explore the contextual prognostic value of cancer immune phenotypes, we applied a multimodal pan-cancer analysis among 31 different histologies (9282 patients), encompassing immune and oncogenic transcriptomic analysis, mutational and neoantigen load and copy number variations.We demonstrated that the favorable prognostic connotation conferred by the presence of a Th-1 immune response was abolished in tumors displaying specific tumor-cell intrinsic attributes such as high transforming growth factor-beta (TGF-β) signaling and low proliferation capacity. This observation was independent of mutation rate. We validated this observation in the context of immune checkpoint inhibition. WNT-β catenin, barrier molecules, Notch, hedgehog, mismatch repair, telomerase activity and AMPK signaling were the pathways most coherently associated with an immune silent phenotype together with mutations of driver genes including IDH1/2, FOXA2, HDAC3, PSIP1, MAP3K1, KRAS, NRAS, EGFR, FGFR3, WNT5A and IRF7.This is the first systematic study demonstrating that the prognostic and predictive role of a bona fide favorable intratumoral immune response is dependent on the disposition of specific oncogenic pathways. This information could be used to refine stratification algorithms and prioritize hierarchically relevant targets for combination therapies.

Dendritic cells (DCs) have been shown to enhance anti-tumor immune responses in several preclinical models. Furthermore, DC-like function can be elicited from peripheral blood monocytes cultured in vitro with interleukin-4 and granulocyte-macrophage colony-stimulating factor. For this reason, a phase 1 study was initiated at the Surgery Branch of the National Cancer Institute to test the toxicity and biological activity of the intravenous administration of peripheral blood monocyte-derived DCs. The DCs were generated by 5- to 7-day incubation in interleukin-4 (1,000 U/mL) and granulocyte-macrophage colony-stimulating factor (1,000 U/mL) of peripheral blood monocytes obtained by leukapheresis. Before administration, the DCs were pulsed separately with the HLA-A*0201-associated melanoma epitopes MART-1(27-35) and gp-100-209-2M. The DCs were administered four times at 3-week intervals. A first cohort of patients (n = 3) was treated with 6 x 10(7) DCs and a second cohort (n = 5) with 2 x 10(8) DCs (in either case, one half of the DCs were pulsed with MART-1(27-35) and the other half was pulsed with gp-100-209-2M). In a final cohort under accrual (n = 2) 2 x 10(8) DCs were administered in combination with interleukin-2 (720,000 IU/kg every 8 hours). The recovery of DCs after in vitro culture ranged from 3% to 35% (mean, 15%) of the original peripheral blood monocytes. Administration of DCs caused no symptoms at any of the doses, and the concomitant administration of interleukin-2 did not cause toxicity other than that expected for interleukin-2 alone. Monitoring of patients' cytotoxic T lymphocyte reactivity before and after treatment revealed enhancement of cytotoxic T lymphocyte reactivity only in one of five patients tested. Of seven patients evaluated for response, one had a transient partial response with regression of pulmonary and cutaneous metastases. A relatively large number of DCs can be safely administered intravenously. The poor clinical outcome of this study perhaps could be explained by the type of protocol used for DC maturation, the route of administration, or both. For this reason, this clinical protocol was interrupted prematurely, whereas other strategies for DC preparation and route of administration are being investigated at the authors' institution.

Interleukin-2 (IL-2) has direct pluripotent effects on cells with immune and inflammatory function. Which of these effects has a critical role in mediating tumor regression remains enigmatic. In this study, we compared early changes in transcriptional profiles of circulating mononuclear cells with those occurring within the microenvironment of melanoma metastases following systemic IL-2 administration.The results suggest that the immediate effects of IL-2 administration on the tumor microenvironment is transcriptional activation of genes predominantly associated with monocyte cell function; minimal effects were noted on migration, activation and proliferation of T cells. However, production of chemokines and markers of adhesion and migration within few hours of IL-2 administration may be responsible for a secondary recruitment of immune cells to the tumor site later.Our results suggest that IL-2 induces inflammation at tumor sites with three predominant secondary effects: activation of antigen-presenting monocytes; massive production of chemoattractants that may recruit other immune cells to the tumor (including MIG and PARC, which are specific for T cells); and activation of cytolytic mechanisms in monocytes (calgranulin, grancalcin) and NK cells (NKG5, NK4).

Melanoma antigen (MA)-specific vaccination strongly enhances antitumor reactivity in vivo and is capable of producing strong cytotoxic T lymphocyte responses in vitro. Furthermore, specific human leukocyte antigen-restricted T cell activation is hypothesized to occur in response to peptide-based immunotherapy, which may lead to the preferential killing of tumor cells bearing the relevant MA. The development of melanoma antigen-loss variants may subsequently occur in vivo.Analysis of 532 melanoma lesions from 204 patients was performed on fine-needle aspiration biopsy specimens. Lesions were graded for the expression of the MAs gp100 and MART-1 with use of immunocytochemistry. A total of 351 melanoma lesions were divided into cohorts on the basis of the treatment received. The pretreatment group (n = 175) consisted of lesions obtained before any form of gp100 immunotherapy, with the posttreatment group (n = 176) consisting of lesions obtained after vaccination with a modified gp100 epitope, gp209-2M +/- interleukin 2 (IL-2).The percentage of lesions not expressing the gp100 antigen is greater than the percentage not expressing MART-1 (26% vs 14%). The frequency of lesions with high expression (> 75%) of gp100 significantly decreased with therapy (47% vs 34%) and conversely negative lesions increased (18% vs 29%). Treatment of lesions with peptide alone (no IL-2) revealed a significant decrease in gp100 expression (47% vs 32%), enhanced with the addition of IL-2 to therapy (47% vs 35%). The expression of MART-1 remained essentially unchanged unless IL-2 was added (54% vs 54%, MART-1 peptide alone, 54% vs 43%, MART-1 peptide + IL-2). Of 94 patients (181 lesions) assessed for gp100 expression before treatment, 10 patients responded to therapy. Pretreatment lesions in responding patients expressed some level of gp100 in all cases compared with 27% of nonresponding lesions, which were negative for gp100 expression. CONCLUSIONS. This study indirectly demonstrates that vaccination with an MA-derived peptide can result in immune selection in vivo. Furthermore, it provides strong immunologic evidence for the specificity of MA vaccines and to the relevance of MA expression in predicting the response to vaccination.

Epithelial ovarian cancer (EOC) is a significant cause of cancer related morbidity and mortality in women. Preferential involvement of peritoneal structures contributes to the overall poor outcome in EOC patients. Advances in biotechnology, such as cDNA microarray, are a product of the Human Genome Project and are beginning to provide fresh opportunities to understand the biology of EOC. In particular, it is now possible to examine in depth, at the molecular level, the complex relationship between the tumor itself and its surrounding microenvironment.This review focuses on the anatomy, physiology, and current immunobiologic research of peritoneal structures, and addresses certain potentially useful animal models. Changes in both the inflammatory and non-inflammatory cell compartments, as well as alterations to the extracellular matrix, appear to be signal events that contribute to the remodeling effects of the peritoneal stroma and surface epithelial cells on tumor growth and spread. These alterations may involve a number of proteins, including cytokines, chemokines, growth factors, either membrane or non-membrane bound, and integrins. Interactions between these molecules and molecular structures within the extracellular matrix, such as collagens and the proteoglycans, may contribute to a peritoneal mesothelial surface and stromal environment that is conducive to tumor cell proliferation and invasion. These alterations need to be examined and defined as possible prosnosticators and as therapeutic or diagnostic targets.

Immunotherapy for the treatment of metastatic melanoma remains a major clinical challenge. The melanoma microenvironment may lead to local T cell tolerance in part through downregulation of costimulatory molecules, such as B7.1 (CD80). We report the results from the first clinical trial, to our knowledge, using a recombinant vaccinia virus expressing B7.1 (rV-B7.1) for monthly intralesional vaccination of accessible melanoma lesions. A standard 2-dose-escalation phase I clinical trial was conducted with 12 patients. The approach was well tolerated with only low-grade fever, myalgias, and fatigue reported and 2 patients experiencing vitiligo. An objective partial response was observed in 1 patient and disease stabilization in 2 patients, 1 of whom is alive without disease 59 months following vaccination. All patients demonstrated an increase in postvaccination antibody and T cell responses against vaccinia virus. Systemic immunity was tested in HLA-A*0201 patients who demonstrated an increased frequency of gp100 and T cells specific to melanoma antigen recognized by T cells 1 (MART-1), also known as Melan-A, by ELISPOT assay following local rV-B7.1 vaccination. Local immunity was evaluated by quantitative real-time RT-PCR, which suggested that tumor regression was associated with increased expression of CD8 and IFN-gamma. The local delivery of vaccinia virus expressing B7.1 was well tolerated and represents an innovative strategy for altering the local tumor microenvironment in patients with melanoma.

Extensive clinical experience has shown that systemic interleukin (IL)-2 administration can induce complete or partial regression of renal cell cancer (RCC) metastases in 15 to 20 % of patients. Since IL-2 has no direct anti-cancer effects, it is believed that cancer regression is mediated either by a direct modulation of immune cell effector functions or through the mediation of soluble factors released as a result of IL-2 administration. We previously observed that transcriptional and protein changes induced by systemic IL-2 administration affect predominantly mononuclear phagocytes with little effect, particularly within the tumor microenvironment, on T cell activation, localization and proliferation. It further appeared that mononuclear phagocyte activation could be best explained by the indirect mediation of a secondary release of cytokines by IL-2 responsive cells either in the circulation or in peripheral tissues. To better characterize the cytokine outburst that follows systemic IL-2 administration we followed the serum levels of 68 soluble factors in ten patients with RCC undergoing high dose (720,000 IU/kg intravenously every 8 hours) IL-2 therapy. Serum was collected before therapy, 3 hours after the 1st and 4th dose and assayed on a multiplexed protein array platform. This study demonstrated that 1) the serum concentration of more than half the soluble factors studied changed significantly during therapy; 2) changes became more dramatic with increasing doses; 3) subclasses of soluble factors displayed different kinetics and 4) cytokine patterns varied quantitatively among patients. This study shows that the cytokine storm that follows systemic IL-2 administration is complex and far-reaching inclusive of soluble factors with disparate, partly redundant and partly contrasting effects on immune function. Therefore comparing in parallel large number of soluble factors, it sets a comprehensive foundation for further elucidation of "cytokine storm" in larger patient pools. Based on this analysis, we propose a prospective collection of serum samples in a larger cohort of patients undergoing IL-2 administration with the purpose of discerning patterns predictive of clinical outcome and toxicity.

Abstract Epithelial ovarian cancer (EOC) represents the most frequent cause of death in the United States from a cancer involving the female genital tract. Contributing to the overall poor outcome in EOC patients, are the metastases to the peritoneum and stroma that are common in this cancer. In one study, cDNA microarray analysis was performed on fresh tissue to profile gene expression in patients with EOC. This study showed a number of genes with significantly altered expression in the pelvic peritoneum and stroma, and in the vicinity of EOC implants. These genes included those encoding coagulation factors and regulatory proteins in the coagulation cascade and genes encoding proteins associated with inflammatory responses. In addition to promoting the formation of blood clots, coagulation factors exhibit many other biologic functions as well as tumorigenic functions, the later including tumor cell proliferation, angiogenesis, invasion, and metastasis. Coagulation pathway proteins involved in tumorigenesis consist of factor II (thrombin), thrombin receptor (protease-activated receptors), factor III (tissue factor), factor VII, factor X and factor I (fibrinogen), and fibrin and factor XIII. In a recent study we conducted, we found that factor XII, factor XI, and several coagulation regulatory proteins, including heparin cofactor-II and epithelial protein C receptor (EPCR), were also upregulated in the peritoneum of EOC. In this review, we summarize evidence in support of a role for these factors in promoting tumor cell progression and the formation of ascites. We also discuss the different roles of coagulation factor pathways in the tumor and peritumoral microenvironments as they relate to angiogenesis, proliferation, invasion, and metastasis. . Since inflammatory responses are another characteristic of the peritoneum in EOC, we also discuss the linkage between the coagulation cascade and the cytokines/chemokines involved in inflammation. Interleukin-8, which is considered an important chemokine associated with tumor progression, appears to be a linkage point for coagulation and inflammation in malignancy. Lastly, we review findings regarding the inflammatory process yielded by certain clinical trials of agents that target members of the coagulation cascade in the treatment of cancer. Current data suggest that disrupting certain elements of the coagulation and inflammation processes in the tumor microenvironment could be a new biologic approach to cancer therapeutics.

Imiquimod is a Toll-like receptor-7 agonist capable of inducing complete clearance of basal cell carcinoma (BCC) and other cutaneous malignancies. We hypothesized that the characterization of the early transcriptional events induced by imiquimod may provide insights about immunological events preceding acute tissue and/or tumor rejection. We report a paired analysis of adjacent punch biopsies obtained pre- and post-treatment from 36 patients with BCC subjected to local application of imiquimod (n = 22) or vehicle cream (n = 14) in a blinded, randomized protocol. Four treatments were assessed (q12 applications for 2 or 4 days, or q24 hours for 4 or 8 days). RNA was amplified and hybridized to 17.5 K cDNA arrays. All treatment schedules similarly affected the transcriptional profile of BCC; however, the q12 × 4 days regimen, associated with highest effectiveness, induced the most changes, with 637 genes unequivocally stimulated by imiquimod. A minority of transcripts (98 genes) confirmed previous reports of interferon-α involvement. The remaining 539 genes portrayed additional immunological functions predominantly involving the activation of cellular innate and adaptive immune-effector mechanisms. Importantly, these effector signatures recapitulate previous observations of tissue rejection in the context of cancer immunotherapy, acute allograft rejection and autoimmunity. This study, based on a powerful and reproducible model of cancer eradication by innate immune mechanisms, provides the first insights in humans into the early transcriptional events associated with immune rejection. This model is likely representative of constant immunological pathways through which innate and adaptive immune responses combine to induce tissue destruction.

Current clinical and histopathological criteria used to define the prognosis of melanoma patients are inadequate for accurate prediction of clinical outcome. We investigated whether genome screening by means of high-throughput gene microarray might provide clinically useful information on patient survival.Forty-three tumor tissues from 38 patients with stage III and stage IV melanoma were profiled with a 17,500 element cDNA microarray. Expression data were analyzed using significance analysis of microarrays (SAM) to identify genes associated with patient survival, and supervised principal components (SPC) to determine survival prediction.SAM analysis revealed a set of 80 probes, corresponding to 70 genes, associated with survival, i.e. 45 probes characterizing longer and 35 shorter survival times, respectively. These transcripts were included in a survival prediction model designed using SPC and cross-validation which allowed identifying 30 predicting probes out of the 80 associated with survival.The longer-survival group of genes included those expressed in immune cells, both innate and acquired, confirming the interplay between immunological mechanisms and the natural history of melanoma. Genes linked to immune cells were totally lacking in the poor-survival group, which was instead associated with a number of genes related to highly proliferative and invasive tumor cells.

Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize peripheral blood stem cells by different mechanisms. A rhesus macaque model was used to compare plerixafor and G-CSF-mobilized CD34(+) cells. Three peripheral blood stem cell concentrates were collected from 3 macaques treated with G-CSF, plerixafor, or plerixafor plus G-CSF. CD34(+) cells were isolated by immunoselection and were analyzed by global gene and microRNA (miR) expression microarrays. Unsupervised hierarchical clustering of the gene expression data separated the CD34(+) cells into 3 groups based on mobilization regimen. Plerixafor-mobilized cells were enriched for B cells, T cells, and mast cell genes, and G-CSF-mobilized cells were enriched for neutrophils and mononuclear phagocyte genes. Genes up-regulated in plerixafor plus G-CSF-mobilized CD34(+) cells included many that were not up-regulated by either agent alone. Two hematopoietic progenitor cell miR, miR-10 and miR-126, and a dendritic cell miR, miR-155, were up-regulated in G-CSF-mobilized CD34(+) cells. A pre-B-cell acute lymphocytic leukemia miR, miR-143-3p, and a T-cell miR, miR-143-5p, were up-regulated in plerixafor plus G-CSF-mobilized cells. The composition of CD34(+) cells is dependent on the mobilization protocol. Plerixafor-mobilized CD34(+) cells include more B-, T-, and mast cell precursors, whereas G-CSF-mobilized cells have more neutrophil and mononuclear phagocyte precursors.

Abstract There is an increasing interest in complementing conventional histopathologic evaluation with molecular tools that could increase the sensitivity and specificity of cancer staging for diagnostic and prognostic purposes. This study strove to identify cancer-specific markers for the molecular detection of a broad range of cancer types. We used 373 archival samples inclusive of normal tissues of various lineages and benign or malignant tumors (predominantly colon, melanoma, ovarian, and esophageal cancers). All samples were processed identically and cohybridized with an identical reference RNA source to a custom-made cDNA array platform. The database was split into training (n = 201) and comparable prediction (n = 172) sets. Leave-one-out cross-validation and gene pairing analysis identified putative cancer biomarkers overexpressed by malignant lesions independent of tissue of derivation. In particular, seven gene pairs were identified with high predictive power (87%) in segregating malignant from benign lesions. Receiver operator characteristic curves based on the same genes could segregate malignant from benign tissues with 94% accuracy. The relevance of this study rests on the identification of a restricted number of biomarkers ubiquitously expressed by cancers of distinct histology. This has not been done before. These biomarkers could be used broadly to increase the sensitivity and accuracy of cancer staging and early detection of locoregional or systemic recurrence. Their selective expression by cancerous compared with paired normal tissues suggests an association with the oncogenic process resulting in stable expression during disease progression when the presently used differentiation markers are unreliable. (Cancer Res 2006; 66(6): 2953-61)

Combination of chemotherapy with cancer vaccines is currently regarded as a potentially valuable therapeutic approach for the treatment of some metastatic tumors, but optimal modalities remain unknown. We designed a phase I/II pilot study for evaluating the effects of dacarbazine (DTIC) on the immune response in HLA-A2(+) disease-free melanoma patients who received anticancer vaccination 1 day following chemotherapy (800 mg/mq i.v.). The vaccine, consisting of a combination of HLA-A2 restricted melanoma antigen A (Melan-A/MART-1) and gp100 analog peptides (250 microg each, i.d.), was administered in combination or not with DTIC to 2 patient groups. The combined treatment is nontoxic. The comparative immune monitoring demonstrates that patients receiving DTIC 1 day before the vaccination have a significantly improved long-lasting memory CD8(+) T cell response. Of relevance, these CD8(+) T cells recognize and lyse HLA-A2(+)/Melan-A(+) tumor cell lines. Global transcriptional analysis of peripheral blood mononuclear cells (PBMC) revealed a DTIC-induced activation of genes involved in cytokine production, leukocyte activation, immune response and cell motility that can favorably condition tumor antigen-specific CD8(+) T cell responses. This study represents a proof in humans of a chemotherapy-induced enhancement of CD8(+) memory T cell response to cancer vaccines, which opens new opportunities to design novel effective combined therapies improving cancer vaccination effectiveness.

Mammalian stem cells are difficult to access experimentally; model systems that can regenerate offer an alternative way to characterize stem cell related genes. Planarian regeneration depends on adult pluripotent stem cells - the neoblasts. These cells can be selectively destroyed using X-rays, enabling comparison of organisms lacking stem cells with wild-type worms. Using a genomic approach we produced an oligonucleotide microarray chip (the Dj600 chip), which was designed using selected planarian gene sequences. Using this chip, we compared planarians treated with high doses of X-rays (which eliminates all neoblasts) with wild-type worms, which led to identification of a set of putatively neoblast-restricted genes. Most of these genes are involved in chromatin modeling and RNA metabolism, suggesting that epigenetic modifications and post-transcriptional regulation are pivotal in neoblast regulation. Comparing planarians treated with low doses of X-rays (after which some radiotolerant neoblasts re-populate the planarian body) with specimens irradiated with high doses and unirradiated control worms, we identified a group of genes that were upregulated as a consequence of low-dose X-ray treatment. Most of these genes encode proteins that are known to regulate the balance between death and survival of the cell; our results thus suggest that genetic programs that control neoblast cytoprotection, proliferation, and migration are activated by low-dose X-rays. The broad differentiation potential of planarian neoblasts is unparalleled by any adult stem cells in the animal kingdom. In addition to our validation of the Dj600 chip as a valuable platform, our work contributes to elucidating the molecular mechanisms that regulate the self-renewal and differentiation of neoblasts.

The advent of high-throughput technology challenges the traditional histopathological classification of cancer, and proposes new taxonomies derived from global transcriptional patterns. Although most of these molecular re-classifications did not endure the test of time, they provided bulk of new information that can reframe our understanding of human cancer biology. Here, we focus on an immunologic interpretation of cancer that segregates oncogenic processes independent from their tissue derivation into at least two categories of which one bears the footprints of immune activation. Several observations describe a cancer phenotype where the expression of interferon stimulated genes and immune effector mechanisms reflect patterns commonly observed during the inflammatory response against pathogens, which leads to elimination of infected cells. As these signatures are observed in growing cancers, they are not sufficient to entirely clear the organism of neoplastic cells but they sustain, as in chronic infections, a self-perpetuating inflammatory process. Yet, several studies determined an association between this inflammatory status and a favorable natural history of the disease or a better responsiveness to cancer immune therapy. Moreover, these signatures overlap with those observed during immune-mediated cancer rejection and, more broadly, immune-mediated tissue-specific destruction in other immune pathologies. Thus, a discussion concerning this cancer phenotype is warranted as it remains unknown why it occurs in immune competent hosts. It also remains uncertain whether a genetically determined response of the host to its own cancer, the genetic makeup of the neoplastic process or a combination of both drives the inflammatory process. Here we reflect on commonalities and discrepancies among studies and on the genetic or somatic conditions that may cause this schism in cancer behavior.

BackgroundCytokines are humoral molecules that elicit regulatory function in immunologic pathways. The level and type of cytokine production has become critical in distinguishing physiologic from pathologic immune conditions. Cytokine profiling has become an important biomarker discovery tool in monitoring of the immune system. However, the variations in cytokine levels in individual subjects over time in healthy individuals have not been extensively studied. In this study, we use multiplex bead arrays to evaluate 27 analytes in paired serum samples taken seven days apart from 144 healthy individuals in order to assess variations over a short time period. MethodsFluorescent bead-based immunoassay (Luminex) was used to measure 27 analytes in serum samples. Measurements were performed on matched samples from 144 healthy donors. To assess inter-plate variability, one arbitrarily selected serum sample was analyzed on each of the first ten plates as bridge sample. ResultsUsing the bridge sample, we showed minimal inter-plate variations in the measurement of most analytes. In measurement of cytokines from the 144 patients at two time points, we found that three cytokines (IL-2, IL-15 and GM-CSF) were undetectable and five analytes (RANTES, MCP-1, VEGF, MIP-1β and PDGF-BB) showed significant difference in concentrations at Day 0 compared to Day 7. ConclusionsThe current study demonstrated higher variations in cytokine levels among individuals than were observed for samples obtained one week apart from identical donors. These data suggest that a serum sample from each subject for use as a baseline measurement is a better control for clinical trials rather than sera from a paired cohort.

Abstract In humans, the role and relationship between molecular pathways that lead to tissue destruction during acute allograft rejection are not fully understood. Based on studies conducted in humans, we recently hypothesized that different immune-mediated tissue destruction processes (i.e. cancer, infection, autoimmunity) share common convergent final mechanisms. We called this phenomenon the "Immunologic Constant of Rejection (ICR)." The elements of the ICR include molecular pathways that are consistently described through different immune-mediated tissue destruction processes and demonstrate the activation of interferon-stimulated genes (ISGs), the recruitment of cytotoxic immune cells (primarily through CXCR3/CCR5 ligand pathways), and the activation of immune effector function genes (IEF genes; granzymes A/B, perforin, etc.). Here, we challenge the ICR hypothesis by using a meta-analytical approach and systematically reviewing microarray studies evaluating gene expression on tissue biopsies during acute allograft rejection. We found the pillars of the ICR consistently present among the studies reviewed, despite implicit heterogeneity. Additionally, we provide a descriptive mechanistic overview of acute allograft rejection by describing those molecular pathways most frequently encountered and thereby thought to be most significant. The biological role of the following molecular pathways is described: IFN-γ, CXCR3/CCR5 ligand, IEF genes, TNF-α, IL-10, IRF-1/STAT-1, and complement pathways. The role of NK cell, B cell and T-regulatory cell signatures are also addressed.

The hypothesis that most cancers are of monoclonal origin is often accepted as a fact in the scientific community. This dogma arose decades ago, primarily from the study of hematopoietic malignancies and sarcomas, which originate as monoclonal tumors. The possible clonal origin of malignant mesothelioma (MM) has not been investigated. Asbestos inhalation induces a chronic inflammatory response at sites of fiber deposition that may lead to malignant transformation after 30-50 years latency. As many mesothelial cells are simultaneously exposed to asbestos fibers and to asbestos-induced inflammation, it may be possible that more than one cell undergoes malignant transformation during the process that gives rise to MM, and result in a polyclonal malignancy.To investigate the clonality patterns of MM, we used the HUMARA (Human Androgen Receptor) assay to examine 16 biopsies from 14 women MM patients. Out of 16 samples, one was non-informative due to skewed Lyonization in its normal adjacent tissue. Fourteen out of the 15 informative samples revealed two electrophoretically distinct methylated HUMARA alleles, the Corrected Allele Ratio (CR) calculated on the allele peak areas indicating polyclonal origin MM.Our results show that MM originate as polyclonal tumors and suggest that the carcinogenic "field effect" of mineral fibers leads to several premalignant clones that give rise to these polyclonal malignancies.

We present the results of a comparative gene expression analysis of 15 metastases (10 regressing and 5 progressing) obtained from 2 melanoma patients with mixed response following different forms of immunotherapy. Whole genome transcriptional analysis clearly indicate that regression of melanoma metastases is due to an acute immune rejection mediated by the upregulation of genes involved in antigen presentation and interferon mediated response (STAT-1/IRF-1) in all the regressing metastases from both patients. In contrast, progressing metastases showed low transcription levels of genes involved in these pathways. Histological analysis showed T cells and HLA-DR positive infiltrating cells in the regressing but not in the progressing metastases. Quantitative expression analysis of HLA-A,B and C genes on microdisected tumoral regions indicate higher HLA expression in regressing than in progressing metastases. The molecular signature obtained in melanoma rejection appeared to be similar to that observed in other forms of immune-mediated tissue-specific rejection such as allograft, pathogen clearance, graft versus host or autoimmune disease, supporting the immunological constant of rejection. We favor the idea that the major factor determining the success or failure of immunotherapy is the nature of HLA Class I alterations in tumor cells and not the type of immunotherapy used. If the molecular alteration is reversible by the immunotherapy, the HLA expression will be upregulated and the lesion will be recognized and rejected. In contrast, if the defect is structural the MHC Class I expression will remain unchanged and the lesion will progress.

We have previously shown that within tumors, recombinant interleukin-2 (rIL-2, aldesleukin) consistently activates tumor-associated macrophages and upregulates IFN-stimulated genes while inducing minimal migration, activation, or proliferation of T cells. These effects are independent of tumor response to treatment. Here, we prospectively evaluated transcriptional alterations induced by rIL-2 in peripheral blood mononuclear cells (PBMC) and within melanoma metastases.We evaluated gene expression changes by serially comparing pre- to posttreatment samples in 13 patients and also compared transcriptional differences among lesions displaying different responsiveness to therapy, focusing on 2 lesions decreasing in size and 2 remaining stable (responding lesions) compared with nonresponding ones.As previously described, the effects of rIL-2 were dramatic within PBMCs, whereas effects within the tumor microenvironment were lesion specific and limited. However, distinct signatures specific to response could be observed in responding lesions pretreatment that were amplified following rIL-2 administration. These signatures match the functional profile observed in other human or experimental models in which immune-mediated tissue-specific destruction (TSD) occurs, underscoring common pathways leading to rejection. Moreover, the signatures observed in pretreatment lesions were qualitatively similar to those associated with TSD, underlining a determinism to immune responsiveness that depends upon the genetic background of the host or the intrinsic genetic makeup of individual tumors.This is the first prospectively collected insight on global transcriptional events occurring during high-dose rIL-2 therapy in melanoma metastases responding to treatment.

Abstract Background: Epidemiologic studies have reported that frequent consumption of quercetin-rich foods is inversely associated with lung cancer incidence. A quercetin-rich diet might modulate microRNA (miR) expression; however, this mechanism has not been fully examined. Methods: miR expression data were measured by a custom-made array in formalin-fixed paraffin-embedded tissue samples from 264 lung cancer cases (144 adenocarcinomas and 120 squamous cell carcinomas). Intake of quercetin-rich foods was derived from a food-frequency questionnaire. In individual-miR–based analyses, we compared the expression of miRs (n = 198) between lung cancer cases consuming high versus low quercetin-rich food intake using multivariate ANOVA tests. In family-miR–based analyses, we used Functional Class Scoring (FCS) to assess differential effect on biologically functional miR families. We accounted for multiple testing using 10,000 global permutations (significance at Pglobal &amp;lt; 0.10). All multivariate analyses were conducted separately by histology and by smoking status (former and current smokers). Results: Family-based analyses showed that a quercetin-rich diet differentiated miR expression profiles of the tumor suppressor let-7 family among adenocarcinomas (PFCS &amp;lt; 0.001). Other significantly differentiated miR families included carcinogenesis-related miR-146, miR-26, and miR-17 (PFCS &amp;lt; 0.05). In individual-based analyses, we found that among former and current smokers with adenocarcinoma, 33 miRs were observed to be differentiated between highest and lowest quercetin-rich food consumers (23 expected by chance; Pglobal = 0.047). Conclusions: We observed differential expression of key biologically functional miRs between high versus low consumers of quercetin-rich foods in adenocarcinoma cases. Impact: Our findings provide preliminary evidence on the mechanism underlying quercetin-related lung carcinogenesis. Cancer Epidemiol Biomarkers Prev; 21(12); 2176–84. ©2012 AACR.

Abstract Activated T cells polarize mesenchymal stromal cells (MSCs) to a proinflammatory Th1 phenotype which likely has an important role in amplifying the immune response in the tumor microenvironment. We investigated the role of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), two factors produced by activated T cells, in MSC polarization. Gene expression and culture supernatant analysis showed that TNF-α and IFN-γ stimulated MSCs expressed distinct sets of proinflammatory factors. The combination of IFN-γ and TNF-α was synergistic and induced a transcriptome most similar to that found in MSCs stimulated with activated T cells and similar to that found in the inflamed tumor microenvironment; a Th1 phenotype with the expression of the immunosuppressive factors IL-4, IL-10, CD274/PD-L1 and indoleamine 2,3 dioxygenase (IDO). Single cell qRT-PCR analysis showed that the combination of IFN-γ and TNF-α polarized uniformly to this phenotype. The combination of IFN-γ and TNF-α results in the synergist uniform polarization of MSCs toward a primarily Th1 phenotype. The stimulation of MSCs by IFN-γ and TNF-α released from activated tumor infiltrating T cells is likely responsible for the production of many factors that characterize the tumor microenvironment.

We have identified eight genes whose expression in human melanoma metastases and ovarian cancers is associated with a lack of Th1 immune signatures. They encode molecules with mechanical barrier function in the skin and other normal tissues and include filaggrin (FLG), tumor-associated calcium signal transducer 2 (TACSTD2), and six desmosomal proteins (DST, DSC3, DSP, PPL, PKP3, and JUP). This association has been validated in an independent series of 114 melanoma metastases. In these, DST expression alone is sufficient to identify melanomas without immune signatures, while FLG and the other six putative barrier molecules are overexpressed in a different subset of melanomas lacking immune signatures. Similar associations have been identified in a set of 186 ovarian cancers. RNA-seq data from 471 melanomas and 307 ovarian cancers in the TCGA database further support these findings and also reveal that overexpression of barrier molecules is strongly associated with early patient mortality for melanoma (p = 0.0002) and for ovarian cancer (p < 0.01). Interestingly, this association persists for FLG for melanoma (p = 0.012) and ovarian cancer (p = 0.006), whereas DST overexpression is negatively associated with CD8+ gene expression, but not with patient survival. Thus, overexpression of FLG or DST identifies two distinct patient populations with low immune cell infiltration in these cancers, but with different prognostic implications for each. These data raise the possibility that molecules with mechanical barrier function in skin and other tissues may be used by cancer cells to protect them from immune cell infiltration and immune-mediated destruction.

The immunologic constant of rejection (ICR) is a broad phenomenon of Th-1 immunity-mediated, tissue-specific destruction.We tested the prognostic value of a 20-gene ICR expression signature in 8766 early breast cancers.Thirty-three percent of tumours were ICR1, 29% ICR2, 23% ICR3, and 15% ICR4. In univariate analysis, ICR4 was associated with a 36% reduction in risk of metastatic relapse when compared with ICR1-3 (p = 2.30E-03). In multivariate analysis including notably the three major prognostic signatures (Recurrence score, 70-gene signature, ROR-P), ICR was the strongest predictive variable (p = 9.80E-04). ICR showed no prognostic value in the HR+/HER2- subtype, but prognostic value in the HER2+ and TN subtypes. Furthermore, in each molecular subtype and among the tumours defined as high risk by the three prognostic signatures, ICR4 patients had a 41-75% reduction in risk of relapse as compared with ICR1-3 patients. ICR added significant prognostic information to that provided by the clinico-genomic models in the overall population and in each molecular subtype. ICR4 was independently associated with achievement of pathological complete response to neoadjuvant chemotherapy (p = 2.97E-04).ICR signature adds prognostic information to that of current proliferation-based signatures, with which it could be integrated to improve patients' stratification and guide adjuvant treatment.

Selective blunting of the status of activation of circulating tumor-specific T cells was invoked to explain their paradoxical coexistence with unhampered tumor growth. By analogy, lack of tumor regression in the face of observable melanoma vaccine-induced T cell responses might be attributed to their status of activation. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited T cell precursor frequency directly in PBMC of patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209-217(210 M) epitope (g209-2 M). Furthermore, we tested by intracellular (IC)-FACS analysis and quantitative real-time PCR (qRT-PCR) the ability of postvaccination PBMC to produce cytokine in response to challenge with vaccine-related epitopes or vaccine-matched (HLA-A*0201) melanoma cells. Vaccine-induced enhancement of T cell precursor frequency could be detected with tHLA in PBMC from six of eight patients studied at frequencies ranging between 0.3 and 2.3% of the total CD8+ population. Stimulation with vaccine-related epitopes induced IFN-gamma expression detectable by IC-FACS or qRT-PCR, respectively, in five and six of these patients. Furthermore, down-regulation of tHLA staining was noted upon cognate stimulation that could be utilized as an additional marker of T cell responsiveness. Finally, we observed in six patients an enhancement of reactivity against vaccine-matched tumor targets that was partly independent of documented vaccine-specific immune responses. A strong correlation was noted between tHLA staining of postvaccination PBMC and IFN-gamma expression by the same samples upon vaccine-relevant stimulation and assessed either by IC-FACS or qRT-PCR. Thus, blunting of the status of T cell activation on itself cannot easily explain the lack of clinical responses observed with vaccination.

We amplified RNAs from 63 fine needle aspiration (FNA) samples from 37 s.c. melanoma metastases from 25 patients undergoing immunotherapy for hybridization to a 6108-gene human cDNA chip. By prospectively following the history of the lesions, we could correlate transcript patterns with clinical outcome. Cluster analysis revealed a tight relationship among autologous synchronously sampled tumors compared with unrelated lesions (average Pearson's r = 0.83 and 0.7, respectively, P < 0.0003). As reported previously, two subgroups of metastatic melanoma lesions were identified that, however, had no predictive correlation with clinical outcome. Ranking of gene expression data from pretreatment samples identified approximately 30 genes predictive of clinical response (P < 0.001). Analysis of their annotations denoted that approximately half of them were related to T-cell regulation, suggesting that immune responsiveness might be predetermined by a tumor microenvironment conducive to immune recognition.

The pathology of humans, in contrast to that of inbred laboratory animals faces the challenge of diversity addressed in genetic terms as polymorphism. Thus, unsurprisingly, treatment modalities that successfully can be applied to carefully-selected pre-clinical models only sporadically succeed in the clinical arena. Indeed, pre-fabricated experimental models purposefully avoid the basic essence of human pathology: the uncontrollable complexity of disease heterogeneity and the intrinsic diversity of human beings. Far from pontificating on this obvious point, this review presents emerging evidence that the study of complex system such as the cytokine network is further complicated by inter-individual differences dictated by increasingly recognized polymorphisms. Polymorphism appears widespread among genes of the immune system possibly resulting from an evolutionary adaptation of the organism facing an ever evolving environment. We will refer to this high variability of immune-related genes as immune polymorphism. In this review we will briefly highlight the possible clinical relevance of immune polymorphism and suggest a change in the approach to the study of human pathology, from the targeted study of individual systems to a broader view of the organism as a whole through immunogenetic profiling.

Abstract Purpose: Epithelial ovarian cancer (EOC) is characterized by early peritoneal involvement ultimately contributing to morbidity and mortality. To study the role of the peritoneum in fostering tumor invasion, we analyzed differences between the transcriptional repertoires of peritoneal tissue lacking detectable cancer in patients with EOC versus benign gynecologic disease. Experimental Design: Specimens were collected at laparotomy from patients with benign disease (b) or malignant (m) ovarian pathology and comprised primary ovarian tumors, paired bilateral specimens from adjacent peritoneum and attached stroma (PE), subjacent stroma (ST), peritoneal washes, ascites, and peripheral blood mononuclear cells. Specimens were immediately frozen. RNA was amplified by in vitro transcription and cohybridized with reference RNA to a custom-made 17.5k cDNA microarray. Results: Principal component analysis and unsupervised clustering did not segregate specimens from patients with benign or malignant pathology. Class comparison identified differences between benign and malignant PE and ST specimens deemed significant by permutation test (P = 0.027 and 0.012, respectively). A two-tailed Student's t test identified 402 (bPE versus mPE) and 663 (mST versus bST) genes differentially expressed at a significance level of P2 ≤ 0.005 when all available paired samples from each patient were analyzed. The same comparison using one sample per patient reduced the pool of differentially expressed genes but retained permutation test significance for bST versus mST (P = 0.031) and borderline significance for bPE versus mPE (P = 0.056) differences. Conclusions: The presence of EOC may foster peritoneal implantation and growth of cancer cells by inducing factors that may represent molecular targets for disease control.

The functional status of circulating vaccine-induced, tumor-specific T cells has been questioned to explain their paradoxical inability to inhibit tumor growth. We enumerated with HLA-A*0201/peptide tetramers (tHLA) vaccine-elicited CD8(+) T cell precursor frequency among PBMC in 13 patients with melanoma undergoing vaccination with the HLA-A*0201-associated gp100:209-217(210 M) epitope. T cell precursor frequency increased from undetectable to 12,400 +/- 3,600 x 10(6) CD8(+) T cells after vaccination and appeared heterogeneous according to previously described functional subtypes: CD45RA(+)CD27(+) (14 +/- 2.6% of tHLA-staining T cells), naive; CD45RA(-)CD27(+) (14 +/- 3.2%), memory; CD45RA(+)CD27(-) (43 +/- 6%), effector; and CD45RA(-)CD27(-) (30 +/- 4.1%), memory/effector. The majority of tHLA(+)CD8(+) T cells displayed an effector, CD27(-) phenotype (73%). However, few expressed perforin (17%). Epitope-specific in vitro stimulation (IVS) followed by 10-day expansion in IL-2 reversed this phenotype by increasing the number of perforin(+) (84 +/- 3.6%; by paired t test, p < 0.001) and CD27(+) (from 28 to 67%; by paired t test, p = 0.01) tHLA(+) T cells. This conversion probably represented a change in the functional status of tHLA(+) T cells rather than a preferential expansion of a CD27(+) (naive and/or memory) PBMC, because it was reproduced after IVS of a T cell clone bearing a classic effector phenotype (CD45RA(+)CD27(-)). These findings suggest that circulating vaccine-elicited T cells are not as functionally active as inferred by characterization of IVS-induced CTL. In addition, CD45RA/CD27 expression may be more informative about the status of activation of circulating T cells than their status of differentiation.

Abstract In a human melanoma model of tumor antigen (TA)–based immunization, we tested the functional status of TA-specific CD8+ cytotoxic T lymphocytes. A “quiescent” phenotype lacking direct ex vivo cytotoxic and proliferative potential was identified that was further characterized by comparing its transcriptional profile to that of TA-specific T cells sensitized in vitro by exposure to the same TA and the T-cell growth factor interleukin 2 (IL-2). Quiescent circulating tumor-specific CD8+ T cells were deficient in expression of genes associated with T-cell activation, proliferation, and effector function. This quiescent status may explain the observed lack of correlation between the presence of circulating immunization-induced lymphocytes and tumor regression. In addition, the activation of TA-specific T cells by in vitro antigen recall and IL-2 suggests that a complete effector phenotype might be reinstated in vivo to fulfill the potential of anticancer vaccine protocols.

CD8-expressing cytotoxic T cell (CTL) interactions with APCs and helper T cells determine their function and ability to survive. In this study, we describe a novel interaction independent of Ag presentation between activated CTLs and bystander CD19-expressing B lymphocytes. Ag-stimulated CTLs serially engage autologous B lymphocytes through CD27/CD70 contact that promotes their survival and proliferation. Moreover, these interactions induce the release of proinflammatory cytokines that follows two general patterns: 1) an epitope-dependent enhancement of cytokine release, and 2) a previously undiscovered coordinate release of cytokines independent of epitope exposure. The latter includes chemoattractants targeting activated T cells. As a result, activated T cells are attracted to B cells, which exert a "helper" role in lymphatic organs or in areas of inflammation. This observation provides a mechanistic explanation to previously reported experimental observations suggesting that B cells are required for T cell priming in vivo.

Abstract Background Mobilized-peripheral blood hematopoietic stem cells (HSCs) have been used for transplantation, immunotherapy, and cardiovascular regenerative medicine. Agents used for HSC mobilization include G-CSF and the CXCR4 inhibitor AMD3100 (plerixafor). The HSCs cells mobilized by each agent may contain different subtypes and have different functions. To characterize mobilized HSCs used for clinical applications, microRNA (miRNA) profiling and gene expression profiling were used to compare AMD3100-mobilized CD133+ cells from 4 subjects, AMD3100 plus G-CSF-mobilized CD133+ cells from 4 subjects and G-CSF-mobilized CD34+ cells from 5 subjects. The HSCs were compared to peripheral blood leukocytes (PBLs) from 7 subjects. Results Hierarchical clustering of miRNAs separated HSCs from PBLs. miRNAs up-regulated in all HSCs included hematopoiesis-associated miRNA; miR-126, miR-10a, miR-221 and miR-17-92 cluster. miRNAs up-regulated in PBLs included miR-142-3p, -218, -21, and -379. Hierarchical clustering analysis of miRNA expression separated the AMD3100-mobilized CD133+ cells from G-CSF-mobilized CD34+ cells. Gene expression analysis of the HSCs naturally segregated samples according to mobilization and isolation protocol and cell differentiation status. Conclusion HSCs and PBLs have unique miRNA and gene expression profiles. miRNA and gene expression microarrays maybe useful for assessing differences in HSCs.

Abstract Background GLV-1h68 is an attenuated recombinant vaccinia virus (VACV) that selectively colonizes established human xenografts inducing their complete regression. Results Here, we explored xenograft/VACV/host interactions in vivo adopting organism-specific expression arrays and tumor cell/VACV in vitro comparing VACV replication patterns. There were no clear-cut differences in vitro among responding and non-responding tumors, however, tumor rejection was associated in vivo with activation of interferon-stimulated genes (ISGs) and innate immune host's effector functions (IEFs) correlating with VACV colonization of the xenografts. These signatures precisely reproduce those observed in humans during immune-mediated tissue-specific destruction (TSD) that causes tumor or allograft rejection, autoimmunity or clearance of pathogens. We recently defined these common pathways in the "immunologic constant of rejection" hypothesis (ICR). Conclusion This study provides the first prospective validation of a universal mechanism associated with TSD. Thus, xenograft infection by oncolytic VACV, beyond offering a promising therapy of established cancers, may represent a reliable pre-clinical model to test therapeutic strategies aimed at modulating the central pathways leading to TSD; this information may lead to the identification of principles that could refine the treatment of cancer and chronic infection by immune stimulation or autoimmunity and allograft rejection through immune tolerance.

We previously showed that tumor-free peritoneum of patients with epithelial ovarian cancer (EOC) exhibited enhanced expression of several inflammatory response genes compared to peritoneum of benign disease. Here, we examined peritoneal inflammatory cell patterns to determine their concordance with selected enhanced genes.Expression patterns of selected inflammatory genes were mined from our previously published data base. Bilateral pelvic peritoneal and subjacent stromal specimens were obtained from 20 women with EOC and 7 women with benign pelvic conditions. Sections were first stained by indirect immunoperoxidase and numbers of monocytes/macrophages (MO/MA), T cells, B cells, and NK cells counted. Proportions of CD68+ cells and CD3+ cells that coexpressed MO/MA differentiation factors (CD163, CCR1, CXCR8, VCAM1, and phosphorylated cytosolic phospholipase A2 [pcPLA2]), which had demonstrated expression in EOC peritoneal samples, were determined by multicolor immunofluorescence.MO/MA were present on both sides of the pelvic peritoneum in EOC patients, with infiltration of the subjacent stroma and mesothelium. CD68+ MO/MA, the most commonly represented population, and CD3+ T cells were present more often in EOC than in benign pelvic tumors. NK cells, B cells, and granulocytes were rare. CXCL8 (IL-8) and the chemokine receptor CCR1 were coexpressed more frequently on MO/MA than on CD3+ cells contrasting with CD68+/CD163+ cells that coexpressed CXCL8 less often. An important activated enzyme in the eicosanoid pathway, pcPLA2, was highly expressed on both CD68+ and CD163+ cells. The adherence molecule Vascular Cell Adhesion Molecule-1 (VCAM1) was expressed on CD31+ endothelial cells and on a proportion of CD68+ MO/MA but rarely on CD3+ cells.The pelvic peritoneum in EOC exhibits a general pattern of chronic inflammation, represented primarily by differentiated MO/MA, and distinct from that in benign conditions concordant with previous profiling results.

Type-1 T cells are critical for effective anti-tumor immune responses. The recently discovered microRNAs (miRs) are a large family of small regulatory RNAs that control diverse aspects of cell function, including immune regulation. We identified miRs differentially regulated between type-1 and type-2 T cells, and determined how the expression of such miRs is regulated.We performed miR microarray analyses on in vitro differentiated murine T helper type-1 (Th1) and T helper type-2 (Th2) cells to identify differentially expressed miRs. We used quantitative RT-PCR to confirm the differential expression levels. We also used WST-1, ELISA, and flow cytometry to evaluate the survival, function and phenotype of cells, respectively. We employed mice transgenic for the identified miRs to determine the biological impact of miR-17-92 expression in T cells.Our initial miR microarray analyses revealed that the miR-17-92 cluster is one of the most significantly over-expressed miR in murine Th1 cells when compared with Th2 cells. RT-PCR confirmed that the miR-17-92 cluster expression was consistently higher in Th1 cells than Th2 cells. Disruption of the IL-4 signaling through either IL-4 neutralizing antibody or knockout of signal transducer and activator of transcription (STAT)6 reversed the miR-17-92 cluster suppression in Th2 cells. Furthermore, T cells from tumor bearing mice and glioma patients had decreased levels of miR-17-92 when compared with cells from non-tumor bearing counterparts. CD4+ T cells derived from miR-17-92 transgenic mice demonstrated superior type-1 phenotype with increased IFN-gamma production and very late antigen (VLA)-4 expression when compared with counterparts derived from wild type mice. Human Jurkat T cells ectopically expressing increased levels of miR-17-92 cluster members demonstrated increased IL-2 production and resistance to activation-induced cell death (AICD).The type-2-skewing tumor microenvironment induces the down-regulation of miR-17-92 expression in T cells, thereby diminishing the persistence of tumor-specific T cells and tumor control. Genetic engineering of T cells to express miR-17-92 may represent a promising approach for cancer immunotherapy.

Abstract Purpose: Abnormalities in the constitutive and IFN-γ–inducible HLA class I surface antigen expression of tumor cells is often associated with an impaired expression of components of the antigen processing machinery (APM). Hence, we analyzed whether there exists a link between the IFN-γ signaling pathway, constitutive HLA class I APM component expression, and IFN-γ resistance. Experimental Design: The basal and IFN-γ–inducible expression profiles of HLA class I APM and IFN-γ signal transduction cascade components were assessed in melanoma cells by real-time PCR (RT-PCR), Western blot analysis and/or flow cytometry, the integrity of the Janus activated kinase (JAK) 2 locus by comparative genomic hybridization. JAK2 was transiently overexpressed in JAK2− cells. The effect of IFN-γ on the cell growth was assessed by XTT [2,3-bis(2-methoxy-4-nitro-S-sulfophenynl)-H-tetrazolium-5-carboxanilide inner salt] assay. Results: The analysis of 8 melanoma cell lines linked the IFN-γ unresponsiveness of Colo 857 cells determined by lack of inducibility of HLA class I surface expression on IFN-γ treatment to a deletion of JAK2 on chromosome 9, whereas other IFN-γ signaling pathway components were not affected. In addition, the constitutive HLA class I APM component expression levels were significantly reduced in JAK2− cells. Furthermore, JAK2-deficient cells were also resistant to the antiproliferative effect of IFN-γ. Transfection of wild-type JAK2 into JAK2− Colo 857 not only increased the basal APM expression but also restored their IFN-γ sensitivity. Conclusions: Impaired JAK2 expression in melanoma cells leads to reduced basal expression of MHC class I APM components and impairs their IFN-γ inducibility, suggesting that malfunctional IFN-γ signaling might cause HLA class I abnormalities. Clin Cancer Res; 17(9); 2668–78. ©2011 AACR.

The identification of tumor antigens recognized by T cells led to the design of therapeutic strategies aimed at eliciting adaptive immune responses. The last decade of experience has shown that, although active immunization can induce enhancement of anticancer T-cell precursors (easily detectable in standard assays), most often they are unable to induce tumor regression and, consequently, have scarcely any impact on overall survival. Moreover, in the few occasions when tumor rejection occurs, the mechanisms determining this phenomenon remain poorly understood, and data derived from in vivo human observations are rare. The advent of high-throughput gene-expression analysis (microarrays) has cast new light on unrecognized mechanisms that are now deemed to be central for the development of efficient immune-mediated tumor rejection. The aim of this article is to review the data on the molecular signature associated with this process. We believe that the description of how the mechanism of immune-mediated tissue destruction occurs would contribute to our understanding of why it happens, thereby allowing us to develop more effective immune therapeutic strategies.

Abstract Background - Traditional Chinese Medicine (TCM) has been used for thousands of years to treat or prevent diseases, including cancer. Good manufacturing practices (GMP) and sophisticated product analysis (PhytomicsQC) to ensure consistency are now available allowing the assessment of its utility. Polychemical Medicines, like TCM, include chemicals with distinct tissue-dependent pharmacodynamic properties that result in tissue-specific bioactivity. Determining the mode of action of these mixtures was previously unsatisfactory; however, information rich RNA microarray technologies now allow for thorough mechanistic studies of the effects complex mixtures. PHY906 is a long used four herb TCM formula employed as an adjuvant to relieve the side effects associated with chemotherapy. Animal studies documented a decrease in global toxicity and an increase in therapeutic effectiveness of chemotherapy when combined with PHY906. Methods - Using a systems biology approach, we studied tumor tissue to identify reasons for the enhancement of the antitumor effect of CPT-11 (CPT-11) by PHY906 in a well-characterized pre-clinical model; the administration of PHY906 and CPT-11 to female BDF-1 mice bearing subcutaneous Colon 38 tumors. Results - We observed that 1) individually PHY906 and CPT-11 induce distinct alterations in tumor, liver and spleen; 2) PHY906 alone predominantly induces repression of transcription and immune-suppression in tumors; 3) these effects are reverted in the presence of CPT-11, with prevalent induction of pro-apoptotic and pro-inflammatory pathways that may favor tumor rejection. Conclusions - PHY906 together with CPT-11 triggers unique changes not activated by each one alone suggesting that the combination creates a unique tissue-specific response.

T cells can mediate remarkable tumor regressions including complete cure in patients with metastatic cancer. Genetic alterations in an individual's cancer cells (the mutanome) encode unique peptides (m-peptides) that can be targets for T cells. The recent advances in next-generation sequencing and computation prediction allows, for the first time, the rapid and affordable identification of m-peptides in individual patients. Despite excitement about the extended spectrum of potential targets in personalized immunotherapy, there is no experience or consensus on the path to their successful clinical application. Major questions remain, such as whether clinical responses to cytokine therapy, T cell transfer, and checkpoint blockade are primarily mediated by m-peptide-specific reactivity, whether m-peptides can be effectively used as vaccines, and which m-peptides are most potently recognized. These and other technological, immunological and translational questions will be explored during a 1-day Workshop on Personalized Cancer Immunotherapy by the Society for Immunotherapy of Cancer, directly before the Annual Meeting, on November 7, 2013 at the National Harbor, MD near Washington, DC.

Abstract Purpose: Chemokines are implicated in T-cell trafficking. We mapped the chemokine landscape in advanced stage ovarian cancer and characterized the expression of cognate receptors in autologous dendritic cell (DC)–vaccine primed T cells in the context of cell-based immunotherapy. Experimental Design: The expression of all known human chemokines in patients with primary ovarian cancer was analyzed on two independent microarray datasets and validated on tissue microarray. Peripheral blood T cells from five HLA-A2 patients with recurrent ovarian cancer, who previously received autologous tumor DC vaccine, underwent CD3/CD28 costimulation and expansion ex vivo. Tumor-specific T cells were identified by HER2/neu pentamer staining and were evaluated for the expression and functionality of chemokine receptors important for homing to ovarian cancer. Results: The chemokine landscape of ovarian cancer is heterogeneous with high expression of known lymphocyte-recruiting chemokines (CCL2, CCL4, and CCL5) in tumors with intraepithelial T cells, whereas CXCL10, CXCL12, and CXCL16 are expressed quasi-universally, including in tumors lacking tumor-infiltrating T cells. DC-vaccine primed T cells were found to express the cognate receptors for the above chemokines. Ex vivo CD3/CD28 costimulation and expansion of vaccine-primed Tcells upregulated CXCR3 and CXCR4, and enhanced their migration toward universally expressed chemokines in ovarian cancer. Conclusions: DC-primed tumor-specific T cells are armed with the appropriate receptors to migrate toward universal ovarian cancer chemokines, and these receptors are further upregulated by ex vivo CD3/CD28 costimulation, which render T cells more fit for migrating toward these chemokines. Clin Cancer Res; 21(12); 2840–50. ©2015 AACR.

In a recent adoptive cell therapy (ACT) clinical trial using autologous tumor-infiltrating lymphocytes (TILs) in patients with metastatic melanoma, we found an association between CD8+ T cells expressing the inhibitory receptor B- and T-lymphocyte attenuator (BTLA) and clinical response. Here, we further characterized this CD8+BTLA+ TIL subset and their CD8+BTLA− counterparts. We found that the CD8+ BTLA+ TILs had an increased response to IL-2, were less-differentiated effector-memory (TEM) cells, and persisted longer in vivo after infusion. In contrast, CD8+BTLA− TILs failed to proliferate and expressed genes associated with T-cell deletion/tolerance. Paradoxically, activation of BTLA signaling by its ligand, herpes virus entry mediator (HVEM), inhibited T-cell division and cytokine production, but also activated the Akt/PKB pathway thus protecting CD8+BTLA+ TILs from apoptosis. Our results point to a new role of BTLA as a useful T-cell differentiation marker in ACT and a dual signaling molecule that curtails T-cell activation while also conferring a survival advantage for CD8+ T cells. These attributes may explain our previous observation that BTLA expression on CD8+ TILs correlates with clinical response to adoptive T-cell therapy in metastatic melanoma.

The immune system has a powerful ability to recognize and kill cancer cells, but its function is often suppressed within tumors, preventing clearance of disease. Functionally diverse innate and adaptive cellular lineages either drive or constrain immune reactions within tumors. The transcription factor (TF) BACH2 regulates the differentiation of multiple innate and adaptive cellular lineages, but its role in controlling tumor immunity has not been elucidated. Here, we demonstrate that BACH2 is required to establish immunosuppression within tumors. Tumor growth was markedly impaired in Bach2-deficient mice and coincided with intratumoral activation of both innate and adaptive immunity. However, augmented tumor clearance in the absence of Bach2 was dependent upon the adaptive immune system. Analysis of tumor-infiltrating lymphocytes from Bach2-deficient mice revealed high frequencies of rapidly proliferating effector CD4+ and CD8+ T cells that expressed the inflammatory cytokine IFN-γ. Effector T cell activation coincided with a reduction in the frequency of intratumoral Foxp3+ Tregs. Mechanistically, BACH2 promoted tumor immunosuppression through Treg-mediated inhibition of intratumoral CD8+ T cells and IFN-γ. These findings demonstrate that BACH2 is a key component of the molecular program of tumor immunosuppression and identify therapeutic targets for the reversal of immunosuppression in cancer.

Purpose: Adoptive cell therapy with autologous tumor-infiltrating lymphocytes (TIL) is a therapy for metastatic melanoma with response rates of up to 50%. However, the generation of the TIL transfer product is challenging, requiring pooled allogeneic normal donor peripheral blood mononuclear cells (PBMC) used in vitro as “feeders” to support a rapid-expansion protocol. Here, we optimized a platform to propagate TIL to a clinical scale using K562 cells genetically modified to express costimulatory molecules such as CD86, CD137-ligand, and membrane-bound IL-15 to function as artificial antigen-presenting cells (aAPC) as an alternative to using PBMC feeders. Experimental Design: We used aAPC or γ-irradiated PBMC feeders to propagate TIL and measured rates of expansion. The activation and differentiation state was evaluated by flow cytometry and differential gene expression analyses. Clonal diversity was assessed on the basis of the pattern of T-cell receptor usage. T-cell effector function was measured by evaluation of cytotoxic granule content and killing of target cells. Results: The aAPC propagated TIL at numbers equivalent to that found with PBMC feeders, whereas increasing the frequency of CD8+ T-cell expansion with a comparable effector-memory phenotype. mRNA profiling revealed an upregulation of genes in the Wnt and stem-cell pathways with the aAPC. The aAPC platform did not skew clonal diversity, and CD8+ T cells showed comparable antitumor function as those expanded with PBMC feeders. Conclusions: TIL can be rapidly expanded with aAPC to clinical scale generating T cells with similar phenotypic and effector profiles as with PBMC feeders. These data support the clinical application of aAPC to manufacture TIL for the treatment of melanoma.

// Simon Jasinski-Bergner 1 , Adi Reches 2 , Christine Stoehr 3 , Chiara Massa 1 , Evamaria Gonschorek 1 , Stefan Huettelmaier 4 , Juliane Braun 4 , Sven Wach 5 , Bernd Wullich 5 , Verena Spath 2 , Ena Wang 6 , Francesco M. Marincola 6 , Ofer Mandelboim 2 , Arndt Hartmann 3 , Barbara Seliger 1 1 Institute of Medical Immunology, Martin Luther University Halle-Wittenberg, Halle, Germany 2 Faculty of Medicine, The Hebrew University of Jerusalem, Ein Kerem, Jerusalem, Israel 3 Institute of Pathology, Friedrich Alexander University Erlangen-Nuremberg, Erlangen, Germany 4 Institute of Molecular Medicine, Martin Luther University Halle-Wittenberg, Halle, Germany 5 Clinic of Urology, Friedrich Alexander University Erlangen-Nuremberg, Erlangen, Germany 6 Sidra Medical and Research Center, Qatar Foundation, Doha, Qatar Correspondence to: Barbara Seliger, e-mail: Barbara.Seliger@uk-halle.de Keywords: non-classical HLA class I molecules, microRNA, HLA-G, immune escape, renal cell carcinoma Received: October 06, 2015 Accepted: March 11, 2016 Published: April 4, 2016 ABSTRACT The non-classical human leukocyte antigen G (HLA-G) is expressed at a high frequency in renal cell carcinoma (RCC) and is associated with a higher tumor grade and a poor clinical outcome. This might be caused by the HLA-G-mediated inhibition of the cytotoxicity of T and NK cells. Therefore a selective targeting of HLA-G might represent a powerful strategy to enhance the immunogenicity of RCC lesions. Recent studies identified a number of HLA-G-regulating microRNAs (miRs) and demonstrated an inverse expression of some of these miRs with HLA-G in RCC in vitro and in vivo . However, it was postulated that further miRs might exist contributing to the tightly controlled selective HLA-G expression. By application of a miR enrichment assay (miTRAP) in combination with in silico profiling two novel HLA-G-regulatory miRs, miR-548q and miR-628-5p, were identified. Direct interactions of both miRs with the 3' untranslated region of HLA-G were confirmed with luciferase reporter gene assays. In addition, qPCR analyses and immunohistochemical staining revealed an inverse, expression of miR-628-5p, but not of miR-548q to the HLA-G protein in primary RCC lesions and cell lines. Stable overexpression of miR-548q and miR-628-5p caused a downregulation of HLA-G mRNA and protein. This leads in case of miR-548q to an enhanced NK cell-mediated HLA-G-dependent cytotoxicity, which could be reverted by ILT2 blockade suggesting a control of the immune effector cell activity at least by this miR. The identification of two novel HLA-G-regulatory miRs extends the number of HLA-G-relevant miRs tuning the HLA-G expression and might serve as future therapeutic targets.

Optimal approaches to induce T cell infiltration of tumors are not known. Chemokines CXCL9, CXCL10, and CXCL11 support effector T cell recruitment and may be induced by IFN. This study tests the hypothesis that intratumoral administration of IFNγ will induce CXCL9–11 and will induce T cell recruitment and anti-tumor immune signatures in melanoma metastases. Nine eligible patients were immunized with a vaccine comprised of 12 class I MHC-restricted melanoma peptides and received IFNγ intratumorally. Effects on the tumor microenvironment were evaluated in sequential tumor biopsies. Adverse events (AEs) were recorded. T cell responses to vaccination were assessed in PBMC by IFNγ ELISPOT assay. Tumor biopsies were evaluated for immune cell infiltration, chemokine protein expression, and gene expression. Vaccination and intratumoral administration of IFNγ were well tolerated. Circulating T cell responses to vaccine were detected in six of nine patients. IFNγ increased production of chemokines CXCL10, CXCL11, and CCL5 in patient tumors. Neither vaccination alone, nor the addition of IFNγ promoted immune cell infiltration or induced anti-tumor immune gene signatures. The melanoma vaccine induced circulating T cell responses, but it failed to infiltrate metastases, thus highlighting the need for combination strategies to support T cell infiltration. A single intratumoral injection of IFNγ induced T cell-attracting chemokines; however, it also induced secondary immune regulation that may paradoxically limit immune infiltration and effector functions. Alternate dosing strategies or additional combinatorial treatments may be needed to promote trafficking and retention of tumor-reactive T cells in melanoma metastases.

ABSTRACT The size of lentiviral DNA reservoirs reflects the effectiveness of immune responses against lentiviruses. So far, abundant information has been gathered on the control of HIV-1 replication. Understanding the innate mechanisms contributing to containment of the HIV DNA reservoir, however, are only partly clarified and are relevant to guiding interventions for reservoir containment or eradication. We studied the contribution of natural killer (NK) cell functional features in HIV patients controlling replication either spontaneously (HIV controllers [HIC]) or after progression and antiretroviral treatment (progressor patients [PP]). An inverse correlation between HIV DNA copy numbers (either total or integrated) in circulating CD4 + cells and NK cell function was observed. Induced interferon gamma (IFN-γ) production and NKp46/NKp30 activating receptor-induced expression correlated inversely with reservoir size. The correlation was present not only for a homogeneous cohort of HIC patients but also when PP were included in the analysis. Adaptive (NKG2C + CD57 + ) NK cell features were not associated with reservoir size. However, a distinct set of 370 differentially expressed transcripts was found to underlie functional differences in NK cells controlling HIV DNA reservoir size. In proof-of-principle in vitro experiments of CD4 + cell infection with HIV-1, purified NK cells with the above-mentioned functional/transcriptional features displayed 10- and 30-fold higher abilities to control HIV replication and DNA burdens in vitro , respectively, than those of other NK cells. Thus, NK cells with a specific functional and transcriptional signature contribute to control of the HIV reservoir in CD4 + cells. Their selection, expansion, and/or adoptive transfer may support strategies to eradicate HIV-1 infection or to safely deescalate antiretroviral treatment. IMPORTANCE The most relevant feature of HIV-1 infection is represented by its DNA reservoir size in the body, which guarantees lifelong infection and resumption of virus replication after antiretroviral treatment interruption. So far, there has been little success in the identification of factors contributing to HIV-1 reservoir containment. In this study, by studying quantitative total and integrated HIV-1 DNA levels and NK cells in HIV-1 patients with either progressive or nonprogressive disease, we observed that inducible IFN-γ and natural cytotoxicity receptor (NCR) expression in a specific subset of NK cells with a characteristic transcriptional signature represents a correlate for HIV-1 reservoir control. This represents an advance in our understanding of the mechanism(s) that controls the lentivirus reservoir. Monitoring, selection, expansion, and adoptive transfer of these NK cells may allow monitoring of treatment efficacy and the likelihood of reservoir control and may support protocols for HIV-1 eradication.

With the advent of checkpoint inhibition, immunotherapy has revolutionized the clinical management of several cancers, but has demonstrated limited efficacy in mammary carcinoma. Transcriptomic profiling of cancer samples defined distinct immunophenotypic categories characterized by different prognostic and predictive connotations. In breast cancer, genomic alterations leading to the dysregulation of mitogen-activated protein kinase (MAPK) pathways have been linked to an immune-silent phenotype associated with poor outcome and treatment resistance. These aberrations include mutations of MAP3K1 and MAP2K4, amplification of KRAS, BRAF, and RAF1, and truncations of NF1. Anticancer therapies targeting MAPK signaling by BRAF and MEK inhibitors have demonstrated clear immunologic effects. These off-target properties could be exploited to convert the immune-silent tumor phenotype into an immune-active one. Preclinical evidence supports that MAPK-pathway inhibition can dramatically increase the efficacy of immunotherapy. In this review, we provide a detailed overview of the immunomodulatory impact of MAPK-pathway blockade through BRAF and MEK inhibitions. While BRAF inhibition might be relevant in melanoma only, MEK inhibition is potentially applicable to a wide range of tumors. Context-dependent similarities and differences of MAPK modulation will be dissected, in light of the complexity of the MAPK pathways. Therapeutic strategies combining the favorable effects of MAPK-oriented interventions on the tumor microenvironment while maintaining T-cell function will be presented. Finally, we will discuss recent studies highlighting the rationale for the implementation of MAPK-interference approaches in combination with checkpoint inhibitors and immune agonists in breast cancer.

The melanoma patient's immune response to tumor has been extensively studied. Yet, the frequently observed coexistence of tumor-associated Ag (TAA)-specific T cells with their target cells in vivo remains unexplained. Loss of TAA expression might contribute to this paradox. We studied TAA expression in metastases by obtaining fine-needle aspirations from 52 tumor lesions in 30 patients with melanoma before and soon after immunotherapy. Limitations due to low amounts of starting material were overcome with a high fidelity antisense RNA amplification method. TAA expression was measured by quantitative real-time PCR of anti-sense RNA. Decrease in gp100/Pmel-17 TAA preceded tumor disappearance in several instances and could be best explained by immune selection because most patients had received gp100/Pmel-17-specific vaccination. Conversely, immune selection was absent in nonregressing lesions. These observations suggest that vaccination, when successful, triggers a broad inflammatory reaction that can lead to tumor destruction despite immune selection. Additionally, lack of clinical response might be attributed to lack of this initiating event rather than immune escape. This study provides an insight into the natural history of tumors and defines a strategy for the characterization of gene expression in tumors during therapy.

Lymphocytes expanded from excised specimens can be used to characterize intratumoral T cell responses. These analyses, however, are limited to one time point in the natural history of the removed tumor. The expansion of autologous tumor cells and tumor-infiltrating lymphocytes (TIL) from fine needle aspirates (FNA) of tumors potentially allows a dynamic evaluation of T cell responses within the same lesion at moments relevant to the disease course or response to therapy. Fourteen TIL cultures and 8 tumor cell lines were generated from 18 FNA (12 patients). Five of six TIL that could be tested against autologous tumor demonstrated specific reactivity. Two additional TIL for which no autologous tumor was available demonstrated recognition of HLA-matched melanoma cell lines. Serial FNA of the same lesions were performed in five HLA-A*0201 patients vaccinated with the emulsified melanoma Ag (MA) epitopes: MART-1:27-35; tyrosinase:368-376(370D); gp100:280-288(288V); and gp100:209-217 (210M). FNA material was separately cultured for a short time in IL-2 (300 IU/ml) after stimulation with irradiated autologous PBMC pulsed with each peptide or FluM1:58-66 (1 micromol/ml). No peptide-specific TIL could be expanded from prevaccination FNA. However, after vaccination, TIL specific for gp100:280(g280), gp100:209 (g209), and MART-1:27-35 (MART-1)-related epitopes were identified in three, three, and two patients, respectively. No Flu reactivity could be elicited in TIL, whereas it was consistently present in parallel PBMC cultures. This excluded PBMC contamination of the FNA material. This analysis suggests the feasibility of TIL expansion from minimal FNA material and localization of vaccine-specific T cells at the tumor site.