Tumor Immunology

Great progress has been made in the field of tumor immunology in the past decade, but optimism about the clinical application of currently available cancer vaccine approaches is based more on surrogate endpoints than on clinical tumor regression. In our cancer vaccine trials of 440 patients, the objective response rate was low (2.6%), and comparable to the results obtained by others. We consider here results in cancer vaccine trials and highlight alternate strategies that mediate cancer regression in preclinical and clinical models.

Complete list of abbreviations of tumor antigens 707-AP = 707 alanine proline Ÿ AFP = alpha (α)-fetoprotein Ÿ ART-4 = adenocarcinoma antigen recognized by T cells 4 Ÿ BAGE = B antigen; β-catenin/m, β-catenin/mutated Ÿ Bcr-abl = breakpoint cluster region-Abelson Ÿ CAMEL = CTL-recognized antigen on melanoma Ÿ CAP-1 = carcinoembryonic antigen peptide -1 Ÿ CASP-8 = caspase-8 Ÿ CDC27m = cell-divisioncycle 27 mutated Ÿ CDK4/m = cycline-dependent kinase 4 mutated Ÿ CEA = carcinoembryonic antigen Ÿ CT = cancer/testis (antigen) Ÿ Cyp-B = cyclophilin B Ÿ DAM = differentiation antigen melanoma (the epitopes of DAM-6 and DAM-10 are equivalent, but the gene sequences are different. DAM-6 is also called MAGE-B2 and DAM-10 is also called MAGE-B1) Ÿ ELF2M = elongation factor 2 mutated Ÿ ETV6-AML1 = Ets variant gene 6/acute myeloid leukemia 1 gene ETS Ÿ G250 = glycoprotein 250 Ÿ GAGE = G antigen Ÿ GnT-V = N-acetylglucosaminyltransferase V Ÿ Gp100 = glycoprotein 100 kD Ÿ HAGE = helicose antigen Ÿ HER-2/neu = human epidermal receptor-2/neurological Ÿ HLA-A*0201-R170I = arginine (R) to isoleucine (I) exchange at residue 170 of the αhelix of the α2-domain in the HLA-A2 gene Ÿ HPV-E7 = human papilloma virus E7 Ÿ HSP70-2M = heat shock protein 70 -2 mutated Ÿ HST-2 = human signet ring tumor -2 Ÿ hTERT or hTRT = human telomerase reverse transcriptase Ÿ iCE = intestinal carboxyl esterase Ÿ KIAA0205 = name of the gene as it appears in databases Ÿ LAGE = L antigen Ÿ LDLR/FUT = low density lipid receptor/GDP-L-fucose: β-D-galactosidase 2-α-Lfucosyltransferase Ÿ MAGE = melanoma antigen Ÿ MART-1/Melan-A = melanoma antigen recognized by T cells-1/Melanoma antigen A Ÿ MC1R = melanocortin 1 receptor Ÿ Myosin/m = myosin mutated Ÿ MUC1 = mucin 1 Ÿ MUM-1, -2, -3 = melanoma ubiquitous mutated 1, 2, 3 Ÿ NA88-A = NA cDNA clone of patient M88 Ÿ NY-ESO-1 = New York -esophageous 1 Ÿ P15 = protein 15 Ÿ p190 minor bcr-abl = protein of 190 3 KD bcr-abl Ÿ Pml/RARα = promyelocytic leukaemia/retinoic acid receptor α Ÿ PRAME = preferentially expressed antigen of melanoma Ÿ PSA = prostate-specific antigen Ÿ PSM = prostate-specific membrane antigen Ÿ RAGE = renal antigen Ÿ RU1 or RU2 = renal ubiquitous 1 or 2 Ÿ SAGE = sarcoma antigen Ÿ SART-1 or SART-3 = squamous antigen rejecting tumor 1 or 3 Ÿ TEL/AML1 = translocation Ets-family leukemia/acute myeloid leukemia 1 Ÿ TPI/m = triosephosphate isomerase mutated Ÿ TRP-1 = tyrosinase related protein 1, or gp75 Ÿ TRP-2 = tyrosinase related protein 2 Ÿ TRP-2/INT2 = TRP-2/intron 2 Ÿ WT1 = Wilms' tumor gene Abbreviations used ALL acute lymphoblastic leukemia Ÿ AML = acute myeloid leukemia Ÿ APL = acute promyelocytic leukemia Ÿ CTL, cytotoxic T lymphocytes Ÿ CML = chronic myelogenous leukemia Ÿ Ets = E-26 transforming specific (family of transcription factors) Ÿ H/N = heand and neck Ÿ MHC = major histocompatibility complex Ÿ NSCLC = non-small cell lung carcinoma Ÿ ORF = open reading frame Ÿ RCC = renal cell carcinoma Ÿ SCC = squamous cell carcinoma Ÿ TSTA = tumor-specific transplantation antigens 4

Most pockets in the human leukocyte antigen-group DR (HLA-DR) groove are shaped by clusters of polymorphic residues and, thus, have distinct chemical and size characteristics in different HLA-DR alleles. Each HLA-DR pocket can be characterized by "pocket profiles," a quantitative representation of the interaction of all natural amino acid residues with a given pocket. In this report we demonstrate that pocket profiles are nearly independent of the remaining HLA-DR cleft. A small database of profiles was sufficient to generate a large number of HLA-DR matrices, representing the majority of human HLA-DR peptide-binding specificity. These virtual matrices were incorporated in software (TEPITOPE) capable of predicting promiscuous HLA class II ligands. This software, in combination with DNA microarray technology, has provided a new tool for the generation of comprehensive databases of candidate promiscuous Tcell epitopes in human disease tissues. First, DNA microarrays are used to reveal genes that are specifically expressed or upregulated in disease tissues. Second, the prediction software enables the scanning of these genes for promiscuous HLA-DR binding sites. In an example, we demonstrate that starting from nearly 20,000 genes, a database of candidate colon cancer-specific and promiscuous T-cell epitopes could be fully populated within a matter of days. Our approach has implications for the development of epitope-based vaccines.

Mesenchymal stem cells (MSCs), a non-hematopoietic stem cell population first discovered in bone marrow, are multipotent cells capable of differentiating into mature cells of several mesenchymal tissues, such as fat and bone. As common progenitor cells of adipocytes and osteoblasts, MSCs are delicately balanced for their differentiation commitment. Numerous in vitro investigations have demonstrated that fat-induction factors inhibit osteogenesis, and, conversely, bone-induction factors hinder adipogenesis. In fact, a variety of external cues contribute to the delicate balance of adipo-osteogenic differentiation of MSCs, including chemical, physical, and biological factors. These factors trigger different signaling pathways and activate various transcription factors that guide MSCs to commit to either lineage. The dysregulation of the adipo-osteogenic balance has been linked to several pathophysiologic processes, such as aging, obesity, osteopenia, osteopetrosis, and osteoporosis. Thus, the regulation of MSC differentiation has increasingly attracted great attention in recent years. Here, we review external factors and their signaling processes dictating the reciprocal regulation between adipocytes and osteoblasts during MSC differentiation and the ultimate control of the adipo-osteogenic balance.

Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinaserelated protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-K b binding motif, TRP-2 181-188 , was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.

Tumor manipulation of host immunity is important for tumor survival and invasion. Many cancers secrete CCL21, a chemoattractant for various leukocytes and lymphoid tissue inducer cells, which drive lymphoid neogenesis. CCL21 expression by melanoma tumors in mice was associated with an immunotolerant microenvironment, which included the induction of lymphoid-like reticular stromal networks, an altered cytokine milieu, and the recruitment of regulatory leukocyte populations. In contrast, CCL21-deficient tumors induced antigen-specific immunity. CCL21-mediated immune tolerance was dependent on host rather than tumor expression of the CCL21 receptor, CCR7, and could protect distant, coimplanted CCL21-deficient tumors and even nonsyngeneic allografts from rejection. We suggest that by altering the tumor microenvironment, CCL21-secreting tumors shift the host immune response from immunogenic to tolerogenic, which facilitates tumor progression.

Myeloid derived suppressor cells (MDSC) have been described as a heterogeneous cell population with potent immune suppressor function in mice. Limited data are available on MDSC in human diseases. Interpretation of these data is complicated by the fact that different markers have been used to analyze human MDSC subtypes in various clinical settings. Human MDSC are CD11b + , CD33 + , HLA-DR neg/low and can be divided into granulocytic CD14 − and monocytic CD14 + subtypes. Interleukin 4Rα, VEGFR, CD15 and CD66b have been suggested to be more specific markers for human MDSC, however these markers can only be found on some MDSC subsets. Until today the best marker for human MDSC remains their suppressor function, which can be either direct or indirect through the induction of regulatory T cells. Immune suppressor activity has been associated with high arginase 1 and iNOS activity as well as ROS production by MDSC. Not only in murine models, but even more importantly in patients with cancer, different drugs have been shown to either reverse the immune suppressor function of MDSC or directly target these cells. Systemic treatment with all-transretinoic acid has been shown to mature human MDSC and reverse their immune suppressor function. Alternatively, MDSC can be targeted by treatment with the multi-targeted receptor tyrosine kinase inhibitor sunitinib. This review will provide a comprehensive summary of the recent literature on human MDSC.

The hypothesis of immunologic surveillance of neoplasia is predicated on the theory that the immune system is capable of discriminating self from foreign antigens, and that tumor-specific antigens are regarded by the immune system as nonself. We propose here an alternate view, that the immune system has evolved to detect danger by employing 'professional' antigen-presenting cells as sentinels of tissue distress. In this model, cancers do not appear dangerous to the immune system, so that the default response of T cells to tumors is to be turned off. We discuss the implications for immunotherapy of malignancy.

Background: CpG-oligonucleotides (CpG-ODN), which induce signaling through Toll-like receptor 9 (TLR9), are currently under investigation as adjuvants in therapy against infections and cancer. CpG-ODN function as Th-1 adjuvants and are able to activate dendritic cells. In humans TLR9 has been described to be strongly expressed in B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells. We determined whether a direct interaction of bacterial DNA with the tumor cells themselves is possible and investigated the expression and function of TLR9 in human malignant solid tumors and cell lines. TLR9 expression by malignant tumor cells, would affect treatment approaches using CpG-ODN on the one hand, and, on the other hand, provide additional novel information about the role of tumor cells in tumor-immunology.

Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes. Over the past decades, the molecular and cell biological events associated with efferocytosis have been rigorously studied, and many eat-me signals and receptors have been identified. The externalization of phosphatidylserine (PS) is arguably the most emblematic eat-me signal that is in turn bound by a large number of serum proteins and opsonins that facilitate efferocytosis. Under physiological conditions, externalized PS functions as a dominant and evolutionarily conserved immunosuppressive signal that promotes tolerance and prevents local and systemic immune activation. Pathologically, the innate immunosuppressive effect of externalized PS has been hijacked by numerous viruses, microorganisms, and parasites to facilitate infection, and in many cases, establish infection latency. PS is also profoundly dysregulated in the tumor microenvironment and antagonizes the development of tumor immunity. In this review, we discuss the biology of PS with respect to its role as a global immunosuppressive signal and how PS is exploited to drive diverse pathological processes such as infection and cancer. Finally, we outline the rationale that agents targeting PS could have significant value in cancer and infectious disease therapeutics.

Interleukin-22 (IL-22) is an IL-10 family cytokine produced by T cells and innate lymphoid cells. The IL-22 signaling pathway orchestrates mucosal immune defense and tissue regeneration through pleiotropic effects including pro-survival signaling, cell migration, dysplasia and angiogenesis. While these functions can prevent initial establishment of tumors, they can also be hijacked by aggressive cancers to enhance tumor growth and metastasis. Thus, the role of the IL-22/IL-22R1 axis in cancer is complex and context-specific. Evidence of IL-22 involvement manifests as dysregulation of IL-22 expression and signaling in patients with many common cancers including those of the gut, skin, lung and liver. Unlike other cancer-associated cytokines, IL-22 has restricted tissue specificity as its unique receptor IL-22R1 is exclusively expressed on epithelial and tissue cells, but not immune cells. This makes it an attractive target for therapy as there is potential achieve anti-tumor immunity with fewer side effects. This review summarizes current findings on functions of IL-22 in association with general mechanisms for tumorigenesis as well as specific contributions to particular cancers, and ponders how best to approach further research in the field.

The greatest risk factor for the development of cervical and other cancers that have been linked to the human papillomavirus (HPV) family is the persistence of the virus. To persist for the decades required to develop HPV-related cancers, the virus must escape host immunity. HPV is a simple DNA virus that has evolved to escape immune attack by a combination of stealth and interference. This review focuses on the mechanisms by which HPV can evade recognition by the host immune system.

Radiotherapy and chemotherapy are two famous modalities in tumor-targeted therapy that lead to systemic and local toxicities for normal tissues. Moreover, several studies have confirmed that exposure of the tumor to radiation or chemotherapy drugs stimulate some signaling pathways in the tumor microenviron-ment (TME), leading to resistance of cancer cells to apoptosis, as well as promoting angiogenesis and tumor growth. Nuclear factor kappa B (NF-κB) plays a central role in the regulation of inflammatory responses in both normal tissues and tumors via the release of several cytokines, regulation of prostaglandins, reduction/oxidation (redox) reactions, angiogenesis, and cell death. Upregulation of NF-κB in normal tissues causes an appearance of inflammatory reactions and oxidative stress, whereas it regulates angiogenesis and suppresses apoptosis, leading to resistance to subsequent doses of radiation or chemotherapy. Selective inhibition of NF-κB in experimental studies has shown promising results for tumor sensitization via apoptosis induction, inhibition of angiogenesis, and increasing delay of tumor growth. The use of some agents for NF-κB inhibition has been shown to alleviate radiation/chemotherapy toxicities in normal cells/ tissues. In this current review, we explained the pivotal role of NF-κB in both normal tissue toxicity and tumor resistance. We also discussed the promising strategies for overcoming these problems with regard to chemotherapy and radiotherapy.

Radiotherapy is one of the most common treatment modalities for controlling a wide range of tumors. However, it has been shown that radiotherapy alone is unable to completely eradicate some tumors and could be associated with a high possibility of tumor recurrence. To date, various experimental and clinical studies have been conducted to explore some efficient targets within tumor microenvironment (TME) to enhance tumor response to radiotherapy; thus help eliminate or eradicate tumors. Although targeting DNA damage responses (DDRs) is associated with severe toxicities, studies in recent decade suggest that inhibition of some apoptosis/survival targets within TME is promising. This is also associated with changes in the numbers of T regulatory cells (Tregs) and cytotoxic T lymphocytes (CTLs). The inhibition of cyclooxygenase-2 (COX-2), phosphoinositide 3-kinase (PI3K), mammalian target of rapamycin (mTOR), mitogen-activated protein kinases (MAPKs) and vascular en-dothelial growth factor (VEGF) have also shown promising results. The combination of receptor tyrosine kinase (RTK) inhibitors with radiotherapy is interesting as well as the clinical use of some drugs and antibodies. Epidermal growth factor receptor (EGFR) inhibitors are the most common RTK inhibitors. Some clinical trials in recent years have shown very interesting results for immune checkpoint inhibitors (ICIs), especially programmed death-ligand 1 (PD-L1) and CTLs-associated antigen 4 (CTLA-4) inhibitors. It has been suggested that administration of ICIs during or after hypofractionated radiotherapy could lead to best results. In this review, we explain TME response to radiotherapy and potential targets for sensitization of cancer cells to radiotherapy.

Gene expression analysis comparing nonpregnant with pregnant bovine uteri, including placenta, was performed with a custom cDNA microarray containing 1,933 independent genes. These genes were classified into six categories according to biological function, as follows: cell and tissue structural dynamics (108 genes), intercellular communication (221), intracellular metabolism , cell cycle and apoptosis (26), regulation of gene expression , expressed sequence tag (EST) and function unknown (617), and uncomplemented genes (583 clones). This array possessed bovine placental/endometrial specificity, as it included many pregnancy-specific molecules, such as pregnancy-associated glycoprotein-1 (PAGs), placental lactogen (PLs), and prolactin-related protein-1 (PRPs). A total of 77 genes were induced and 12 repressed in the placenta/endometrium. Our results point to a fundamental role for bovine placentalspecific genes such as PAGs, PLs, and PRPs, in implantation and placentogenesis, and document that cDNA microarray analysis from bovine placenta/ endometrium is possible and is a specific tool for monitoring genome-wide gene expression during the establishment and maintenance of pregnancy.

Colorectal cancer (CRC) is one of the most common cancer types and represents a major therapeutic challenge. Although initial events in colorectal carcinogenesis are relatively well characterized and treatment for early-stage disease has significantly improved over the last decades, the mechanisms underlying metastasis – the main cause of death – remain poorly understood. Correspondingly, no effective therapy is currently available for advanced or metastatic disease. There is increasing evidence that colorectal cancer is hierarchically organized and sustained by cancer stem cells, in concert with various stromal cell types. Here, we review the interplay between cancer stem cells and their microenvironment in promoting metas-tasis and discuss recent insights relating to both patient prognosis and novel targeted treatment strategies. A better understanding of these topics may aid the prevention or reduction of metastatic burden.

Activating transcription factor-1 (ATF-1) and cAMPresponsive element (CRE)-binding protein (CREB) have been implicated in cAMP and Ca 2+ -induced transcriptional activation. The expression of the transcription factors CREB and ATF-1 is upregulated in metastatic melanoma cells. However, how overexpression of ATF-1/ CREB contributes to the acquisition of the metastatic phenotype remains unclear. Here, the eect of disrupting ATF-1 activity was investigated using intracellular expression of an inhibitory anti-ATF-1 single chain antibody fragment (ScFv). Intracellular expression of ScFv anti-ATF-1 in MeWo melanoma cells caused signi®cant reduction in CRE-dependent promoter activation. In addition, expression of ScFv anti-ATF-1 in melanoma cells suppressed their tumorigenicity and metastatic potential in nude mice. ScFv anti-ATF-1 rendered the melanoma cells susceptible to thapsigargininduced apoptosis in vitro and caused massive apoptosis in tumors transplanted subcutaneously into nude mice, suggesting that ATF-1 and its associated proteins act as survival factor for human melanoma cells. This is the ®rst report to demonstrate the potential of ScFv anti-ATF-1 as an inhibitor of tumor growth and metastasis of solid tumor in vivo.

It is generally accepted that human and experimental tumor cells can lose major histocompatibility complex (MHC) class I molecules. These human leukocyte antigen (HLA) losses are detected when the primary tumor breaks the basal membrane, invades the surrounding tissues, and starts to metastasize. These altered HLA class I phenotypes probably constitute the major tumor escape mechanism facing anti-tumor T-cell mediated responses. Thus, it is important to characterize these phenotypes in clinical tumor samples, analyze the mechanism(s) responsible for them, and counsel patients before and during peptide anti-cancer immunotherapy. The present paper summarizes the most relevant altered HLA class I phenotypes found in human tumor samples, indicates their frequency, and outlines the mechanisms implicated. This review also points out that the natural killer (NK) escape mechanism of HLA class I deficient cancer cells is yet to be defined. Knowledge accumulated to date reveals that HLA class I molecules are an important crossroad in tumor immunology. Human Immunology 61, 65-73 (2000).

Delivery is one of the most critical obstacles confronting nanoparticle use in cancer diagnosis and therapy. For most oncological applications, nanoparticles must extravasate in order to reach tumor cells and perform their designated task. However, little understanding exists regarding the effect of nanoparticle shape on extravasation. Herein we use real-time intravital microscopic imaging to meticulously examine how two different nanoparticles behave across three different murine tumor models. The study quantitatively demonstrates that high-aspect ratio single-walled carbon nanotubes (SWNTs) display extravasa-tional behavior surprisingly different from, and counterintuitive to, spherical nanoparticles although the nanoparticles have similar surface coatings, area, and charge. This work quantitatively indicates that nanoscale extravasational competence is highly dependent on nanoparticle geometry and is heterogeneous.

Tumor microenvironment (TME) is a host for a complex network of heterogeneous stromal cells with overlapping or opposing functions depending on the dominant signals within this milieu. Reciprocal paracrine interactions between cancer cells with cells within the tumor stroma often reshape the TME in favor of the promotion of tumor. These complex interactions require more sophisticated approaches for cancer therapy, and, therefore, advancing knowledge about dominant drivers of cancer within the TME is critical for designing therapeutic schemes. This review will provide knowledge about TME architecture, multiple signaling, and cross communications between cells within this milieu, and its targeting for immunotherapy of cancer.

Tumor-associated eosinophilia has been observed in numerous human cancers and several tumor models in animals; however, the details surrounding this eosinophilia remain largely undefined and anecdotal. We used a B16-F10 melanoma cell injection model to demonstrate that eosinophil infiltration of tumors occurred from the earliest palpable stages with significant accumulations only in the necrotic and capsule regions. Furthermore, the presence of diffuse extracellular matrix staining for eosinophil major basic protein was restricted to the necrotic areas of tumors, indicating that eosinophil degranulation was limited to this region. Antibody-mediated depletion of CD4 ؉ T cells and adoptive transfer of eosinophils suggested, respectively, that the accumulation of eosinophils is not associated with T helper cell type 2-dependent immune responses and that recruitment is a dynamic, ongoing process, occurring throughout tumor growth. Ex vivo migration studies have identified what appears to be a novel chemotactic factor(s) released by stressed/dying melanoma cells, suggesting that the accumulation of eosinophils in tumors occurs, in part, through a unique mechanism dependent on a signal(s) released from areas of necrosis. Collectively, these studies demonstrate that the infiltration of tumors by eosinophils is an early and persistent response that is spatial-restricted. It is more important that these data also show that the mechanism(s) that elicit this host response occur, independent of immune surveillance, suggesting that eosinophils are part of an early inflammatory reaction at the site of tumorigenesis.

Inhibitory receptors mediate CD8 T-cell hyporesponsiveness against cancer and infectious diseases. PD-1 and CTLA-4 have been extensively studied, and blocking antibodies have already shown clinical benefit for cancer patients. Only little is known on extended co-expression of inhibitory receptors and their ligands. Here we analyzed the expression of eight inhibitory receptors by tumor-antigen specific CD8 T-cells. We found that the majority of effector T-cells simultaneously expressed four or more of the inhibitory receptors BTLA, TIM-3, LAG-3, KRLG-1, 2B4, CD160, PD-1 and CTLA-4. There were major differences depending on antigen-specificity, differentiation and anatomical localization of T-cells. On the other hand, naive T-cells were only single or double positive for BTLA and TIM-3. Extended co-expression is likely relevant for effector Tcells, as we found expression of multiple ligands in metastatic lesions of melanoma patients. Together, our data suggest that naive T-cells are primarily regulated by BTLA and TIM-3, whereas effector cells interact via larger numbers of inhibitory receptors. Blocking multiple inhibitory receptors simultaneously or sequentially may improve T-cell based therapies, but further studies are necessary to clarify the role of each receptor-ligand pair.

Here, similar findings are demonstrated for patients with human immunodeficiency virus or chronic hepatitis C virus infection. Furthermore, the presence of type 2 CD8+ Ts cells infiltrating diseased tissues in patients with autoimmune thyroiditis or cancer is shown. Collectively, these findings suggest that type 2 CD8+ Ts cells may be involved in the control of pathologic chronic immune responses, contributing in some cases to the pathogenesis of the disease.

Helmut Sies established the concept of oxidative stress in 1985.
However, it took some time to introduce this concept into
pathology, where investigators count on formalin-fixed
paraffin-embedded tissue sections. I sought out antigens for
this purpose based on an oxidative stress-induced rat renal
carcinogenesis model, which revealed that 8-hydroxy-2′-
deoxyguanosine and 4-hydroxy-2-nonenal-modified proteins
are ideal. These two monoclonal antibodies successfully
revealed the involvement of oxidative stress in numerous human diseases, including carcinogenesis and atherosclerosis.
Shigeru Okada established the aforementioned ferric
nitrilotriacetate (Fe-NTA)-induced rat renal carcinogenesis
model, which thus far has answered many questions regarding
the presence of target genes in oxidative stress-induced carcinogenesis and the sites that are susceptible to oxidative stress in the genome. Particularly, the similarity of genomic alterations between Fe-NTA-induced renal cancer and human cancers suggests that excess iron plays a role also inhuman carcinogenesis. Furthermore, excess iron is amajor pathology in asbestosinduced mesothelioma, including chrysotile. Despite an analogy to asbestos, multi-wall carbon nanotubes were distinct in that diameter is another responsible factor for mesothelial carcinogenesis. Recently, non-thermal plasma emerged as a candidate for medical intervention for wounds and cancers via manipulating oxidative stress. Counteracting excess iron is a promising preventive strategy for major diseases.

The role of cytokines in modulating the formation of new tumors is mediated by their ability to regulate antigen-specific anti-tumor responses and by the activation of non-specific mechanisms, including those involved in the processes of inflammation and innate resistance. Cytokines may influence the growth of tumors by acting directly on tumor cells as growth promoting or growth inhibiting factors or indirectly by attracting inflammatory cell types and affecting angiogenesis. Due to the potency and complexity of cytokine activity against tumor growth, the improvement of cloning techniques and the availability of recombinant forms of different cytokines, a great effort has been made in the recent years to exploit this anti-tumor potential for cancer therapy. This important goal has been difficult to achieve in most cases due to toxicity of most cytokines which could not be dissociated from their anti-tumoral functions. Nevertheless, if well designed, treatment protocols and/or modifications of the cytokine molecules may in some situations augment the anti-tumor effects while limiting the toxicity. One of these molecular approaches could be the design of peptides containing the functional domain of certain cytokines, exemplified by IT9302, a peptide homologous to the functional domain of IL-10, which has demonstrated to increase tumor NK cell sensitivity.

The extrahepatic enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes tryptophan degradation in the first and rate-limiting step towards biosynthesis of the central metabolic co-factor nicotinamide adenine dinucleotide (NAD). While this pathway has been known for decades, the actual physiological role for IDO in mammals remained obscure, because (i.) most cell types do not express the downstream enzymes in the NAD biosynthesis pathway and (ii.) mammals salvage rather than synthesize NAD to meet their metabolic needs. An immunological role for IDO was hinted at with the observation that IDO expression is stimulated by interferonγ and subsequently confirmed by the discovery of its physiological importance in protecting the fetus from maternal immunity. Similarly, elevations in tryptophan catabolism in cancer patients were known since the 1950s, but the basis and meaning of this phenomenon were uncertain until it was shown that IDO, which is commonly elevated in tumors and draining lymph nodes, suppresses T cell immunity in the tumor microenvironment. Indeed, by creating peripheral tolerance to tumor antigens, IDO can undermine immune responses that thwart tumor cell survival in the context of an underlying inflammatory environment that facilitates tumor outgrowth. In preclinical studies, small molecule inhibitors of IDO compromise this mechanism of immunosuppression and strongly leverage the efficacy of a variety of classical chemotherapeutic agents, supporting the clinical development of IDO inhibitors as a therapeutic goal. This essay summarizes key findings that implicate IDO as an important mediator of peripheral tolerance and discusses the development of anti-cancer modalities that incorporate the use of IDO inhibitors.

It is estimated that more than half of cancer patients undergo radiotherapy during the course of their treatment. Despite its beneficial therapeutic effects on tumor cells, exposure to high doses of ionizing radiation (IR) is associated with several side effects. Although improvements in radiotherapy techniques and instruments could reduce these side effects, there are still important concerns for cancer patients. For several years, scientists have been trying to modulate tumor and normal tissue responses to IR, leading to an increase in therapeutic ratio. So far, several types of radioprotectors and radiosensitizers have been investigated in experimental studies. However, high toxicity of chemical sensitizers or possible tumor protection by radioprotectors creates a doubt for their clinical applications. On the other hand, the protective effects of these radioprotectors or sensitizer effects of radiosensitizers may limit some type of cancers. Hence, the development of some radioprotectors without any protective effect on tumor cells or low toxic radiosensitizers can help improve therapeutic ratio with less side effects. Melatonin as a natural body hormone is a potent antioxidant and anti-inflammatory agent that shows some anti-cancer properties. It is able to neutralize different types of free radicals produced by IR or pro-oxidant enzymes which are activated following exposure to IR and plays a key role in the protection of normal tissues. In addition, melatonin has shown the ability to inhibit long-term changes in inflammatory responses at different levels, thereby ameliorating late side effects of radiotherapy. Fortunately, in contrast to classic antioxidants, some in vitro studies have revealed that melatonin has a potent anti-tumor activity when used alongside irradiation. However, the mechanisms of its radiosensitive effect remain to be elucidated. Studies suggested that the activation of pro-apoptosis gene, such as p53, changes in the metabolism of tumor cells, suppression of DNA repair responses as well as changes in biosynthesis of estrogen in breast cancer cells are involved in this process. In this review, we describe the molecular mechanisms for radioprotection and radiosensitizer effects of melatonin. Furthermore, some other proposed mechanisms that may be involved are presented.

Transforming growth factor β1 (TGF-β), enriched in the tumor microenvironment and broadly immunosuppressive, inhibits natural
killer (NK) cell function by yet-unknown mechanisms. Here we
show that TGF-β–treated human NK cells exhibit reduced tumor
cytolysis and abrogated perforin polarization to the immune synapse. This result was accompanied by loss of surface expression of
activating killer Ig-like receptor 2DS4 and NKp44, despite intact
cytoplasmic stores of these receptors. Instead, TGF-β depleted
DNAX activating protein 12 kDa (DAP12), which is critical for surface
NK receptor stabilization and downstream signal transduction.
Mechanistic analysis revealed that TGF-β induced microRNA
(miR)-183 to repress DAP12 transcription/translation. This pathway was confirmed with luciferase reporter constructs bearing
the DAP12 3′ untranslated region as well as in human NK cells
by use of sense and antisense miR-183. Moreover, we documented reduced DAP12 expression in tumor-associated NK cells in lung cancer patients, illustrating this pathway to be consistently perturbed in the human tumor microenvironment.

Head and neck cancer represents a challenging disease. Despite recent treatment advances, which have improved functional outcomes, the long-term survival of head and neck cancer patients has remained unchanged for the past 25 years. One of the goals of adjuvant cancer therapy is to eradicate local regional microscopic and micrometastatic disease with minimal toxicity to surrounding normal cells. In this respect, antigen-specific immunotherapy is an attractive therapeutic approach. With the advances in molecular genetics and fundamental immunology, antigen-specific immunotherapy is being actively explored using DNA, bacterial vector, viral vector, peptide, protein, dendritic cell, and tumor-cell based vaccines. Early phase clinical trials have demonstrated the safety and feasibility of these novel therapies and the emphasis is now shifting towards the development of strategies, which can increase the potency of these vaccines. As the field of immunotherapy matures and as our understanding of the complex interaction between tumor and host develops, we get closer to realizing the potential of immunotherapy as an adjunctive method to control head and neck cancer and improve long-term survival in this patient population.

Interactions with antigen-presenting cells (APCs) interrupt T cell migration through tissues and trigger signaling pathways that converge on the activation of transcriptional regulators, including nuclear factor of activated T cells (NFAT), which control T cell function and differentiation. Both stable and unstable modes of cognate T cell-APC interactions have been observed in vivo, but the functional significance of unstable, serial contacts has remained unclear.

Lung cancer is a major public health problem
causing more deaths than any other cancer. A better understanding
of the biology of this disease and improvements in
treatment are greatly needed. Increasing evidence supports
the concept that a rare and specialized population of cancer
cells, so-called cancer-initiating cells with stem cell-like characteristics,
is responsible for tumor growth, maintenance, and
recurrence. Cancer-initiating cells also exhibit characteristics
that render them resistant to both radiation and chemotherapy,
and therefore they are believed to play a role in treatment
failure. This has led to the hypothesis that traditional therapies
that indiscriminately kill tumor cells will not be as effective as
therapies that selectively target cancer-initiating cells. Investigating
putative cancer-initiating cells in lung cancer will
greatly benefit the understanding of the origins of this disease
and may lead to novel approaches to therapy by suggesting
markers for use in either further isolating this population for
study or for selectively targeting these cells. This review will
discuss (1) lung cancer, (2) stem cells, and the role of cancerinitiating
cells in tumorigenesis; (3) markers and functional
characteristics associated with lung cancer-initiating cells; and
(4) the potential to selectively target this subpopulation of
tumor cells.

Experimental models indicate that tumor cells in suspension, unlike solid tumor fragments, might be unable to produce life-threatening cancer outgrowth when transferred to animal models, irrespective of the number of cells transferred, although they induce specific immune responses. Human tumor cells cultured in three dimensions display increased pro-angiogenic capacities and resistance to interferons, chemotherapeutic agents or irradiation, as compared with cells cultured in twodimensional (2D) monolayers. Tumor cells cultured in three dimensions were also shown to be characterized by defective immune recognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens (TAAs) and by a capacity to inhibit CTL proliferation and dendritic cell (DC) functions. Downregulation of human leukocyte antigen (HLA) or TAA expression and high production of lactic acid might play a role in the elicitation of these effects. Here, we propose that growth in 3D architectures might provide new insights into tumor immunology and could represent an integral missing component in pathophysiological tumor immune escape mechanisms.

Epigenetic modifications, like histone acetylation, are essential for regulating gene expression within cells. Cancer cells acquire pathological epigenetic modifications resulting in gene expression patterns that facilitate and sustain tumorigenesis. Epigenetic manipulation therefore is emerging as a novel targeted therapy for cancer. Histone Acetylases (HATs) and Histone Deacetylases (HDACs) regulate histone acetylation and hence gene expression. Histone deacetylase (HDAC) inhibitors are well known to affect cancer cell viability and biology and are already in use for the treatment of cancer patients. Immunotherapy can lead to clinical benefit in selected cancer patients, especially in patients with limited disease after tumor debulking. HDAC inhibitors can potentially synergize with immunotherapy by elimination of tumor cells. The direct effects of HDAC inhibitors on immune cell function, however, remain
largely unexplored. Initial data have suggested HDAC inhibitors to be predominantly immunosuppressive, but more recent reports have challenged this view. In this review we will discuss the effects of HDAC inhibitors on tumor cells and different immune cell subsets, synergistic interactions and possible mechanisms. Finally, we will address future challenges and potential application of HDAC inhibitors in immunocombination therapy of cancer

CD4(+)CD25(+)FOXP3(+) T regulatory cells (T(regs)) prevent autoimmunity by restricting overexuberant immune responses, but the same subpopulation can incur detrimental effects on antitumor responses. In both cases, the suppressor potential of T(regs) appears to be strongly influenced by their compartmentalization. In myelodysplastic syndromes (MDS), immune deregulation and autoimmunity in the early stages might lead to ineffective hematopoiesis and bone marrow (BM) failure, whereas late-stage disease is characterized by the immune escape of the malignant clone. We show that these two stages of MDS are associated with differential T(reg) activity. Specifically, we found that in early stage MDS, compared with normal hematopoiesis and late stage MDS, T(regs) are dysfunctional and their BM homing through the CXCL12/CXCR4 axis is seriously impaired as a result of CXCR4 downregulation. Conversely, in late stage MDS, T(regs) are systemically and locally expanded and retain their function and migratory capacity. Moreover, T(reg) levels follow the disease course and are significantly reduced in treatment responding patients. Our findings indicate T(reg) involvement in the pathophysiology of MDS; defective suppressor function and BM trafficking of T(regs) may be important in the autoimmune process of early MDS, but increased T(reg) activity could favor leukemic clone progression in late stage disease.

Despite advances in animal studies, where the cure of the majority of mice with pre-established (albeit earlystage) tumors has become almost standard, human clinical trials have been much less successful. Here we describe some of the most recent advances in the specialist field of tumor immunology and immunotherapy, highlighting salient work to identify key problem areas and potential solutions. We make particular note of recent developments in adoptive therapy; whole-cell, DNA, and peptide vaccines; and antibody therapy. We also describe the revival of interest in regulatory T cells and conclude by detailing the need for clinical trial read-out autonomy and methods to predict which patients will respond to a particular treatment.

Programmed cell death-1 ligand-1 (PD-L1) overexpression in cancer cells accelerates tumor progression. PD-L1 possesses two main pro-oncogenic functions. First, PD-L1 is a strong immunosuppressive molecule that inactivates tumor-specific T cells by binding to the inhibitory receptor PD-1. Second, PD-L1 function relies on the delivery of intrinsic intracellular signals that enhance cancer cell survival, regulate stress responses and confer resistance toward pro-apoptotic stimuli, such as interferons. Here, we review the current knowledge on intracellular signal transduction pathways regulated by PD-L1, describe its associated signalosome and discuss potential combinations of targeted therapies against the signalosome with PD-L1/PD-1 blockade therapies.

Nanoparticles have been extensively developed for therapeutic and diagnostic applications. While the focus of nanoparticle trafficking in vivo has traditionally been on drug delivery and organ-level biodistribution and clearance, recent work in cancer biology and infectious disease suggests that targeting different cells within a given organ can substantially affect the quality of the immunological response. Here, we examine the cell-level biodistribution kinetics after administering ultrasmall Pluronic-stabilized poly(propylene sulfide) nanoparticles in the mouse. These nanoparticles depend on lymphatic drainage to reach the lymph nodes and blood, and then enter the spleen rather than the liver, where they interact with monocytes, macrophages and myeloid dendritic cells. They were more readily taken up into lymphatics after intradermal (i.d.) compared to intramuscular administration, leading to ,50% increased bioavailability in blood. When administered i.d., their distribution favored antigen-presenting cells, with especially strong targeting to myeloid cells. In tumor-bearing mice, the monocytic and the polymorphonuclear myeloid-derived suppressor cell compartments were efficiently and preferentially targeted, rendering this nanoparticulate formulation potentially useful for reversing the highly suppressive activity of these cells in the tumor stroma.

A decrease in the expression of HLA antigens is considered a characteristic of tumor progression and is considered an important tumor-escape mechanism. In general, HLA Class I expression is even further decreased on metastases. Tumor cells that loose their HLA Class I antigens become less susceptible to lysis by specific T cells, but may become more sensitive to Natural Killer cells. Loss of HLA Class I can be observed at different levels, i.e. total loss of Class I, loss of expression of one locus or one haplotype, or even one specific allele. We studied HLA expression on human uveal melanoma and observed that loss of expression of a locus or one or more alleles is a common phenomenon. However, in contrast with the commonly accepted paradigm, loss of HLA Class I expression on the uveal melanoma was not associated with tumor cell escape and a worse survival, but with a better survival of the patients involved. We hypothesize that this is due to the route of metastases formation: in uveal melanoma, spreading of metastases is purely hematogeneous, and it is quite possible that NK-cell mediated surveillance of tumor cells in the blood is the underlying mechanism. This is supported by our finding that metastases of uveal melanoma have a high HLA Class I expression, leading to our conclusion that uveal melanoma is an exception to the general rule regarding HLA Class I expression in tumor immunology. Human Immunology 63, 444-451 (2002).

Two main kinds of immune strategy are possible against neoplasia. The first potentiates a selected effector arm. In vitro culture with exogenous interleukin-2 (IL-2) increases the activity of natural killer cells and leads to the expansion ofT cytotoxic lymphocytes. Systemic reinfusion of both of these cells with high doses of IL-2 mediates the regression of a variety of murine and human tumors. In an alternative strategy, a few regulatory lymphocytes turn on immune reactivity by triggering a cascade of interconnected effector functions. The efficacy of this strategy rests on the repertoire of effector mechanisms moved to action. An effective immunoregulatory maneuver is the addition of helper determinants on the surface of tumor cells. Its power can be further increased by the pre-induction of helper T lymphocytes specific to the helper determinants. This approach can be achieved in mice by coupling muramyl dipeptides to tumor cells, along with eliciting T lymphocytes specifically reactive to Bacillus Calmette-Guerin. Noncytotoxic T helper lymphocytes produce factors which recruit nonspecific (macrophages) as well as specific (cytolytic T lymphocytes) anti-tumor attacking cells. In this way protection can be afforded against primary tumors and metastases, as well as leukemia cells. As the activity of helper lymphocytes rests mostly on lymphokine release, the use of molecularly defined lymphokines mimicking T-helper functions has also been attempted. In a few experimental models, the association of low doses of IL-2 with non-reactive lymphocytes from tumor-bearing mice promotes an effective anti-tumor reaction in the host. Moreover, the combination of distinct lymphokines can also build a molecularly defined helper system able to activate in sequence non-specific and specific anti-tumor reactions in vivo. Trials intended to evaluate the clinical impact of these helper approaches in the management of human tumors are being started or are already under way.

Colorectal cancer (CRC) diagnosis often occurs at late stages when tumor cells have already disseminated. Current therapies are poorly effective for metastatic disease, the main cause of death in CRC. Despite mounting evidence implicating the tumor microenvironment in CRC progression and metastasis, clinical practice remains predominantly focused on targeting the epithelial compartment. Because CRCs remain largely refractory to current therapies, we must devise alternative strategies. Transforming growth factor (TGF)-b has emerged as a key architect of the microenvironment in poor-prognosis cancers. Disseminated tumor cells show a strong dependency on a TGF-b-activated stroma during the establishment and subsequent expansion of metastasis. We review and discuss the development of integrated approaches focused on targeting the ecosystem of poor-prognosis CRCs.

Glucocorticoid-induced TNF-related protein (GITR) has been shown to stimulate T cell-mediated antitumor immunity in mice. However, the functional relevance of GITR and its ligand (GITRL) for non-T cells has yet to be fully explored. In addition, recent evidence suggests that GITR plays different roles in mice and humans. We studied the role of GITR-GITRL interaction in human tumor immunology and report for the first time that primary gastrointestinal cancers and tumor cell lines of different histological origin express substantial levels of GITRL. Signaling through GITRL down-regulated the expression of the immunostimulatory molecules CD40 and CD54 and the adhesion molecule EpCAM, and induced production of the immunosuppressive cytokine TGF-␤ by tumor cells. On NK cells, GITR is constitutively expressed and up-regulated following activation. Blocking