Target Discovery and Validation Reviews and Protocols

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14 An Overview of the Immune System and Technical Advances in Tumor Antigen Discovery and Validation Mouldy Sioud Summary The ability of the immune system to distinguish between self- and nonself antigens is controlled by mechanisms of central and peripheral tolerance. Although the induction and maintenance of tolerance is important for preventing autoimmunity, breaking self-tolerance is a crucial constituent for combating cancers. Cancer patients are able to develop spontaneous immune responses to tumors that they bear, however these responses are not suboptimal for eradicating tumors. Moreover, none of the current immune strategies is able to activate the immune system to respond against tumor cells as it responds against infectious agents. These observations have raised the question of how to activate immunity in cancer patients to a threshold required for tumor rejection. Because tolerance is emerging as a central obstacle for immune recognition of human tumor antigens, this chapter describes how T- and B-cells are generated and activated in the periphery. It also outlines the technical advances in tumor antigen discovery and validation. Key Words: B-cell development; B-cell receptor; dendritic cells; genomics; germinal center; immune tolerance; immunoediting; phage display; proteomics; serological analysis of recombinant tumor cDNA expression libraries (SEREX); T-cell development; T-cell receptor; tumor antigens. 1. Introduction The primary function of the immune system is to defend the body against pathogenic microorganisms. The self–nonself (SNS) discrimination model assumes that the “foreign-ness” of a particular entity is what triggers the activation of innate and adaptive responses (1). Given the vast number of genetic and epigenetic changes associated with carcinogenesis, it becomes evident that tumors express many neoantigens that should be recognized by the immune system. In this respect, antibodies against a large repertoire of tumor-associated From: Methods in Molecular Biology, vol. 360, Target Discovery and Validation Reviews and Protocols Volume I, Emerging Strategies for Targets and Biomarker Discovery Edited by: M. Sioud © Humana Press Inc., Totowa, NJ 277
278 Sioud gene products have been described in cancer patients (2,3). Moreover, we and others have shown that a high number of T-cells are present within tumors and that these cells are specifically recruited to tumor beds (4,5). In addition, T-cells reactive with tumor antigens are present in the peripheral blood and lymph nodes of diverse patients. Collectively, these results indicate that the immune system can recognize cancer cells. However, despite their potential immunogenicity, tumor antigens do not induce significant immune responses, which could destroy malignant cells; therefore, a central question is whether recognition of tumor antigens by immune cells would mainly lead to tolerance. The different mechanisms for recognition and elimination of pathogens have been divided in the immune system into “innate immunity,” which encompasses the presumably more primitive elements of the system, including macrophages, natural killer (NK), and antigen-presenting cells, and “adaptive immunity,” which encompasses T- and B-lymphocytes. The activation of innate immunity is mainly mediated by the recognition of potential pathogens by Toll-like receptors (TLRs) that are mainly expressed on immune cells such as macrophages, dendritic cells (DCs), and monocytes (6,7). These receptors function as sensors of infection and induce the activation of innate and adaptive immune responses. Upon recognizing conserved structures, which are referred to as pathogen-associated molecular patterns (PAMPs), TLRs activate host defense responses through their intracellular signaling domain, the Toll/interleukin-1 receptor (TIR) domain, and the downstream adaptor protein MyD88. Although members of the TLRs all signal through MyD88, the signaling pathways induced by individual receptors differ (7). So far, 10 TLRs have been described in humans, and ligands have been defined for nine of these TLRs. TLR1 and -2 are activated by triacylated lipoproteins, TLR3 by double-stranded viral RNA, TLR4 by lipopolysaccharide (LPS), TLR5 by bacterial flagellin, TLR2 and -6 by diacylated lipopeptides, TLR7 and -8 by imidazoquinolines and single-stranded RNA, and TRL9 by unmethylated bacterial CpG DNA sequences (7). Upon activation, TLRs trigger the release of cytokines and the induction of co-stimulatory molecules that can influence the nature of the adaptive T- and or B-cell responses. Activation of TLRs also induces antimicrobial pathways that kill intracellular organisms (6–8). Although the innate response is rapid, the response sometimes damages normal tissues, leading to severe diseases, such a sepsis. Adaptive immunity is the hallmark of the immune system in higher animals. It uses antigen-specific receptors expressed on T- and B-cells. The receptors on B-lymphocytes are Igs, which can be secreted by corresponding plasma cells after antigenic stimulation. Bound and secreted Igs can bind to a variety of soluble as well as cell-bound antigens, such as virus proteins. In contrast to B-cells, T-cells recognize only processed antigens that are presented as peptides by major histocompatibility complex (MHC) class I or II. Despite this
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METHODS IN MOLECULAR BIOLOGY 360 T
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M E T H O D S I N M O L E C U L A R
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© 2007 Humana Press Inc. 999 River
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vi Preface get. Approaches to targe
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viii Contents 10 Transgenic Animal
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x Contributors SAHOHIME MATSUMOTO
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xii Contents of Volume 2 12 Validat
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2 Sioud disease pathogenesis and pe
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4 Sioud A more fruitful approach fo
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6 Sioud both transient and permanen
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8 Sioud serum biomarkers. This syne
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10 Sioud Fig. 2. Schematic of targe
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12 Sioud 13. Magin, T. M. (1998) Le
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Harnessing the Human Genome 15 The
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Harnessing the Human Genome 17 Fig.
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Harnessing the Human Genome 23 The
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Harnessing the Human Genome 27 repr
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Harnessing the Human Genome 29 14.
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Harnessing the Human Genome 31 45.
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34 Imoto et al. (4,5), and Bayesian
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36 Imoto et al. 2. Materials Two ty
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38 Imoto et al. Fig. 3. Graphical v
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40 Imoto et al. Fig. 5. Result of t
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42 Imoto et al. p(D |G) = ∫ p(D,
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44 Imoto et al. β k (k = 1,…,q j
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46 Imoto et al. Fig. 7. Partial res
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48 Imoto et al. Fig. 8. Strategy to
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50 Imoto et al. Fig. 9. (continued)
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52 Imoto et al. Table 3 List of Roo
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54 Imoto et al. 5. De Hoon, M. J. L
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56 Imoto et al. 39. Kamimura, T., S
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58 Beaty et al. cytotoxic drugs and
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60 Beaty et al. Fig. 1. Principles
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62 Beaty et al. oligonucleotide mic
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64 Beaty et al. 3. The 12% polyacry
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66 Beaty et al. 2.3.3. Protein Stai
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68 Beaty et al. 3.1.3. Labeling of
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70 Beaty et al. 1 µL of the anneal
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72 Beaty et al. 4. Phenol:cholorfor
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74 Beaty et al. 19. Wash twice with
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76 Beaty et al. The Following Volum
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78 Beaty et al. using a protein ass
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80 Beaty et al. 6. Shake the gel fo
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82 Beaty et al. Fig. 2. Preparation
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84 Beaty et al. search in. A widely
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86 Beaty et al. 3. Kern, S. E., Hru
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88 Beaty et al. 34. Peters, D. G.,
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5 Molecular Classification of Breas
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Molecular Profiling of Breast Cance
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Fig. 2. (Continued) the right ident
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6 Discovery of Differentially Expre
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Gene Target Discovery 117 In the fi
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Gene Target Discovery 119 sequences
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Gene Target Discovery 121 There is
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Gene Target Discovery 123 digestion
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Gene Target Discovery 125 Fig. 1. P
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Gene Target Discovery 127 5. Sargen
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Gene Target Discovery 129 41. Berry
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132 Matsumoto, Miyagishi, and Taira
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144 Fig. 1. The ribozyme-expression
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146 Sano and Taira 5. 10X L (low-sa
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148 Sano and Taira ribozymes may cl
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150 Fig. 3. A system for the identi
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152 Sano and Taira 4. Notes 1. We r
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9 Production of siRNA- and cDNA-Tra
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Transfected Cell Arrays 157 Fig. 1.
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Transfected Cell Arrays 159 2. The
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Transfected Cell Arrays 161 5. Simp
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164 Houdebine Fig. 1. Different met
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166 Houdebine concentrations become
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168 Houdebine Fig. 2. Major methods
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170 Houdebine integrate into the me
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172 Houdebine 2.2.3. Knock-In Into
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174 Houdebine as insulators. The co
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176 Houdebine Fig. 5. Different met
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178 Houdebine systems. Moreover, st
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180 Houdebine contribute to generat
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184 Houdebine more extensively. Tra
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186 Houdebine and stroma. Several o
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188 Houdebine explained at the mole
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190 Houdebine of the intestinal epi
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192 Houdebine interference of the g
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194 Houdebine 17. Whitelaw, C. B. A
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196 Houdebine 51. Bell, A. C., West
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198 Houdebine 86. Carmichael, G. G.
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200 Houdebine 121. Pailhoux, E., Vi
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202 Houdebine 156. Auerbach, A. B.,
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204 Vijayaraj, Söhl, and Magin 1.
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208 Vijayaraj, Söhl, and Magin fil
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210 Table 1 Orthologous Human and M
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212 Table 2 Orthologous Human and M
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222 Vijayaraj, Söhl, and Magin 1.2
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Page 332 and 333: 15 Potential Target Antigens for Im
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16 Identification of Tumor Antigens
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Immunoproteomics 329 by “shot gun
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Immunoproteomics 331 isoelectric fo
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Immunoproteomics 333 3. 2D-PAGE is
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17 Protein Arrays A Versatile Toolb
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Protein Arrays 337 Table 1 Classifi
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Protein Arrays 339 fractions from c
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Protein Arrays 341 combinatorial sc
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Protein Arrays 343 The ideal proteo
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Protein Arrays 345 18. Cekaite, H.
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Protein Arrays 347 49. Yuko Kawahas
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Index 349 Index A Acetyl-CoA carbox
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Index 351 conditional by using FRT
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Index 353 Recombinant tumor antigen
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