Target Discovery and Validation Reviews and Protocols

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Overview of Immune System 299 (44). Th cells can be further subdivided functionally by the pattern of cytokines they produce (46). Th1 cells produce IL-2 and interferon (IFN)γ, whereas Th2 cells produce IL-4, -5, and -6. Th1-type response plays a major role in cellular immunity, whereas Th2-type response is critical for cellular immunity. In mice, IgG2 B-cell response is associated with a Th1-like immune response and IgG1 with a Th2-like response (45). Human type 1 immunity is usually associated with IgG1 and IgG3 subclasses, and type 2 immunity is associated with the IgG4 isotype and IgE (47). There is one other Th subset, Th3, that secrets mainly the cytokine transforming growth factor (TGF)β, which can have suppressive activity on Th1 and Th2 cells (48,49). These T-cells may derive from either thymic Treg cells or peripheral T CD4+ Th cells under certain stimulation conditions (Fig. 7). CD8+ Tc cells are directly cytotoxic to cells bearing their specific antigen and are expected to play important role in tumor immunology through a process of cell-mediated cytotoxicity that involves Fas, perforin, or both. Perforinmediated lysis requires a cognate interaction between the antigen-specific TCR on a T-lymphocyte and the specific antigen (usually a peptide) presented on an MHC molecule on the target cell’s plasma membrane. Fas-mediated cytotoxicity involves the ligation of Fas on the target cell by Fas ligand on T-cells, but it does not require a cognate recognition interaction via the TCR. Cytotoxicity also may be signaled via the tumor necrosis factor (TNF) receptor on a target cell (8). It has been shown that the CD8+ T-cells also can be subdivided into IFNγproducing Tc1 cells and IL-4–producing Tc2 cells similar to the Th subsets. Innate effector cells, such as DCs, NK, natural killer T-cells (NKT), and γδ T-cells, produce several cytokines, including Th1 and Th2 cytokines. Thus, several immunoregulatory cells in addition to T-cells control the outcome of an immune response. A type 1 immunity, which is regulated by IL-12 and IFNγ, plays a crucial role for eradication of tumors in vivo. 10. Immune Response to Tumors Based upon the immunosurveillance model (1), the immune system can play a protective role in tumor development by recognizing and destroying nascent transformed cells. The first question for many of us was whether tumor-specific antigens exist, and, if so, whether there is immunological recognition of these antigens in humans. Studies in mice showed that the immune system can recognize and reject certain tumors and that immunodeficient mice, lacking INF-γ and RAG-2, have an increased incidence of cancer (50,51). In humans, the incidence of some cancers is increased in immunodeficient patients and is increased with age, owing to the immunosenescence. Evidence provided by autologous typing indicated the presence of patient-restricted antigens and shared antigens. Moreover, the presence of
300 Sioud tumor infiltrating lymphocyte supports the activation of these T-cells by tumor antigens (4,5). Indeed, specific homing of T-cells into tumor tissues compared with the surrounding normal tissues was found (5). The existence of immune response against tumors has now been validated in several studies (see Chapter 1–15, Volume 1). However, in contrast to the notion that the immune system should eliminate tumor cells, the developed responses failed to prevent tumor growth. According to the current view, the immune system also functions to select tumor variants with reduced immunogenicity capable of withstanding the tumor-suppressing activity of the immune response (52). This process, which is referred to as cancer “immunoediting,” is a dynamic process composed of at least three phases: elimination, equilibrium, and escape. To become clinically detectable, cancer cells must circumvent both innate and adaptive immunity. Thus, the host immunological environment selects tumors during tumor development and therefore leads to the generation of the selected tumors that survive with reduced immunogenicity. This heartening truth, which arises because most of the tumor cells are under constant selection pressure for survival, has been underlined by several recent studies (52). 11. Technical Advances in Tumor Antigen Discovery One aim of tumor immunology is to increase the antitumor immune response and to mediate killing of tumor cells by the host’s immune system, in particular, Tc cells (53). Given the prevalence of autoantigens on human cancers and the ability of tumors to induce tolerance to neoantigens acquired during transformation, tolerance is emerging a central obstacle for immune recognition of human cancer antigens. Because tumor cells do not express costimulatory molecules, antigens presented by these cells will induce tolerance despite being tumor specific. Thus, one of the main challenges of this field is to identity tumor antigens capable of inducing protective antitumor immunity as well as to uncover the cellular and molecular mechanisms that lead to the strange behavior of the immune system toward tumor cells. To identify tumor antigens, here referred as tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs), several techniques have been explored. 11.1. Autologous Typing Autologous typing was an early approach of determining whether patients with cancer recognize tumor-specific antigens on the surface of their cancer cells (54). Sera from patients with cancer were tested for reactivity with surface antigens of cultured autologous cancer cells and normal cells. Positive reactivity simply signifies that the antigens being detected on the target cells. Because of the potential presence of alloantibodies directed against alloantigens not present on other autologous cell types, extensive absorption steps are needed to
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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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182 Houdebine Fig.7. Example of a v
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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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206 Vijayaraj, Söhl, and Magin Fig
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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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214 Vijayaraj, Söhl, and Magin ulc
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216 Vijayaraj, Söhl, and Magin of
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218 Vijayaraj, Söhl, and Magin sug
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220 Vijayaraj, Söhl, and Magin abs
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222 Vijayaraj, Söhl, and Magin 1.2
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224 Vijayaraj, Söhl, and Magin Fig
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226 Vijayaraj, Söhl, and Magin gen
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230 Vijayaraj, Söhl, and Magin f.
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234 Vijayaraj, Söhl, and Magin b.
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240 Vijayaraj, Söhl, and Magin alt
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242 Table 3 Time Schedule For Aggre
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Page 332 and 333: 15 Potential Target Antigens for Im
Page 334 and 335: Tumor Antigen Discovery 321 develop
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Page 338 and 339: Tumor Antigen Discovery 325 16. Joc
Page 340 and 341: 16 Identification of Tumor Antigens
Page 342 and 343: Immunoproteomics 329 by “shot gun
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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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