Tumor Immunology

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Tumor Immunology
immunity and lack of tumor growth in the same host if larger doses of the same cells are administered. Autochthonous is an adjective that indicates “pertaining to self,” occuring in the same subject. Also called autologous. Spontaneous remission is the reversal of progressive growth of the neoplasm with inadequate or no treatment. Spontaneous remission occurs only rarely. A benign tumor is an abnormal proliferation of cells that leads to a growth that is localized and contained within epithelial barriers. It does not usually lead to death, in contrast to a malignant tumor. Malignant is an adjective that means leading to death, as by a malignant neoplasm. Metastasis is the transfer of disease from one organ or part to another not directly connected with it. For example, malignant tumors may need anatomical sites distant from the primary tumor’s site of origin, leading to the establishment of secondary tumors. Malignolipin (historical) is a substance claimed in the past to be specific for cancer and to be detectable in the patient’s blood early in the course of the disease. This is no longer considered valid. Malignolipin is comprised of fatty acids, phosphoric acid choline, and spermine. When injected into experimental animals, it can produce profound anemia, leukopoiesis, and cachexia. Oncogenes are genes with the capacity to induce neoplastic transformation of cells. They are derived from either normal genes termed protooncogenes or from oncogenic RNA (oncorna) viruses. Their protein products are critical for regulation of gene expression or growth signal transduction. Translocation, gene amplification, and point mutation may lead to neoplastic transformation of protooncogene. Oncogenes may be revealed through use of viruses that induce tumors in animals or by derivation of tumorcausing genes from cancer cells. There are more than 20 protooncogenes and cellular oncogenes in the human genome. An oncogene alone cannot produce cancer. It must be accompanied by malignant transformation which involves multiple genetic steps. Oncogenes encode four types of proteins that include growth factors, receptors, intracellular transducers, and nuclear transcription factors.

Biologists have long been fascinated with possible differences between neoplastic cells (Figure 22.1 to Figure 22.3) and their so-called normal counterparts or tissues of origin. This led to the search for antigens on tumors that are absent from normal tissues. The aim of finding such immunologic differences would be both for cancer testing and for cancer treatment purposes. This search has met with varying degrees of success. A neoplasm is any new and abnormal growth that may be either a benign or malignant tumor. Cancer is an invasive, metastatic, and highly anaplastic cellular tumor that leads to death. Neoplasms are often divided into two broad categories of carcinoma and sarcoma. Modification of proteins by phosphorylation or specific proteolysis may change their covalent architecture to yield new antigenic determinants or epitopes termed neoantigens. The epitope is newly expressed on cells during development or in neoplasia. Neoantigens include tumorassociated antigens. New antigenic determinants may also emerge when a protein changes conformation or when a molecule is split, exposing previously unexpressed epitopes. A carcinogen is any chemical or physical cancer-producing agent. Carcinogens comprise the epigenetic type that does not damage DNA but causes other physiological alterations that predispose to cancer, and the genotoxic type that reacts directly with DNA or with micromolecules that then react with DNA. A carcinoma is a malignant tumor composed of epithelial cells that infiltrate surrounding tissues and lead to metastases. A choriocarcinoma is an unusual malignant neoplasm of the placenta trophoblast cells in which the fetal neoplastic cells are allogeneic in the host. On rare occasions, these neoplasms have been “rejected” spontaneously by the host. Antimetabolites have been used in the treatment of choriocarcinoma. Sarcomas are tumors arising from connective tissue. “Sneaking through” is the successful growth of a sparse number of transplantable tumor cells that have been inoculated into a host in contrast to the induction of tumor

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FIGURE 22.1 Schematic representation of a tumor cell attached through E-selectin molecules to an endothelial cell surface.

FIGURE 22.3 Schematic representation of a melanoma cell attached to an endothelial cell surface through a VLA-4-VCAM-1 interaction.

Oncomouse is a commercially developed transgenic animal into which human genes have been introduced to make the mouse more susceptible to neoplasia. This transgenic mouse is used for both medical and pharmaceutical research. A protooncogene is a cellular gene that shows homology with a retroviral oncogene. It is found in normal mammalian DNA and governs normal proliferation and probably also differentiation of cells. Mutation or recombination with a viral genome may convert a protooncogene into an oncogene, signifying that it has become activated. Oncogenes may act in the induction and/or maintenance of a neoplasm. Protooncogenes united with control elements may induce transformation of normal fibroblasts into tumor cells. Examples of protooncogenes are c-fos, c-myc, c-myb, c-ras, etc. Alteration of the protooncogenes, leading to synthesis of an aberrant gene product, is believed to facilitate its becoming tumorigenic. An elevation in the quantity of gene product produced is also believed to be associated with protooncogenes becoming tumorigenic. Cellular oncogene: See protooncogene. Ras is one of a group of 21-kDa guanine nucleotidebinding proteins with intrinsic GTPase activity that participates in numerous different signal transduction pathways in a variety of cells. Ras gene mutations may be associated with tumor transformation. Ras is attracted to the plasma membrane by tyrosine phosphorylated adapter proteins during T lymphocyte activation, where GDP–GTP exchange factors are activated. GTP-Ras then activates the

FIGURE 22.2 Schematic representation of a leukemia cell attached to an endothelial cell surface via adhesion molecules.

The oncogene theory is a concept of carcinogenesis that assigns tumor development to latent retroviral gene activation through irradiation or carcinogens. These retroviral genes are considered to be normal constituents of the cell. Following activation, these oncogenes are presumed to govern the neoplasm through hormones that are synthesized and even the possible construction of a complete oncogenic virus. This concept states that all cells may potentially become malignant. Oncogenesis is the process whereby tumors develop.

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MAP kinase cascade that results in fos gene expression and assembly of AP-1 trascription factor. Ras: See small G proteins. Rous sarcoma virus (RSV) is an RNA type C oncovirus that is single stranded and produces sarcomas in chickens. It is the typical acute transforming retrovirus. Within its genome are gag, pol, env, and v-src genes; gag encodes a core protein, pol encodes reverse transcriptase, and env encodes envelope glycoprotein. V-scr is an oncogene associated with the oncogenic capacity of the virus. A promoter is (1) the DNA molecular site where RNA polymerase attaches and the point at which transcription is initiated. The promoter is frequently situated adjacent to the operator, and upstream from it is an operon. A TATA box and a promoter are both required for immunoglobulin gene transcription. (2) In tumor biology, a promoter mediates the second stage or promotion stage in the process of carcinogenesis. It may be a substance that can induce a tumor in an experimental animal that has been previously exposed to a tumor initiator. Yet the promoter alone is not carcinogenic. A v-myb oncogene is a genetic component of an acute transforming retrovirus that leads to avian myeloblastosis. It represents a truncated genetic form of c-myb. Tumor promoter: See phorbol ester(s). Amphiregulin is a glycoprotein member of the epidermal growth factor (EGF) family of proteins. The carboxylterminal amino acid residues of amphiregulin positions 46–84 share much sequence homology with the EGF family of proteins. The actions of amphiregulin are wide ranging, including the stimulation of proliferation of certain tumor cell lines, fibroblasts, and various other normal cells. These actions are mediated by binding to EGF receptors possessing intrinsic tyrosine kinase activity. Tumor imaging is an experimental and clinical medical technique employed to localize neoplastic lesions in a body using a labeled antibody or its fragment. Tumor imaging is based on the presence of an antigen expressed only on a tumor cell or at least has a significant difference in amount and/or distribution between the tumor and normal tissues. Tumor-associated antigens (Figure 22.4) are antigens designated as CA-125, CA-19-9, and CA195, among others, that may be linked to certain tumors such as lymphomas, carcinomas, sarcomas, and melanomas, but the immune response to these tumor-associated antigens is not sufficient to mount a successful cellular or humoral immune response against the neoplasm. Three classes of tumor-associated antigens have been described. Class I

FIGURE 22.4 Schematic representation of tumor-associated antigens (TAA) among normal and neoplastic cells.

antigens are very specific for a certain neoplasm and are absent from normal cells. Class II antigens are found on related neoplasms from separate individuals. Class III antigens are found on malignant as well as normal cells, but show increased expression in the neoplastic cells. Assays of clinical value will probably be developed for Class II antigens, since they are associated with multiple neoplasms and very infrequently found in normal individuals. Tumor antigens are cell surface proteins on tumor cells that can induce a cell-mediated and/or humoral immune response. See also tumor-associated antigens and tumorspecific antigens. Thymicleukemia antigen (TL) is an epitope on thymocyte membrane of TL+ mice. As the T lymphocytes mature, antigen disappears but resurfaces if leukemia develops. TL antigens are specific and are normally present on the cell surface of thymocytes of certain mouse strains. They are encoded by a group of structural genes located at Tla locus, in the linkage group IX, very close to the D pole of the H-2 locus on chromosome 17. There are three structural Tl genes, one of which has two alleles. The TL antigens are numbered from 1 to 4 specifying four antigens: TL.1, TL.2, TL.3, and TL.4. Antigens TL.3 and TL.4 are mutually exclusive. Their expression is under the control of regulatory genes, apparently located at the same Tla locus. Normal mouse thymocytes belong to three phenotypic groups: Tl−, Tl.2, and TL.1, 2, 3. Development of leukemia in the mouse induces a restructuring of the TL surface antigens of thymocytes with expression of TL.1 and TL.2 in TL− cells, expression of TL.1 in TL.2 cells, and expression of TL.4 in both TL− and TL.2 cells. When normal thymic cells leave the thymus, the expression of TL antigen ceases. Thus, thymocytes are TL+ (except the TL−) and the peripheral T cells are TL−. In transplantation experiments TL+ tumor cells undergo antigenic modulation.

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Tumor cells exposed to homologous antibody stop expressing the antigen and thus escape lysis when subsequently exposed to the same antibody plus complement. CD10 (CALLA) is an antigen, also referred to as common acute lymphoblastic leukemia antigen (CALLA), that has a mol wt of 100 kDa. CD10 is now known to be a neutral endopeptidase (enkephalinase). It is present on many cell types, including stem cells, lymphoid progenitors of B and T cells, renal epithelium, fibroblasts, and bile canaliculi. Prostate-specific antigen (PSA) is a marker in serum or tissue sections for adenocarcinoma of the prostate. PSA is a 34-kDa glycoprotein found exclusively in benign and malignant epithelium of the prostate. Men with PSA levels of 0 to 4.0 ng/ml and a nonsuspicious digital rectal examination are generally not biopsied for prostate cancer. Men with PSA levels of 10.0 ng/ml and above typically undergo a prostate biopsy. About one half of these men will be found to have prostate cancer. Certain kinds of PSA, known as bound PSA, link themselves to other proteins in the blood. Other kinds of PSA, known as free PSA, float by themselves. Prostate cancer is more likely to be present in men who have a low percentage of free PSA relative to the total amount of PSA. This finding is especially valuable in helping to differentiate between cancer and other benign conditions, thus eliminating unnecessary biopsies among men in that diagnostic gray zone who have total PSA levels between 4.0 and 10.0 ng/ml. The PSA molecule is smaller than prostatic acid phosphatase (PAP). In patients with prostate cancer, preoperative PSA serum levels are positively correlated with the disease. PSA is more stable and shows less diurnal variation than does PAP. PSA is increased in 95% of new cases for prostatic carcinoma compared with a 60% increase for PAP; PSA is increased in 97% of recurrent cases compared with a 66% increase of PAP. PAP may also be increased in selected cases of benign prostatic hypertrophy and prostatitis, but these elevations are less than those associated with adenocarcinoma of the prostate. It is inappropriate to use either PSA or PAP alone as a screen for asymptomatic males. TUR, urethral instrumentation, prostatic needle biopsy, prostatic infarct, or urinary retention may also result in increased PSA values. PSA is critical for the prediction of recurrent adenocarcinoma in postsurgical patients. PSA is also a useful immunocytochemical marker for primary and metastatic adenocarcinoma of the prostate. Oncofetal antigens (Figure 22.5) are markers or epitopes present in fetal tissues during development but not present, or found in minute quantities, in adult tissues. These cellcoded antigens may reappear in certain neoplasms of adults due to derepression of the gene responsible for their formation. Examples include carcinoembryonic antigen (CEA), which is found in the liver, intestine, and pancreas

FIGURE 22.5 Oncofetal antigen.

of the fetus, and also in both malignant and benign gastrointestinal conditions. Yet it is useful to detect recurrence of adenocarcinoma of the colon based upon demonstration of CEA in the patient’s serum; α-fetoprotein (AFP) is demonstrable in approximately 70% of hepatocellular carcinomas. A fetal or oncofetal antigen that is expressed as a normal constituent of embryos and not in adult tissues. It is reexpressed in neoplasms of adult tissues, apparently as a result of derepression of the gene responsible for its formation. Carcinoma-associated antigens are self antigens whose epitopes have been changed due to effects produced by certain tumors. Self antigens are transformed into a molecular structure for which the host is immunologically intolerant. Examples include the T antigen, which is an MN blood group precursor molecule exposed by the action of bacterial enzymes, and Tn antigen, which is a consequence of somatic mutation in hematopoietic stem cells caused by inhibition of galactose transfer to N-acetyl-D-galactosamine. Embryonic antigens are protein or carbohydrate antigens synthesized during embryonic and fetal life that are either absent or formed in only minute quantities in normal adult subjects. Fetoproteins (AFP) and carcinoembryonic antigen (CEA) are fetal antigens that may be synthesized once again in large amounts in individuals with certain tumors. Their detection and level during the course of the disease and following surgery to remove a tumor reducing the substance may serve as a diagnostic and prognostic indicator of the disease process. Blood group antigens, such as the iI, which are reversed in their levels of expression in the fetus and in the adult, may show a reemergence of i antigen in adult patients with thalassemia and hypoplastic anemia. Cold autoagglutinins specific for it may be found in infectious mononucleosis patients. Common acute lymphoblastic leukemia antigen (CD10) is rarely found on peripheral blood cells of normal subjects, whereas CALLA cells coexpressing IgM and CD19 molecules may be found in fetal bone marrow and peripheral blood samples. CD10 may be expressed in children with common acute lymphoblastic leukemia.

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complemented by morphological studies and evaluation of proper controls. CEA is the abbreviation for carcinoembryonic antigen. SV40 (simian virus 40) (Figure 22.7 and Figure 22.8) is an oncogenic polyoma virus. It multiplies in cultures of rhesus monkey kidney and produces cytopathic alterations in African green monkey cell cultures. Inoculation into newborn hamsters leads to the development of sarcomas. SV40 has 5243 base pairs in its genome. It may follow either of two patterns of life cycle according to the host cell. In permissive cells, such as those from African green monkeys, the virus-infected cells are lysed, causing the escape of multiple viral particles. Lysis does not occur in nonpermissive cells infected with the virus. By contrast, they may undergo oncogenic transformation in which SV40 DNA sequences become integrated into the genome of the host cell. Cells that have become transformed have characteristic morphological features and growth properties. SV40 may serve as a cloning vector. It is a diminutive icosahedral papovavirus that contains double-stranded DNA. It may induce progressive multifocal leukoencephalopathy. SV40 is useful for the in vitro transformation of cells as a type of “permissive” infection ultimately resulting in lysis of infected host cells. Oncogenic virus (Figure 22.9) is any virus, whether DNA or RNA, that can induce malignant transformation of cells. An example of a DNA virus would be human papillomavirus, and an RNA virus would be retrovirus. α-fetoprotein (Figure 22.10) is a principal plasma protein in the α globulin fraction present in the fetus. It bears considerable homology with human serum albumin. It is produced by the embryonic yolk sac and fetal liver and consists of a 590-amino acid residue polypeptide chain structure. It may be elevated in pregnant women bearing fetuses with open neural tube defects, central nervous system defects, gastrointestinal abnormalities, immunodeficiency syndromes, and various other abnormalities. After parturition, the high levels in fetal serum diminish to levels that cannot be detected. α-fetoprotein induces immunosuppression, which may facilitate neonatal tolerance. Based on in vitro studies, it is believed to facilitate suppressor T lymphocyte function and diminish helper T lymphocyte action. Liver cancer patients reveal significantly elevated serum levels of α-fetoprotein. In immunology, however, it is used as a marker of selected tumors such as hepatocellular carcinoma. It is detected by the avidin–biotin–peroxidase complex (ABC) immunoperoxidase technique using monoclonal antibodies. The melanoma antigen-1 gene (MAGE-1) in humans was derived from a malignant melanoma cell line. It encodes for an epitope that a cytotoxic T lymphocyte clone

FIGURE 22.6 Carcinoembryonic antigen (CEA).

Carcinoembryonic antigen (CEA) (Figure 22.6) is a 200-kDa membrane glycoprotein epitope that is present in the fetal gastrointestinal tract in normal conditions. However, tumor cells, such as those in colon carcinoma, may reexpress it. CEA was first described as a screen for identifying carcinoma by detecting nanogram quantities of the antigen in serum. It was later shown to be present in certain other conditions as well. CEA levels are elevated in almost one third of patients with colorectal, liver, pancreatic, lung, breast, head and neck, cervical, bladder, medullarythyroid, and prostatic carcinoma. However, the level may be elevated also in malignant melanoma, lymphoproliferative disease, and smokers. Regrettably, CEA levels also increase in a variety of nonneoplastic disorders, including inflammatory bowel disease, pancreatitis, and cirrhosis of the liver. Nevertheless, determination of CEA levels in the serum is valuable for monitoring the recurrence of tumors in patients whose primary neoplasm has been removed. If the patient’s CEA level reveals a 35% elevation compared with the level immediately following surgery, this may signify metastases. This oncofetal antigen is comprised of one polypeptide chain with one variable region at the amino terminus and six constant region domains. CEA belongs to the immunoglobulin superfamily. It lacks specificity for cancer, thereby limiting its diagnostic usefulness. It is detected with a mouse monoclonal antibody directed against a complex glycoprotein antigen present on many human epithelial-derived tumors. This reagent may be used to aid in the identification of cells of epithelial lineage. The antibody is intended for qualitative staining in sections of formalin-fixed, paraffin-embedded tissue. Anti-CEA antibodies specifically bind to antigens located in the plasma membrane and cytoplasmic regions of normal epithelial cells. Unexpected antigen expression or loss of expression may occur, especially in neoplasms. Occasionally, stromal elements surrounding heavily stained tissue and/or cells show immunoreactivity. Clinical interpretation of any staining or its absence must be

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FIGURE 22.7 SV-40.

FIGURE 22.9 Oncogenic virus.

breast carcinomas, but is not expressed on the majority of normal tissues. Even though MAGE-1 has not been shown to induce tumor rejection, cytotoxic T lymphocytes in melanoma patients manifest specific memory for MAGE1 protein.
FIGURE 22.8 SV-40. Resolution 3.1 Å.

specific for melanoma recognizes. This clone was isolated from a patient bearing melanoma. MAGE-1 protein is found on one half of all melanomas and one fourth of all

Melanoma-associated antigens (MAA) are antigens associated with the aggressive, malignant, and metastatic tumors arising from melanocytes or melanocyte-associated nevus cells. Monoclonal antibodies have identified 40+ separate MAAs. They are classified as MHC molecules,

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FIGURE 22.10 α-fetoprotein.

FIGURE 22.11 Tumor-specific antigens (TSA).

cation-binding proteins, growth-factor receptors, gangliosides, high molecular weight extracellular matrix-binding molecules, and nevomelanocyte differentiation antigens. Some of the antigens are expressed on normal cells, whereas others are expressed on tumor cells. Melanoma patient blood sera often contain anti-MAA antibodies which are, regrettably, not protective. Monoclonal antibodies against MAAs aid studies on the biology of tumor progression, immunodiagnosis, and immunotherapy trials. Modulation: See antigenic modulation. Tumor cells may be subject to alterations in antigenic structure. Antigenic transformation refers to changes in a cell’s antigenic profile as a consequence of antigenic gain, deletion, reversion, or other process. Antigenic gain refers to nondistinctive normal tissue components that are added or increased without simultaneous deletion of other normal tissue constituents. Antigenic deletion describes antigenic determinants that have been lost or masked in the progeny of cells that usually contain them. Antigenic deletion may take place as a consequence of neoplastic transformation or mutation of parent cells resulting in the disappearance or repression of the parent cell genes. Antigenic modulation is the loss of epitopes or antigenic determinants from a cell surface following combination with an antibody. The antibodies either cause the epitope to disappear or become camouflaged by covering it. Antigenic diversion refers to the replacement of a cell’s antigenic profile by the antigens of a different normal tissue cell. Used in tumor immunology, antigenic reversion is the change in antigenic profile characteristic of an adult cell to an antigenic mosaic that previously existed in the immature or fetal cell stage of the species. Antigenic reversion may accompany neoplastic transformation. Tumor cells express tumor-specific determinants or epitopes present on tumor cells but identifiable also in varying quantities and forms on normal cells. Tumor-specific antigen (TSA) (Figure 22.11) are present on tumor cells,

FIGURE 22.12 Tumor-specific transplantation antigens (TSTA).

but not found on normal cells. Murine tumor-specific antigens can induce transplantation rejection in mice. Tumorspecific transplantation antigen (TSTA) (Figure 22.12) are epitopes that induce rejection of tumors transplanted among syngeneic (histocompatible) animals. Tumor rejection antigen is an antigen that is detectable when transplanted tumor cells are rejected. Also called tumor transplant antigen. TATA is the abbreviation for tumor-associated transplantation antigen. Macrophages (Figure 22.13 and Figure 22.14) are mononuclear phagocytic cells derived from monocytes in the blood that were produced from stem cells in the bone marrow. These cells have a powerful although nonspecific role in immune defense. These intensely phagocytic cells contain lysosomes and exert microbicidal action against microbes which they ingest. They also have effective tumoricidal activity. They may take up and degrade both protein and polysaccharide antigens and present them to T lymphocytes in the context of MHC class II molecules.

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FIGURE 22.13 Macrophage-mediated tumor cell lysis is mediated by several mechanisms. Activated macrophages express Fcγ receptors that anchor IgG molecules attached to tumor cells but not normal cells, resulting in the release of lysosomal enzymes and reactive oxygen metabolites that lead to tumor cell lysis. Another mechanism of macrophage-mediated lysis includes the release of the cytokine tumor necrosis factor α that may unite with high affinity TNFα receptors on a tumor cell surface resulting in its lysis, or the effect of TNFα on the small blood vessels and capillaries of vascularized tumors leading to hemorrhagic necrosis producing a localized Shwartzman-like reaction.

FIGURE 22.14 Macrophage-mediated tumor immunity.

They interact with both T and B lymphocytes in immune reactions. They are frequently found in areas of epithelium, mesothelium, and blood vessels. Macrophages have been referred to as adherent cells since they readily adhere to glass and plastic and may spread on these surfaces and manifest chemotaxis. They have receptors for Fc and C3b on their surfaces, stain positively for nonspecific esterase and peroxidase, and are Ia antigen positive when acting as accessory cells that present antigen to CD4+ lymphocytes in the generation of an immune response. Monocytes, which may differentiate into macrophages when they migrate into the tissues, make up 3 to 5% of leukocytes in the peripheral blood. Macrophages that are tissuebound may be found in the lung alveoli, as microglial cells in the central nervous system, as Kupffer cells in the liver,

as Langerhans cells in the skin, as histiocytes in connective tissues, as well as macrophages in lymph nodes and peritoneum. Multiple substances are secreted by macrophages including complement components C1 through C5, factors B and D, properdin, C3b inactivators, and β-1H. They also produce monokines such as interleukin-1, acid hydrolase, proteases, lipases, and numerous other substances. Natural killer (NK) cells (Figure 22.15 and Figure 22.16) attack and destroy tumor cells and certain virus-infected cells. They constitute an important part of the natural immune system, do not require prior contract with antigen, and are not MHC restricted by the MHC antigens. NK cells are lymphoid cells of the natural immune system that express cytotoxicity against various nucleated cells,

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FIGURE 22.15 NK-cell-mediated killing of tumor cells by antibody-dependent cell-mediated cytotoxicity.

cytoplasmic granules. The cells are also called large granular lymphocytes (LGLs). In addition to their ability to kill selected tumor cells and some virus-infected cells, they also participate in ADCC by anchoring antibody to the cell surface through an Fc γ receptor. Thus, they are able to destroy antibody-coated nucleated cells. NK cells are believed to represent a significant part of the natural immune defense against spontaneously developing neoplastic cells and against infection by viruses. NK cell activity is measured by a 51Cr release assay employing the K562 erythroleukemia cell line as a target. NK cells secrete IFN-γ and fail to express antigen receptors such as immunoglobulin receptors or T-cell receptors. Cell-surface stimulatory receptors and inhibitory receptors, which recognize self-MHC molecules, regulate their activation. Designer lymphocytes are lymphocytes into which genes have been introduced to increase the cell’s ability to lyse tumor cells. Tumor-infiltrating lymphocytes transfected with these types of genes have been used in experimental adoptive immunotherapy. Cytotoxic T lymphocytes (CTLs) (Figure 22.17 and Figure 22.18) are specifically sensitized T lymphocytes that are usually CD8+ and recognize antigens through the T cell receptor on cells of the host infected by viruses or that have become neoplastic. CD8+ cell recognition of the target is in the context of MHC class I histocompatibility molecules. Following recognition and binding, death of the target cell occurs a few hours later. CTLs secrete lymphokines that attract other lymphocytes to the area and release serine proteases and perforins that produce ion channels in the membrane of the target, leading to cell lysis. Interleukin-2, produced by CD4+ T cells, activates cytotoxic T cell precursors. Interferon-γ generated from CTLs activates macrophages. CTLs have a significant role in the rejection of allografts and in tumor immunity. A minor population of CD4+ lymphocytes may also be cytotoxic, but they recognize targetcell antigens in the context of MHC class II molecules.

FIGURE 22.16 Proposed mechanism of NK cell cytotoxicity restricted to altered self cells. Kill signal is generated when the NK cell’s NKR-PI receptor interacts with membrane glycoprotein or normal and altered self cells. The kill signal can be countermanded by interaction of the NK cells Ly49 receptor with class I MHC molecules. Thus, MHC Class I expression prevents NK cell killing of normal cells. Diminished Class I expression on altered self cells leads to their destruction.

including tumor cells and virus-infected cells. NK cells, killer (K) cells, or antibody-dependent, cell-mediated cytotoxicity (ADCC) cells induce lysis through the action of antibody. Immunologic memory is not involved, as previous contact with the antigen is not necessary for NK cell activity. The NK cell is approximately 15 µm in diameter and has a kidney-shaped nucleus with several, often three, large

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FIGURE 22.17 Cytotoxic T lymphocyte (CTL)-mediated tumor lysis.

FIGURE 22.18 CTL-mediated killing of tumor cells.

between antibody molecules and immune system cells to produce an effector function. Antibody-directed enzyme prodrug therapy (ADEPT) is a type of treatment in which an antibody is used to target an enzyme to a tumor and unbound reagent is allowed to clear. A nontoxic prodrug is then given, and this is activated by the enzyme to form a cytotoxic drug at the tumor site. An important part of adept is bystander killing. Since the drugs are activated extracellularly by the antibody–enzyme complex, neighboring cells may also be killed by a mechanism that does not require translocation across intracellular membranes. By contrast, immunotoxins kill only the cell to which they bind. A heteroconjugate is a hybrid of two different antibody molecules. Heteroconjugate antibodies (Figure 22.20) are antibodies against a tumor antigen coupled covalently to an antibody specific for a natural killer cell or cytotoxic T lymphocyte surface antigen. These antibodies facilitate binding of cytotoxic effector cells to tumor target cells. Antibodies against effector cell surface markers may also be coupled covalently with hormones that bind to receptors on tumor cells.

FIGURE 22.19 Antibody-dependent cell-mediated cytotoxicity (ADCC).

Tumor-specific IgG antibodies may act in concert with immune system cells to produce antitumor effects. Antibody-dependent cell-mediated cytotoxicity (ADCC) (Figure 22.19) is a reaction in which T lymphocytes and NK cells, including large granular lymphocytes, neutrophils, and macrophages, may lyse tumor cells, infectious agents, and allogeneic cells by combining through their Fc receptors with the Fc region of IgG antibodies bound through their Fab regions to target cell surface antigens. Following linkage of Fc receptors with Fc regions, destruction of the target is accomplished through released cytokines. It represents an example of participation

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FIGURE 22.20 Heteroconjugate antibodies.

Immunosurveillance refers to the policing or monitoring function of immune system cells to recognize and destroy clones of transformed cells prior to their development into neoplasms and to destroy tumors after they develop. Immunosurveillance is believed to be mediated by the cellular limb of the immune response. Indirect evidence in support of the concept includes (1) an increased incidence of tumors in aged individuals who have decreased immune competence, (2) increased tumor incidence in children with T cell immunodeficiencies, and (3) the development of neoplasms (lymphomas) in a significant number of organ or bone marrow transplant recipients who have been deliberately immunosuppressed. Immunoselection is the selective survival of cells due to their diminished cell surface antigenicity. This permits these cells to escape the injurious effects of either antibodies or immune lymphoid cells. Immunological escape is a mechanism of escape in which tumors that are immunogenic continue to grow in immunocompetent syngeneic hosts in the presence of a modest in vivo antitumor immune response. Escape mechanisms may facilitate tumors in evading a fatal tumoricidal response and render them incapable of inducing such a response. Failure of tumor antigen presentation by MHC class I molecules, and lack of costimulation and downregulation of tumor-destructive immune responses by tumor antigens, immune complexes, and molecules, and such as TGF-β and P15E are all believed to contribute to the inefficiency of tumor immunity. Immunologic enhancement (tumor enhancement) (Figure 22.21) describes the prolonged survival, conversely the delayed rejection, of a tumor allograft in a host as a consequence of contact with a specific antibody. Antitumor antibodies may have a paradoxical effect. Instead of eradicating a neoplasm, they may facilitate its survival and progressive growth in the host. Both the peripheral and central mechanisms have been postulated. Coating of tumor cells with antibody was presumed, in the past, to interfere with the ability of specifically reactive lymphocytes to destroy them, but today a central effect in suppressing

FIGURE 22.21 Immunologic enhancement (tumor enhancement).

cell-mediated immunity, perhaps through suppressor T lymphocytes, is also possible. Enhancing antibodies are blocking antibodies that favor survival of tumor or normal tissue allografts. Immunologic facilitation (facilitation immunologique) is the slightly prolonged survival of certain normal tissue allografts, e.g., skin, in mice conditioned with isoantiserum specific for the graft. Immunotherapy employs immunologic mechanisms to combat disease. These include nonspecific stimulation of the immune response with BCG immunotherapy in treating certain types of cancer, and the IL-2/LAK cell adoptive immunotherapy technique for treating selected tumors. Biological response modifiers (BRM) are a wide spectrum of molecules, such as cytokines, that alter the immune response. They include substances such as interleukins, interferons, hematopoietic colony-stimulating factors, tumor necrosis factor, B lymphocyte growth and differentiating factors, lymphotoxins, and macrophageactivating and chemotactic factors, as well as macrophageinhibitory, eosinophils chemotactic, and osteoclast-activating factors, etc. BRM may modulate the immune system of the host to augment antirecombinant DNA technology and are available commercially. An example is α interferon used in the therapy of hairy cell leukemia. α Interferon α (IFN-α) is an immunomodulatory 189amino acid residue glycoproteins synthesized by macrophages and B cells that are able to prevent the replication of viruses, are antiproliferative, and are pyrogenic, inducing fever. IFN-α stimulates natural killer cells and induces expression of class I MHC antigens. It also has an immunoregulatory effect through alteration of antibody responsiveness. The 14 genes that encode IFN-α are positioned on the short arm of chromosome 9 in man. Polyribonucleotides, as well as RNA or DNA viruses, may induce

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IFN-α secretion. Recombinant IFN-α has been prepared and used in the treatment of hairy cell leukemia, Kaposi’s sarcoma, chronic myeloid leukemia, human papilloma virus-related lesions, renal cell carcinoma, chronic hepatitis, and other selected conditions. Patients may experience severe flu-like symptoms as long as the drug is administered. They also have malaise, headache, depression, supraventricular tachycardia, and may possibly develop congestive heart failure. Bone marrow suppression has been reported in some patients. Immunoscintigraphy (Figure 22.22 and Figure 22.23) is the formation of two-dimensional images of the distribution of radioactivity in tissues following the administration of antibodies labeled with a radionuclide that are specific for tissue antigens. A scintillation camera is used to record the images. Immunolymphoscintigraphy is a method used to determine the presence of tumor metastasis to lymph nodes. Antibody fragments or monoclonal antibodies against specific tumor antigens are radiolabeled and then detected by scintigraphy. Radioimmunoscintigraphy is the use of radiolabeled antibodies to localize tumors or other lesions through use of radioactivity scanning following injection in vivo.
FIGURE 22.23 Immunoscintigraphy (nude mouse) with a 131I-labeled monoclonal antibody. The mouse shown bears a human colon carcinoma in its left flank. The scintigrams were recorded 2, 5, and 7 days postinjection. While the second picture shows mainly the blood pool and little of the tumor, the tumor is the major imaged spot in the body after 5 days; after 7 days, only the tumor is recognizable.

FIGURE 22.24 Immunotoxin.

Immunolymphoscintigraphy is a method to determine the presence of tumor metastasis to lymph nodes. Antibody fragments or monoclonal antibodies against specific tumor antigens are radiolabeled and then detected by scintigraphy. An immunotoxin (Figure 22.24) is produced by linking an antibody specific for target cell antigens with a cytotoxic substance such as the toxin ricin. Upon parenteral injection, its antibody portion directs the immunotoxin to the target and its toxic portion destroys target cells on contact. An immunotoxin may also be a monoclonal antibody or one of its fractions linked to a toxic molecule

FIGURE 22.22 Immunoscintigraphy.

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such as a radioisotope, a bacterial or plant toxin, or a chemotherapeutic agent. The antibody portion is intended to direct the molecule to antigens on a target cell such as those of a malignant tumor and the toxic portion of the molecule is for the purpose of destroying the target cell. Contemporary methods of recombinant DNA technology have permitted the preparation of specific hybrid molecules for use in immunotoxin therapy. Immunotoxins may have difficulty reaching the intended target tumor, may be quickly metabolized, and may stimulate the development of antiimmunotoxin antibodies. Crosslinking proteins may likewise be unstable. Among the uses of immunotoxin is the purging of T cells from hematopoietic cell preparations used for bone marrow transplantation. Immunotoxins have potential for antitumor therapy and as immunosuppressive agents. Ricin is a toxic protein found in seeds of Ricinus communis (castor bean) plants. It is a heterodimer comprised of a 30-kDa α chain, which mediates cytotoxicity, and a 30kDa β chain, which interacts with cell surface galactose residues that facilitate passage of molecules into cells in endocytic vesicles. Ricin inhibits protein synthesis by linkage of a dissociated α chain in the cytosol to ribosomes. The ricin heterodimer or its α chain conjugated to a specific antibody serves as an immunotoxin. Ricinus communis: See ricin. Abrin (Figure 22.25) and ricin are examples of immunotoxins. Abrin is a powerful toxin and lectin used in immunological research by Paul Ehrlich (circa 1900). It is extracted from the seeds of the jequirity plant and causes agglutination of erythrocytes. Magic bullet is a term coined by Paul Ehrlich in 1900 to describe what he considered to be the affinity of a drug

for a particular target. He developed “606” (salvarsan), an arsenical preparation, to treat syphilis. In immunology, it describes a substance that could be directed to a target by a specific antibody and injure the target once it arrives. Monoclonal antibodies have been linked to toxins such as diphtheria toxin, or ricin, as well as to cytokines for use as magic bullets. Adoptive immunotherapy (Figure 22.26 and Figure 22.27) is the experimental treatment of terminal cancer patients with metastatic tumors unresponsive to other modes of therapy by the inoculation of lymphokine-activated killer (LAK) cells or tumor-infiltrating lymphocytes (TIL) together with IL-2. This mode of therapy has shown some success in approximately one tenth of treated individuals with melanoma or renal cell carcinoma. Lymphokine-activated killer (LAK) cells are lymphoid cells derived from normal or tumor patients cultured in medium with recombinant IL-2, which become capable of lysing NK-resistant tumor cells as revealed by 51Crrelease cytotoxicity assays. These cells are also referred to as lymphokine-activated killer cells. Most LAK activity is derived from NK cells. The large granular lymphocytes (LGL) contain all LAK precursor activity and all active NK cells. In accord with the phenotype of precursor cells, LAK effector cells are also granular lymphocytes expressing markers associated with human NK cells. The asialo Gm1+ population, known to be expressed by murine NK cells, contains most LAK precursor activity. Essentially all LAK activity resides in the LGL population in the rat. LAK cell and IL-2 immunotherapy has been employed in human cancer patients with a variety of histological tumor types when conventional therapy has been unsuccessful. Approximately one fourth of LAK- and IL-2-treated patients manifested significant responses, and some individuals experienced complete remission. Serious side effects include fluid retention and pulmonary edema attributable to the administered IL-2. LAK cells are lymphokine-activated killer cells. IL-2/LAK cells are interleukin-2/lymphokine-activated killer cells. NK cells, which express only the p70 and not the p55 receptor for IL-2, are incubated with IL-2, converting them into an activated form referred to as LAK cells. The IL-2/LAK combination has been used to treat cancer patients through adoptive immunotherapy, which has been successful in inducing transient regression of tumors in selected cases of melanoma, colorectal carcinoma, non-Hodgkin’s lymphoma, and renal cell carcinoma, as well as regression of metastases in the liver and lung of some patients. There may be transient defective chemotaxis of neutrophils, and patients often develop “capillary leak syndrome,” producing pulmonary edema. Patients may also develop congestive heart failure.

FIGURE 22.25 Abrin – A.

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FIGURE 22.26 Interleukin-2 (IL-2) immunotherapy.

Tumor immunity: Numerous experimentally induced tumors in mice express numerous specific transplantation antigens which can induce an immune response that leads to destruction of neoplastic cells in vivo. Lymphocytes play a critical role in the immunological destruction of many antigenic tumors. Both cell-mediated and antibodymediated immune responses to human neoplasms have been identified and their targets characterized in an effort to develop clinically useful immunotherapy. Tumor-infiltrating lymphocytes (TIL) are lymphocytes isolated from the tumor they are infiltrating. They are cultured with high concentrations of IL-2, leading to expansion of these activated T lymphocytes in vitro. TILs are very effective in destroying tumor cells and have proven much more effective than LAK cells in experimental models. TILs have 50 to 100 times the antitumor activity produced by LAK cells. TILs have been isolated and grown from multiple resected human tumors, including those from kidney, breast, colon, and melanoma. In contrast to the non-B-non-T LAK cells, TILs nevertheless are generated from T lymphocytes and phenotypically resemble cytotoxic T lymphocytes. TILs from malignant mela-

noma exhibit specific cytolytic activity against cells of the tumor from which they were extracted, whereas LAK cells have a broad range of specificity. TILs appear unable to lyse cells of melanomas from patients other than those in whom the tumor originated. TILs may be tagged in order that they may be identified later. TIL is the abbreviation for tumor-infiltrating lymphocytes. Reverse immunology is a process that involves computerized algorithms to predict the likelihood of a particular mutation resulting in a strong antigen. Several of the mutant proteins and peptides have been used to examine the possibility of inducing tumor-specific immunity. Hybrid resistance is the resistance of members of an F1 generation of animals to growth of a transplantable neoplasm from either one of the parent strains. Concomitant immunity is resistance to a tumor that has been transplanted into a host already bearing that tumor. Immunity to the reinoculated neoplasm does not inhibit growth of the primary tumor.

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FIGURE 22.27 Interleukin-12 (IL-12) immunotherapy.

CA-15-3 is an antibody specific for an antigen frequently present in the blood serum of metastatic breast carcinoma patients. CA-19-9 is a tumor-associated antigen found on the Lewis A blood group antigen that is sialylated or in mucincontaining tissues. In individuals whose serum levels exceed 37 U/ml, 72% have carcinoma of the pancreas. In individuals whose levels exceed 1000 U/ml, 95% have pancreatic cancer. Anti-CA-19-9 monoclonal antibody is useful to detect the recurrence of pancreatic cancer following surgery and to distinguish between neoplastic and benign conditions of the pancreas. However, it is not useful for pancreatic cancer screening. CA-125 is a mucinous ovarian carcinoma cell surface glycoprotein detectable in the patient’s blood serum. Increasing serum concentrations portend a grave prognosis. It may also be found in the blood sera of patients with other adenocarcinomas such as breast, gastrointestinal tract, uterine cervix, and endometrium. CALLA is common acute lymphoblastic leukemia antigen. Also known as CD10.

Calcitonin is a hormone that influences calcium ion transport. Immunoperoxidase staining demonstrates calcitonin in thyroid parafollicular or C cells. It serves as a marker characteristic of medullary thyroid carcinoma and APUD neoplasms. Lung and gastrointestinal tumors may also form calcitonin. Blocking factors are agents such as immune complexes in the serum of tumor-bearing hosts that interfere with the capacity of immune lymphoid cells to mediate cytotoxicity of tumor target cells. Antimalignin antibodies are specific for the 10-kDa protein malignin comprised of 89 amino acids. These antibodies are claimed to be increased in cancer patients without respect to tumor cell type. It has been further claimed that antibody levels are related to survival. These claims will require additional confirmation and proof to be accepted as fact. The Winn assay is a method to determine the ability of lymphoid cells to inhibit the growth of transplantable tumors in vivo. Following incubation of the lymphoid cells and tumor cells in vitro, the mixture is injected into the

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FIGURE 22.28 Tumor necrosis factor (TNF)-mediated immune reaction.

skin of X-irradiated mice. Growth of the transplanted cells is followed. T lymphocytes that are specifically immune to the tumor cells will inhibit tumor growth and provide information related to tumor immunity. α Tumor necrosis factor α (TNF-α) (Figure 22.28 and Figure 22.29) is a cytotoxic monokine produced by macrophages stimulated with bacterial endotoxin. TNF-α participates in inflammation, wound healing, and remodeling of tissue. TNF-α, which is also called cachectin, can induce septic shock and cachexia. It is a cytokine comprised of 157 amino acid residues. It is produced by numerous types of cells including monocytes, macrophages, T lymphocytes, B lymphocytes, NK cells, and other types of cells stimulated by endotoxin or other microbial products. The genes encoding TNF-α and TNF-β (lymphotoxin) are located on the short arm of chromosome 6 in man in the MHC region. High levels of TNF-α are detectable in the blood circulation very soon following administration of endotoxins or microorganisms. The administration of recombinant TNF-α induces shock, organ failure, and hemorrhagic necrosis of tissues in experimental animals including rodents, dogs, sheep, and rabbits, closely resembling the effects of lethal endotoxemia. TNF-α is produced during the

first 3 d of wound healing. It facilitates leukocyte recruitment, induces angiogenesis, and promotes fibroblast proliferation. It can combine with receptors on selected tumor cells and induce their lysis. TNF mediates the antitumor action of murine natural cytotoxic (NC) cells, which distinguishes their function from that of NK and cytotoxic T cells. TNF-α was termed “cachectin” because of its ability to induce wasting and anemia when administered on a chronic basis to experimental animals. Thus it mimics the action in cancer patients and in those with chronic infection with HIV or other pathogenic microorganisms. It can induce anorexia which may lead to death from malnutrition. Tumor necrosis factor-β (TNF-β) is a 25-kDa protein synthesized by activated lymphocytes. It can kill tumor cells in culture, induce expression of genes, stimulate proliferation of fibroblasts, and can mimic most of the actions of TNF-α (cachectin). It participates in inflammation and graft rejection and was previously termed “lymphotoxin.” TNF-β and TNF-α have approximately equivalent affinity for TNF receptors. Both 55-kDa and 80-kDa TNF receptors bind TNF-β. TNF-β has diverse effects that include killing of some cells and causing proliferation of others. It is the mediator whereby cytolytic T cells, natural killer

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FIGURE 22.29 Tumor necrosis immunotherapy.

cells, lymphokine-activated killer cells, and “helperkiller” T cells induce fatal injury to their targets. TNF-β and TNF-α have been suggested to play a role in AIDS, possibly contributing to its pathogenesis.

Tumor necrosis factor receptor is a receptor for tumor necrosis factor that is comprised of 461 amino acid residues and which possesses an extracellular domain that is rich in cysteine.

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