《免疫学》（英文版） Chapter 23 Experimental Systems

ART TK Experimental chapter 23 Systems XPERIMENTAL SYSTEMS OF VARIOUS TYPES ARE ART TK used to unravel the complex cellular interactions of the immune response In vivo systems, which involve the whole animal, provide the most natural experi mental conditions. However, in vivo systems have a myriad of unknown and uncontrollable cellular interactions that add ambiguity to the interpretation of data. At the other extreme are in vitro systems, in which defined populations of lymphocytes are studied under controlled and conse- quently repeatable conditions; in vitro systems can be sim- Addition of Expression Profile of Diffuse Large B-cell Lymphoma plified to the extent that individual cellular interactions can be studied effectively. Yet they have their own limitations, the Experimental Animal Model most notable of which is their artificiality. For example, pro- iding antigen to purified B cells in vitro does not stimulate a Cell-Culture Systems maximal antibody production unless T cells are present. a Protein Biochemistry Therefore a study of antibody production in an artificial in vitro system that lacks T cells could lead to the incorrect con- a Recombinant DNA Technology clusion that B cells do not synthesize high levels of antibod- Analysis of DNA Regulatory Sequene ies. One must ask whether a cellular response observed in vitro reflects reality or is a product of the unique conditions Gene Transfer into Mammalian Cells of the in vitro system itself. a Microarrays-An Approach for Analyzing Patterns This chapter describes some of the experimental systen of Gene Expression outinely used to study the immune system. It also covers some recombinant DNA techniques that have revolution- ized the study of the immune system in the past decade orso Other chapters also cover experimental systems and tech ues in detail. Table 23-1 lists them and directs the reader to the appropriate location for a description are genetically well characterized, and have a rapid breeding cycle. The immune system of the mouse has been character ized more extensively than that of any other species. The Experimental Animal Models value of basic research in the mouse system is highlighted the enormous impact this research has had on clinical inter The study of the immune system in vertebrates requires suit- vention in human disease able els. The choice of an animal depends on suitability for attaining a particular research goal. If large Inbred Strains Can Reduce Experimental amounts of antiserum are sought, a rabbit, goat, sheep, or Variation horse might be an appropriate experimental animal. If the goal is development of a protective vaccine, the animal cho- To control experimental variation caused by differences in sen must be susceptible to the infectious agent so that the the genetic backgrounds of experimental animals, immu efficacy of the vaccine can be assessed Mice or rabbits can be nologists often work with inbred strains-that is, genetically used for vaccine development if they are susceptible to the identical animals produced by inbreeding. The rapid breed pathogen. But if growth of the infectious agent is limited to ing cycle of mice makes them particularly well suited for the humans and primates, vaccine development may require the production of inbred strains, which are developed by re use of monkeys, chimpanzees, or baboons peated inbreeding between brother and sister littermates. In For most basic research in immunology, mice have been this way the heterozygosity of alleles that is normally found he experimental animal of choice. They are easy to handle, in randomly outbred mice is replaced by homozygosity at all

■ Experimental Animal Models ■ Cell-Culture Systems ■ Protein Biochemistry ■ Recombinant DNA Technology ■ Analysis of DNA Regulatory Sequences ■ Gene Transfer into Mammalian Cells ■ Microarrays—An Approach for Analyzing Patterns of Gene Expression Addition of Expression Profile of Diffuse Large B-cell Lymphoma. Experimental Systems       used to unravel the complex cellular interactions of the immune response. In vivo systems, which involve the whole animal, provide the most natural experi￾mental conditions. However, in vivo systems have a myriad of unknown and uncontrollable cellular interactions that add ambiguity to the interpretation of data. At the other extreme are in vitro systems, in which defined populations of lymphocytes are studied under controlled and conse￾quently repeatable conditions; in vitro systems can be sim￾plified to the extent that individual cellular interactions can be studied effectively. Yet they have their own limitations, the most notable of which is their artificiality. For example, pro￾viding antigen to purified B cells in vitro does not stimulate maximal antibody production unless T cells are present. Therefore a study of antibody production in an artificial in vitro system that lacks T cells could lead to the incorrect con￾clusion that B cells do not synthesize high levels of antibod￾ies. One must ask whether a cellular response observed in vitro reflects reality or is a product of the unique conditions of the in vitro system itself. This chapter describes some of the experimental systems routinely used to study the immune system. It also covers some recombinant DNA techniques that have revolution￾ized the study of the immune system in the past decade or so. Other chapters also cover experimental systems and tech￾niques in detail. Table 23-1 lists them and directs the reader to the appropriate location for a description. Experimental Animal Models The study of the immune system in vertebrates requires suit￾able animal models. The choice of an animal depends on its suitability for attaining a particular research goal. If large amounts of antiserum are sought, a rabbit, goat, sheep, or horse might be an appropriate experimental animal. If the goal is development of a protective vaccine, the animal cho￾sen must be susceptible to the infectious agent so that the efficacy of the vaccine can be assessed. Mice or rabbits can be used for vaccine development if they are susceptible to the pathogen. But if growth of the infectious agent is limited to humans and primates, vaccine development may require the use of monkeys, chimpanzees, or baboons. For most basic research in immunology, mice have been the experimental animal of choice. They are easy to handle, are genetically well characterized, and have a rapid breeding cycle. The immune system of the mouse has been character￾ized more extensively than that of any other species. The value of basic research in the mouse system is highlighted by the enormous impact this research has had on clinical inter￾vention in human disease. Inbred Strains Can Reduce Experimental Variation To control experimental variation caused by differences in the genetic backgrounds of experimental animals, immu￾nologists often work with inbred strains—that is, genetically identical animals produced by inbreeding. The rapid breed￾ing cycle of mice makes them particularly well suited for the production of inbred strains, which are developed by re￾peated inbreeding between brother and sister littermates. In this way the heterozygosity of alleles that is normally found in randomly outbred mice is replaced by homozygosity at all chapter 23 ART TK E ART TK

526 PART IV The Immune System in Health and Disease Adoptive- Transfer Systems Permit the in Vivo TABLE 23-1 described in other chapters Examination of Isolated Cell Populations Method ocation In some experiments, it is important to eliminate the im mune responsiveness of the syngeneic host so that the re- Bone-marrow transplantation Ch. 2 Clinical Focus sponse of only the transferred lymphocytes can be studied in Preparation of immunotoxins Fig.4-22 isolation. This can be accomplished by a technique called Genetic engineering of Fig 5-20 and adoptive transfer: first, the syngeneic host is exposed to dimeric mouse-human ch 5 Clinical Focus X-rays that kill its lymphocytes then the donor immune cells monoclonal antibodies are introduced. Subjecting a mouse to high doses of x-ray Determination of antibody affinity Fig 6.2 (650-750 rads) can kill 99.99% of its lymphocytes, after by equilibrium dialysis which the activities of lymphocytes transplanted from th Precipitation reactions Fig spleen of a syngeneic donor can be studied without int mmunodiffusion and Figs. 6.5 and 6.6 ference from host lymphocytes. If the host s hematopoietic immunoelectrophoresis ells might influence an adoptive-transfer experiment, then Fig. 6.7 higher x-ray levels(900-1000 rads)are used to eliminate the Radioimmunoassay(RIA) Fig 6.9 entire hematopoietic system. Mice irradiated with such doses ELISA assays Fg.610 will die unless reconstituted with bone marrow from a syn ELISPOT assay Fig 6.11 geneic donor Fig.6.12 The adoptive-transfer system has enabled immunologists Immunoprecipitation Fig.6.13 to study the development of injected lymphoid stem cells in Fig.6.14 various organs of the recipient, and have facilitated the study Flo Fg.615 of various populations of lymphocytes and of the cellular in Production of congenic mice teractions required to generate an immune response. Such ex periments, for instance, first enabled immunologists to show Mixed lymphocyte reaction Fig.14-16 that a T helper cell is necessary for B-cell activation in the Cell-mediated lympholysis humoral response. In these experiments, adoptive transfer of Fig.14-17 purified B cells or purified T cells did not produce antibody in Production of vaccinia vector the irradiated host. Only when both cell populations were Fig 18-5 transferred was antibody produced in response to antigen. Production of multivalent Fg.187 subunit vaccines SCID Mice and scld-Human mice HLA typing Fig.21-4 Are a valuable animal model for Immunodeficiency An autosomal recessive mutation resulting in severe com bined immunodeficiency disease(SCID)developed sponta neously in a strain of mice called CB-17. These CB-17 SCID loci. Repeated inbreeding for 20 generations usually yields an mice fail to develop mature T and B cells and consequently inbred strain whose progeny are homozygous at more than are severely compromised immunologically. This defect is 98%of all loci. More than 150 different inbred strains of due to a failure in V(D)J recombination SCID mice must be mice are available, each designated by a series of letters and/ housed in a sterile(germ-free)environment, because they or numbers(Table 23-2). Most strains can be purchased by cannot fight off microorganisms of even low pathogenicity immunologists from such suppliers as the Jackson Labora- The absence of functional T and B cells enables these mice to tory in Bar Harbor, Maine. Inbred strains have also been pro- accept foreign cells and grafts from other strains of mice c duced in rats, guinea pigs, hamsters, rabbits, and domestic even from other species fowl Because inbred strains of animals are genetically identi- Apart from their lack of functional T and B cells, SCID mice cal(syngeneic) within that strain, their immune responses appear to be normal in all respects. When normal bone can be studied in the absence of variables introduced by indi- marrow cells are injected into SCID mice, normal T and B cells vidual genetic differences-an invaluable property. With develop, and the mice are cured of their immunodeficiency. inbred strains, lymphocyte subpopulations isolated from one This finding has made SCID mice a valuable model system for animal can be injected into another animal of the same strain the study of immunodeficiency and the process of differen without eliciting a rejection reaction. This type of experi- tion of bone-marrow stem cells into mature T or Bcells. mental system permitted immunologists to first demonstrate Interest in SCiD mice mushroomed when it was found that lymphocytes from an antigen-primed animal could trans- that they could be used to study the human immune system. fer immunity to an unprimed syngeneic recipient In this system, portions of human fetal liver, adult thymus

loci. Repeated inbreeding for 20 generations usually yields an inbred strain whose progeny are homozygous at more than 98% of all loci. More than 150 different inbred strains of mice are available, each designated by a series of letters and/ or numbers (Table 23-2). Most strains can be purchased by immunologists from such suppliers as the Jackson Labora￾tory in Bar Harbor, Maine. Inbred strains have also been pro￾duced in rats, guinea pigs, hamsters, rabbits, and domestic fowl. Because inbred strains of animals are genetically identi￾cal (syngeneic) within that strain, their immune responses can be studied in the absence of variables introduced by indi￾vidual genetic differences—an invaluable property. With inbred strains, lymphocyte subpopulations isolated from one animal can be injected into another animal of the same strain without eliciting a rejection reaction. This type of experi￾mental system permitted immunologists to first demonstrate that lymphocytes from an antigen-primed animal could trans￾fer immunity to an unprimed syngeneic recipient. Adoptive-Transfer Systems Permit the in Vivo Examination of Isolated Cell Populations In some experiments, it is important to eliminate the im￾mune responsiveness of the syngeneic host so that the re￾sponse of only the transferred lymphocytes can be studied in isolation. This can be accomplished by a technique called adoptive transfer: first, the syngeneic host is exposed to x-rays that kill its lymphocytes; then the donor immune cells are introduced. Subjecting a mouse to high doses of x-rays (650–750 rads) can kill 99.99% of its lymphocytes, after which the activities of lymphocytes transplanted from the spleen of a syngeneic donor can be studied without inter￾ference from host lymphocytes. If the host’s hematopoietic cells might influence an adoptive-transfer experiment, then higher x-ray levels (900–1000 rads) are used to eliminate the entire hematopoietic system. Mice irradiated with such doses will die unless reconstituted with bone marrow from a syn￾geneic donor. The adoptive-transfer system has enabled immunologists to study the development of injected lymphoid stem cells in various organs of the recipient, and have facilitated the study of various populations of lymphocytes and of the cellular in￾teractions required to generate an immune response. Such ex￾periments, for instance, first enabled immunologists to show that a T helper cell is necessary for B-cell activation in the humoral response. In these experiments, adoptive transfer of purified B cells or purified T cells did not produce antibody in the irradiated host. Only when both cell populations were transferred was antibody produced in response to antigen. SCID Mice and SCID-Human Mice Are a Valuable Animal Model for Immunodeficiency An autosomal recessive mutation resulting in severe com￾bined immunodeficiency disease (SCID) developed sponta￾neously in a strain of mice called CB-17. These CB-17 SCID mice fail to develop mature T and B cells and consequently are severely compromised immunologically. This defect is due to a failure in V(D)J recombination. SCID mice must be housed in a sterile (germ-free) environment, because they cannot fight off microorganisms of even low pathogenicity. The absence of functional T and B cells enables these mice to accept foreign cells and grafts from other strains of mice or even from other species. Apart from their lack of functional T and B cells, SCID mice appear to be normal in all respects. When normal bone￾marrowcells are injected into SCID mice, normal T and B cells develop, and the mice are cured of their immunodeficiency. This finding has made SCID mice a valuable model system for the study of immunodeficiency and the process of differentia￾tion of bone-marrow stem cells into mature T or B cells. Interest in SCID mice mushroomed when it was found that they could be used to study the human immune system. In this system, portions of human fetal liver, adult thymus, 526 PART IV The Immune System in Health and Disease TABLE 23-1 Immunological methods described in other chapters Method Location Bone-marrow transplantation Ch. 2 Clinical Focus Preparation of immunotoxins Fig. 4-22 Genetic engineering of Fig. 5-20 and chimeric mouse-human Ch 5 Clinical Focus monoclonal antibodies Determination of antibody affinity Fig. 6.2 by equilibrium dialysis Precipitation reactions Fig. 6.4 Immunodiffusion and Figs. 6.5 and 6.6 immunoelectrophoresis Hemagglutination Fig. 6.7 Radioimmunoassay (RIA) Fig. 6.9 ELISA assays Fig. 6.10 ELISPOT assay Fig. 6.11 Western blotting Fig. 6.12 Immunoprecipitation Fig. 6.13 Immunofluorescence Fig. 6.14 Flow cytometry Fig. 6.15 Production of congenic mice Fig. 7-3 Mixed lymphocyte reaction (MLR) Fig. 14-16 Cell-mediated lympholysis (CML) Fig. 14-17 Production of vaccinia vector vaccine Fig. 18-5 Production of multivalent Fig. 18-7 subunit vaccines HLA typing Fig. 21-4

Experimental Systems CHAPTER 23 527 TABLE 23-2 Some inbred mouse strains commonly used in immunology Common substrains Characteristics A/He High incidence of mammary tumors in some substrains High incidence of leukemia AKR/Cum Thy 1. 2 allele in AKR/Cum, and Thy 1. 1 allele in other substrains(Thy gene encodes BALB/c BALB/cj Sensitivity to radiation BALB/C AnN Used in hybridoma technology BALB/CBy Many myeloma cell lines were generated in these mic CBA/ Gene(rd) causing retinal degeneration in CBA/ Gene (xid)causing X-linked immunodeficiency in CBA/N C3H/He Gene(rd) causing retinal degeneration C3H/He」 High incidence of mammary tumors in many substrains(these carry a C3H/HeN mammary-tumor virus that is passed via maternal milk to offspring C57BL/6J High incidence of hepatomas after irradiation C57BL/6B High complement activity C57BL/10 Very close relationship to C57BL/6 but differences in at least two loci 57BL/10ScSn Frequent partner in preparation of congenic mice C57BR 57BR/cdj High frequency of pituitary and liver tumors Very resistant to x-irradiation C57L/J usceptibility to experimental autoimmune encephalomyelitis(EAE) C57L/N High frequency of pituitary and reticular cell tumors C58 C58/ High incidence of leukemia C5B/LWN DBA1」 High incidence of mammary tumors DBA/IN DBA/ DBA/2J Low immune response to some antigens DBA/2N Low response to pneumococcal polysaccharide type Hairless(hr) gene, usually in heterozygous state immune hemolytic anemia and lupus-like nephi Autoimmune disease similar to systemic lupus erythematosus(SLE) in F, progeny from crosses with NZW NZW/N SLE-type autoimmune disease in Fi progeny from crosses with NZB High incidence of leukemia High level of aggression and severe fighting to the point of death, especially in males ndency to develop certain autoimmune diseases, most susceptible to EAE Tendency to develop several autoimmune diseases, especially EAE 129/Sy SOURCE: Adapted from Federation of American Societies for Eperimental Biology, 1979, Biological Handbooks, VoL. llL: Inbred and Genetically Defined Strains of Laboratory Animals. and adult lymph nodes are implanted into SCID mice(Fig- where they mature into T and B cells, producing a SCID ure 23-1).Because the mice lack mature T and B cells of their human mouse. Because the human lymphocytes are exposed own, they do not reject the transplanted human tissue. The to mouse antigens while they are still immature, they later implanted human fetal liver contains immature lymphocytes recognize mouse cells as self and do not mount an immuno- stem cells), which migrate to the implanted human tissues, logic response against the mouse host

and adult lymph nodes are implanted into SCID mice (Fig￾ure 23-1). Because the mice lack mature T and B cells of their own, they do not reject the transplanted human tissue. The implanted human fetal liver contains immature lymphocytes (stem cells), which migrate to the implanted human tissues, where they mature into T and B cells, producing a SCID￾human mouse. Because the human lymphocytes are exposed to mouse antigens while they are still immature, they later recognize mouse cells as self and do not mount an immuno￾logic response against the mouse host. Experimental Systems CHAPTER 23 527 TABLE 23-2 Some inbred mouse strains commonly used in immunology Strain Common substrains Characteristics A A/He High incidence of mammary tumors in some substrains A/J A/WySn AKR AKR/J High incidence of leukemia AKR/N AKR/Cum Thy 1.2 allele in AKR/Cum, and Thy 1.1 allele in other substrains (Thy gene encodes a T-cell surface protein) BALB/c BALB/cj Sensitivity to radiation BALB/c AnN Used in hybridoma technology BALB/cBy Many myeloma cell lines were generated in these mice CBA CBA/J Gene (rd) causing retinal degeneration in CBA/J CBA/H CBA/N Gene (xid) causing X-linked immunodeficiency in CBA/N C3H C3H/He Gene (rd) causing retinal degeneration C3H/HeJ High incidence of mammary tumors in many substrains (these carry a C3H/HeN mammary-tumor virus that is passed via maternal milk to offspring) C57BL/6 C57BL/6J High incidence of hepatomas after irradiation C57BL/6By High complement activity C57BL/6N C57BL/10 C57BL/10J Very close relationship to C57BL/6 but differences in at least two loci C57BL/10ScSn C57BL/10N Frequent partner in preparation of congenic mice C57BR C57BR/cdj High frequency of pituitary and liver tumors Very resistant to x-irradiation C57L C57L/J Susceptibility to experimental autoimmune encephalomyelitis (EAE) C57L/N High frequency of pituitary and reticular cell tumors C58 C58/J High incidence of leukemia C58/LwN DBA/1 DBA/1J High incidence of mammary tumors DBA/1N DBA/2 DBA/2J Low immune response to some antigens DBA/2N Low response to pneumococcal polysaccharide type III HRS HRS/J Hairless (hr) gene, usually in heterozygous state NZB NZB/BINJ High incidence of autoimmune hemolytic anemia and lupus-like nephritis NZB/N Autoimmune disease similar to systemic lupus erythematosus (SLE) in F1 progeny from crosses with NZW NZW NZW/N SLE-type autoimmune disease in F1 progeny from crosses with NZB P P/J High incidence of leukemia SJL SJL/J High level of aggression and severe fighting to the point of death, especially in males Tendency to develop certain autoimmune diseases, most susceptible to EAE SWR SWR/J Tendency to develop several autoimmune diseases, especially EAE 129 129/J High incidence of spontaneous teratocarcinoma 129/SvJ SOURCE: Adapted from Federation of American Societies for Experimental Biology, 1979, Biological Handbooks, Vol. III: Inbred and Genetically Defined Strains of Laboratory Animals

528 PART IV The Immune System in Health and Disease then be grown in a chemically defined basal medium(on- SCID mouse taining saline, sugars, amino acids, vitamins, trace elements, and other nutrients) to which various serum supplements are added. For some experiments, serum-free culture condi tions are employed. Because in vitro culture techniques re o Transplant human thymus quire from 10-to 100-fold fewer lymphocytes than do typical and lymph-node tissue in vivo techniques, they have enabled immunologists to under kidney capsule the functional properties of I lymphocytes. It was by means of cell-culture techniques, liver cells(stem cells) example, that immunologists were first able to define the functional differences between CD4* T helper cells and CD8 T cytotoxic cells. Cell-culture techniques have also been used to identify numerous cytokines involved in the activation, growth, and differentiation of various cells involved in the immune re- nents showed that media conditioned, 米a④ Human thymus releases or modified, by the growth of various lymphocytes or antigen- presenting cells would support the growth of other lymphoid of--A cells Conditioned media contain the secreted products from actively growing cells. Many of the individual cytokines that characterized various conditioned media have subsequently been identified and purified, and in many cases the genes that encode them have been cloned. These cytokines, which play a SCID-human mouse central role in the activation and regulation of the immune response, are described in Chapter 12 and elsewhere FIGURE 23-1 Production of SCID-human mouse. This system Cloned Lymphoid Cell Lines permits study of human lymphocytes within an animal model. In this example, human T-cells are transferred to SCID mouse, but B-cells A primary lymphoid cell culture comprises a heterogeneous also can be transferred by the use of bone-marrow precursors group of cells that can be propagated only for a limited time This heterogeneity can complicate the interpretation of perimental results. To avoid these problems, immunologists use cloned lymphoid cell lines and hybrid cells. Normal mammalian cells generally have a finite life span in The beauty of the SCID-human mouse is that it enables culture; that is, after a number of population doublings char one to study human lymphocytes within an animal model. acteristic of the species and cell type, the cells stop dividing. In This valuable system has proved useful in research on the contrast, tumor cells or normal cells that have undergone development of various lymphoid cells and also as an impor- transformation induced by chemical carcinogens or viruses cytes cannot be infected with HIV, whereas the lymphocytes said to be immortal. Such cells are referred to as cell ir / e tant animal model in AIDS research, since mouse lympho- can be propagated indefinitely in tissue culture; thus, they are of a SCiD-human mouse are readily infected. The first cell line-the mouse fibroblast l cell-was de. ived in the 1940s from cultured mouse subcutaneous con- nective tissue by exposing the cultured cells to a chemical carc Cell-Culture Systems nogen, methylcholanthrene, over a 4-month period. In the 1950s, another important cell line, the Hela cell was de The complexity of the cellular interactions that generate an rived by culturing human cervical cancer cells. Since these immune response has led immunologists to rely heavily on early studies, hundreds of cell lines have been established,each various types of in vitro cell-culture systems. A variety of cells consisting of a population of genetically identical (syngeneic) can be cultured, including primary lymphoid cells, cloned cells that can be grown indefinitely in culture lymphoid cell lines, and hybrid cells. Table 23-3 lists some of the cell lines used in immunologic research and briefly describes their properties. Some were Primary Lymphoid Cell Cultures derived from spontaneously occurring tumors of lympho- cytes, macrophages, or other cells involved in the im Primary lymphoid cell cultures can be obtained by isolating mune response. In other cases, the cell line was induced by lymphocytes directly from blood or lymph or from various transformation of normal lymphoid cells with viruses such as lymphoid organs by tissue dispersion. The lymphocytes can Abelson's murine leukemia virus(A-MLV), simian virus 40

The beauty of the SCID-human mouse is that it enables one to study human lymphocytes within an animal model. This valuable system has proved useful in research on the development of various lymphoid cells and also as an impor￾tant animal model in AIDS research, since mouse lympho￾cytes cannot be infected with HIV, whereas the lymphocytes of a SCID-human mouse are readily infected. Cell-Culture Systems The complexity of the cellular interactions that generate an immune response has led immunologists to rely heavily on various types of in vitro cell-culture systems. A variety of cells can be cultured, including primary lymphoid cells, cloned lymphoid cell lines, and hybrid cells. Primary Lymphoid Cell Cultures Primary lymphoid cell cultures can be obtained by isolating lymphocytes directly from blood or lymph or from various lymphoid organs by tissue dispersion. The lymphocytes can then be grown in a chemically defined basal medium (con￾taining saline, sugars, amino acids, vitamins, trace elements, and other nutrients) to which various serum supplements are added. For some experiments, serum-free culture condi￾tions are employed. Because in vitro culture techniques re￾quire from 10- to 100-fold fewer lymphocytes than do typical in vivo techniques, they have enabled immunologists to assess the functional properties of minor subpopulations of lymphocytes. It was by means of cell-culture techniques, for example, that immunologists were first able to define the functional differences between CD4+ T helper cells and CD8+ T cytotoxic cells. Cell-culture techniques have also been used to identify numerous cytokines involved in the activation, growth, and differentiation of various cells involved in the immune re￾sponse. Early experiments showed that media conditioned, or modified, by the growth of various lymphocytes or antigen￾presenting cells would support the growth of other lymphoid cells. Conditioned media contain the secreted products from actively growing cells. Many of the individual cytokines that characterized various conditioned media have subsequently been identified and purified, and in many cases the genes that encode them have been cloned. These cytokines, which play a central role in the activation and regulation of the immune response, are described in Chapter 12 and elsewhere. Cloned Lymphoid Cell Lines A primary lymphoid cell culture comprises a heterogeneous group of cells that can be propagated only for a limited time. This heterogeneity can complicate the interpretation of ex￾perimental results. To avoid these problems, immunologists use cloned lymphoid cell lines and hybrid cells. Normal mammalian cells generally have a finite life span in culture; that is, after a number of population doublings char￾acteristic of the species and cell type, the cells stop dividing. In contrast, tumor cells or normal cells that have undergone transformation induced by chemical carcinogens or viruses can be propagated indefinitely in tissue culture; thus, they are said to be immortal. Such cells are referred to as cell lines. The first cell line—the mouse fibroblast L cell—was de￾rived in the 1940s from cultured mouse subcutaneous con￾nective tissue by exposing the cultured cells to a chemical carcinogen, methylcholanthrene, over a 4-month period. In the 1950s, another important cell line, the HeLa cell, was de￾rived by culturing human cervical cancer cells. Since these early studies, hundreds of cell lines have been established, each consisting of a population of genetically identical (syngeneic) cells that can be grown indefinitely in culture. Table 23-3 lists some of the cell lines used in immunologic research and briefly describes their properties. Some were derived from spontaneously occurring tumors of lympho￾cytes, macrophages, or other accessory cells involved in the im￾mune response. In other cases, the cell line was induced by transformation of normal lymphoid cells with viruses such as Abelson’s murine leukemia virus (A-MLV), simian virus 40 528 PART IV The Immune System in Health and Disease SCID mouse Transplant human thymus and lymph-node tissue under kidney capsule Inject with human fetal liver cells (stem cells) Stem cells migrate to the human thymus Human thymus releases mature human T cells into circulation SCID–human mouse FIGURE 23-1 Production of SCID-human mouse. This system permits study of human lymphocytes within an animal model. In this example, human T-cells are transferred to SCID mouse, but B-cells also can be transferred by the use of bone-marrow precursors

Experimental Systems CHAPTER 23 529 Cell lines commonly used in grown for extended periods in tissue culture, enabling im TABLE 23-3 immunologic research munologists to obtain large numbers of homogeneous cells in culture Cell line Description Until the late 1970s, immunologists did not succeed maintaining normal T cells in tissue culture for extended Mouse fibroblast cell line often used in periods. In 1978, a serendipitous finding led to the observa DNA transfection studies and to assay tion that conditioned medium containing a T-cell growth tumor necrosis factor (NF) factor was required. The essential component of the condi sP2/0 Nonsecreting mouse myeloma; often ioned medium turned out to be interleukin 2(IL-2). By cul- used as a fusion partner turing normal T lymphocytes with antigen in the presence of hybridoma secretion L-2, clones of antigen-specific T lymphocytes could be P3X63-Ag8653 Nonsecreting mouse myeloma; often sed as a fusion partner for lated. These individual clones could be propagated and stud- hybridoma secretion ied in culture and even frozen for storage. After thawing, the MPC 11 IgG2b-secreting myeloma clones continued to grow and express their original antigen- specific functions P3X63Ag8 Mouse lgG1-secreting myeloma Development of cloned lymphoid cell lines has enabled MOPC 315 Mouse IgA-secreting myeloma munologists to study a number of events that previously could not be examined. For example, research on the molec 70Z/3 Mouse pre-B-cell lymphoma; used to ular events involved in activation of naive lymphocytes by study earby events in B-cell differentiation antigen was hampered by the low frequency of naive B and BCL 1 Mouse B-cell leukemia lymphoma that T cells specific for a particular antigen; in a heterogeneous expresses membrane IgM and IgD and population of lymphocytes, the molecular changes occurring can be activated with mitogen to in one responding cell could not be detected against a back ground of 10-10 nonresponding cells. ClonedT-and B-cell CTLL-2 Mouse T-cell line whose growth is lines with known antigenic specificity have provided immu pendent on IL-2; often used to assay IL-2 production nologists with large homogeneous cell populations in which to study the events involved in antigen recognition. Similarly, Jurkat Human T-cell leukemia that secretes IL-2 the genetic changes corresponding to different maturational Do1110 Mouse T-cell hybridoma with specificity stages can be studied in cell lines that appear to be"frozenat different stages of differentiation. Cell lines have also bee Mouse monocyte-macrophage line useful in studying the soluble factors produced by lymphoid P338D1 Mouse monocyte-macrophage line that cells. Some cell lines secrete large quantities of various cyto- secretes high levels of IL-1 kines; other lines express membrane receptors for particular WEH 265.1 Mouse monocyte line tokines. These cell lines have been used by immunologists Mouse mastocytoma cells; often used as to purify various cytokines and their receptors and eventu- target to assess killing by cytotoxic ally to clone their T lymphocytes(CTLs With the advantages of lymphoid cell lin es come a num YAC-1 Mouse lymphoma cells; often used as of limitations. Variants arise spontaneously in the course of arget for NK cells cloning to limit Human myeloid-leukemia cell line the cellular heterogeneity that can develop. If variants are African green monkey kidney cells selected in subcloning, it is possible that two subclones derived transformed by SV40; often used in rom the same parent clone may represent different subpopu- DNA transfection studies lations. Moreover, any cell line derived from tumor cells or characteristic of the tumor or of the transformed state thus, researchers must be cautious when extrapolating results ob- tained with cell lines to the normal situation in vivo neverthe. (SV40), Epstein-Barr virus(EBV), or human T-cell leukemia less, transformed cell lines have made a major contribution to the study of the immune response, and many molecular events Lymphoid cell lines differ from primary lymphoid cell discovered in experiments with transformed cell lines have cultures in several important ways: They survive indefinitely been shown to take place in normal lymphocytes. in tissue culture, show various abnormal growth properties, with more or less than the normal diploid number of chro. Hybrid Lymphoid Cell Lines mosomes for a species are said to be aneuploid. The big In somatic-cell hybridization, immunologists fuse normal B advantage of cloned lymphoid cell lines is that they can be or T lymphocytes with tumor cells, obtaining hybrid cells, or

(SV40), Epstein-Barr virus (EBV), or human T-cell leukemia virus type 1(HTLV-1). Lymphoid cell lines differ from primary lymphoid cell cultures in several important ways: They survive indefinitely in tissue culture, show various abnormal growth properties, and often have an abnormal number of chromosomes. Cells with more or less than the normal diploid number of chro￾mosomes for a species are said to be aneuploid. The big advantage of cloned lymphoid cell lines is that they can be grown for extended periods in tissue culture, enabling im￾munologists to obtain large numbers of homogeneous cells in culture. Until the late 1970s, immunologists did not succeed in maintaining normal T cells in tissue culture for extended periods. In 1978, a serendipitous finding led to the observa￾tion that conditioned medium containing a T-cell growth factor was required. The essential component of the condi￾tioned medium turned out to be interleukin 2 (IL-2). By cul￾turing normal T lymphocytes with antigen in the presence of IL-2, clones of antigen-specific T lymphocytes could be iso￾lated. These individual clones could be propagated and stud￾ied in culture and even frozen for storage. After thawing, the clones continued to grow and express their original antigen￾specific functions. Development of cloned lymphoid cell lines has enabled immunologists to study a number of events that previously could not be examined. For example, research on the molec￾ular events involved in activation of naive lymphocytes by antigen was hampered by the low frequency of naive B and T cells specific for a particular antigen; in a heterogeneous population of lymphocytes, the molecular changes occurring in one responding cell could not be detected against a back￾ground of 103 –106 nonresponding cells. Cloned T- and B-cell lines with known antigenic specificity have provided immu￾nologists with large homogeneous cell populations in which to study the events involved in antigen recognition. Similarly, the genetic changes corresponding to different maturational stages can be studied in cell lines that appear to be “frozen” at different stages of differentiation. Cell lines have also been useful in studying the soluble factors produced by lymphoid cells. Some cell lines secrete large quantities of various cyto￾kines; other lines express membrane receptors for particular cytokines. These cell lines have been used by immunologists to purify various cytokines and their receptors and eventu￾ally to clone their genes. With the advantages of lymphoid cell lines come a number of limitations. Variants arise spontaneously in the course of prolonged culture, necessitating frequent subcloning to limit the cellular heterogeneity that can develop. If variants are selected in subcloning, it is possible that two subclones derived from the same parent clone may represent different subpopu￾lations. Moreover, any cell line derived from tumor cells or transformed cells may have unknown genetic contributions characteristic of the tumor or of the transformed state; thus, researchers must be cautious when extrapolating results ob￾tained with cell lines to the normal situation in vivo. Neverthe￾less, transformed cell lines have made a major contribution to the study of the immune response, and many molecular events discovered in experiments with transformed cell lines have been shown to take place in normal lymphocytes. Hybrid Lymphoid Cell Lines In somatic-cell hybridization, immunologists fuse normal B or T lymphocytes with tumor cells, obtaining hybrid cells, or Experimental Systems CHAPTER 23 529 TABLE 23-3 Cell lines commonly used in immunologic research Cell line Description L-929 Mouse fibroblast cell line; often used in DNA transfection studies and to assay tumor necrosis factor (TNF) SP2/0 Nonsecreting mouse myeloma; often used as a fusion partner for hybridoma secretion P3X63-Ag8.653 Nonsecreting mouse myeloma; often used as a fusion partner for hybridoma secretion MPC 11 Mouse IgG2b-secreting myeloma P3X63-Ag8 Mouse IgG1-secreting myeloma MOPC 315 Mouse IgA-secreting myeloma J558 Mouse IgA-secreting myeloma 7OZ/3 Mouse pre–B-cell lymphoma; used to study early events in B-cell differentiation BCL 1 Mouse B-cell leukemia lymphoma that expresses membrane IgM and IgD and can be activated with mitogen to secrete IgM CTLL-2 Mouse T-cell line whose growth is dependent on IL-2; often used to assay IL-2 production Jurkat Human T-cell leukemia that secretes IL-2 DO11.10 Mouse T-cell hybridoma with specificity for ovalbumin PU 5-1.8 Mouse monocyte-macrophage line P338 D1 Mouse monocyte-macrophage line that secretes high levels of IL-1 WEHI 265.1 Mouse monocyte line P815 Mouse mastocytoma cells; often used as target to assess killing by cytotoxic T lymphocytes (CTLs) YAC-1 Mouse lymphoma cells; often used as target for NK cells HL-60 Human myeloid-leukemia cell line COS-1 African green monkey kidney cells transformed by SV40; often used in DNA transfection studies

530 PARt IV The Immune System in Health and Disease heterokaryons, containing nuclei from both parent cells. Ran Chromosomes dom loss of some chromosomes and subsequent cell prolifer ation yield a clone( contain a single nucleus with chromosomes fro of the fused cells such a clone is called a hybridoma. Historically, cell fusion was promoted with Sendai virus, but now it is generally done with polyethylene glycol. Normal antigen-primed B cells can be fused with cancerous plasm Normal t or B cell ancerous t or b cell cells, called myeloma cells( Figure 23-2). The hybridoma 7-10 (grows continuously thus formed continues to express the antibody genes of the normal B lymphocyte but is capable of unlimited growth, a characteristic of the myeloma cell. B-cell hybridomas that secrete antibody with a single antigenic specificity, called monoclonal antibody, in reference to its derivation from a single clone, have revolutionized not only immunology but biomedical research as well as the clinical laboratory. Chapter 4 describes the production and uses of monoclonal antibod- ies in detail (see Figures 4-21) Nucleus Nucleus of of normal T-cell hybridomas can also be obtained by fusing T lym- lymphocyte phocytes with cancerous T-cell lymphomas. Again, the result ing hybridoma continues to express the genes of the normal Tcell but acquires the immortal-growth properties of the can- cerous T lymphoma cell. Immunologists have generated a Heterokaryon number of stable hybridoma cell lines representing T-helper and T-cytotoxic lineages Random chromosomal loss Protein Biochemistry The structures and functions of many important molecules of the immune system have been determined with the tech- niques of protein biochemistry, and many of these tech- niques are in constant service in experimental immunology For example, fluorescent and radioactive labels allow immu nologists to localize and visualize molecular activities, and the ability to determine such biochemical characteristics of a (expresses some normal protein as its size, shape, and three-dimensional struct grows indefinitely like a cancer cell) provided essential information for understanding the tions of immunologically important molecules. Radiolabeling Techniques Allow Sensitive detection of antio Monoclonal or Antibodies antibody 2(12) Radioactive labels on antigen or antibody are extremely sen sitive markers for detection and quantification. There are a number of ways to introduce radioactive isotopes into pro- B- teins or peptides. For example, tyrosine residues may be labeled with radioiodine by chemical or enzymatic proce dures. These reactions attach an iodine atom to the phenol FIGURE 23-2 Production of B-cell and T-cell hybridomas by ring of the tyrosine molecule. One of the enzymatic iodin- somatic-cell hybridization. The resulting hybridomas express some tion techniques, which uses lactoperoxidase, can label pro- of the genes of the original normal B or T cell but also exhibit the teins on the plasma membrane of a live cell without labeling immortal-growth properties of the tumor cell. This procedure is used proteins in the cytoplasm, allowing the study of cell-surface to produce B-cell hybridomas that secrete monoclonal antibody and roteins without isolating them from other cell constituents. T-cell hybridomas that secrete various growth factors

heterokaryons, containing nuclei from both parent cells. Ran￾dom loss of some chromosomes and subsequent cell prolifer￾ation yield a clone of cells that contain a single nucleus with chromosomes from each of the fused cells; such a clone is called a hybridoma. Historically, cell fusion was promoted with Sendai virus, but now it is generally done with polyethylene glycol. Normal antigen-primed B cells can be fused with cancerous plasma cells, called myeloma cells (Figure 23-2). The hybridoma thus formed continues to express the antibody genes of the normal B lymphocyte but is capable of unlimited growth, a characteristic of the myeloma cell. B-cell hybridomas that secrete antibody with a single antigenic specificity, called monoclonal antibody, in reference to its derivation from a single clone, have revolutionized not only immunology but biomedical research as well as the clinical laboratory. Chapter 4 describes the production and uses of monoclonal antibod￾ies in detail (see Figures 4-21). T-cell hybridomas can also be obtained by fusing T lym￾phocytes with cancerous T-cell lymphomas. Again, the result￾ing hybridoma continues to express the genes of the normal T cell but acquires the immortal-growth properties of the can￾cerous T lymphoma cell. Immunologists have generated a number of stable hybridoma cell lines representing T-helper and T-cytotoxic lineages. Protein Biochemistry The structures and functions of many important molecules of the immune system have been determined with the tech￾niques of protein biochemistry, and many of these tech￾niques are in constant service in experimental immunology. For example, fluorescent and radioactive labels allow immu￾nologists to localize and visualize molecular activities, and the ability to determine such biochemical characteristics of a protein as its size, shape, and three-dimensional structure has provided essential information for understanding the func￾tions of immunologically important molecules. Radiolabeling Techniques Allow Sensitive Detection of Antigens or Antibodies Radioactive labels on antigen or antibody are extremely sen￾sitive markers for detection and quantification. There are a number of ways to introduce radioactive isotopes into pro￾teins or peptides. For example, tyrosine residues may be labeled with radioiodine by chemical or enzymatic proce￾dures. These reactions attach an iodine atom to the phenol ring of the tyrosine molecule. One of the enzymatic iodina￾tion techniques, which uses lactoperoxidase, can label pro￾teins on the plasma membrane of a live cell without labeling proteins in the cytoplasm, allowing the study of cell-surface proteins without isolating them from other cell constituents. 530 PART IV The Immune System in Health and Disease Polyethylene glycol Chromosomes Normal T or B cell (dies after 7–10 days in culture) Cancerous T or B cell (grows continuously in culture) Heterokaryon Nucleus of cancer cell Nucleus of normal lymphocyte Random chromosomal loss Hybridoma (expresses some normal B-cell or T-cell genes but grows indefinitely like a cancer cell) B-cell hybridoma T-cell hybridoma Monoclonal antibody Interleukin 2 (IL-2) FIGURE 23-2 Production of B-cell and T-cell hybridomas by somatic-cell hybridization. The resulting hybridomas express some of the genes of the original normal B or T cell but also exhibit the immortal-growth properties of the tumor cell. This procedure is used to produce B-cell hybridomas that secrete monoclonal antibody and T-cell hybridomas that secrete various growth factors

Experimental Systems CHAPTER 23 531 Radioisotopes commonly used may cause denaturation and loss of activity. A convenient la- TABLE23-4in immunology laboratories beling system has been developed which may be used in con- junction with the ELISA and ELISPOT assays described in Radiation type Autoradiography Chapter 6. This labeling technique exploits the high affinity of the reaction between the vitamin biotin and avidin, a large molecule that may be labeled with radioactive isotopes, with 6. 8 da fluorescent molecules, or with enzymes. Biotin is a small 5'Cr 27.8 da molecule(mol wt. 244)that can be coupled to an antibody (or 143da to any protein molecule) by a gentle chemical reaction that causes no loss of antibody activity. After the biotin-coupled 874da 14C5730y 235y the avidin molecule ( figure 23-3). The reaction between bio- tin and avidin is highly specific and of such high affinity that y(gamma)radiation may be detected in a solid scintillation counter. the bond between the two molecules under most assay condi B(beta)radiation is detected in a liquid scintillation counter by its ability to convert energy to photons of light in a solution containing phosphorescent tions is virtually irreversible 可dMm级 Proteins by Size and Charge Gel Electrophoresis Separates normal autoradiographic techniques. When subjected to an electric field in an electrophoresis chamber, a charged molecule will move toward the oppo- sitely charged electrode. The rate at which a charged mole- cule moves in a stable field (its electrophoretic mobility) A general radiolabeling of cell proteins may be carried out depends upon two factors specific to the molecule: one is the by growing the cells in a medium that contains one or more sign and magnitude of its net electrical charge, and the other radiolabeled amino acids. The amino acids selected for this is its size and shape. All other factors being equal, if mole application are those most resistant to metabolic modification cules are of equal size the one with higher net charge will during cell growth so that the radioactive label will appear in move faster in an applied electrical field due to the molecular the cell protein rather than in all cell constituents. Leucine seiving properties of the solid medium. It also follows that marked with"C or H, and cysteine or methionine labeled small molecules will move faster than large ones of the same withS, are the most commonly used amino acids for meta- net charge. Although there are exceptions in which the shape bolic labeling of proteins. Table 23-4 lists some properties of of a molecule may increase or decrease its frictional drag and the radioisotopes used in immunologic research cause atypical migration behavior, these general principles Biotin Labels facilitate detection Most electrophoretic separations are not conducted in of small Amounts of proteins free solution but rather in a stable supporting medium, such as a gel. The most popular in reseach laboratories is a poly- In some instances direct labeling of proteins, especially with merized and crosslinked form of acrylamide. Separation on enzymes or other large molecules, as described in Chapter 6, polyacrylamide gels, commonly referred to as Biotin active ester Labeled avidin biotinylated Ab FIGURE23-3Labeling of antibody with biotin. An antibody prepara- antibody, the bound antibody can be detected with labeled avidin.The tion is mixed with a biotin ester, which reacts with the antibody. The avidin can be radioactively labeled or linked to an enzyme that catalyzes biotin-labeled antibody can be used to detect antigens on a solid sub- a color reaction, as in ELISA procedures(see Figure 6-10) strate such as the well of a microtiter plate. After washing away unbound

A general radiolabeling of cell proteins may be carried out by growing the cells in a medium that contains one or more radiolabeled amino acids. The amino acids selected for this application are those most resistant to metabolic modification during cell growth so that the radioactive label will appear in the cell protein rather than in all cell constituents. Leucine marked with 14C or 3 H, and cysteine or methionine labeled with 35S, are the most commonly used amino acids for meta￾bolic labeling of proteins. Table 23-4 lists some properties of the radioisotopes used in immunologic research. Biotin Labels Facilitate Detection of Small Amounts of Proteins In some instances direct labeling of proteins, especially with enzymes or other large molecules, as described in Chapter 6, may cause denaturation and loss of activity. A convenient la￾beling system has been developed which may be used in con￾junction with the ELISA and ELISPOT assays described in Chapter 6. This labeling technique exploits the high affinity of the reaction between the vitamin biotin and avidin, a large molecule that may be labeled with radioactive isotopes, with fluorescent molecules, or with enzymes. Biotin is a small molecule (mol. wt. 244) that can be coupled to an antibody (or to any protein molecule) by a gentle chemical reaction that causes no loss of antibody activity. After the biotin-coupled antibody has reacted in the assay system, the labeled avidin is introduced and binding is measured by detecting the label on the avidin molecule (Figure 23-3). The reaction between bio￾tin and avidin is highly specific and of such high affinity that the bond between the two molecules under most assay condi￾tions is virtually irreversible. Gel Electrophoresis Separates Proteins by Size and Charge When subjected to an electric field in an electrophoresis chamber, a charged molecule will move toward the oppo￾sitely charged electrode. The rate at which a charged mole￾cule moves in a stable field (its electrophoretic mobility) depends upon two factors specific to the molecule: one is the sign and magnitude of its net electrical charge, and the other is its size and shape. All other factors being equal, if mole￾cules are of equal size the one with higher net charge will move faster in an applied electrical field due to the molecular seiving properties of the solid medium. It also follows that small molecules will move faster than large ones of the same net charge. Although there are exceptions in which the shape of a molecule may increase or decrease its frictional drag and cause atypical migration behavior, these general principles underlie all electrophoretic separations. Most electrophoretic separations are not conducted in free solution but rather in a stable supporting medium, such as a gel. The most popular in reseach laboratories is a poly￾merized and crosslinked form of acrylamide. Separation on polyacrylamide gels, commonly referred to as polyacrylamide Experimental Systems CHAPTER 23 531 TABLE 23-4 Radioisotopes commonly used in immunology laboratories Isotope Half-life Radiation type* Autoradiography† 125I 60.0 da
+ 131I 6.8 da
+ 51Cr 27.8 da
– 32P 14.3 da + 35S 87.4 da + 14C 57.30 yrs + 3 H 12.35 yrs – *
(gamma) radiation may be detected in a solid scintillation counter. (beta) radiation is detected in a liquid scintillation counter by its ability to convert energy to photons of light in a solution containing phosphorescent compounds. † Radiation may also be detected by exposure to x-ray film. 35S and 14C must be placed in direct contact with film for detection. 3 H cannot be detected by normal autoradiographic techniques. Labeled avidin Ag Ab Biotin active ester Biotinylated Ab Avidin bound to biotinylated Ab FIGURE 23-3 Labeling of antibody with biotin. An antibody prepara￾tion is mixed with a biotin ester, which reacts with the antibody. The biotin-labeled antibody can be used to detect antigens on a solid sub￾strate such as the well of a microtiter plate. After washing away unbound antibody, the bound antibody can be detected with labeled avidin. The avidin can be radioactively labeled or linked to an enzyme that catalyzes a color reaction, as in ELISA procedures (see Figure 6-10).

532 Iv The Immune System in Health and disease To ple Buffer 30 Plastic 29 Buffer Bottom Relative mo FIGURE 23-4 Gel electrophoresis(a)A standard PAGE apparatus with cathode at the top and anode at the bottom. Samples are loaded on the top of the gel in sample wells and electrophoresis is accom- Stable plished by running a current from the cathode to the anode.(b)The obility, or distance traveled by a species during SDS. Molecules PAGE, is inversely proportional to the log of its molecular weight. The migrate to 7.0 molecular weight of a protein is readily determined by the log of its migration distance with a standard curve that plots the migration dis which their 6.0 tances of the set of standard proteins against the logs of their molecu lar weights.( c)Isoelectric focusing, or IEF, separates proteins solely by is zer 5.0 charge. Proteins are placed on a stable pH gradient and subjected to electrophoresis. Each protein migrates to its isoelectric point, the point at which its net charge is zero. Part (b) after K. Weber and M. Osbom, 975, The Proteins, 3rd ed, vol. 1, p. 179. Academic Press, gel electrophoresis(PAGE), may be used for analysis of pro- arate the components of a mixture of proteins according to teins or nudeic acids( Figure 23-4a) molecular weight. Second, because the electrophoretic mobil- In one common application, the electrophoresis of pro- ity, or distance traveled by a species during SDS-PAGE, is in- teins through a polyacrylamide gel is carried out in the pres- versely proportional to the logarithm of its molecular weight, ence of the detergent sodium dodecyl sulfate(SDS). This that distance is a measure of its molecular weight The gel is method, known as SDS-PAGE, provides a relatively simple stained with a dye that reacts with protein to visualize the and highly effective means of separating mixtures of proteins locations of the proteins. The migration distance of a protein on the basis of size. SDS is a negatively charged detergent that in question is then compared with a plot of the distances binds to protein in amounts proportional to the length of the migrated by a set of standard proteins( Figure 23-4b) protein. This binding destroys the characteristic tertiary and Another electrophoretic technique, isoelectric focusing secondary structure of the protein, transforming it into a (EF), separates proteins solely on the basis of their char negatively charged rod. A protein binds so many negatively This method is based on the fact that a molecule will move in harged SDS molecules that its own intrinsic charge becomes an electric field as long as it has a net positive or negative insignificant by comparison with the net charge of the SDs charge; molecules that bear equal numbers of positive and molecules. Therefore, treatment of a mixture of proteins with negative charges and therefore have a net charge of zero will SDS transforms them into a collection of rods whose electric not move. At most pH values, proteins(which characteristi charges are proportional to their molecular weights. This has cally bear a number of both positive and negative charges) two extremely useful consequences. First, it is possible to sep- have either a net negative or a net positive charge. However

gel electrophoresis (PAGE), may be used for analysis of pro￾teins or nucleic acids (Figure 23-4a). In one common application, the electrophoresis of pro￾teins through a polyacrylamide gel is carried out in the pres￾ence of the detergent sodium dodecyl sulfate (SDS). This method, known as SDS-PAGE, provides a relatively simple and highly effective means of separating mixtures of proteins on the basis of size. SDS is a negatively charged detergent that binds to protein in amounts proportional to the length of the protein. This binding destroys the characteristic tertiary and secondary structure of the protein, transforming it into a negatively charged rod. A protein binds so many negatively charged SDS molecules that its own intrinsic charge becomes insignificant by comparison with the net charge of the SDS molecules. Therefore, treatment of a mixture of proteins with SDS transforms them into a collection of rods whose electric charges are proportional to their molecular weights. This has two extremely useful consequences. First, it is possible to sep￾arate the components of a mixture of proteins according to molecular weight. Second, because the electrophoretic mobil￾ity, or distance traveled by a species during SDS-PAGE, is in￾versely proportional to the logarithm of its molecular weight, that distance is a measure of its molecular weight. The gel is stained with a dye that reacts with protein to visualize the locations of the proteins. The migration distance of a protein in question is then compared with a plot of the distances migrated by a set of standard proteins (Figure 23-4b). Another electrophoretic technique, isoelectric focusing (IEF), separates proteins solely on the basis of their charge. This method is based on the fact that a molecule will move in an electric field as long as it has a net positive or negative charge; molecules that bear equal numbers of positive and negative charges and therefore have a net charge of zero will not move. At most pH values, proteins (which characteristi￾cally bear a number of both positive and negative charges) have either a net negative or a net positive charge. However, 532 PART IV The Immune System in Health and Disease Apparent mass (kd) 70 10 20 30 40 50 60 0.2 0.4 0.6 0.8 1.0 Relative mobility Anode Cathode Sample wells Sample Buffer Gel Plastic frame − + + Top Mass (kd) Stable pH gradient Bottom 200 100 68 43 36 29 17 12 (a) (c) (b) Buffer Molecules migrate to position at which their net charge is zero − − − −− − + − + +− − − + pH 7.0 6.0 5.0 − 7.0 6.0 5.0 − + ++ − − − − + + + − Direction of electrophoresis FIGURE 23-4 Gel electrophoresis. (a) A standard PAGE apparatus with cathode at the top and anode at the bottom. Samples are loaded on the top of the gel in sample wells and electrophoresis is accom￾plished by running a current from the cathode to the anode. (b) The electrophoretic mobility, or distance traveled by a species during SDS￾PAGE, is inversely proportional to the log of its molecular weight. The molecular weight of a protein is readily determined by the log of its migration distance with a standard curve that plots the migration dis￾tances of the set of standard proteins against the logs of their molecu￾lar weights. (c) Isoelectric focusing, or IEF, separates proteins solely by charge. Proteins are placed on a stable pH gradient and subjected to electrophoresis. Each protein migrates to its isoelectric point, the point at which its net charge is zero. [Part (b) after K. Weber and M. Osborn, 1975, The Proteins, 3rd ed., vol. 1, p. 179. Academic Press.]

Experimental Systems CHAPTER 23 533 for each protein there is a particular pH, called its isoelectricAcidic point(pD), at which that protein has equal numbers of posi- tive and negative charges. Isoelectric focusing makes use of a gel containing substances, called carrier ampholytes, that al range themselves into a continuous pH gradient when sub- jected to an electric field. When a mixture of proteins is ap to such a gel and subjected to electrophoresis, each ein moves until it reaches that point in the gradient the pH of the gel is equal to its isoelectric point. It then stops moving because it has a net charge of zero. Isoelectric focusing is an extremely gentle and effective way of separat ing different proteins(Figure 23-4c) A method known as two-dimensional gel electrophoresis (2D gel electrophoresis) combines the advantages of SDS- PAGE and isoelectric focusing in one of the most sensitive and discriminating ways of analyzing a mixture of proteins In this method, one first subjects the mixture to isoelectric focusing on an IEf tube gel, which separates the molecules on the basis of their isoelectric points without regard to mol- ecular weight. This is the first dimension. In the next step, FIGURE 23-5 Two-dimensional gel electrophoresis of a5s-methionine one places the IEF gel lengthwise across the top of an sDs. labeled total cell proteins from murine thymocytes. These proteins polyacrylamide slab(that is, in place of the sample wells in were first subjected to isoelectric focusing(direction of migration indi- Figure 23-4a)and runs SDS-PAGE Preparatory to this step, SDSPAG arrow) and then the focused proteins were separated by all proteins have been reacted with SDS and therefore mi- SDS-PAGE(direction of migration indicated by blue arrow). The gel grate out of the IEF gel and through the SDS-PAGE slab ac- was exposed to xray film to detect the labeled proteins. [Courtesy af cording to their molecular weights. This is the second dimen- B A Osborne sion. The position of the proteins in the resulting 2D gel car be visualized in a number of ways. In the least sensitive the gel is stained with a protein-binding dye(such as Coomassie blue). If the proteins have been radiolabeled, the more sensi- silver staining is a method odphy can be used. Alternatively, microscope, the theoretical limit of resolution of the electron silver staining is a method of great sensitivity that takes ad- microscope is about 0.002 nm. If it were possible to build an vantage of the capacity of proteins to reduce silver ions to an instrument that could actually approach this limit, the elec- easily visualized deposit of metallic silver. Finally, immuno- tron microscope could readily be used to determine the blotting-blotting of proteins onto a membrane and detec- detailed atomic arrangement of biological molecules, since tion with antibody (see Figure 6-13)-can be used as a way of the constituent atoms are separated by distances of 0. 1 nm to locating the position of specific proteins on 2D gels if an ap- 0.2 nm. In practice, aberrations inherent in the operation of propriate antibody is available. Figure 23-5 shows an autora- the magnetic lenses that are used to image the electron beam diograph of a two-dimensional gel of labeled proteins from limit the resolution to about 0. 1 nm(1A). This practical limit murine thymocytes. can be reached in the examination of certain specimens, par- ticularly metals. Other considerations, however, such as X-Ray Crystallography Provides specimen preparation and contrast, limit the resolution for Structural Information biological materials to about 2 nm(20 A). To determine the arrangement of a molecule's atoms, then, we must turn to A great deal of information about the structure of cells, parts x-rays, a form of electromagnetic radiation that is readily of cells, and even molecules has been obtained by light micro- generated in wavelengths on the order of size of interatomic scopy. The microscope uses a lens to focus radiation to form distances. Even though there are no microscopes with lenses an image after it has passed through a specimen. However, a that can focus x-rays into images, x-ray crystallography can practical limitation of light microscopy is the limit of resolu- reveal molecular structure at an extraordinary level of detail. tion Radiation of a given wavelength cannot resolve struc- X-ray crystallography is based on the analysis of the diffrac tural features less than about 1/2 its wavelength. Since the tion pattern produced by the scattering of an x-ray beam as it shortest wavelength of visible light is around 400 nm, even passes though a crystal. The degree to which a particular atom the very best light microscopes have a theoretical limit of res- scatters x-rays depends upon its size. Atoms such as carbon, olution of no less than 200 nm. oxygen, or nitrogen, scatter x-rays more than do hydrogen Because of the much shorter wavelength(0.004 nm)of atoms, and larger atoms, such as iron, iodide, or mercury give the electron at the voltages normally used in the electron intense scattering X-rays are a form of electromagnetic waves:

for each protein there is a particular pH, called its isoelectric point (pI), at which that protein has equal numbers of posi￾tive and negative charges. Isoelectric focusing makes use of a gel containing substances, called carrier ampholytes, that ar￾range themselves into a continuous pH gradient when sub￾jected to an electric field. When a mixture of proteins is ap￾plied to such a gel and subjected to electrophoresis, each protein moves until it reaches that point in the gradient where the pH of the gel is equal to its isoelectric point. It then stops moving because it has a net charge of zero. Isoelectric focusing is an extremely gentle and effective way of separat￾ing different proteins (Figure 23-4c). A method known as two-dimensional gel electrophoresis (2D gel electrophoresis) combines the advantages of SDS￾PAGE and isoelectric focusing in one of the most sensitive and discriminating ways of analyzing a mixture of proteins. In this method, one first subjects the mixture to isoelectric focusing on an IEF tube gel, which separates the molecules on the basis of their isoelectric points without regard to mol￾ecular weight. This is the first dimension. In the next step, one places the IEF gel lengthwise across the top of an SDS￾polyacrylamide slab (that is, in place of the sample wells in Figure 23-4a) and runs SDS-PAGE. Preparatory to this step, all proteins have been reacted with SDS and therefore mi￾grate out of the IEF gel and through the SDS-PAGE slab ac￾cording to their molecular weights. This is the second dimen￾sion. The position of the proteins in the resulting 2D gel can be visualized in a number of ways. In the least sensitive the gel is stained with a protein-binding dye (such as Coomassie blue). If the proteins have been radiolabeled, the more sensi￾tive method of autoradiography can be used. Alternatively, silver staining is a method of great sensitivity that takes ad￾vantage of the capacity of proteins to reduce silver ions to an easily visualized deposit of metallic silver. Finally, immuno￾blotting—blotting of proteins onto a membrane and detec￾tion with antibody (see Figure 6-13)—can be used as a way of locating the position of specific proteins on 2D gels if an ap￾propriate antibody is available. Figure 23-5 shows an autora￾diograph of a two-dimensional gel of labeled proteins from murine thymocytes. X-Ray Crystallography Provides Structural Information A great deal of information about the structure of cells, parts of cells, and even molecules has been obtained by light micro￾scopy. The microscope uses a lens to focus radiation to form an image after it has passed through a specimen. However, a practical limitation of light microscopy is the limit of resolu￾tion. Radiation of a given wavelength cannot resolve struc￾tural features less than about 1/2 its wavelength. Since the shortest wavelength of visible light is around 400 nm, even the very best light microscopes have a theoretical limit of res￾olution of no less than 200 nm. Because of the much shorter wavelength (0.004 nm) of the electron at the voltages normally used in the electron microscope, the theoretical limit of resolution of the electron microscope is about 0.002 nm. If it were possible to build an instrument that could actually approach this limit, the elec￾tron microscope could readily be used to determine the detailed atomic arrangement of biological molecules, since the constituent atoms are separated by distances of 0.1 nm to 0.2 nm. In practice, aberrations inherent in the operation of the magnetic lenses that are used to image the electron beam limit the resolution to about 0.1 nm (1Å). This practical limit can be reached in the examination of certain specimens, par￾ticularly metals. Other considerations, however, such as specimen preparation and contrast, limit the resolution for biological materials to about 2 nm (20 Å). To determine the arrangement of a molecule’s atoms, then, we must turn to x-rays, a form of electromagnetic radiation that is readily generated in wavelengths on the order of size of interatomic distances. Even though there are no microscopes with lenses that can focus x-rays into images, x-ray crystallography can reveal molecular structure at an extraordinary level of detail. X-ray crystallography is based on the analysis of the diffrac￾tion pattern produced by the scattering of an x-ray beam as it passes though a crystal. The degree to which a particular atom scatters x-rays depends upon its size. Atoms such as carbon, oxygen, or nitrogen, scatter x-rays more than do hydrogen atoms, and larger atoms, such as iron, iodide, or mercury give intense scattering. X-rays are a form of electromagnetic waves; Experimental Systems CHAPTER 23 533 Acidic Basic FIGURE 23-5 Two-dimensional gel electrophoresis of 35S-methionine labeled total cell proteins from murine thymocytes. These proteins were first subjected to isoelectric focusing (direction of migration indi￾cated by red arrow) and then the focused proteins were separated by SDS-PAGE (direction of migration indicated by blue arrow). The gel was exposed to x-ray film to detect the labeled proteins. [Courtesy of B. A. Osborne.]

534 PART I The Immune System in Health and Disease as the scattered waves overlap, they alternately interfere with (a) and reinforce each other. An appropriately placed detector records a pattern of spots( the diffraction pattern) whose dis- tribution and intensities are determined by the structure of the diffracting crystal. This relationship between crystal structure and diffraction pattern is the basis of x-ray crystallographic analysis. Here is an overview of the procedures used OBTAIN CRYSTAIS OF THE PROTEIN OF INTEREST To those who Diffracted beams have not experienced the frustrations of crystallizing proteins, this may seem a trivial and incidental step of an otherwise Detector (e ., film) highly sophisticated process. It is not. There is great variation from protein to protein in the conditions required to produce crystals that are of a size and geometrical formation appro- priate for x-ray diffraction analysis. For example, myoglobin (b) formed crystals over the course of several days at pH 7 in a 3 M solution of ammonium sulfate. but 1.5 m ammonium sulfate at pH 4 worked well for a human IgG1. There is no set formula that can be applied, and those who are consistently successful are persistent, determined, and, like great chefs, have a knack for making just the right"sauce SELECTION AND MOUNTING. Crystal specimens must be at least 0. 1 mm in the smallest dimension and rarely exceed a few millimeters in any dimension. Once chosen, a crystal is vested into a capillary tube along with the solution from which the crystal was grown(the mother liquor"). This keeps the crys- tal from drying and maintains its solvent content, an important consideration for maintaining the internal order of the speci men. The capillary is then mounted in the diffractionapparatus GENERATING AND RECORDING A DIFFRACTION PATTERN The precisely positioned crystal is then irradiated withx-rays of a known wavelength produced by accelerating electrons against the copper target of an x-ray tube. When the x-ray beam strikes (c) the crystal, some of it goes straight through and some is scat- tered; sensitive detectors record the position and intensity of Tyr 100H the scattered beam as a pattern of spots(Figure 23-6a, b) INTERPRETING THE DIFFRACTION PATTERN. The core of diffraction analysis is the mathematical deduction of the detailed structure that would produce the diffraction pattern observed. One must calculate to what extent the waves scat tered by each atom have combined to reinforce or cancel each other to produce the net intensity observed for each spot in Asp the array. a difficulty arises in the interpretation of complex diffraction patterns because the waves differ with respect to phase, the timing of the period between maxima and min ima. Since the pattern observed is the net result of the inter action of many waves, information about phase is critical to calculating the distribution of electron densities that is re- sponsible. The solution of this"phase problem"looms as a FIGURE 23-6 X-ray crystallography.(a)Schematic diagram of an major obstacle to the derivation of a high-resolution struc- x-ray crystallographic experiment in which an x-ray beam bombards ure of any complex molecule. the crystal and diffracted rays are detected. (b) Section of x-ray dif- The problem is solved by derivatizing the protein-mod- fraction pattern of a crystal of murine IgG2a.(c) Section from the ifying it by adding heavy atoms, such as mercury, and then electron-density map of murine IgG2a. / Part (a)from L. Stryer, 1995, obtaining crystals that have the same geometry as(are iso- Biochemistry, 4th ed. parts(b)and (c) courtesy of A McPherson./

as the scattered waves overlap, they alternately interfere with and reinforce each other. An appropriately placed detector records a pattern of spots (the diffraction pattern) whose dis￾tribution and intensities are determined by the structure of the diffracting crystal. This relationship between crystal structure and diffraction pattern is the basis of x-ray crystallographic analysis. Here is an overview of the procedures used: OBTAIN CRYSTALS OF THE PROTEIN OF INTEREST. To those who have not experienced the frustrations of crystallizing proteins, this may seem a trivial and incidental step of an otherwise highly sophisticated process. It is not. There is great variation from protein to protein in the conditions required to produce crystals that are of a size and geometrical formation appro￾priate for x-ray diffraction analysis. For example, myoglobin formed crystals over the course of several days at pH 7 in a 3 M solution of ammonium sulfate, but 1.5 M ammonium sulfate at pH 4 worked well for a human IgG1. There is no set formula that can be applied, and those who are consistently successful are persistent, determined, and, like great chefs, have a knack for making just the right “sauce.” SELECTION AND MOUNTING. Crystal specimens must be at least 0.1 mm in the smallest dimension and rarely exceed a few millimeters in any dimension. Once chosen, a crystal is har￾vested into a capillary tube along with the solution from which the crystal was grown (the “mother liquor”).This keeps the crys￾tal from drying and maintains its solvent content, an important consideration for maintaining the internal order of the speci￾men.The capillary is then mounted in the diffraction apparatus. GENERATING AND RECORDING A DIFFRACTION PATTERN. The precisely positioned crystal is then irradiated with x-rays of a known wavelength produced by accelerating electrons against the copper target of an x-ray tube. When the x-ray beam strikes the crystal, some of it goes straight through and some is scat￾tered; sensitive detectors record the position and intensity of the scattered beam as a pattern of spots (Figure 23-6a,b). INTERPRETING THE DIFFRACTION PAT TERN. The core of diffraction analysis is the mathematical deduction of the detailed structure that would produce the diffraction pattern observed. One must calculate to what extent the waves scat￾tered by each atom have combined to reinforce or cancel each other to produce the net intensity observed for each spot in the array. A difficulty arises in the interpretation of complex diffraction patterns because the waves differ with respect to phase, the timing of the period between maxima and min￾ima. Since the pattern observed is the net result of the inter￾action of many waves, information about phase is critical to calculating the distribution of electron densities that is re￾sponsible. The solution of this “phase problem” looms as a major obstacle to the derivation of a high-resolution struc￾ture of any complex molecule. The problem is solved by derivatizing the protein—mod￾ifying it by adding heavy atoms, such as mercury, and then obtaining crystals that have the same geometry as (are iso- 534 PART IV The Immune System in Health and Disease X-ray source X-ray beam Crystal Detector (e.g., film) Diffracted beams (a) (b) Tyr 100H Gly 97 Gly 96 Asp 101 Tyr 102 Tyr 100I Ala 100J Met 100K Trp 103 (c) FIGURE 23-6 X-ray crystallography. (a) Schematic diagram of an x-ray crystallographic experiment in which an x-ray beam bombards the crystal and diffracted rays are detected. (b) Section of x-ray dif￾fraction pattern of a crystal of murine IgG2a. (c) Section from the electron-density map of murine IgG2a. [Part (a) from L. Stryer, 1995, Biochemistry, 4th ed.; parts (b) and (c) courtesy of A. McPherson.]