《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