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