7
Human Immune-System Biologic Markers of Immunotoxicity

This chapter deals with markers that could be useful for assessing immunotoxicity in the human immune system. It also discusses currently available clinical tests, and a clinical testing regimen is proposed.

Immunity results from many complex interacting mechanisms that involve antigenspecific and antigen-nonspecific elements. The specific immune system employs two broad classes of cells that react with antigens: B cells and T cells. B cells are precursors of the antibody-secreting plasma cells of the humoral immune system, which in turn produce the five major classes of immunoglobulin molecules. T cells consist of an array of subtypes of cells: those that mediate important immunoregulatory functions, such as help or suppression; those involved in effector functions, such as the direct destruction of antigen-bearing cells; and those that make soluble products called lymphokines. Together, these T cells play a central role in delayed hypersensitivity or cellular immune response. In addition to the antigen-specific elements, there are numerous populations of cells (e.g., monocytes and natural killer cells) and factors (e.g., complement or interferon) that act in conjunction with specific mechanisms of immunity.

TESTS FOR ASSESSING IMMUNITY

An unusual susceptibility to infection and sometimes to autoimmune phenomena or allergy is a characteristic of a meaningful defect in immunity, whether primary or secondary (Waldmann, 1988). The first clue to the nature of the immunologic defect is often provided by the kind of infection. In general, patients with impaired humoral immunity have an increased incidence of recurrent infections with high-grade encapsulated bacterial pathogens, such as Pneumococcus and Hemophilus influenzae, that lead to chronic sinopulmonary infection, meningitis, and bacteremia. When cellular immunity is intact in such patients, they have less severe problems with fungal and viral infections. Abnormalities of T cells and thus of cell-mediated immunity predispose individuals to infection by a wide variety of agents, including viruses that lead to disseminated infections, particularly herpes simplex, varicella zoster, and cytomegalovirus; fungi that lead especially to mucocutaneous candidiasis; and parasitic organisms, including the protozoan Pneumocystis carinii. Infection, of course, can cause as well as result from immunodeficiency. A variety of infectious agents, such as the human immunodeficiency



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Biologic Markers in Immunotoxicology 7 Human Immune-System Biologic Markers of Immunotoxicity This chapter deals with markers that could be useful for assessing immunotoxicity in the human immune system. It also discusses currently available clinical tests, and a clinical testing regimen is proposed. Immunity results from many complex interacting mechanisms that involve antigenspecific and antigen-nonspecific elements. The specific immune system employs two broad classes of cells that react with antigens: B cells and T cells. B cells are precursors of the antibody-secreting plasma cells of the humoral immune system, which in turn produce the five major classes of immunoglobulin molecules. T cells consist of an array of subtypes of cells: those that mediate important immunoregulatory functions, such as help or suppression; those involved in effector functions, such as the direct destruction of antigen-bearing cells; and those that make soluble products called lymphokines. Together, these T cells play a central role in delayed hypersensitivity or cellular immune response. In addition to the antigen-specific elements, there are numerous populations of cells (e.g., monocytes and natural killer cells) and factors (e.g., complement or interferon) that act in conjunction with specific mechanisms of immunity. TESTS FOR ASSESSING IMMUNITY An unusual susceptibility to infection and sometimes to autoimmune phenomena or allergy is a characteristic of a meaningful defect in immunity, whether primary or secondary (Waldmann, 1988). The first clue to the nature of the immunologic defect is often provided by the kind of infection. In general, patients with impaired humoral immunity have an increased incidence of recurrent infections with high-grade encapsulated bacterial pathogens, such as Pneumococcus and Hemophilus influenzae, that lead to chronic sinopulmonary infection, meningitis, and bacteremia. When cellular immunity is intact in such patients, they have less severe problems with fungal and viral infections. Abnormalities of T cells and thus of cell-mediated immunity predispose individuals to infection by a wide variety of agents, including viruses that lead to disseminated infections, particularly herpes simplex, varicella zoster, and cytomegalovirus; fungi that lead especially to mucocutaneous candidiasis; and parasitic organisms, including the protozoan Pneumocystis carinii. Infection, of course, can cause as well as result from immunodeficiency. A variety of infectious agents, such as the human immunodeficiency

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Biologic Markers in Immunotoxicology virus (HIV), can have specific and nonspecific effects on the immune system. Many tests have been developed to assess immunity (Bentwich et al., 1982, 1988; Rosen et al., 1986). A systemic approach to the evaluation of immunocompetence, which is based on simple screening procedures followed by appropriate specialized tests of immune function, usually permits the definition of the immune defect. Such an approach should include evaluation of the humoral immune system (B-cell system), of the cellular immune system (T-cell system), and of nonspecific resistance (polymor-phonuclear leukocytes, natural killer cells, immune complement). It should be emphasized that although some exogenous agents can alter several elements of the human immune system, others have a primary effect only on a single element. For example, low doses of cyclosporin A selectively affect T cells by acting on lymphokine (interleukin-2) production. Conversely, anticonvulsive drugs, such as phenytoin, act primarily on the humoral immune system, leading to selective deficiency of IgA. Many of the screening tests were developed to define the position of the defect in the events of cellular maturation and regulatory cellular interaction that lead to profound hereditary immunodeficient states. These tests are not sensitive enough to detect modest immunodeficiency caused by toxic agents. It should be emphasized that normal individuals show a wide range of responses in the tests discussed below. Thus, when studying one person, it should not be concluded that a modest variation from the normal range for an immunologic test is caused by a putative toxic agent. There are numerous testing methods and variations for many immunologic factors and these are constantly evolving. Many of the standard tests are discussed by Reese and Betts (1991) and Aloisi (1988). TESTS OF THE HUMORAL IMMUNE SYSTEM The evaluation of the human humoral immune system involves the measurement of serum concentrations of immunoglobulins, the assessment of antibody formation after immunization, the measurement of "natural antibodies," and the enumeration of circulating B cells. Immunoglobulin Concentration There are five major classes of immunoglobulin: IgM, IgG, IgA, IgD, and IgE. There are two subclasses of IgA: 1 and 2; and four subclasses of IgG: 1-4. Several methods are available for measuring serum immunoglobulin concentration, including single-radial diffusion, double diffusion in agar gel, immunoelectrodiffusion, radiommunoassay, enzyme-linked immunosorbent assay (ELISA), and automated laser nephelometry. Electrophoresis is not satisfactory for the quantitation of immunoglobulins, but it is useful in the detection of monoclonal immunoglobulins (M-components). The single-radial-diffusion assay is widely used. Gel diffusion methods are very sensitive to differences in diffusion constants and thus to differences in molecular size. It is not possible to measure the concentration of immunoglobulin in body fluids unless the molecules measured in the fluid are the same size as those in the standards. Thus, reliable measurements cannot be made of such proteins as low-molecular-weight IgM in abnormal plasma or IgA in external secretions—where it appears as a dimer rather than as the monomer present in the standard sera—unless special standard preparations that contain immunoglobulins of the same size are used. Furthermore, the use of goat or sheep antisera can give spuriously high estimates for serum IgA in patients with selective IgA deficiency because many of

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Biologic Markers in Immunotoxicology these patients have circulating antibodies to the ruminant proteins. The serum concentration of each of the major immunoglobulin classes, with the exception of IgD, which appears predominantly on the cell surface, should be determined. Furthermore, IgG subclass determinations are of increasing importance. The determinations, however, must be well standardized because antisera vary in quality. Because serum immunoglobulin concentrations vary with age and environment, appropriate norms must be used. Patients can manifest a deficiency in all immunoglobulin classes (common variable hypogammaglobulinemia or X-linked lymphoproliferative syndrome after infection by Epstein-Barr virus) or, alternatively, they can have a deficiency of only a single class (IgA in selective-IgA deficiency as a primary defect or after phenytoin therapy). They can even have reduced amounts of only a single subclass (IgG2 deficiency in some persons who have recurrent Hemophilus influenzae infections). The concentration of immunoglobulins cannot be used as the sole criterion for the diagnosis of immunodeficiency. Diminished immunoglobulin concentrations can result from loss into the gastrointestinal tract as well as from decreased synthesis. An indication of loss can be obtained by measuring serum albumin, which usually is lost concomitantly. Normal total immunoglobulin concentrations or even total immunoglobulin subclass concentrations do not exclude humoral immune deficiency. The response to antigenic stimulation with both protein and polysaccharide antigens must be defined if immunodeficiency is strongly suspected. Failure to respond to one or more classes of antigens has been observed in patients with normal or high levels of immunoglobulin classes, regardless of whether they have isolated immunoglobulin class or subclass deficiency. Specifically, patients with the Wiskott-Aldrich syndrome can exhibit normal or even elevated immunoglobulin concentration, yet have multiple infections because of their failure to make specific immune responses, especially when they are exposed predominantly to polysaccharide antigens. Antibody Formation Antibody-mediated immunity (humoral immunity) can be assessed by specific antibody responses to specific antigens to which the population is commonly exposed. Humoral immunity after immunization can be assessed the same way. Protein and polysaccharide antigens should be used. Live vaccines (bacillus Calmette-Guérin) including those for poliomyelitis, rubella, and mumps should not be given when a profound primary or secondary immunodeficiency is suspected. Tests for the following are recommended: Natural Antibodies: Isohemagglutinins are natural antibodies to blood group A or B antigens found in all normal individuals except those with type AB red cells. By 3 years of age, 98% of normal persons with type A, B, or O blood have isohemagglutinin titers of at least 1:16. Patients with the Wiskott-Aldrich syndrome can have normal immunoglobulin levels yet lack isohemag-glutinins as an indicator of their antibody-deficient state. Other natural antibodies that can be assayed include heterolysins (e.g., against sheep or rabbit red blood cells), antistreptolysin, and bactericidal antibodies against Escherichia coli. Antibody Response to Immunization: Commercial diphtheria-tetanus (DT) vaccine can be given in recommended doses. Blood is taken 2 weeks after each injection and tetanus and diphtheria antibodies are determined. Three doses of killed poliomyelitis vaccine (1.0 mL intramuscularly, at intervals of 2 weeks) can be used. Blood is taken 2 weeks after the last injection and antibody is

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Biologic Markers in Immunotoxicology usually determined by virus neutralization. In patients who have been immunized with DT or diphtheria-pertussis-tetanus (DPT) vaccine, one booster injection is given, followed by determination of antibodies. Additional Active Immunization: Polyribose phosphate (PRP), the Hemophilus influenzae capsular polysaccharide, is a potent but harmless antigen. A single dose (0.05 mg subcutaneously) is sufficient to immunize a healthy person. Immunization of young children with PRP conjugated to a protein carrier is now becoming a standard practice. Consequently, to measure antibody responses purely to carbohydrate antigens, pneumococcal or meningococcal polysaccharides free of carrier proteins should be used. Blood is drawn after 2 weeks and antibody is determined. These and other pure polysaccharides are not useful (and could be contraindicated) in children under 1 year of age. Furthermore, interpretation of results in children under 5 years is difficult. Keyhole limpet hemocyanin (KLH) is another useful protein antigen. B Cells B cells are counted by immunofluorescence detection of membrane-bound immunoglobulin or B-cell = specific antigens (CD19 and CD20). Monocytes can be distinguished from B cells by peroxidase or esterase staining, by ingestion of IgG-coated latex particles, or by the use of monoclonal antibodies specific for monocytes. Precursor B cells can be identified among bone marrow cells with purified fluorochrome-labeled antibodies to detect cytoplasmic µ heavy chains in cells that do not have demonstrable surface immunoglobulin or cytoplasmic light chains. Further information about the nature of the defects in immunoglobulin production can be obtained by the use of in vitro immunoglobulin biosynthesis studies. Several such procedures have been developed to study defects in the maturation of B cells into immunoglobulin-secreting plasma cells. Although some antigen-specific systems have been proposed, in most cases, the peripheral blood mononuclear cells from the patient to be studied are cultured with a polyclonal activator of B cells, such as pokeweed mitogen or Staphylococcus aureus Cowen strain activator. Immunoglobulins synthesized and secreted by the plasmacytoid cells generated can be determined by specific radioimmunoassay or ELISA of the supernatant fluid or by defining the number of immunoglobulin-secreting cells by a reverse hemolytic plaque assay. Modifications of the reverse hemolytic plaque assay have been used in attempts to define helper and suppressor T-cell activity. A description of the details of any specific approach is beyond the scope of this report, and there is no consensus that any one procedure reflects in vivo regulatory T-cell function in all cases. CELLULAR IMMUNE SYSTEM Several tests are commonly used to assess cell-mediated immunity, including those that enumerate T cells and T-cell subsets, identify delayed skin reactions, and measure in vitro stimulation of lymphocytes to proliferate and form blast cells. Other in vitro tests measure T-cell effector or regulatory function. As is the case for humoral immunity, a series of simple tests is available to screen for defects in cell-mediated immunity. A white blood cell count and differential should be obtained, and the absolute lymphocyte count should be calculated by multiplying the total white cell count by the percentage of lymphocytes. Children have higher absolute lymphocyte counts than adults do, so age must be considered in evaluating lymphocyte numbers. Lymphocyte counts consistently below 1,500/mm3 indicate lymphocytopenia and can signify a defect in the T-cell system. The proportion of circulating T cells in the mononuclear cell preparation can be determined

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Biologic Markers in Immunotoxicology by the sheep red blood cell rosette method or, more usually, by immunofluorescence with the use of CD2 or CD3 monoclonal antibodies. Normally, T cells constitute 55-80% of peripheral blood lymphocytes. Normal values reported for absolute numbers of circulating T cells are 1,620-4,320/mm3 for the first week to 18 months of life and 590-3,090/mm3 after 18 months of age (Fleisher et al., 1975). Some sets of T cells have been defined through the use of monoclonal antibodies. The association of a particular T-cell subset defined with a monoclonal antibody with a given function has caused some confusion in the analysis of immunologic data in immunodeficiency states. For example, CD4-positive cells have commonly been associated with helper functions and CD8 cells have been associated with cytotoxic functions. This dichotomy is an oversimplification. The CD4 population has been shown to contain not only helper cells but also memory cells and cytotoxic cells for targets bearing class II MHC (major histocompatibility complex) molecules and suppressor-inducer cells (Engleman et al., 1981; Meuer et al., 1983). The CD8 population contains cells that can recognize antigen presented by macrophages, and they can augment and amplify the interaction of CD4 cells with B cells. Thus, describing CD4 cells as helper cells and CD8 cells as suppressor-cytotoxic cells is not justified. More important, it has become clear that the CD4 cells and CD8 cells recognize foreign antigens in the context of distinct major histocompatibility antigens. CD4 cells recognize antigen in the association with class II MHC human leukocyte antigen, (HLA-D) molecules, and CD8 cells recognize antigens in association with class I MHC (HLA-A, HLA-B, and HLA-C) (Engleman et al., 1981; Meuer et al., 1983). Abnormalities in the number of CD4 or CD8 cells can be associated with abnormalities in the ability to recognize antigens and regulatory functions of T cells that can lead to immunoincompetence or to autoimmunity. Skin Testing The ability of patients to manifest preexisting T-cell immunity has been evaluated in vivo using a series of skin test antigens that normally produce a response. The prototype is the tuberculin skin test. Because delayed cutaneous hypersensitivity, a localized immunologic skin response, depends on functional thymus-derived lymphocytes, it is used in screening for T-cell-mediated immunodeficiency. The antigens generally used are mumps, trichophyton, purified protein derivative (PPD), Candida or Monilia, tetanus, and diphtheria. At least five of the antigens listed below must be used to ascertain defective cell-mediated immunity. All skin tests are by intradermal injection of 0.1 mL of appropriate dilutions of the antigen and should be read in 48-72 hours for maximal diameter of induration. A negative test is not informative in young children because they might not have acquired immunity. Tuberculin: 0.1 mL, containing 2-10 international units (IU) of Tween-stabilized soluble PPD. If negative, the test should be repeated using 50 IU. Candida or Monilia: Initially test at 1:100 dilution. If no reaction, test at 1:10 dilution. Trichophyton: Use at 1:30 dilution. Mumps: Use undiluted; read at 6-8 hours for early Arthus reaction (antibody mediated) and then at 48 hours for delayed cellular hypersensitivity. Tetanus and diphtheria fluid toxoids: Use at 1:100 dilution. KLH: Use 100 µg in 0.1 mL intradermally 2 weeks after immunization with 2.5 mg KLH subcutaneously. In Vitro Stimulation of Lymphocytes A common test of lymphocyte function is

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Biologic Markers in Immunotoxicology to determine the capacity of such cells to enlarge and convert into blastlike cells that synthesize DNA and incorporate thymidine after in vitro stimulation. Lymphocytes can be activated by mitogens, such as phytohemagglutinin (PHA), concanavalin A (Con A), or pokeweed mitogen (PWM). PPD, candidin, streptokinase, tetanus, and diphtheria also can activate lymphocytes if the patient has already encountered these antigens. Allogeneic cells used in the one-way mixed-lymphocyte culture (MLC) and antibodies to T-cell surface molecules, such as CD3, CD2, and CD43, involved in signal transduction also stimulate T-cell proliferation. T-cell lymphocytic blastic transformation can be assessed directly by measuring blastogenesis and proliferation of cells; expression of activation antigens such as CD69 or CD25, the IL-2 receptor; and release of mediators. The blastogenic response is assessed by 3H-or 14C-labeled thymidine incorporation for 16-24 hours, followed by DNA extraction techniques or cell precipitation on filter paper and subsequent liquid scintillation counting. The interpretation of mitogenic responses to various stimuli must be made with caution regarding the type of responding cell. For example, PHA stimulates T cells but it also can stimulate B cells when it is bound to particulate matter. PWM stimulates a response in T and B cells, although T cells must be present for B cells to be stimulated. The MLC reaction is the result of T-cell reactivity to MHC-encoded peptides displayed on the surface of B cells and monocytes. It should be noted that the T cells in the population of normal irradiated or mitomycin-C-treated lymphocytes used as the stimulators can secrete factors that induce blastogenesis by the patient's lymphocytes. Because this can be misleading, it is preferable to use B-cell lines or T-cell-depleted normal cells as the stimulators. Activated T cells express IL-2 receptors, transferrin receptors, and class II MHC molecules not present or present in low numbers on resting T cells. T-cell populations to be assessed for their capacity to express these receptors are stimulated with a soluble lectin, such as PHA, and examined 3 days after stimulation by direct or indirect immunofluorescence using monoclonal antibodies to the IL-2 (CD25) or to transferrin receptors or to class II MHC molecules. For indirect immunofluorescence, an irrelevant mouse monoclonal immunoglobulin and a fluorochrome-labeled antimouse immunoglobulin are used as a control for potential Fc binding of mouse monoclonal cells. Fc is the fragment of an antibody that binds to antibody receptors on cells and to C1q, the subunit of the first component of complement. Activated T cells and monocytes synthesize and secrete IL-2, -4, -5, -6, -7, -8, interferon, and other cytokines. The supernatants of peripheral-blood mononuclear cells stimulated by soluble PHA can be assessed for IL-2 by determining their capacities to stimulate 3H-thymidine uptake by mouse IL-2-dependent, cultured T-cell lines. There are specific in vitro systems to assay the other cytokines. OTHER TESTS There are several assay systems for biologic markers of nonspecific immunity. As noted before, all patients should have an absolute peripheral white blood cell count as well as a differential test to define the proportions of the white blood cell types. Lymphocytopenia can be associated with primary immunodeficiency diseases but can also occur secondarily to viral infections, malnutrition, stress, and autoimmune diseases or to hematopoietic malignancy. Neutropenia has many causes and often is associated with bacterial abscesses. Bone marrow aspiration or biopsy is important for exclusion of other diseases, for identification of plasma cells and pre-B cells, and for diagnosis of obscure infections. NK (natural killer) cells are large

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Biologic Markers in Immunotoxicology granular lymphocytes. They are cytotoxic cells that are effective without prior sensitization. The proportion of NK cells can be identified with appropriate monoclonal antibodies, including CD16, which identifies a protein of 50-60 kilodaltons (kD) molecular weight on large granular lymphocytes and granulocytes and CD57. Functional assays of NK activity involve the ability of the appropriate mononuclear cells to kill specific NK targets, such as the K562 cell in which the cell-mediated cytolysis in vitro is quantitated by 51Cr release from the target cells. Apart from neutropenia, there are defects of phagocytic function that affect polymorphonuclear or mononuclear phagocytes. Neutrophil function depends on movement in response to chemotactic stimulus, adherence, endocytosis, and killing or destruction of the ingested particles. Phagocyte mobility depends on the integrity of the cytoskeleton and contractile system. Directional mobility can be mediated by receptors. Endocytosis depends on the expression of membrane receptors for IgG, C3b, and iC3b and on the fluidity of the membrane. Defects in intracellular killing of ingested microorganisms usually result from failure of the ''respiratory burst" that is critical to production of superoxide radicals and hydrogen peroxide. The organisms cultured from lesions of patients with this type of defect are generally catalase positive and include staphylococcus, Escherichia coli, Serratia marcescens, fungi, and nocardia. Patients with defects in mobility and in adherence and endocytosis usually have infections of the skin, periodontitis, and intestinal or perianal fistulae. On the other hand, patients with normal endocytosis and defective killing tend to have chronic granulomas. The measurement of the nitroblue tetrazolium dye reduction by actively phagocytosing leukocytes has been accepted as a standard measure for the adequacy of the respiratory burst. Assays for bacterial killing yield highly variable results, depending on the bacterial species used in the assay. Chemotaxis and contractibility of phagocytes can be assessed. The classic complement system consists of nine components (C 1-9) and a series of regulatory proteins (C1 inhibitor, C4 binding protein, and properdin factors H and I). Many biologic activities important in the inflammatory response and in host resistance to infection take place at various points in the classical or alternative pathways of complement activation. Three clinical states should raise the suspicion of a deficiency of a complement component: systemic lupus erythematosus, recurrent infections of the type seen in hypogammaglobulinemia in a patient with normal immunoglobulin levels, and severe neisserial infection. In the laboratory, the measurement of serum hemolytic complement (CH50) is an important test. In inherited complement deficiencies, with the exception of hypercatabolism of C3, serum hemolytic complement is usually absent and rarely above 10% of the normal value. More detailed analysis of complement components requires functional and antigenic measurements of the individual components, usually best performed in laboratories that focus on the complement system. OPPORTUNITIES FOR DEVELOPMENT OF BIOLOGIC MARKERS THAT ASSESS THE EFFECT OF IMMUNOTOXICANTS Primary Humoral Immune Responses Although the tests for humoral, cellular, and nonspecific immunity have been of great value in studying profound hereditary immunodeficiency states, they are not sensitive enough to meaningfully detect modest immunodeficiency in populations of individuals exposed to immunotoxic agents. Furthermore, the available tests usually are directed toward evaluating a complete immune response, such as cell-mediated immunity, without defining the nature of the immunologic

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Biologic Markers in Immunotoxicology damage (antigen recognition, lymphokine production, lymphokine receptor expression, effector response). Finally, most of the available procedures, including tests for serum immunoglobulin levels, isohemagglutinin titers, response-to-recall skin test antigens, and proliferative responses to recall antigens, tend to assess the capacity of the individual to make a secondary recall response rather than to make a primary response to a new antigen. The ability to develop an immune response to a new agent is a more sensitive measure of immune impairment than is the inducement of secondary immune response. Furthermore, a test that requires a primary immune response permits a thorough analysis of the events of antigen processing and initial recognition. This section discusses the scientific basis for finding biologic markers that can be used in assessing the effects of toxic agents on the immune system. The primary response to KLH is one such marker. The individual to be studied is immunized subcutaneously with 2.5 mg of KLH. Two weeks later, delayed hypersensitivity responses are assessed in an intradermal skin test with 100 µg KLH. The antibody response to KLH is determined in serum obtained 2 weeks after the KLH immunization. It would be of value to develop other agents for primary immunization to extend the repertoire beyond KLH. However, the subcommittee does not recommend the use of dinitrochlorobenzene for skin testing because it is mutagenic and causes necrosis. Activation Antigens on Lymphocyte Surfaces and in Serum The success of the human immune response requires that T and B cells change from a resting to an activated state when resting cells first encounter a foreign pathogen. Appropriately processed and presented antigens react with antigen receptors on the lymphocyte surfaces to trigger T-or B-cell activation. As part of the activation step, the lymphocyte expresses an array of cell surface antigens that are, for the most part, induced receptors for growth factors. These are not found on the surface of resting cells. The activation antigens that have been extensively studied on the surface of T cells include the 55-kD IL-2 receptor peptide defined by CD25 antibodies, the transferrin receptor, the insulin receptor, and class II MHC molecules (Waldmann, 1986). Additional activation antigens have been identified, including those designated CD30 and CD69 by the Nomenclature Committee of the Fourth Conference on Human Leukocyte Differentiation Antigens (Knapp et al., 1989). CD69 identifies a homodimer that appears within the first few hours after lymphocyte activation. B and T cells display surface activation. For example, the antigen defined by CD23, the low-affinity Fc-∈ receptor is absent on resting cells but is expressed on activated B cells. The ability of a person to express specific growth factor receptors on appropriately activated B and T cells can be determined by immunofluorescence using appropriate specific monoclonal antibodies in conjunction with other studies of the in vitro stimulation of lymphocytes. For example, PHA-stimulated peripheral blood mononuclear cells can be assessed with a CD25 antibody 48-72 hours after stimulation with PHA for the cells' expression of the 55-kD peptide of the IL-2 receptor. A series of cell surface receptors is released into the body's fluids, including the serum. The concentrations of the released receptors in the serum can be assessed using an appropriate ELISA technique with two monoclonal antibodies that recognize distinct epitopes on the peptide. Released forms of the activation antigens (antigens that are expressed on activated but not normal resting cells), the 55-kD IL-2 receptor (CD25), CD8, CD23, CD30, and the transferrin receptor have been identified in the serum (Rubin et al., 1985). The levels of some of these receptors in the serum have been

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Biologic Markers in Immunotoxicology correlated with antigenic exposure and with specific disease states. For example, normal individuals have measurable amounts of IL-2 receptors in their serum, and some patients with lymphoreticular malignancies or selected autoimmune diseases and individuals receiving allografts have elevated serum levels of this receptor. The cell surface expression and subsequent release into the body fluids, including serum, of soluble IL-2 receptors appear to be a consequence of cellular activation of various cell types that could play a role in the regulation of the immune response. Thus, the analysis of serum levels of IL-2 receptors and other activation antigens could provide a new approach to the in vivo analysis of lymphocyte activation. Theoretically, elevated levels of these activation antigens result from exposure to viruses and chemicals that are toxic to the immune system. Synthesis and Secretion of Lymphokines After Lymphocyte Activation After activation, the T and B cells in peripheral-blood mononuclear cells express the genes that encode a series of lymphokine molecules (IL-1 to IL-10) and colony-stimulating factors. As a consequence, the cells synthesize and secrete measurable quantities of these lymphokines that are involved in the control of T and B cells and in eosinophil and basophil growth and differentiation. Biologic assays (such as IL-2-dependent, cultured-T-cell line proliferation in response to IL-2), radioimmunoassays, and ELISA procedures have been developed to quantitate the concentration of lymphokines and colony-stimulating factors. Furthermore, with the molecular cloning of genes that encode each of these lymphokines, one can quantitate messenger RNA transcription for each of the lymphokines after appropriate lymphocyte activation. In general, for the lymphokines produced by T cells, peripheral-blood mononuclear or T-cell populations are activated with Con A, PWM, or insolubilized CD3 antibodies, and then the appropriate assays are used to quantitate the specific lymphokines produced and secreted into the media. Different patterns of lymphokine secretion have been observed in different subsets of T cells, with γ interferon and IL-2 produced by one subset and IL-4 and IL-5 by another subset of long-term cloned CD4 T-cell lines from mice. Thus, an assay of the pattern of increased lymphokine production could be of value in pinpointing the action of an immunotoxicant on a particular subset of immune-system cells. Furthermore, one can see depressed production of a lymphokine at doses of an immunomodulatory agent that do not affect other aspects of the human immune response. For example, at appropriate doses cyclosporin A inhibits the production of IL-2 without abrogating the induction of IL-2 receptor expression. Proliferative Responses to Super-Antigens A series of antigens, the "super-antigens," including Staphylococcus enterotoxin A and Staphylococcus enterotoxin B, have been defined by White et al. (1989). These antigens are recognized by all T cells that use a particular variable-region β-T-cell receptor. For example, Staphylococcus enterotoxin is recognized by subsets of murine T cells that use the Vß3 and Vß8 T-cell receptor genes, whereas Staphylococcus enterotoxin A is recognized by populations of Vß11-bearing T cells. The enterotoxins are called superantigens because they can interact with large numbers of T-cell receptors based only on Vß usage and because of a relaxed MHC restriction pattern of recognition. Used in proliferation assays, these agents could be of value in assessing the effect of xenobiotics on the human cellular immune system.

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Biologic Markers in Immunotoxicology Self-HLA-Restricted Cell-Mediated Cytotoxicity Immunization with antigens elicits cytotoxic T-lymphocytes (CTLs), which are antigen-specific and restricted to lyse target cells that share MHC gene products with the CTLs. The CTLs are believed to be important in recovery from viral infections. T cells develop receptor specificity for the host MHC gene products expressed on the thymic epithelium prior to antigenic exposure. T-cell receptors that express specificities for foreign antigens and MHC class I gene products are selected when an antigen is encountered on the host's antigen-presenting cells. The maturation of such CTLs requires helper T (amplifier) cells and could be regulated by suppressor T cells. Self-restricted CTLs specific to influenza A-Hong Kong virus can be generated in vitro from human peripheral blood mononuclear cells of patients who have been exposed to influenza virus. The generation of CTLs depends on T cells and monocytes in culture. The peripheral-blood mononuclear cells can be assessed for the patient's capacity to generate self-HLA-restricted, cell-mediated cytotoxicity to viral antigens. When this approach is used in patients with common variable hypogammaglobulinemia, the capacity to produce virus-specific self-restricted CTL is variable but usually relatively normal. In contrast, patients with the immunodeficiency states, ataxia-telangiectasia and the Wiskott-Aldrich syndrome, are unable to produce a significant increase of virus-specific CTLs in vitro. CTL enumeration might be of value as a biologic marker for assessing the effect of immunotoxic agents on the human immune system. TABLE 7-1 Tier 1 (All Persons Exposed to Immunotoxicants) I. Humoral immunity   Immunoglobulin class concentrations in serum (IgM, IgG, IgA, IgE) and immunofixation electrophoresis.   Natural immunity: Antibody levels to ubiquitous antigens (e.g., anti-A and anti-B group substances in individuals of non-AB blood type).   Secondary antibody responses to proteins (e.g., diphtheria, tetanus, poliomyelitis) and polysac-charides (e.g., pneumococcal, meningococcal).   Note: In immunization studies, live microorganisms should not be given to persons suspected of being severely immunocompromised. II. Lymphocytes   Enumeration of B and T cells in blood.   Surface analysis of CD3, CD4, CD8, CD20.   Secondary delayed-type hypersensitivity reaction (e.g., candida, diphtheria, tetanus).   Alternative: Multiple antigen skin test kit. III. Autoantibody titers (to red blood cells, nuclei [ANA], DNA, mitochondria, IgE [rheumatoid factor]).

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Biologic Markers in Immunotoxicology PROPOSED TESTING REGIMEN Because the immune system has a large functional reserve, toxic damage is likely to be measurable only after significant impairment. Nevertheless, it is possible to propose a series of well established or relatively well established procedures that can be used to follow individuals or groups known or suspected to have been exposed to an immunotoxicant or potential immunotoxicant. Other less well established but promising tests can be expected to become available and should be considered according to circumstances. All tests should be performed once or twice a year by a physician on persons who are suspected of exposure to immunotoxicants so that latency and changes over time can be assessed. Finally, aliquots of fresh serum and pelleted white blood cells should be stored at -70°C for each individual studied at each time. For group studies, a tiered approach, a series of tests done in sequence, is recommended. Tier 1 (Table 7-1) is proposed for evaluation of individual persons exposed or potentially exposed to an immunotoxicant. The combined Tier 1 and Tier 2 (Table 7-2) TABLE 7-2 Tier 2 (All Persons with Abnormal Tier 1 Test Results and a Fraction of the Total Exposed Population To Be Determined by Statistician) I. Humoral Immunity   Primary antibody response to protein and polysaccharide antigens.   Note: There is a need to develop a panel of antigens that can be used in sequential studies on a given individual since a particular antigen can be used only once to assess a primary response. II. Cellular immunity   Proliferative response to mitogens (PHA, Con A) and possible antigens such as tetanus.   Primary DTH reaction (KLH).   Note: Here, too, there is a need for a panel of standard antigens for sequential testing.   These could be the same as those used to assess primary antibody responses. III. NK cells, monocytes, and other T-and B-cell markers   CD5, CD11, CD16, CD19, CD23, CD64; class II MHC on T cells by two-color flow cytometry for coexpression of class II and a T-cell marker such as CD3. IV. Serum levels of cytokines (e.g., IL-1, IL-2, IL-6) and of shed or secreted cellular activation markers and receptors (e.g., CD25). V. Class I and II MHC antigen typing.

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Biologic Markers in Immunotoxicology regimens should be followed in those exposed and showing abnormalities in Tier 1. Tier 3 (Table 7-3) should be considered for those persons who exhibit abnormalities in Tier 2 tests or for a random fraction of population tested using Tier 2. SUMMARY Toxic agents can injure the immune system in any of its broad capabilities. Affected persons run the risk of developing diseases, such as serious infections and neoplasia. A broad series of tests has been developed to assess humoral (antibody mediated) and cellular (T-cell mediated) immunity as well as nonspecific resistance (mediated by NK cells, complement, etc.). A testing regimen that involves a series of currently recommended assays is proposed that can be used to follow individuals or groups known or suspected to have been exposed to a potential immunotoxicant. A tiered approach is proposed, beginning in Tier 1 with a series of simple tests to be done on all individuals to screen for immunodeficiency. In this regimen, Tier 2 tests would be performed on individuals with abnormal Tier 1 tests and on a fraction of the total exposed population. Tier 3 would be used for those who test positive in Tier 2 and for a random fraction of the Tier 2 population. Although the tests for humoral, cellular, and nonspecific immunity have great value in studies of profound hereditary immunodeficiency states, they have not been evaluated for their ability to detect more modest immunodeficiencies that might be observed in individuals exposed to immunosuppressive immunotoxic agents. Therefore, the tests for immune-system biologic markers that have been proposed here for risk assessment should be validated prospectively in populations exposed to putative immunotoxicants and in control groups for their ability to predict the development of disease associated with immunodeficiency. To accomplish this, efforts should be made on an individual patient basis to establish the degree of exposure to specific immunotoxicants. This could be done with retrospective exposure analysis, but the use of markers of exposure—blood concentrations and tissue concentrations—might be a more TABLE 7-3 Tier 3 (To Be Considered for Those with Abnormalities in Tier 2 Tests or for a Random Fraction of the Entire Population in Tier 2) If a proportion of CD 16 cells of Tier 2, III is abnormal: nonspecific killing of a tumor cell line to test for NK function. If primary DTH reaction in Tier 2, II is abnormal: cell proliferation in response to phorbol ester and calcium ionophore, anti-CD3 antibody, and a staphylococcal enterotoxin B (experimental). Generation of secondary cell-mediated immune reactions (proliferation and MHC-restricted cytotoxicity) in vivo, e.g., with influenza virus (experimental). Immunoglobulin subclass levels in serum (IgA1, IgA2, IgG1-4). Antiviral titers (e.g., influenza, parainfluenza, cytomegalovirus, HIV) in serum (no deliberate immunization).

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Biologic Markers in Immunotoxicology appropriate approach. Nevertheless, it is imperative that the dose-response relation ship be examined. RECOMMENDATIONS Because available tests can lack the sensitivity required to detect modest immunodeficiency, a major focus should be on devising more sensitive tests for markers of immune impairment. Biologic marker tests that focus on the ability to develop a primary response to a new antigen should be developed and validated. Such tests could be more sensitive than are the available tests that examine secondary recall responses. Furthermore, a test requiring a primary immune response permits an analysis of the events of antigen processing and recognition. Tests should be developed that examine the expression of activation antigens, including lymphokine receptors (such as CD25, the IL-2 receptor) expressed on the surface of lymphocytes. An analysis of serum concentrations of the released form of these antigens also should be performed. Theoretically, the expression of such cell surface molecules that are not present on normal resting cells but are expressed following lymphocyte activation could be induced as a consequence of exposure to immunotoxicants.

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