Sunday, July 27, 2008

Clinical Immunology

CLINICAL IMMUNOLOGY

(Current Medical Diagnosis & Treatment 2008 )

Cells Involved in Immunity

Development of T & B Lymphocytes

Thymus-derived cells (T lymphocytes) mediate cellular immune responses; bone marrow-derived cells (B lymphocytes) are involved in humoral immunity. Both T and B lymphocytes are derived from precursor or stem cells in the marrow. Precursors of T cells migrate to the thymus, where they develop some of the functional and cell surface characteristics of mature T cells. Through positive and negative selection, clones of autoreactive T cells are eliminated, and mature T cells migrate to the peripheral lymphoid tissues. There they enter the pool of long-lived lymphocytes that recirculate from the blood to the lymph.

B cell maturation proceeds in antigen-independent and antigen-dependent stages. Antigen-independent maturation includes development from precursor cells in the marrow through the naive B cell (a cell that has not been exposed to antigen previously) found in the peripheral lymphoid tissues. Antigen-dependent maturation occurs following the interaction of antigen with naive B cells. The final products of B cell development are circulating long-lived memory B cells and plasma cells found predominantly in primary follicles and germinal centers of the lymph nodes and spleen. Plasma cells are terminally differentiated B cells responsible for synthesis and secretion of immunoglobulin.

Subpopulations of T Cells

T lymphocytes are heterogeneous with respect to their cell surface features (Table 1) and functional characteristics. At least three subpopulations of T cells are now recognized.

Table 1. Selected Surface Antigens on Immune Cells Detected by Monoclonal Antibodies.

Cluster of Differentiation

Primary Cellular Distribution

Function

CD2

T cells, NK cells

Adhesion molecule

CD3

Pan-T cell marker

T cell receptor

CD4

T helper-inducer cells, macrophage

Binds to MHC class II

CD5

T cells, B cell subset


CD7

T cells


CD8

T cytotoxic-suppressor cells

Binds MHC class I

CD10

Immature B cells

CALLA; also found in ALL

CD11a

Leukocytes

Adhesion molecule

CD11b



CD11c



CD13/33

Granulocytes

Granulocyte marker

CD14

Monocytes

Monocyte marker

CD16/56

NK cells

NK cell markers; CD16 is low-affinity Fcγ receptor

CD19

Pan-B cell marker

Appears early in B cell maturation

CD20/21/22

B cell markers

Appear after CD19; CD21 is complement receptor (CR2)

CD23

Activated B cells, macrophages

Low-affinity Fcε receptor

CD25

Activated T, B cells and macrophages

IL-2 receptor; activation marker

CD28

T cells

Costimulatory receptor

CD34

Hematopoietic progenitor cells

"Stem cell" marker

CD38

Plasma cells


CD45

Leukocytes

Panleukocyte marker

CD45RA/RO

T cells

CD45RA on "naive" T cells; CD45RO on "memory" T cells


Helper-Inducer T Cells

These cells help amplify the production of antibody-forming cells from B lymphocytes after interaction with antigen. Helper (CD4) T cells also amplify the production of effector T cells that mediate cytotoxicity. Activated CD4 T cells regulate immune responses by two mechanisms: through cell-to-cell contact and through elaboration of soluble factors or cytokines. Two subsets of helper T cells can be identified on the basis of their pattern of cytokine production. The subsets are called type 1 T helper (TH1) cells, which produce interleukin (IL)-2 and gamma interferon; and type 2 T helper (TH2) cells, which produce interleukins-4, -5, and -6, among others. Both subsets produce IL-3 and GM-CSF. The TH1 subset of CD4 T cells provides cellular immune responses to intracellular pathogens and underlies the pathogenesis of delayed-type hypersensitivity.

TH2 helper T cells play a central role in immediate hypersensitivity and humoral immune responses, since IL-4 promotes IgE production and IL-5 is an eosinophil proliferation and differentiation factor (Table 2).

Table 2. Major activities of selected cytokines.

Cytokine

Primary Biologic Activity

IL-1

Major source is activated macrophages. Enhances T and B cell activation. Endogenous pyrogen and major inflammatory mediator.

IL-2

Autocrine and paracrine T cell activation and growth factor.

IL-3

Multilineage hematopoietic growth factor.

IL-4

T and B cell growth factor. Induces IgE isotype switching; TH2 CD4 cell and mast cell growth factor.

IL-5

Promotes eosinophil growth and differentiation and IgA synthesis.

IL-6

B cell differentiation factor; acute phase reactant.

IL-7

Growth factor for very early B and T lymphocytes.

IL-8

Chemotactic factor neutrophils, lymphocytes. Up-regulates adhesion molecule expression.

IL-10

Down-regulates cellular activation. Inhibits production of proinflammatory cytokines by monocytes and macrophages.

IL-12

Augments IFN-γ production, enhances TH1 CD4 response.

IL-13

Functions overlap with those of IL-4.

TNF

Overlaps with activity with IL-1 but has more antitumor activity. Mediates host response to gram-negative bacteria and systemic toxicity of LPS.

IFN-γ, -β, -α

Antiviral and antitumor activities. Activates macrophages. Enhances cytotoxic lymphocyte and NK activity.

GM-CSF

Growth factor for granulocytes, macrophages, and eosinophils. Activates neutrophil phagocytosis. Enhances eosinophil-mediated cytotoxicity. Promotes basophil histamine release.

TGF-β

Major regulatory factor, inhibiting leukocyte growth, proliferation and proinflammatory cytokines.

Cytotoxic or Killer T Cells

These cells are generated after mature T cells interact with certain foreign antigens. They are responsible for defense against intracellular pathogens (eg, viruses), tumor immunity, and organ graft rejection. Most killer T cells express the CD8 phenotype, though in certain circumstances CD4 T cells can be cytotoxic. Cytotoxic T cells may kill their target through osmotic lysis, by secretion of tumor necrosis factor (TNF), or by induction of apoptosis, ie, programmed cell death.

Suppressor T Cells

Suppressor T cells are CD8 regulatory cells that modulate antibody formation and cellular immunity in an antigen-specific manner. It is unclear, however, whether suppressor T cells are a distinct T cell phenotype or if their inhibitory functions merely reflect the profile of the factors they secrete.

B Lymphocytes

The majority of B cells express both IgM and IgD on the surface and are derived from pre-B cells found mainly in the bone marrow. Pre-B cells contain intracytoplasmic IgM but do not express surface immunoglobulin.

B cells have been commonly identified by other surface markers in addition to immunoglobulins. These include the receptor for the Fc portion of immunoglobulins, B cell-specific antigens CD19 and CD20, and surface antigens coded for by the HLA-D genetic region in humans. All mature B cells bear surface immunoglobulin that is the antigen-specific receptor. The major role of B cells is differentiation to antibody-secreting plasma cells. However, B cells may also release cytokines and function as antigen-presenting cells.

Other Cells Involved in Immune Responses

Macrophages

Macrophages are involved in the ingestion, processing, and presentation of antigens for interaction with lymphocytes. These CD14 cells play an important role in T and B lymphocyte cooperation in the induction of antibody responses. In addition, they are effector cells for certain types of tumor immunity.

NK (Natural Killer) Cells

These lymphocytic cells, which are indirectly related to the T cell lineage, can kill a wide spectrum of target cells. They are recognized by the presence of specific surface antigens (CD16 or CD56) and Fc receptors. Many appear as large granular lymphocytes. Their role in host defense is probably the killing of virally infected cells and tumor cells in the absence of prior sensitization and without MHC restriction.

Cytokines

Many T cell functions are mediated by cytokines, humoral factors secreted by immunologically active cells. Cytokines are secreted when cells are activated by antigens or other cytokines. Table 45–2 lists some examples of cytokines and their functions. The cytokines can be functionally organized into groups according to their major activities: (1) those that promote and mediate natural immunity, such as IL-1, IL-6, interferon (IFN)-γ, and IL-8; (2) those that support allergic inflammation, such as IL-4, IL-5, and IL-13; (3) those controlling lymphocyte regulatory activity, such as IL-10, produced by the TH2 helper T cell, and IFN-γ and IL-12, which are produced by the TH1 T helper cell; and (4) those that act as hematopoietic growth factors (IL-3, IL-7, and GM-CSF). This complicated network of interacting cytokines functions to modulate and regulate cellular function in such a way that the host is able to survive in a hostile environment.

Alpdogan O et al. IL-7 and IL-15: therapeutic cytokines for immunodeficiency. Trends Immunol. 2005 Jan;26(1):56–64. [PMID: 15629410]

Hallett WH et al. Natural killer cells: biology and clinical use in cancer therapy. Cell Mol Immunol. 2004 Feb;1(1):12–21. [PMID: 16212916]

Smyth MJ et al. Activation of NK cell cytotoxicity. Mol Immunol. 2005 Feb;42(4):501–10. [PMID: 15607806]

Viau M et al. B-lymphocytes, innate immunity, and autoimmunity. Clin Immunol. 2005 Jan;114(1):17–26. [PMID: 15596405]

Yokoyama WM et al. How do natural killer cells find self to achieve tolerance? Immunity. 2006 Mar;24(3):249–57. [PMID: 16546094]

Tests for Cellular Immunity

Leukocyte Immunophenotyping by Flow Cytometry

Utilizing fluorescent-labeled monoclonal antibodies directed against specific cell surface antigens, or clusters of differentiation (CD), leukocytes can be immunophenotyped and enumerated by flow cytometry. During lymphocyte development, different patterns of CD markers are expressed at various stages of maturation (Table 1). Flow cytometry segregates populations of leukocytes for analysis by cellular size and complexity. By these parameters, polymorphonuclear cells and monocytes can be enumerated and analyzed separately from peripheral blood lymphocyte populations.

In normal individuals, roughly 75% of circulating lymphocytes are T cells (CD3), two-thirds of which are CD4 T helper-inducer cells and one-third CD8 suppressor or cytotoxic T cells. CD19 B cells make up 7–24% of circulating lymphocytes, and the remainder are CD16/CD56 NK cells. Lymphocyte immunophenotyping can be used in suspected cases of immunodeficiency or lymphoproliferative syndromes or following organ transplantation.

Thymic hypoplasia (DiGeorge syndrome) is associated with a marked decrease in the number of T cells in the peripheral blood; the absence of B cells in the blood is a feature of X-linked agammaglobulinemia. Marked reductions of both T and B cells occur in severe combined immunodeficiency disease (SCID). Patients with AIDS have reduced numbers of T cells and reduced CD4:CD8 ratios.

Many hematologic neoplasms can express aberrant CD markers or lose characteristic phenotypic patterns. B cell lymphomas can express inappropriate maturational markers or even T cell- or granulocyte-associated clusters of differentiation. In lymphoproliferative diseases, the suspicious lymphocyte population may demonstrate a monoclonal pattern by bearing surface immunoglobulin of a single isotype (IgM, IgG, or IgA) or a single immunoglobulin light chain (κ or λ). Immunophenotyping may be used in conjunction with histopathologic analysis of bone marrow biopsies and fine-needle aspirates from suspicious lymph nodes or peripheral blood. In poorly differentiated leukemias and lymphomas, it can also provide prognostic information and help optimize treatment regimens.

T Cell Antigen Receptors

The structure of T cell antigen receptors and the genes that encode these glycoproteins have been defined. The receptor structure is a complex of two molecules, one containing variable α/β or γ/β chains and the other the monomorphic CD3 molecule. Genes encoding the β chain are homologous with immunoglobulin genes. Rearrangement of T cell receptor genes proceeds during T cell development to generate diversity for antigen recognition in a fashion similar to that of immunoglobulin genes in B cells. Lymphoid malignancies often feature chromosomal translocations that occur disproportionately in or near the T cell receptor genes. Identification of T cell receptor gene rearrangements has been used to distinguish clonal T cell leukemias and lymphomas from reactive processes.

Functional Testing of Cell-Mediated Immunity

Delayed-Type Hypersensitivity Skin Testing

Cell-mediated immune function can be assessed qualitatively by evaluating skin reactivity following intradermal injection of a battery of recall antigens to which humans are frequently sensitized (ie, streptokinase, streptodornase, purified protein derivative, trichophyton, dermatophyton, mumps, tetanus, or candida). Intradermal injections of 0.1 mL of recommended test strengths are observed for maximal induration and erythema at 24 and 48 hours. A positive reaction varies in size with particular antigens but is generally 5–10 mm in diameter. Anergy or lack of skin reactivity to all of these substances indicates a depression of cell-mediated immunity. Delayed hypersensitivity skin tests depend on complex interactions of T cells, macrophages, and other immunoreactants; thus, failure to respond cannot identify the exact cellular defect.

Patch testing can be clinically indicated for the diagnosis of suspected allergic contact dermatitis. The patch test is performed by topical application of the suspected contactant allergen. A positive test at 48–72 hours consists of erythema, swelling, and papules. Concentrations of allergens for patch testing must be screened in nonallergic subjects to avoid false-positive irritant responses.

In Vitro Lymphocyte Proliferation after Stimulation with Mitogens or Antigens

T lymphocytes are transformed to blast cells upon short-term incubation with mitogens or recall antigens in vitro. Mitogens stimulate lymphocytes nonspecifically. Phytohemagglutinin (PHA), pokeweed mitogen (PWM), and concanavalin A (ConA) can all be used in clinical laboratory assays. T cell activation is determined quantitatively by following the cellular uptake and incorporation of [3H]thymidine introduced into the culture medium. The uptake indicates T cell function and correlates well with other manifestations of cell-mediated immunity as measured by skin tests. These tests can detect abnormalities in T cells despite normal or slightly reduced cell counts, particularly following bone marrow transplantation or in congenital immunodeficiency diseases. Stimulation of recipients' lymphocytes or donor lymphocytes (the mixed lymphocyte reaction) is a critical test for determining histocompatibility, especially after renal and bone marrow transplantation.

Goleva E et al. Factors that regulate naturally occurring T regulatory cell-mediated suppression. J Allergy Clin Immunol. 2005 Nov;116(5):1094–100. [PMID: 16275382] ]

Krogsgaard M et al. How T cells 'see' antigen. Nat Immunol. 2005 Mar;6(3):239–45. [PMID: 15716973]

Xu D. Dual surface immunoglobulin light-chain expression in B-cell lymphoproliferative disorders. Arch Pathol Lab Med. 2006 Jun;130(6):853–6. [PMID: 16740039]

Zingoni A et al. NK cell regulation of T cell-mediated responses. Mol Immunol. 2005 Feb;42(4):451–4. [PMID: 15607797]

IMMUNOGLOBULIN STRUCTURE & FUNCTION

Disorders of immune function are the cause of many human illnesses. The basic unit of all immunoglobulins consists of four polypeptide chains linked by disulfide bonds. There are two identical heavy chains and two identical light chains. Both heavy and light chains have a carboxyl terminal constant (c) region and an amino terminal variable (V) region. A hypervariable portion of the V regions of heavy and light chains folded together in a three-dimensional conformation forms the combining site, which is responsible for the specific interaction with antigen.

Antibodies contain one of five classes of heavy chains (γ, α, µ, σ, and ε) and one of two types of light chains (κ and λ). About 10 million different antibody specificities are thought to exist in a given individual.

Immunoglobulin Classes

Immunoglobulin M (IgM)

The IgM molecule is found predominantly in the intravascular compartment and on the surface of B lymphocytes and does not normally cross the placenta. IgM antibody predominates in early, primary immune responses; carbohydrate antigens such as blood group antigens stimulate IgM.

Immunoglobulin A (IgA)

IgA is present in blood and in relatively high concentrations in saliva, colostrum, tears, and secretions of the bronchi and gastrointestinal tract. Secretory IgA plays an important role in host defense against viral and bacterial infections by blocking transport of microbes across mucosa.

Immunoglobulin G (IgG)

IgG comprises about 70% of total serum immunoglobulins and is distributed in the extracellular fluid; it is the only immunoglobulin that normally crosses the placenta. Antigen-bound IgG fixes complement via the Fc region of the constant chain. Immune effector cells express Fc receptors and complement receptors that facilitate phagocytosis and cytolysis. Immune complex activation of the classic complement pathway also generates soluble factors that chemoattract neutrophils, increase vascular permeability, and amplify the inflammatory response.

Immunoglobulin E (IgE)

IgE is present in serum in very low concentrations as a single immunoglobulin unit with ε heavy chains. Fifty percent of patients with allergic diseases have increased serum IgE levels. The specific interaction between antigen and mast cell-bound IgE results in the release of histamine, leukotrienes, proteases, chemotactic factors, and cytokines. These mediators can produce bronchospasm, vasodilation, increased vascular permeability, smooth muscle contraction, and chemoattraction of other inflammatory and immune cells.

Immunoglobulin D (IgD)

IgD is present in the serum in very low concentrations and is found on the surface of most B lymphocytes in association with IgM, where it probably serves as a receptor for antigen.

Tests for Immunoglobulins

In some diseases, increased serum immunoglobulins, especially monoclonal types, are critical for diagnosis. Chronic liver diseases, chronic infection, or idiopathic inflammatory states can cause polyclonal or oligoclonal increases in immunoglobulins, which are incidental or of unknown significance. If immunodeficiency is suspected in the presence of recurrent bacterial infections, measurement of serum immunoglobulin levels provides an essential test of B cell and plasma cell function. In acquired immune deficiencies such as AIDS, paradoxical increases in immunoglobulins can occur.

Antibodies and immunoglobulins can be measured in three ways: (1) by quantitative and qualitative determinations of serum immunoglobulins; (2) by determination of isohemagglutinin and febrile agglutinin titers; and (3) by determination of antibody titers following immunization with tetanus toxoid, diphtheria toxoid, or pneumococcal polysaccharide vaccines. The first method tests for the presence of serum immunoglobulins but not for the functional adequacy of the immunoglobulins. The second tests for functional antibodies present in the serum of almost all individuals as a consequence of exposure to blood group antigens (ABO blood groups) or infection. The third method examines functional humoral immunity in the serum after intentional immunization.

Protein Electrophoresis & Immunoelectrophoresis

Serum protein electrophoresis is a test to measure semiquantitatively various proteins in serum or urine. Proteins are electrically separated on a strip of cellulose acetate, on the basis of charge, into albumin, α1, α2, β, and γ globulins. This test is useful to screen for diseases with excess or deficiency of immunoglobulins.

Immunoelectrophoresis is used to identify the specific immunoglobulin class in a body fluid. Serum, for example, is separated electrophoretically and then reacted with appropriate antisera directed against IgG, IgA, or IgM. The resulting patterns allow identification of abnormal immunoglobulins such as myeloma (M) proteins. This method is also useful in differentiation of monoclonal from polyclonal increases in immunoglobulins. It is only semiquantitative and thus cannot be used to determine immunoglobulin levels precisely, eg, in Waldenström's macroglobulinemia.

A similar technique called immunofixation electrophoresis has to a large extent replaced immunoelectrophoresis. Serum proteins are separated electrophoretically in a gel and then immunoprecipitated in situ with monospecific antisera. This method has the advantages of more rapid results and slightly higher resolution of low levels of monoclonal immunoglobulin chains. If protein electrophoresis is normal despite suspicion of an M protein, immunoelectrophoresis or immunofixation electrophoresis of both serum and concentrated urine should be performed because of the greater sensitivity of these tests combined.

Quantitative Immunoglobulin Determinations

Quantitative determinations of total serum IgG, IgA, IgE, and IgM levels can be made rapidly and accurately by nephelometry. Nephelometry detects scattered light when specific antiserum immunoprecipitates soluble antigen. Antiserum is available for a variety of antigens, including all immunoglobulin isotypes. Measurement of serum IgD levels has no recognized clinical use, and antigen-specific IgE levels should be measured with more sensitive techniques such as in vitro RAST or in vivo allergy skin testing.

Functional Testing of Humoral Immunity

Laboratory evaluation of humoral function should include assessment of specific antibody responses. Isohemagglutinins are naturally occurring IgM antibodies against ABO blood groups and can be detected in most patients depending on blood type. Humoral responses can also be assessed by immunization with protein and carbohydrate antigens. This is most easily accomplished by measuring anti-tetanus, anti-diphtheria, and anti-pneumococcal antibody titers, before and 3–4 weeks after vaccination with diphtheria-tetanus (dT) and polyvalent pneumococcal vaccine. In this context, a fourfold increase in antibody titer is considered normal. In patients suspected to be suffering from humoral immunodeficiency, quantification of IgG subclasses may be appropriate, particularly when total IgG immunoglobulins are at the low end of the normal range.

Cunningham-Rundles C et al. Molecular defects in T- and B-cell primary immunodeficiency diseases. Nat Rev Immunol. 2005 Nov;5(11):880–92. [PMID: 16261175]

Yonekawa K et al. Targeting leukocyte integrins in human diseases. J Leukoc Biol. 2005 Feb;77(2):129–40. [PMID: 15548573]

Immunogenetics & Transplantation

Genetic Control of the Immune Response

The ability to mount a specific immune response is under the direct control of genes for the major transplantation antigens (major histocompatibility complex; MHC). These MHC molecules play a critical role in the positive and negative selection of thymocytes during T cell development, the process of antigen presentation between immunocompetent cells, and transplantation rejection reactions to foreign tissue. In a rare immunodeficiency disorder, "bare lymphocyte syndrome," the surface expression of HLA molecules is deficient, leading to impaired immune defenses and recurrent infection.

In humans, this genetic region has been designated the HLA complex because these antigens were first detected on peripheral blood lymphocytes. The complex includes antigens HLA-A, -B, -C, -DR, and others, each with many alleles. The HLA region has been localized to chromosome 6 and codes for over 200 genes. In nearly all instances, the HLA complex is inherited intact as two haplotypes (one from each parent), and within any particular family, therefore, the number of different combinations found is 4 (ie, siblings have a 1:4 chance or 25% of being HLA-identical). In contrast, the number of antigen combinations among unrelated individuals is enormous, resulting in probabilities of less than one in several thousand, depending on the phenotype involved, of finding HLA-compatible individuals in a random donor pool. This is particularly important when compatible donors are needed for allosensitized patients requiring platelet transfusions or organ transplantation. Family members have the highest likelihood of being compatible donors, whereas compatibility between unrelated individuals has a low probability. HLA-A and -B typing or cross-matching is used for selection of compatible donors for platelet transfusions to allosensitized, thrombocytopenic recipients. Typing for these class I antigens as well as for HLA-D (class II) antigens is important in determining compatibility for organ transplantation. Typing for HLA markers is of value in studying associations between the HLA system and genetic control of disease susceptibility.

Ferry H et al. B-cell tolerance. Transplantation. 2006 Feb 15;81(3):308–15. [PMID: 16477212]

Fujii S. Application of natural killer T-cells to posttransplantation immunotherapy. Int J Hematol. 2005 Jan;81(1):1–5. [PMID: 15717680]

Rifle G et al. Donor-specific antibodies in allograft rejection: clinical and experimental data. Transplantation. 2005 Feb 15;79(3 Suppl):S14–8. [PMID: 15699738]

Rose ML. Activation of autoimmune B cells and chronic rejection. Transplantation. 2005 Feb 15;79(3 Suppl):S22–4. [PMID: 15699740]

Wood KJ. Is B cell tolerance essential for transplantation tolerance? Transplantation. 2005 Feb 15;79(3 Suppl):S40–2. [PMID: 15699748]

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