Blood Immune System Notes

Immune System Protection

• The immune system protects us through two main systems:
  • Innate immune system: Responds rapidly to features present in many pathogens.
  • Adaptive immune system: Responds to specific features present only in a given pathogen.
• Both systems identify features on disease-causing organisms and then work to eliminate or neutralize them.

Innate vs. Adaptive Immunity

• Innate Immunity (rapid response):
  • Cells involved: Mast cells, macrophages, natural killer cells, dendritic cells.
  • Also involves complement proteins. These proteins enhance (complement) the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism, promote inflammation, and attack the pathogen's cell membrane.
• Adaptive Immunity (slow response):
  • Cells involved: T cells, B cells.
  • Includes natural killer T cells.
  • Involves antibodies and cytokines. Cytokines are a broad and loose category of small proteins that are important in cell signaling. They are released by cells and affect the behavior of other cells.

Innate Immune System

• Ancient defense system; the first line of defense against foreign pathogens.
• Components:
  • Epithelial lining that surrounds host cells. This barrier prevents pathogen entry. Includes physical barriers such as skin and mucous membranes, chemical barriers such as antimicrobial substances in saliva, sweat, and tears, and biological barriers such as beneficial microorganisms.
  • Phagocytes, which ingest and destroy pathogens.
  • Toll-like receptors (TLRs). These are a class of proteins that play a key role in the innate immune system. They are single, membrane-spanning, non-catalytic receptors usually expressed in cells such as macrophages and dendritic cells that recognize structurally conserved molecules derived from microbes.

Toll-Like Receptors (TLRs)

• TLRs are a family of receptors that recognize pathogens.
• Different TLRs are located on the cell membrane and endosomes.
  • Cell membrane: TLR1, TLR2, TLR4, TLR5, TLR6
  • Endosome: TLR3, TLR7, TLR8, TLR9

TLR Structure

• Extracellular region:
  • Leucine-rich repeats (LRR). These repeats mediate protein-protein interactions and are involved in pathogen recognition.
  • Cysteine-rich regions (CRR). CRRs stabilize the receptor structure and are involved in ligand binding.
• Intracellular region:
  • Toll/IL-1R receptor (TIR) domain. The TIR domain is essential for initiating downstream signaling cascades that activate immune responses.

Lipopolysaccharide (LPS)

• Injection of less than 1 mg of LPS into a human being produces a fever and other signs of inflammation. LPS is a major component of the outer membrane of Gram-negative bacteria, important for the structural integrity of the bacteria, and is a potent activator of the immune system.

Pathogen-Associated Molecular Patterns (PAMPs) Recognized by Human TLRs

TLRs Recognize PAMPS

• TLRs recognize PAMPs through leucine-rich repeats.

TLR Signaling

• TLRs recognize various molecules:
  • Peptidoglycan, Lipoteichoic acid, Lipoproteins (TLR1, TLR2)
  • LPS (TLR4)
  • Flagella (TLR5)
  • ssRNA (TLR7)
  • dsRNA (TLR3)
  • CpG DNA (TLR9)
• MyD88 is a common adaptor protein used in TLR signaling. MyD88 interacts with the intracellular TIR domain of the TLRs.
• Signaling leads to activation of transcription factors such as AP-1, NF-κB, and IRFs, resulting in the production of cytokines like TNF-α, IL-1β, and IL-18. These cytokines mediate inflammation and regulate the immune response.

TLR Signaling and Innate Immune Responses

• TLR signaling is divergent and plays essential roles in innate immune responses.
• TLRs activate downstream signaling pathways, leading to the induction of innate immune responses by producing inflammatory cytokines, type I interferon (IFN), and other mediators.
• TLRs recruit TIR domain-containing adaptor proteins such as MyD88 and TRIF, which initiate signal transduction pathways that culminate in the activation of NF-κB, IRFs, or MAP kinases to regulate the expression of cytokines, chemokines, and type I IFNs that ultimately protect the host from microbial infection.

Adaptive Immune System

• Humoral and cellular immune responses.
• Humoral immune response: antibodies (immunoglobulins). This involves B cells and antibody production.
• Antigen: A foreign macromolecule. Antigens can be proteins, polysaccharides, lipids, or nucleic acids.
• Immunogen: If the binding of the foreign molecule stimulates an immune response. Not all antigens are immunogens.
• Epitope or antigenic determinant: Specific affinity of an antibody for the antigen. This is the part of the antigen that is recognized by the antibody.
• Cellular immune response: cytotoxic T lymphocytes (killer T cells). This involves T cells that directly kill infected cells.
• Major histocompatibility complex (MHC): A group of genes that code for proteins found on the surfaces of cells that help the immune system recognize foreign substances. MHC proteins present antigens to T cells.
• Helper T lymphocytes: stimulates the differentiation and proliferation of B and cytotoxic T cells. These cells are crucial for orchestrating the adaptive immune response.

Adaptive Immunity

• Antibody-Mediated Immunity:
  • B cells recognize pathogens and produce antibodies. Antibodies neutralize pathogens, mark them for destruction by phagocytes, and activate the complement system.
• Cell-Mediated Immunity:
  • T cells attack infected cells displaying antigens. T cells recognize and kill infected cells, preventing the spread of infection.
• Both lead to the formation of memory B and T cells. Memory cells provide long-lasting immunity and allow for a rapid response upon subsequent exposure to the same antigen.

Cell-Mediated Immunity

• Deals with intracellular pathogens.
• Intracellular microorganisms leave traces on the surface of their host cells.
• Vertebrates use an ingenious mechanism to display these traces.
• All vertebrate cells exhibit on their surfaces a sample of peptides derived from digestion of proteins on their cytoplasm.
• These proteins are displayed by integral membrane proteins that are encoded by the major histocompatibility complex (MHC).
• Peptides derived from cytoplasmic proteins are bound to and displayed by class I MHC proteins.
• Dendritic cells migrate to lymphatic tissue, where they use an MHC-like mechanism.

Cell-Mediated Immunity Definition

• Immune response that does not involve antibodies.
• Involves activation of phagocytes, antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to antigen.
• T cells continually scan the surfaces of all cells and kill those that exhibit foreign markings.

Peptide Generation and Delivery

• Foreign peptides bound to class I MHC proteins signal that a cell is infected and mark it for destruction by cytotoxic T cells.

T-Cell Receptor (TCR)

• A protein complex found on the surface of T cells (T lymphocytes).
• Responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.

Classical MHC Molecules

• MHC Class I and Class II present peptides to immune cells as part of routine immune surveillance.
• MHC Class I:
  • Comprised of three alpha subunits and beta macroglobulin.
  • The binding groove is deep, with closed ends, and binds peptides of 8-10 amino acids in length.
• MHC Class II:
  • A heterodimer of a 2-unit alpha chain and a 2-unit beta chain.
  • The binding groove is shallow and open at each end, allowing binding of peptides 13-17 amino acids in length.

MHC Diversity

• MHC proteins are remarkably diverse in the human population.
• Each person expresses as many as six distinct class I MHC proteins.
• Human MHC class I protein: human leukocyte antigen A2 (HLA-A2).
• Side chains from the MHC molecule interact in the anchor residues. Anchor residues are specific amino acids in the peptide that bind to the MHC molecule.
• The other residues are highly variable.
• The identities of only two of the nine residues are crucial for binding.
• MHC-peptide complexes have kinetic stability; once bound, a peptide is not released, even after a period of days.

Differences Between Class I and Class II MHC

Antigen Presentation

• Infected cells present antigens via Class I MHC molecules to T cells.
• Antigen-presenting cells present antigens via Class II MHC molecules to T cells. Antigen-presenting cells (APCs) include dendritic cells, macrophages, and B cells.

Cytotoxic T Cells and Helper T Cells

• Cytotoxic T cell: Kills cancer cells, infected cells (particularly with viruses), or damaged cells. Cytotoxic T cells recognize and kill target cells by releasing cytotoxic granules.
• Helper T cells: Required for almost all adaptive immune responses; help activate B cells to secrete antibodies and macrophages to destroy ingested microbes and activate cytotoxic T cells to kill infected target cells. Helper T cells secrete cytokines that activate other immune cells.

T Cell Receptors (TCR)

• T cell receptors recognize target peptides presented by MHC Class I molecules.

T Cell Target Recognition

• The variable regions of the α and β chains of the T-cell receptor form a binding site that recognizes a combined epitope – foreign peptide bound to an MHC protein.
• Neither the foreign peptide alone nor the MHC protein alone forms a complex with the T-cell receptor.
• Fragments of an intracellular pathogen are presented in a context that allows their detection, leading to the initiation of an appropriate response.
• Cytotoxic T cells initiate apoptosis in cells to which they bind through interactions between T-cell receptors and class I MHC–peptide complexes. Apoptosis is programmed cell death.

Components of the Immune System

• B lymphocytes (from bone marrow cells): Responsible for the synthesis of immunoglobulins. B cells differentiate into plasma cells that secrete antibodies.
• T lymphocytes (are of thymic origin): involved in cell-mediated immunologic processes. T cells include helper T cells and cytotoxic T cells.
• Innate immune system: Defends against infection in a nonspecific manner and is not adaptive. The innate immune system responds rapidly to pathogens without prior exposure.
  • Consists of cells: phagocytes, neutrophils, natural killer cells.

Plasma Cells

• Synthesize and secrete plasma immunoglobulins in plasma in response to exposure to a variety of antigens.

Immunoglobulin Structure

• Composed of light and heavy chains.
• Antigen-binding sites are located on the Fab fragment. The Fab fragment is the region of an antibody that binds to antigens.
• The Fc fragment is involved in complement and cell binding. The Fc fragment mediates effector functions by binding to Fc receptors on immune cells and complement proteins.
• Variable and constant domains. Variable domains are responsible for antigen recognition, while constant domains determine the class of the antibody.
• Hinge region allows for flexibility. The hinge region allows the antibody to bind to antigens that are spaced differently on the target cell.

B Cell and T Cell Receptors

• B cell receptor: Contains antigen-binding site, light chain, heavy chain, variable regions, constant regions, and transmembrane region. The B cell receptor is a membrane-bound antibody molecule.
• T cell receptor: Contains antigen-binding site. The T cell receptor recognizes peptide antigens presented by MHC molecules.

Immunoglobulin Classification

• Classes: IgG, IgD, IgE, IgM, IgA.
• Measured by total light chain assays (FLC kappa and FLC lambda). FLC assays measure the concentration of free light chains in the serum.
• IgA contains a joining chain and secretory protein. The joining chain (J chain) links IgA monomers together, and the secretory component protects IgA from degradation in mucosal secretions.

Types of Antibodies

• IgA: Secreted into mucous, saliva, tears, colostrum, tags pathogens for destruction. IgA neutralizes pathogens and prevents them from adhering to mucosal surfaces.
• IgD: B-cell receptor, stimulates release of IgM. The precise function of IgD is not fully understood.
• IgE: Binds to mast cells and basophils, allergy and antiparasitic activity. IgE triggers the release of histamine and other inflammatory mediators.
• IgG: Binds to phagocytes, main blood antibody for secondary responses, crosses placenta. IgG opsonizes pathogens, activates complement, and neutralizes toxins.
• IgM: Fixes complement, main antibody of primary responses, B-cell receptor, immune system memory. IgM is the first antibody produced during an immune response.

Immunoglobulin Functions

Primary vs. Secondary Immune Response

• Primary immune response: Occurs when an antigen comes in contact to the immune system for the first time; the immune system learns to recognize antigen and makes antibody against it. The primary response is characterized by a slow increase in antibody titers and the production of IgM antibodies.
• Secondary immune response: Occurs when the person is exposed to the same antigen a second time; immunological memory has been established, and the immune system can start making antibodies immediately. The secondary response is faster and stronger than the primary response, with a rapid increase in antibody titers and the production of IgG antibodies.

Antibody Variable Regions

• Hypervariable regions in heavy and light chains form the antigen-binding site.
• These are also called complementarity-determining regions (CDRs).

Antibody Variability

• V(L) and V(H) domains are quite heterogeneous.
• No two variable regions from different humans have been found to have identical amino acid sequences.
• Variable regions comprise relatively invariable regions and other hypervariable regions.
• L chains have three hypervariable regions, and H chains have four.
• These hypervariable regions comprise the antigen-binding site and dictate the amazing specificity of antibodies.
• Hypervariable regions are also termed complementary-determining regions (CRDs).
• The interaction between antibodies and antigens involve noncovalent forces and bonds. These include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.

IgA and IgM

• Serum IgA: Monomer.
• Secretory IgA: Dimer with J chain and secretory component.
• IgM: Pentamer with J chain.

Properties of Human Immunoglobulins

Immunoglobulin Genes

• Each light chain is the product of three separate structural genes: a variable region (VL) gene, a joining region (J) gene, and a constant region (CL) gene.
• Each heavy chain is the product of four different genes: a variable region (VH) gene, a diversity region (D) gene, a joining region (J) gene, and a constant region (CH) gene.

Class or Isotype Switching

• Antibodies with identical specificity but of different classes are generated in a specific chronologic order in response to the immunogen.
• The switch is designated “class or isotype switching.”
• Three classes (IgM, IgG, and IgA) of immunoglobulin molecules against the same antigen have identical variable domains of both their light (VL) chains and heavy (VH) chains and are said to share an idiotype.
• Idiotypes are the antigenic determinants formed by the specific amino acids in the hypervariable regions.
• The different classes of these three immunoglobulins (called isotypes) are thus determined by their different CH regions, which are combined with the same antigen-specific VH regions.

Immunoglobulin Heavy Chain Isotype Switching

• B cells undergo isotype switching with the help of helper T cells.
• Cytokines influence the isotype switch: IgM, IgG, IgE, and IgA are produced depending on the cytokines present.

Immunoglobulin Disorders

• Increased production:
  • Multiple myeloma: Abnormal plasma cells build up in the bone marrow and form tumors, leading to increased antibody production.
• Decreased production:
  • Agammaglobulinemia: Inherited immune deficiencies characterized by a low concentration of antibodies in the blood due to a lack of particular lymphocytes.

Cytokines: Messengers of the Immune System

• Cell signaling molecules that aid cell-to-cell communication in immune responses and stimulate the movement of cells towards sites of inflammation, infection, and trauma.

Cytokines Overview

• Cytokines regulate a network of responses designed to kill invading microorganisms.
• Classes of cytokines: interleukins, tumor necrosis factor, interferons, and colony-stimulating factors.
• Current terminology: immunomodulating agents.
• Cytokines are essential regulators of both the innate and adaptive immune response. They play a crucial role in regulating the intensity and duration of immune responses.

Phagocytosis

• Phagocytosis is the process of a cell engulfing a large particle ( ≥ 0.5 μm), giving rise to an internal compartment called the phagosome.

Pinocytosis and Receptor-Mediated Endocytosis

• Pinocytosis: A cell absorbs small particles outside the cell and brings them inside.
• Receptor-mediated endocytosis (RME): Cells absorb metabolites, hormones, proteins, and viruses by the inward budding of the plasma membrane (invagination).

Phagocytosis by White Blood Cells

• Ingestion of microorganisms, foreign particles, and cellular debris by cells such as neutrophils and macrophages (monocytes).
• Neutrophils and monocytes are armed with both oxygen-independent and oxygen-dependent mechanisms to kill bacteria.
• Oxygen-independent mechanisms: electrically charged proteins, lysosomes, lactoferrins, proteases, and hydrolytic enzymes. These mechanisms do not require oxygen to kill bacteria.

Oxygen-Dependent System

• Enzymes: NADPH oxidase and myeloperoxidase (MPO).
• MPO system is the most potent of the bactericidal mechanisms. It catalyzes the production of hypochlorous acid (HOCl), a potent oxidant and disinfectant.
• NADPH oxidase is the membrane-associated complex containing flavocytochrome plus additional peptides that translocate from the cytoplasm upon activation of the leukocyte. It generates superoxide radicals, which are converted to other reactive oxygen species.
• Deficiencies in MPO do not confer increased susceptibility to infection because peroxide from NADPH oxidase is bactericidal.

Phagocytosis Steps

1. Attachment of the pathogen to a phagocytic cell. This is often mediated by opsonins, such as antibodies and complement proteins.
2. Ingestion of the microorganism, leading to vacuole formation. The vacuole is called a phagosome.
3. Destruction of the microorganism via respiratory burst (NADPH oxidase). The respiratory burst generates reactive oxygen species that kill the pathogen.

Chronic Granulomatous Disease (CGD)

• A diverse group of hereditary diseases in which certain cells of the immune system have difficulty forming reactive oxygen compounds (most importantly the superoxide radical due to defective phagocyte NADPH oxidase) used to kill certain ingested pathogens.
• Leads to the formation of granulomata (nodular areas of inflammation) in many organs. Granulomas are formed when the immune system attempts to wall off substances it perceives as foreign but is unable to eliminate.
• Affects about 1 in 200,000 people in the United States, with about 20 new cases diagnosed each year.

CGD Symptoms and Treatment

• Associated Symptoms:
  • Severe Acne
  • Excessive Granulomata - often in GI tract
  • Lupus
  • Chorioretinitis
• Diagnosis:
  • Nitroblue Tetrazolium Test (NBT). The NBT test measures the ability of neutrophils to produce superoxide radicals. In CGD, neutrophils are unable to reduce NBT, resulting in a negative test.
• Treatment :
  • Antibacterial and antifungal prophylaxis. Prophylactic antibiotics and antifungals help prevent infections.
  • Interferon Gamma. Interferon gamma enhances the ability of phagocytes to kill pathogens.
  • Stem cell or Bone Marrow Transplant