Chapter 3 - Recognition and Response

Common Features of Receptor-Ligand Interactions

• Receptor-ligand binding occurs through multiple noncovalent bonds.
• Individual bonds might be weak, but collectively they provide a strong binding affinity.
• Many such bonds exist between receptors and ligands, leading to significant cumulative bond strength.
• Meaningful receptor-ligand interactions result from the total bonding interactions that hold surfaces together with sufficient binding energy over time, which is necessary for a signal to be sent.
• Ligand-receptor binding induces molecular changes in the receptor:
  • Conformational changes
  • Dimerization/clustering
  • Altered location in the membrane
  • Covalent modifications
• Receptor alterations trigger cascades of intracellular events:
  • Enzyme activation
  • Changes in intracellular locations of molecules

Combinatorial Protein Chain Use and Receptor Variability

• Cells use receptors to bind to a diverse range of antigens (Ags).
• A single protein chain combined with multiple partners creates variation in binding sites.
• This results in different 3D shapes for the ligand-binding site (e.g., cytokine receptors).
• Multiple protein chains contribute to the ligand-binding site.
  • Low-affinity binding (single protein) cannot transduce an activation signal from a cytokine into the cell.
  • High-affinity binding (multiple subunits) produces a dimeric receptor for signal transduction.

Ligand Binding and Signal Transduction

• Aggregation due to ligand binding enhances ligand binding.
• Non-Ag-activated lymphocytes express heterodimeric receptors.
• Ag-activated lymphocytes (BCR or TCR) increase expression of the 3rd chain of the IL-2 receptor.
• Extended contact facilitates signal transduction and the exchange of cytokine signals.
• This conserves energy and prevents accidental initiation of an immune response.

Immunoglobulin (Ig) Domains

• Immune receptors contain immunoglobulin (Ig) domains.
• Each Ig domain has:
  • A parallel pair of β-sheets.
  • Hydrophobic amino acid (AA) residues facing each other.
  • Hydrophilic AA residues interacting with the environment.
  • Forms a “hydrophobic sandwich” stabilized by intrachain disulfide bonds.
  • Polypeptide regions link β-sheets together.
  • These regions can vary in sequence length and structure for variability.
  • Variations in polypeptide regions result in the Immunoglobulin superfamily.

Immune Antigen Receptor Molecules (Antibody)

• Three AA hypervariable regions are found in variable heavy ($V_H$) and variable light ($V_L$) regions.
• These regions combine to form the antibody combining site.
• The regions are complementarity-determining regions (CDR1, CDR2, and CDR3) in $V_H$ and $V_L$.
• Near each CDR is an invariant amino acid that forms the framework region and is responsible for the folding of CDRs to form the antibody combining site.

Antibody Structure

• Each antibody (Ab) has two binding sites.
• Antigen specificity is determined by the interaction between light- and heavy-chain variable regions.
• Antibody effector activity (e.g., phagocytosis and complement fixation) is a function of the interaction of the constant regions of the heavy chain.
  • $F_c$ region (Fragment crystallizable region):
  • Tail region of Ab
  • Non-Ag binding region
  • Fab region (Fragment Ag-binding region):
  • Recognizes and binds to specific Ag
  • Antigen-binding portion

Antibody Structure in Detail

• Antibodies are quaternary proteins with two identical heavy (H) chains and two identical light (L) chains.
• Disulfide bonds connect H-L chains and H-H chains at cysteine residues.
• L chains have two Ig domains.
• H chains have 4-5 Ig domains.
• Ag-binding sites are made up of H and L chains.
• Have variable (V regions) at amino-terminal domains.
• Have constant (C regions) at carboxyl-terminal regions.
  • $V_L$ and $C_L$ (light chains)
  • $V_H$ and $C_H$ (heavy chains)

Classes of Antibodies

• Constant regions of heavy chains identify five distinct classes of antibody called isotypes:
  • IgA: alpha (α) heavy chain
  • IgD: delta (δ) heavy chain
  • IgE: epsilon (ε) heavy chain
  • IgG: gamma (γ) heavy chain
  • IgM: mu (μ) heavy chain
• Light-chain isotypes are:
  • kappa (κ)
  • lambda (λ)
• L chains (light colors)
• H chains (dark colors)
• J chain: polypeptide linked by two disulfide bonds to Fc region in two monomers.

BCRs Require Coreceptors

• Antibody molecules form a B-cell receptor (BCR) complex with molecules involved in signal transduction.
• Require Igα and Igβ for signal transduction via ITAMs.
• ITAM (immunoreceptor tyrosine-based activation motif):
  • Short AA sequences
  • Act as docking sites for binding downstream signaling molecules
  • Intracytoplasmic

BCRs Require Coreceptors in Detail

• Coreceptors: CD19, CD21, and CD81 (AKA TAPA-1).
• Interaction of CD21 (on B cell) and Ag-associated C3d increases the strength of BCR-Ag binding.
• The B cell receptor complex functions as two components:
  • Recognition with BCR and CD21
  • Signal transduction component (Igα and Igβ)

T-Cell Antigen Receptors and MHC Proteins

• Most naïve T cells recognize specific Ags after processing by a host cell.
• Host cell processing results in short Ag peptides presented on the surface of professional antigen-presenting cells (pAPCs).
• T-cell receptor (TCR) specificity is for Ag-derived peptides only when in complex with plasma-bound MHC proteins.
• This allows T cells to browse for cells with complimentary MHC-peptide Ags.
• TCRs are only membrane-bound and never secreted in soluble form.
• pAPC: Professional antigen-presenting cells

TCR Structure

• TCR chains have two Ig domains.
  • One variable at the N terminus serves as an Ag-binding site.
  • One constant that lifts the Ag-binding site away from the plasma membrane.
• Held together by a disulfide bond between the C domain and the plasma membrane.

TCR Coreceptors

• The T-cell receptor (TCR) complexes with coreceptors involved in antigen recognition.
• CD3 contains ITAMs that transmit the signal to the cell.
  • ITAMs are signaling components in immune cells
• CD4 and CD8 function to increase the avidity of peptide binding by TCR.
  • CD4 → MHC class II
  • CD8 → MHC class I

TCR Coreceptors in Detail

• Full activation of a naïve T cell requires the CD28 coreceptor an APC.
• CD28 must simultaneously engage a ligand on APC and MHC-peptide complex.
• This forms a safeguard against inappropriate activation to induce an autoimmune response.
• CD28 does not interact with the MHC-peptide complex.
• CD80 and CD86 ligands are on APCs.
• The expression level is enhanced with Ag presentation by dendritic cells (activated APCs).
• CD3 complex: Transduction of signals received by the receptor into the cytoplasm of the cell

T-Cell Accessory Molecules

• CD4: binds to Class II MHC and functions in adhesion and signal transduction and is a member of the Ig superfamily
• CD8: binds to Class I MHC and functions in adhesion and signal transduction and is a member of the Ig superfamily
• CD2 (LFA-2): binds to CD58 (LFA-3) and functions in adhesion and signal transduction and is a member of the Ig superfamily
• CD28: binds to CD80 and CD86 and functions in signal transduction and is a member of the Ig superfamily
• CTLA-4: binds to CD80 and CD86 and functions in signal transduction
• CD45R: binds to CD22 and functions in adhesion and signal transduction and is a member of the Ig superfamily
• CD5: binds to CD72 and functions in signal transduction

Cytokines

• Cytokines: Secreted, low-molecular-weight (MW) proteins for communication between cells of the immune system.
• Regulate the intensity and duration of immune responses.
• Signals are usually generated from ligand binding with a complementary cell-bound receptor.
• The sensitivity of a target cell to a specific cytokine is determined by the presence of specific cytokine receptors.
• Cytokine secretion usually occurs close to the receptor.
• Few cytokine molecules are needed to mediate biological effects.

Cytokine Receptors and Their Effects

• Cytokine-receptor binding is noncovalent and may be of high or low affinity.
• Activated cytokine receptors:
  • Cause changes in the expression of adhesion molecules and chemokine receptors on target membranes.
  • Signal for increased or decreased enzyme activity.
  • Change transcription for proliferation, differentiation, or changes to effector functions.
  • Induce apoptosis (usually in intracellularly infected cells).

The Six Major Cytokine Families

• Interleukin-1 family:
  • Representative members: IL-1α, IL-1β, IL-1Rα, IL-18, IL-33
  • Comments: IL-1 was the first non-interferon cytokine to be identified; members of this family include important inflammatory mediators.
• Class 1 (hematopoietin) cytokine family:
  • Representative members: IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-13, IL-15, IL-21, IL-23, GM-CSF, G-CSF, growth hormone, prolactin, erythropoietin/hematopoietin
  • Comments: Members of this large family of small cytokine molecules exhibit striking sequence and functional diversity.
• Class 2 (interferon) cytokine family:
  • Representative members: IFN-α, IFN-β, IFN-γ, IL-10, IL-19, IL-20, IL-22, IL-24
  • Comments: While the IFNs have important roles in antiviral responses, all are important modulators of immune responses.
• Tumor necrosis factor family:
  • Representative members: TNF-α, TNF-β, CD40L, Fas (CD95), BAFF, APRIL, LT-β
  • Comments: Members of this family may be either soluble or membrane-bound; they are involved in immune system development, effector functions, and homeostasis.
• Interleukin-17 family:
  • Representative members: IL-17 (IL-17A), IL-17B, IL-17C, IL-17D, IL-17F
  • Comments: This is the most recently discovered family; members function to promote neutrophil accumulation and activation and are proinflammatory.
• Chemokines:
  • Representative members: IL-8, CCL19, CCL21, RANTES, CCL2 (MCP-1), CCL3 (MIP-1α)
  • Comments: All serve chemoattractant function.
  • Interleukins: Cytokines secreted by leukocytes that primarily affect the growth and differentiation of some hematopoietic and immune system cells.
  • Chemokines: Cytokines that mediate chemotaxis, especially leukocytes via receptors to regions where chemokine concentration is highest.

Characteristics of Cytokines

• Pleiotropy: Cytokines can have pleiotropic effects and induce different biological effects depending on the target cell.
• Redundancy: Two or more cytokines may be redundant and mediate similar effects on target cells.
• Synergy: Synergy occurs when the activity of two cytokines together is greater than their additive effects.
• Antagonism: A cytokine can have antagonistic activity and inhibit the action of another cytokine.
• Cascade Induction: Cascade induction occurs when the action of a cytokine on a target cell causes the cell to produce one or more additional cytokines.

IL-1 Family Cytokines

• Secreted primarily by macrophages and dendritic cells.
• Promotes fever, inflammation, vasodilation, and pain early in the immune response.
• IL-1 secretion is stimulated by viral, parasitic, or bacterial Ags.
• IL-1 helps to activate B- and T cells (adaptive immune system).
• Promotes destruction of viral RNA.

Class 1 Cytokines

• Largest family of cytokines.
• Are a single protein family with a defining structure.
• Most class 1 cytokines are made up of multiple subunits.
• Four-helix bundle motif arranged antiparallel.
• Mediate diverse effects including proliferation, differentiation, and Ab secretion.
• Signal onset of B- and T cell proliferation (IL-2).
• Regulate $T_H$ functions (IL-4).
• B cell differentiation to plasma cells and Ab secretion (IL-6).
• Initiate differentiation of specific leukocyte lineages (GM-CSF, G-CSF).
  • GM-CSF: Granulocyte-macrophage colony-stimulating factor
  • G-CSF: Granulocyte-stimulating factor

Class 2 Cytokines

• Three subfamilies of interferons (IFNs)
• Type I interferons
  • IFN-α and IFN-β
  • Secreted by activated macrophages, dendritic cells, and virally infected cells after PRR-mediated recognition
  • Bind to plasma membrane IF receptors on different cell types
  • Induce the production of ribonucleases to destroy viral RNA and inhibit cellular protein synthesis
  • Prevent virally-infected cells from replicating or making new viral particles to limit the spread of infection

Type II interferon (IFN-γ)

• Produced by activated T- and NK cells, released as a dimer
• Induces activation of macrophages
  • Destruction of intracellular pathogens
• Stimulates differentiation of $T_C$ cells
• Key cytokine made by $T_H$1 cells (subset of $T_H$ cells) that support adaptive immunity

TNF Family Cytokines

• Tumor necrosis factor (TNF) regulates the development, effector function, and homeostasis of cells of the skeletal, neuronal, and immune system
• Both soluble proteins and transmembrane proteins
• Have short intracytoplasmic N-terminal regions and longer extracellular C-terminal regions
• The extracellular region interacts with cytokine receptors
• Some TNF are both soluble and membrane-bound

IL-17 Family Cytokines

• IL-17 family cytokines
  • Released by activated T cells
  • Receptors found on neutrophils, keratinocytes, nonlymphoid cells
• IL-17 binds to $T_H$17 subset cells
  • Induces secretion of cytokines that support the proinflammatory state
  • $T_H$17 subset cells act at the interface of innate and adaptive immunity
• All are transmembrane proteins

Chemokines

• Structurally related family of small cytokines
• Bind to cell surface receptors and induce chemotaxis toward the chemokine source
• Chemoattractants: Molecules that elicit movement
• Some chemokines have an affinity for glycosaminoglycans on endothelial cell membranes
  • Allows chemokines to bind to the inner surface of blood vessels and set up a chemoattractant gradient
  • Directs movement to infection sites
• The tertiary structure of chemokines constrains highly conserved disulfide bonds

Receptors and Cell Signaling

• A cellular signal is any event that instructs a cell to change its metabolic or proliferative state
• Signals are usually generated by the binding of a ligand to a complementary cell-bound receptor
• A cell can become more or less susceptible to the actions of a ligand by increasing or decreasing the expression of the receptor for that ligand
• Cell signaling often induces a change in the transcriptional program of the target cell
• Sometimes, multiple signals through multiple receptors are required to effect particular outcomes
• The integration of all signals received by a cell occurs at the molecular level inside the recipient cell

Concepts in Lymphocyte Signaling

• Ligand-receptor binding leads to a change in cellular function by the transduction of energy into biochemical changes, including
  • Transcription factor activity
  • Changes in intracellular, membrane-bound, or secreted proteins
  • Alterations of protease activity
  • Fluctuations of secretory or phagocytic activity
  • Cell metabolic activity to ready for division, differentiation, and death

Class 1 Receptor Signaling Pathways

• Class 1 receptors utilize the JAK/signal transducer and activator of transcription (STAT) pathway for signal transmission to the cellular cytoplasm
• Transmits external signals across cell membranes to the nucleus → results in changes to gene expression
• JAK: Janus Kinase
• enzymes associated with cytokine receptors
• STAT: Signal Transducer and Activator of Transcription

BCR and TCR receptors and Lipid Rafts

• BCRs and TCRs have intracytoplasmic regions
  • Too small for interactions with kinases
• BCR and TCR ligand binding induces receptors to oligomerize (noncovalent binding) on the mb surface → movement to areas of lymphocyte mb known as lipid rafts
• Lipid rafts: Highly ordered, detergent-insoluble, sphingolipid, and cholesterol-rich regions
• The movement of receptors and coreceptors into lipid rafts makes them susceptible to enzyme activity associated with the rafts
• Lyn: tyrosine kinase

### Src-Family Kinases and Immune Cell Activation