Immunology

For Professor Lu's BICD 140 course (Recommendation: Study cards to memorize individual concepts, practice connecting them by writing/drawing)

How Does the Immune System Affect Our Lives? | Good Way

• Infectious Diseases: Protection from almost any deficiency in immunity

• Cancer: Immune system functions in tumor surveillance

How Does the Immune System Affect Our Lives? | Bad Way

• Autoimmune Diseases: Type I diabetes, lupus, etc.

• Hypersensitivity Diseases: Allergy incidence rose 6-20%, Asthma up 3-8%

• Transplantation: Immune response from the recipient is key if transplants are accepted or rejected

• Heart Disease: Chronic inflammation disease

• Obsessive Compulsive Disorder (OCD): Cured OCD in mice with bone marrow transplant

What is Immunity? | Self vs. Non-Self

Protection from infectious diseases

What is Immunity? | Healthy vs. Sick

Immunopathology: Mechanisms that eliminate pathogens may also cause tissue damage

What is Immunity? | Primary vs. Secondary

Immunity may infer that an individual is capable of resolving an infection after an initial encounter or protected from reinfection with that same pathogen

4 Pathogen Classes

• May always cause overt disease or be opportunistic pathogens that strike when the immune system is weakened

• All classes can be found inside or outside of the cell, but proliferate in specific areas

4 Pathogen Classes | Parasites

Proliferate intracellularly AND extracellularly

4 Pathogen Classes | Fungus

Proliferate extracellularly

4 Pathogen Classes | Bacteria

Proliferate intracellularly AND extracellularly

4 Pathogen Classes | Viruses

Proliferate intracellularly

3 Big Killers

• Do not have a perfect vaccine that confers full immunity

  • Malaria

  • HIV

  • Tuberculosis

New Diseases, New Battles

• SARS: 2003 in Hong Kong/China

• Ebola

• MERS

• Covid-19

Virulence Theory

• Relative pathogenicity or the relative ability to do damage to the host of an infectious agent

  • Pathogen is selected to carry out replication and transmission, not selected to be more destructive (calibrate virulence based on the host)

    • If it kills the host before it is transmitted, then the pathogen dies out

  • More hosts available = Better for parasitic transmission and vice versa

How is the Immune System Selected?

The immune system does not prevent disease, but it is selected to be slightly different, usually more effective

Innate Immunity

• Manifest in virtually all cells in the body

• Ready to go at all times w/ immediate response

• Limited specificity in recognizing the different classes of pathogens

  • Molecular patterns as unmethylated DNA, dsRNA, cell wall components, etc.

• The specificities of the innate pathogen-receptors are encoded

Adaptive Immunity

• Only exists in vertebrates

• Have specialized immune cells (B/T Lymphocytes)

• “Right” cells are selected from a lymphocyte pool; slower response but can provide long-lasting protection

  • Highly specific response to unique components of pathogen

• Specificities of the adaptive pathogen receptors are acquired through gene rearrangement during the organism’s lifetime

Various Ways to Prevent Bugs from Crossing Epithelia

• Three Methods:

  • Mechanical: Usually flow of fluid, mucus, etc.

  • Chemical: Enzymes, antimicrobial peptides, high acidity

  • Microbiological: Normal flora of microbiome

Not All Bugs are Bad

Sometimes antibiotics are given, but they wipe out good and bad bugs which can have a higher risk of pathogenic microbes invading

All of the Cells of the Immune System

These cells all come from hematopoietic stem cells in the bone marrow, and there are 4 major types to know

Cells of the Immune System | Neutrophils

• Phagocytosis

• Reactive Oxygen/Nitrogen species

• Antimicrobial Peptides: Trap pathogens w/ sticky DNA then kill themselves

Cells of the Immune System | Macrophages

• Garbage Collectors who eat dead pathogens

  • Phagocytosis

  • Inflammatory mediators

  • Cytokines

  • Reactive Oxygen/Nitrogen Species

Cells of the Immune System | Dendritic Cells

• Detective cells who eat dead cells but also go back to the T Cells and report what pathogen is present

  • T cells cannot directly recognize pathogens unlike innate immune cells

  • Can present antigens to B cells as well

• Highly phagocytic

• Costimulatory signals

• Antigen presentation

• Link innate and adaptive immunity

• Act as APCs (antigen presenting cells)

Ralph Steinmann

Discovered dendritic cells, got Nobel Prize posthumously

Cells of the Immune System | Natural Killer Cells

• Border Patrol cells that do well fighting intracellular pathogens

• Macrophage activation

• Lysis of viral-infected cells

• Have two types of receptors:

  • Inhibitory Receptor: Tells them to not kill uninfected cells due to the detection of MHC Class I produced by healthy cells

  • Activating Receptor: Tells them to kill target because MHC Class I production is inhibited in diseased cells

Main Innate Immune Mechanisms | Direct Killing

• Phagocytosis: Eat and digest

• Secrete antimicrobial peptides (antibiotics)

• Lysis of microbes

• Perforation of pathogen cell membrane through complement protein

Main Innate Immune Mechanisms | Accessory Role

• Opsonization: Increase pathogen uptake via Complement

• Recruit more immune cells (Complement and Chemokines)

• Activate more immune cells (Cytokines)

• Induce systemic inflammatory responses like fever (Cytokines)

What are Cytokines?

Small cell-signaling protein molecules secreted by numerous cells to affect the behavior of other cells

What are Interleukins?

Group of cytokines first seen expressed by white blood cells (leukocytes)

What are Chemokines?

Induce directed chemotaxis in nearby responsive cells (chemotactic cells)

What is Phagocytosis?

• Eat and digest!

• Bacterium or pathogen is phagocytosed by a neutrophil

• The neutrophil ultimately dies (apoptosis) and is consumed by a macrophage

Secreting Anti-Microbial Properties

• Two methods:

  • Transmembrane pore-forming

  • Modes of intracellular killing

Lysis of Microbes

• Perforation of pathogen cell membranes

  • C5b: Subunit required for perforation

What is Complement?

• System of plasma proteins made by the liver that play multiple roles in innate immunity

• C3 and C5: Continuously made by the liver

  • C3 is in its inactive form until it binds to bacteria, changing its conformation and initiating auto-cleavage

    • C3a: Released

    • C3b: On surface of pathogen (IMPORTANT for cleavage of C5, generating C5b and C5a)

Opsonization

• For large molecules

• C3b facilitates phagocytosis by attaching to the bacterial cell surface

  • CR1 on the macrophage binds C3b on bacterium (C3b is an opsonin)

  • Opsonins: Any molecule that enhances phagocytosis by marking an antigen for an immune response

    • Wagyu analogy: Wagyu is good on its own, but seasoning makes it more delicious/irresistible (opsonins are the “seasoning” for bacteria)

Recruiting More Immune Cells: Inflammation

• C3a/C5a increase vascular permeability, recruiting inflammatory cells

  • Can cause heat, pain, swelling, and redness!

Complement Activation Pathway (practice drawing the full pathway)

Three Pathways for Complement Activation | Alternative Pathway

• First to act

• Pathogen surface creates local environment conducive to complement activation

Three Pathways for Complement Activation | Lectin Pathway

• Second to act

• Mannose-binding lectin binds to pathogen surface

Three Pathways for Complement Activation | Classical Pathway

• Third to act

  • Sometimes acts second, happens simultaneously with the lectin pathway

• C-Reactive protein or antibody binds to specific antigen on pathogen surface (antibodies needed too)

  • Needs IL-6 signaling for CRP production

Innate Immune Cells Able to Produce Cytokines After Encountering Pathogens: IL-6

• Cytokines can induce production of proteins in liver

  • IL-6: Informs liver to make mannose-binding lectin and C-reactive proteins

    • C-Reactive: Binds phosphocholins on bacterial surfaces

    • Mannose-Binding Lectin: Binds to carbohydrates on bacterial surface

Interferons

• Virus-infected cells lead to interferon response (IFN-α and IFN-β)

  • Induce resistance to viral replication

  • Increase expression of ligands

  • Activate NK cells to kill infected cells

    • Innate immune cells like NK cells always ready to kill, but their effector functions increased 20-100 fold when stimulated w/ cytokines made by macrophages

Effector Cells

Mature, activated cells

Naïve Cells

Fully developed, not activated cells (have not encountered any pathogens)

TNFα

• TNFα: Get more neutrophils out of the bone marrow

• Chemokines recruit cells from distal locations, provide directional signals for migrating cells

IL-1

• Induce systemic inflammatory response (systemic response)

  • IL-1: Secreted by phagocytes travels in blood to hypothalamus, muscle

  • Increased body temperature to point set by the hypothalamic thermostat

  • Decreased viral/bacterial replication

Overview of Cytokine Functions

Septic Shock

• Too much systemic innate response can be deadly

• TNFα made by macrophages causes cells to make platelet activation factor which normally prevents pathogens from entering the blood

  • Septic Shock: Systemic edema followed by excessive coagulation and organs are starved and shut down

PAMPs

Pathogen-Associated Molecular Patterns: Molecules associated w/ groups of pathogens that are recognized by cells of the innate immune system

PRRs

Pattern Recognition Receptor: Innate immune receptors recognize PAMPs

Where Our Immune Cells Find Pathogens | Extracellular

• Sites of Infection: Interstitial Spaces, Blood, Lymph Nodes

  • Defense: Have complement macrophages and neutrophils

• Sites of Infection: Epithelial surfaces (Have TLR4/5 Receptors)

  • Defense: Antimicrobial peptides

Where Our Immune Cells Find Pathogens | Intracellular

• Sites of Infection: Cytoplasmic (RIG-I, cGAS, and Nod2 receptors)

  • Defense: NK cells

• Sites of Infection: Vesicular (Have TLR 3, 7, and 9 receptors)

  • Defense: Activated Macrophages

Different Receptor Systems

• 3 to know:

  • On Surface: Toll-like receptor

  • Endosomal: Toll-like receptor

  • Cytoplasm: Nod-like sensors for intracellular bacterial detection

    • CARD-family sensors for virus detection

TLRs

Toll-Like Receptors: Similar to the protein produced by Drosophila’s Toll genes (susceptible to fungal infections)

Toll Processors

Charles A. Janeway Jr. and Ruslan Medzhitov identified these receptors, caused a large drama in 2011 Nobel Prize decision

Localization of Different TLRS

• Surface: TLR4-LPS and TLR5-Flagellum

  • Endosomal:

    • TLR3: dsRNA

    • TLR7: ssRNA

    • TLR9: CpGDNA

LPS (Lipopolysaccharide)

• Component of cell wall in gram- bacteria

• Endotoxin: Kept “within” bacterial cells; presence of endotoxins in the blood can cause unwanted inflammatory response → Septic Shock

Multiple TLRs May Recognize Different Structures in the Same Pathogen

• LPS recognized by TLR4

  • Recognition requires LPS Binding Receptors CD14 w/ TLR4

    • CD: Cluster of Designation is a protocol used for the identification and investigation of cell surface molecules providing targets for immunophenotyping of cell (KNOW CD14 FOR EXAM)

    • MyD88 binds TLR4 and activates IRAK4 to phosphorylate TRAF6

    • Leads to release of NFKB, activating transcription of genes for inflammatory response

MyD88 Pathways

• Two cytoplasmic pathways:

  • Dependent: Uses IRAK4 as adaptor protein

    • Trigger NFKB Pathway

    • Produce inflammatory cytokines (IL-1, IL-6, TNFα)

  • Independent: Use TRIF as an adaptor protein triggered by TLR4

    • Trigger IRF3 pathway

    • Produce IFNs to fight viral infections (IFNα/β)

    • Will continue to function if the Dependent pathway is shut down

IRFs

• Interferon Regulatory Factor proteins that regulate transcription of interferons

• Nod-like receptor can sense bacterial infection in cytoplasm and induced NFKB-mediated inflammation

• CARD-family sensors (RIG-1) can recognize cytoplasmic dsRNA (and ssRNA) viral infection, use interferons to respond

Cyclic GMP-AMP Synthase

cGAS: Recognize cytoplasmic DNA viral infection

Dendritic Cells

• Highly phagocytic, link innate and adaptive immunity

  • Professional antigen presenting cells (APCs)

  • Antigen: Part of pathogen that antibodies bind to

    • Adaptive immune cells (ie T Cells) do not recognize pathogens unlike innate immune cells

Ralph Steinmann

Contributed to dendritic cell discovery/function

What is the Immune System?

• Varying locations depending on whether it’s adaptive or innate

  • Innate Immune System:

    • Tissue (mostly exists here!)

    • Lymphoid Organs, Blood

  • Adaptive Immune System:

    • Lymphoid Organs (mostly exists here!)

    • Blood, Tissue

Lymph

Plasma that has leaked from the blood into the tissues, collected through lymphatic vessels

Lymphoid Organs

• Contain lymphocytes, but also other types of cells and structure to support the production, maintenance, and circulation of lymphocytes

  • Lymphatic vessels collect lymph to carry back to lymphoid organs

Primary Lymphoid Organs

• Central Lymphoid Organ: Where lymphocytes are generated (from immature progenitor cells)

• From bone marrow

• T Cells go to the Thymus to finish maturation

Secondary Lymphoid Organs

Peripheral Lymphoid Organs: When mature naïve lymphocytes reside and an adaptive immune response is initiated

Lymphocytes Continuously Survey the Secondary Lymphoid Organs for Evidence of Infection

• Lymphocytes are unique as they travel through blood and lymph

• Secondary lymphoid organs compartmentalize the infection and provide a meeting place for the cells of the adaptive immune response

• T-Cell Area: Mostly T-Cells

  • Exist in Lymph Nodes

• Arterial Vein: “I-5” that lets them travel to rest of body

• Efferent Lymphatic Vessel

Lymphoid Follicle

Dedicated area for B Cells in lymph nodes

Spleen

• Deals with pathogens that make it to the blood

  • No connection to lymphatics

  • Key Difference: Only connected via blood vessels unlike LN, where both pathogens and lymphocytes enter spleen via the blood

Gut-Associated Lymphoid Tissue

• “The adjacent city”: Only separated w/ epithelial cells from the gut

• Similar microanatomy to spleen and LN, but differ in:

  • Route of pathogen entry (direct delivery across mucosa)

  • Migration pattern of lymphocytes after activation (tend to stay within mucosal system)

• M Cells: Create a “window” in the epithelium that allows for immune cells, especially dendritic cells to reach out and detect pathogens

The Adaptive Response is Specific to the Current Infection

• Antibodies made during infection w/ vaccine bind to the virus and prevent reinfection w/ virus

  • Ex: Antibodies made for measles don’t bind to influenza

The Adaptive Immune Response

• Diversity and Clonal Selection

  • Only adaptive immune population w/ receptors that recognize the specific pathogen will respond during a particular infection

  • T-Cells: Activated by dendritic cells, only specific few

Clonal Selection

• Four Principles (KNOW FOR EXAM):

  • Every lymphocyte bears a single type of receptor with a unique specificity

  • Lymphocyte Activation: Interaction between a foreign molecule and a lymphocyte receptor capable of binding that molecule w/ high affinity

  • Differential effector cells derived from an activated lymphocyte will bear receptors of identical specificity to those of the parental cell

  • Lymphocytes bearing receptors specific for ubiquitous self-molecules are deleted at an early stage in lymphoid cell development (deleted from mature repertoire)

Effectors of Adaptive Immunity

• Two types:

  • B Cells: Secrete their antigen receptor (antibody)

    • Humoral immunity: Involves substances found in the humours (body fluids)

    • Attack external bacteria (via antibodies)

  • T Cells: Do not secrete their antigen receptor

    • Cell-Mediated Immunity: Activation of macrophages lead to microbial killing and lysis of infected cells

    • Attack virus-infected cells and phagocytosed microbes in macrophages

Isotype Switching

The constant region of the heavy chain of an antibody changes to interact with different effector molecules, which may increase the affinity the antibody has for a specific antigen (antigen specificity DOES NOT change)

T Helper Lymphocytes

• CD4: Helper T Cells

  • Interact w/ macrophages to activate them (TH1)

Cytotoxic Lymphocytes

CD8: Kill virus-infected cells

Antigen Receptors

• Recognition of Pathogens

  • B Cell Receptor: Recognize pathogens in native form

    • Bound to B Cell: Y shape w/ two binding sites (antibody)

  • T Cells: Need APCs to process and present antigens to them

TCR

• Binds to antigen derived peptides bound to MHC (Major Histocompatibility Complex) molecules

  • CD8 T cells cannot “see” antigen w/o MHC Class I

  • CD4 T cells need MHC Class II on surface of dendritic cell

Functional Outcome of B Cell Activation is Antibody Production

• Antibodies bind directly to toxin/pathogens

  • Neutralization: Pathogen cannot invade cells

  • Opsonization: Increase phagocytosis

  • Complement Deposition

    • Ultimately leads to degradation by a macrophage

Functional Outcome of T Helper Cell Activation is the Activation of Other Cell Types

• TH1: Recognizes complex of peptide antigen w/ MHC Class II and activate macrophages

• TH2: Recognizes complex of peptide antigen w/ MHC Class II and activates B Cell (and CD4-TH2 cells for full B Cell Activation)

  • Poor quality antibodies created without T Cells, so B Cells’ response is limited

Functional Outcome of Cytotoxic T Cell Activation is the Lysis of Infected Cells

• Peptide fragments of viral proteins bound by MHC Class I in ER, transported to cell surface where cytotoxic T Cells recognize complex of virus infection

  • NK cells are Plan B when these cells fail to kill infected cells

Principle Antigen Presenting Cells (APCs) for T Cells

• Dendritic cells found in the lymphoid organs, skin, and connective tissue

  • Macrophages found everywhere in body

  • Circulating Monocytes: Can turn into macrophages or dendritic cells early on

  • B Cells: Present antigens bound to antibody receptors

Flow Cytometry Applications

• Visualization of samples on a single cell level

• Can find a rare cell type amongst a sample comprised of non-relevant counts

• Sorting of distinct populations within a heterogeneous sample

• Widely used in immunology

How Does Flow Cytometry Work?

• The Basics: A sample of cells is labeled with antibodies conjugated with fluorochromes via incubation at low temp

  • Antibodies specific for cell surface receptors, intracellular receptors

• Cell suspension is forced into a tiny stream of liquid so that only one cell enters the machine at a time

  • Each cell and any antibody attached to it intercept intercept the laser beam and excited to a higher energy state

• Energy is released as a photon of light (w/ fluorochromes, photon has distinct spectral properties)

Interpreting Graphs: Scatter

• Cells are not opaque, they can create side scatter due to granularity

  • Granularity: Number of things in the cell

  • Low side scatter can indicate that the cells are lymphocytes

• Forward scatter: Due to size

• Density: Color brightness

  • Identify populations by high or low, not by the specific number

• Location on graph is relative

• KNOW GRAPH SHOWN FOR EXAM

Translating Flow Cytometry Graphs to Peaks

• Darker color: Less cells

• x/y axis: Fluorescent Intensity = Expression level

• Bottom left corner: Double negative population (does not have high levels of either cell population listed on axes)

Differentiating Between CD4 (Helper) and CD8 (Cytotoxic) T Cells

• Thymus: High population of mature T cells here

• Spleen: Secondary lymph organ with mature but naïve T cells

Flow Cytometry Overview (KNOW FOR EXAM)

INF-α and INF-β

• Three functions:

  • Slow down viral replication

  • Make cells a better target for NK cells

  • Can activate NK cells directly

    • In covid, viral load was reduced and symptoms decreased w/ MORE interferons present, and vice versa

    • Type I interferons have delayed response, caused more immunopathology any many deaths (increase pathology later on)

GALT

Gut-associated lymphoid tissue is a specialized immune system in our digestive tract

Tolerance

Our body needs tolerance against things that are not dangerous (ie commensal bacteria in our gut) to avoid constantly attacking everything (leads to autoimmune disease, allergies otherwise)

How do We Produce an Infinite Variety of Antibodies?

Each B Cell expresses a unique antibody receptor that is selected by antigen

Plasma Cells

Effector B Cells that secrete large volumes of antibodies

Antibody Structure and Generation of B Cell Receptor/Antibody

• Antibodies (Ab) are globular proteins that specifically bind to foreign molecules

  • Made by B Cells, differentiated to become plasma cells

  • That ONE antibody is a copy of its B Cell receptor, so antibodies are secreted B Cell receptors

  • Antigens are anything that bind to an antibody

• Fc and Fc receptor interaction facilitates antibody-mediated opsonization!

Antibody Structure | Heavy Chain

5 different constant regions (IgM, IgG (1-4), IgA (1-2), IgD, IgE…) with 9 total in humans

Antibody Structure | Light Chain

• 5 total in humans

  • Igκ and Igλ (1-4) in humans

Antibody Structure | Variable Region

• Antigen recognition/binding site with 10,000,000,000,000 possible variable regions

• Unlimited diversity

Antibody Structure | Constant Region

Biological activity occurs on heavy chains in constant region