IMMUNOLOGY

• Immunity
• Latin term Immunis = exempt

History

• Discipline of immunology grew out of observation that individuals who recovered from infectious diseases were protected from disease

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• 15^th Century
• Chinese and Turks tried to prevent smallpox
• Dried crust from pustules were inhaled or inserted into small cuts
• 1718
• Lady Montagu had that technique done in her children

History

• 1798
• Edward Jenner
• Noticed that milkmaids that contracted cowpox were immune to smallpox
• Innoculated small boy with fluid from cowpox pustule
• He then intentionally infected the boy with smallpox – the child did not develop smallpox
• 1881
• Louis Pasteur
• Vaccinated sheep with heat-attenuated anthrax
• Then infected sheep with virulent strain of anthrax – they did not develop anthrax

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History

• 1883
• Metchnikoff demonstrated that certain white blood cells were able to phagocytize microorganisms
• 1901
• Von Behring and Kitasato
• Demonstrated that serum (noncellular component of blood) from animals immunized to diptheria could transfer that immunity to non-immunized animals

A Little Bit of History

• Couple of terms we need to be familiar with:
• Pathogen – something that causes disease
• Antigen – any foreign substance that binds specifically to an antibody or T cell receptor
• Immunogen – a substance capable of eliciting an immune response
• All immunogens are antigens but not all antigens are immunogens (i.e. haptens)
• Epitope – portion of the antigen that is recognized by an antibody or T cell receptor

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• Immune system evolved to protect multicellular organisms from pathogens
• Does this by 2 related activities
• Recognition and response

2 Systems of Immunity

• Innate Immunity
• 1^st line of defense
• Molecular and cellular mechanisms deployed before an infection
• Distinguishes between self and pathogens but not specialized to distinguish small differences in the foreign particles
• Adaptive Immunity
• Develops in response to infection
• Adapts to recognize, eliminate, and remember pathogen

Innate Immunity

• Less specific
• 1^st line of defense
• Barriers that protect host
• Skin
• Acidity of stomach
• Lysozymes in fluids
• Phagocytic cells
• Antimicrobial peptides (interferons, complement)
• Temperature

Adaptive Immunity

• Highly specific
• Characteristic attributes
• Antigenic specificity
• Antibodies can distinguish between 2 proteins that differ in only 1 amino acid
• Diversity
• Immunologic memory
• Self-nonself recognition

Innate Immune Response

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Overview of the Innate Immune Response

Physical Barriers to Infection

Kuby, 8^th Edition, Figure 4-2

Physical Barriers – Antimicrobial Peptides

Kuby, 7^th Edition, Table 5-2

Physical Barriers – Antimicrobial Peptides

Kuby, 8^th Edition, Figure 4-4

Physical Barrier Innate Immunity

Defensins and cathelicidins.

• Major families of antimicrobial peptides.
• Widely in a variety of epithelial cells and sometimes in leukocytes.
• Role in the innate immune system through antimicrobial, chemotactic, and regulatory activities.
• Protect against bacteria, fungi, viruses, and parasites.
• Ancient arm of the innate immune system that evolved to directly neutralize invading microbes.
• 100s of defensin proteins have been identified from amoeba, plants, birds, mammals, etc.

• Classified based on their secondary structural features.
• Cathelicidins (CATionic HELIcal bacteriCIDal proteIN) are α-helical peptides
• Human cathelicidin LL37 is highly expressed by PMNs and numerous mucosal and epithelial cell types.
• Defensins are β-strand peptides connected by disulfide bonds
• Most are short peptides (<100 amino acids) and carry a positive charge
• AKA – “cationic antimicrobial peptides”
• Interact with microbial cell membrane components to increase cellular permeability resulting in cell death. They also act to modulate the inflammatory response.

Innate Immune System Summary

Innate immune responses do not depend on immune recognition by lymphocytes but have co-evolved with and are functionally integrated with the adaptive elements of the immune system.

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For example a macrophage:

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1. displays the very primitive immune defense of phagocytosis; but also

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• expresses MHC molecules and acts as an antigen-presenting cell, a function that makes sense only in relation to the evolution of T cells.

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Innate Immunity:

What the Body is Trying to Do?

The innate immune response recognizes a few molecular motifs that are found on pathogens but not in the host (us).

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Specifically, the innate immune response recognizes three sorts of things in order to trigger phagocytosis, cell-mediated killing, and cytokine release:

1. Foreign molecular structures call pathogen-associated molecular patterns (PAMPs)
2. Stress or damage indicators expressed by body cells, referred to as damage-associated molecular patterns (DAMPs)
3. The absence of certain “self” marker molecules.
1. This is done by NK cells.

Phagocytosis

The binding of microbes—bacteria, fungi, protozoan parasites, and viruses—to phagocytes via pattern recognition receptors or opsonins and opsonin receptors activates signaling pathways. These signaling pathways trigger actin polymerization, resulting in membrane extensions around the microbe particles and their internalization, forming phagosomes. The phagosomes then fuse with lysosomes and, in neutrophils, with preformed primary and secondary granules. The resulting phagolysosomes contain an arsenal of antimicrobial agents that then kill and degrade the internalized microbes. These agents include antimicrobial proteins and peptides (including defensins and cathelicidins), low pH, acid-activated hydrolytic enzymes (including lysozyme and proteases), and specialized molecules that mediate oxidative attack.

Receptors that Trigger Phagocytosis

Kuby, 7^th Edition, Table 5-3

Phagocytosis – Killing Mechanisms

Oxidative attack on the phagocytosed microbes, which occurs in neutrophils, macrophages, and dendritic cells, employs highly toxic reactive oxygen species (ROS) and reactive nitrogen species (RNS), which damage intracellular components. The reactive oxygen species are generated by the phagocytes’ unique NADPH oxidase enzyme complex (also called phagosome oxidase).

Kuby, 8^th Edition, Figure 4-5

Toll like receptors (TLRs)

TLRs are membrane-spanning proteins that share a common structural element in their extracellular region called leucine-rich repeats (LRRs); multiple LRRs make up the horseshoe-shaped extracellular ligand-binding domain of the TLR polypeptide chain.

Kuby, 7^th Edition, Figure 5-10

PAMPs and PRRs

Kuby, 8^th Edition, Figure 4-6

TLRs…..

Kuby, 7^th Edition, Table 5-4

TLRs and their Ligands

Inflammation

Inflammation is the body’s response to tissue damage. The way the body responds depends on:

• What has caused the damage
• Its location
• Its severity

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• Damage can be caused by physical means or by infection.
• If an infection is present, the body’s innate systems for limiting damage and repairing tissues work in concert with the adaptive immune responses.
• This involves a number of overlapping stages, which typically take place over a number of days or weeks.

Hallmarks of inflammation:

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• Influx of fluid (edema)

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• Increased temperature (hyperthermia)

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• Decreased oxygenation (local hypoxia)

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• Influx of white blood cells (extravasation)

Inflammation

Acute inflammation during infection: recruit / activate leukocytes, eliminate the pathogen, resolve the damage, disappearance of leukocytes from the tissue, regenerate tissue function.

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Three principle changes in tissue during acute inflammation:

• Increased blood supply to the affected area.
• Increase capillary permeability (allows for large serum molecules to enter the tissue).
• Increase in leukocyte migration into the affected tissue.

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Chronic inflammation during infection:

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• Most pathogenic organisms have developed systems to deflect immune responses that would eliminate them.

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• In this case the body often tries to contain the infection or minimize the damage it causes.

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• Persistent antigenic stimulus and the cytotoxic effects of the unresolved infection leads to ongoing chronic inflammation.

The Inflammatory Response

Kuby, 7^th Edition, Figure 5-17

Overview of Innate Immune Response

Kuby, 8^th Edition, Overview Figure 4-15

Adaptive Immunity

• Effective adaptive immune response involves 2 groups of cells
• Lymphocytes
• B cells
• T cells
• Antigen-presenting cells

Adaptive Immunity - Lymphocytes

• B cells
• Mature in bone marrow
• Antigen binding receptor – Antibody
• Glycoproteins

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Adaptive Immunity - Antibodies

• Glycoproteins
• Structure
• 2 identical polypeptides – heavy chains
• 2 shorter identical polypeptides – light chains

Antibodies

• Antigen coated by antibody is eliminated in several ways
• Antibody can cross-link several antigens, making it easier to be ingested by phagocytic cells
• Activate complement system resulting in lysis of microorganism

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Adaptive Immunity - Lymphocytes

• T cells
• Arise in bone marrow but mature in thymus
• 2 well define subpopulations of T cells
• T helper cells
• T cytotoxic cells

• T cells recognize antigen presented in MHC molecule
• MHC = Major Histocompatibility Complex
• MHC Class I – found on all of our nucleated cells
• Cytotoxic T cells recognize this
• MHC Class II – found on antigen presenting cells (B cells, dendritic cell and macrophages)
• Helper T cells recognize this

• T cells
• Cytokines secreted by T_H cells can activate phagocytic cells
• T_C cells can kill altered self-cells
• Cells infected by viruses
• Tumor cells

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• Antigen presenting cell associating with T cell

• Initial encounter with antigen causes primary response
• Later contact with antigen will result in more rapid response – secondary response

Immune Dysfunction

• Allergies and Asthma
• Graft rejection
• Autoimmune Disease
• Immunodeficiency

OVERVIEW

Bridge to the Adaptive Immune System

Kuby, 8^th Edition, Figure 4-23

Antigens

• Substances which can be recognized by Ig of B cells and TCR’s of T cells (when accompanied by MHC)

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• B and T cells also differ in the way they recognize Ag

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Immunogenicity vs. Antigenicity

• Immunogenicity = ability to induce a humoral or cell-mediated IR

Ex: B cells + Ag* 🡪 Effector and Memory B cells

T cells + Ag* 🡪 Effector and Memory T cells

*these substances more appropriately called immunogens.

• Antigenicity = ability to combine specifically with products of the above responses (i.e., antibodies and/or cell-surface receptors)
• All substances which are immunogenic are also antigenic; not the reverse
• Some small molecules (Haptens) are antigenic but not capable of inducing a specific IR; they lack immunogenicity

Types of Antigens

• Autoantigens-”self”
• Alloantigens-”same species”
• Heteroantigens-”different species”
• T-cell dependent antigens-Requires T cell involvement; proteins
• T-cell independent antigens-Does not require T cell involvement; polysaccharides

Factors influencing immunogenicity

• Our IS recognizes only small parts of parasites
• Particular macromolecules such as proteins (#1) and polysaccharides (#2)
• Lipids and nucleic acids do NOT, by themselves, stim IR unless they’re attached to proteins or polysacch’s

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• Immunogenicity is not an intrinsic property of the Ag, but depends on certain biological factors relative to the organism in which it is located

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The Nature of Immunogens

• Determined by 4 properties:

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• Degree of Foreignness
• Molecular size
• Chemical structure + heterogeneity
• Ability to be processed and presented by an APC

1) Degree of Foreignness

• The body must be able to distinguish “self” from “non-self”
• the greater the phylogenetic distance between 2 organisms, the greater the structural differences, hence foreignness (Ex: BSA ->rabbits; chicks vs goats)
• Some macromolecules show conservancy of structure across phyla (e.g., collagen and cytochrome C)
• Other macromolecules “outside” an organism’s system can be immunogenic! (e.g., cornea and sperm cells)

2) Molecular size

• Most immunogens are ≥ 100,000 daltons (Da)
• Most molecules < 5-10,000 are poor ones

3) Chemical structure/heterogeneity

• All 4 levels of protein structure contribute to structural complexity…1°, 2°, 3°, 4° and contribute to immunogenicity
• Lipids can induce IR if presented properly
• Lipids are typically ‘haptens’ carried by proteins (Ab’s are produced vs the lipid portion)
• Ab’s can form vs steroids & fatty acid derivatives…
• Several clinical assays use Ab’s to check for these subst
• Ab’s vs leukotrienes 🡪 for evaluation of asthma
• Ab’s vs steroids -> to measure amts in patient’s circulation
• TCR recognize lipid Ag assoc with CD1(resembles MHC I)
• T cells recog vs lipids of Mycobacterium

B cell response

4) Ability to be processed/presented

*Development of both Humoral and Cell-mediated IR requires T cell recognition of processed/ presented Ag

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*large, insoluble macromolecules and polymers are better immunogens than small and soluble

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*those molecules resistant to enzyme degradation (esp. D-amino acids) are poor immunogens

The biological system contributes to immunogenicity

1. Genotype of recipient– genetic makeup of person is important

-there is a strong genetic link to immune response

-e.g., MHC gene products, genes encoding B/T receptors

2) Dosage and route of Ag admin – exp’tl evidence indicates a dose-response curve to every immunogen

-insufficient doses 🡪 nonresponse or tolerance

-single doses 🡪 insignificant response

-“booster” shots are required for many immunizations

-route affects which immune organ/cells involved…

Adjuvants (L. adjuvare = “to help”)

• Substance which, when added to Ag, enhances its immunogenicity; used for immunizations

how this works is not entirely understood, but they appear to help by:

• Persistence of Ag in tissue
• Enhancement of co-stimulatory triggers (B7 molecules)
• Increased local inflammation
• Nonspecific increase of lymphocytes

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E.g. Aluminum potassium sulfate (alum)

Commonly used adjuvants:

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• Alum - aluminum potassium sulfate - precipitates the antigen, resulting in increased persistence of the antigen. Increases “size” of antigen ↑ phagocytosis.

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• Incomplete Freund’s adjuvant - mineral oil-based - increases persistence of the antigen, mild granuloma.

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• Complete Freund’s Adjuvant - mineral oil-based adjuvant containing dead Mycobacterium - increases persistence of the antigen, stimulates a chronic inflammatory response (granuloma), and co-stimulatory signals. Activates macrophages and DCs.

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• Bacterial Lipopolysaccharides - stimulate nonspecific lymphocyte activation and proliferation, and costimulatory signals.

Epitope or Antigenic Determinant - the region of an antigen that binds to a T cell receptor or a B cell receptor (antibody).

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- Since an epitope is the part of the antigen that binds to the B cell or T cell receptor, it is the part that determines the antigenicity of the antigen - thus the term “antigenic determinant”.

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-T and B cells recognize different epitopes on an antigen

Haptens

• haptens, small organic molecules that are antigenic but not immunogenic.
• Chemical coupling of a hapten to a large protein, called a carrier, yields an immunogenic hapten-carrier conjugate. �