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Introduction
25 January 2016

11:04

Why do you need an immune system?
• Pathogens, parasites, viruses
• Body is a good environment
• Immunity required for suvival
• Extreme measures required for children with a non-functional IS
○ Mutation
• Defensive system
○ Diverse and complex
○ Overlapping and partially redundant
• More than just erythrocytes and antibodies
○ Diverse range of cells and molecules
• Pathogens lead to a specific type of immune response
• Innate and adaptive
○ Overlap
○ Roles, cell types, molecules
• Innate
○ Ancient, conserved
○ Plants, invertebrates, vertebrates
○ Non-specific, no memory response
• Adaptive
○ Vertebrates only
○ Specific, memory response

Immunology Page 1

Innate Immune Response
25 January 2016

11:11

Barrier
• Standing defences
○ Physical barriers (walls)
○ Inhospitable environments (moat)
○ Eyelids
○ Mucus and cilia
○ Earwax (antimicrobials)
○ Gut
 Low pH, mucus, proteases, microflora
• Innate antimicrobials
○ Lysozymes
 Attack peptidoglycans in gram+ bacterial cell walls
 Expose lipid bilayer
○ Defensins
 Small proteins
 Lungs, skin, tears
 Viruses, fungi, bacteria
 Attack lipid bilayer
□ Charged
□ Create pores/punctures
• Breached barrier ---> identification
Identify
• When and what to attack
• Distinguish self/non-self
• Antibody molecules - some are unique to non-self
• Pattern Recognition Receptors (PRR)
○ Innate cell receptors that recognise non-self molecules
○ Lipopolysaccharide (gram- only)
• Pathogen-Associated Molecular Patterns (PAMPs)
○ Highly conserved, hard to evade recognition
○ Toll-like receptors
 Multiple
 Range of PAMPs
 TLR9 - viral/bacterial DNA
 Found in drosophila, sheep, horses, humans etc
...

 Range of PPRs to detect pathogens
○ Dendritic cells
 Langerhans in skin
 Range of PPRs
 Activation leads to inflammation
 Release of immune molecules
□ Cytokines
 Small, soluble
 Intercellular messengers
 e
...
interleukins, interferons
 Bind to specific receptors on immune cells
□ Chemokines
 Specialised cytokines - chemoattractant
 Attracts immune cells to tissues or within tissues
 Mediate immune responses
 Does not kill pathogens
 Takes in and presents pathogenic antigens
 Attract other immune cells
○ Macrophages
 Range of PRR
 Phagocytosis after PRR activation
 Can kill pathogens
 Critical for adaptive response
Killing
1
...
Transported by phagosome into cell
3
...
Pathogen destroyed
• Acification, oxygen derive toxins, antimicrobial peptides (defensins etc
...

○ Multiple antigens on each pathogen
• Pattern Recognition Receptors PRRs
○ Innate recognition
○ No recognition means infection
○ B/T cells have different PRRs - at least 1 will successfully recognise
 Random DNA rearrangement of BCR and TCR
B Cell Receptor
• BCR interact with antigen
• 200-500,000 identical BCRs on surface
• BCRs can be shed into the blood and tissue fluid and antibodies
○ Antibodies = soluble BRRs
• Antibody structure
○ 2 heavy (50kDa), 2 light (25kDa) chains
○ Disulphide bonds between H and L

Immunology Page 5

○

○ Variable region different on each naïve B cell
• Activation
○ Cross-linked BCRs - multiple recognition
○ Release cytokines and antibodies
○ Endocytose, process, present antigens to T cells via MHC class II
 Only way T cells can recognise
T Cell Receptor
• Interact with antigenic peptide in MHC molecules
○ Presented by B cells, dendritic, macrophage etc
...
Activates
programmed cell death
○ Both result in apoptosis
Memory
• During infection, T and B cells that are activated divide
• Cytokine interleukin IL-2 released and IL-2 receptor expressed
• Memory T cells/B cells/plasma cells
○ Most T and B cells die off
○ Memory cells survive for lifetime
• Faster, larger response to subsequent infection
○ More efficient and effective
○ Higher affinity BCR and TCR

Immunology Page 6

Lymphoid Tissues
01 February 2016

11:02

• Every area of the body can be infected
• Immune cells must be everywhere and traffic quickly
• Different sites require different responses
○ Retinal cells cannot be replaced
○ Gut cells constantly replaced
• Specific tissues for appropriate responses
• Sentinel cells throughout
○ Dendritic, macrophages, etc
...
2o in all
 Lymphoglandular complexes
□ Caecum in horses, ruminants etc
...
B cells proliferate and generate high affinity B cells

Immunology Page 7

□ 1o lymphoid follicle - B cells
□ Paracortical area - T cells
□ Medullary cortex - macrophages
 Naïve lymphocytes constantly circulating
 Antigens and APCs move from infected tissues to lymph nodes
○ Spleen
 Filter microbes, antigens from blood only
 APCs and lymphocytes
 Same activation role as lymph nodes
 Red pulp
□ RBC destruction and storage - non-immune
 White pulp
□ Mature DCs and macrophages migrate from tissues to spleen via blood
 Reside in marginal zone
□ Interact with lymphocytes in spleen
○ MALT - Mucosa Associated Lymphoid Tissues
 Gut Associated Lymphoid Tissues - GALT
 Nasal Associated Lymphoid Tissues - NALT
 Bronchus Associated Lymphoid Tissues - BALT
 Skin Associated Lymphoid Tissues - SALT
 Different environments
 Most exposure to pathogens at mucosal surfaces
 Higher lymphocytes concentration than the rest of the body
 Specialised lymphocytes
 Lots of IgA antibodies
□ Protected from mucosal proteases

 GALT
□ Peyers patches

Immunology Page 8








Small intestine
M-cells sampling antigens at the surface
Dome below containing T cells, B cells, dendritic, and macrophages
Activates lymphocytes
Germinal centre
Prevents inappropriate activation
◊ Food allergy
◊ Inflammatory Bowel Disease
◊ cytokines interleukin-10, TGFβ inhibit inflammatory response
and induce IgA production

Immunology Page 9

Antibodies
03 February 2016

10:58

• Critical to most immunity
• Produced by B cells and plasma cells (modified B cells)
Generic Structure
• 2 heavy chains (50kDa), 2 light chains (25kDa)
• Disulphide bonds between
• 2 identical binding sites
○ Can lead to agglutination - clumping of antibody-antigen complexes
• Bind to conformational shapes on antigen surfaces (epitopes)
• Antigens can have multiple epitopes
• In the innate immune response have a new PRR to recognise a new PAMP
• Not 1000s of genes for different antigens
Receptors
• Every cell expressing an insulin receptor has the same receptor
• Every cell expressing TLR4 receptors have the same receptor
• BCRs (and TCRs) are different
○ Different B cells express different BCRs
• Naïve B cells genetically different
○ Genes that create antibodies
• Parts of DNA different at L chain and H chain genes
Antibody Formation
• Initially each pre-B cell is genetically identical
• 2 L chain genes k and λ
• These genes have multiple options
• Heavy-chain locus has multiple variable V, diversity D and junction J segments
• Light-chain locus only has multiple V D and J segments
• Rearrangement of V D J segments in pre-B cells gives them their diversity
• At least 1011 possibilities
• Can recognise nearly anything
• DNA is cut by recombinase RAG1 and RAG2
○ Removes non-selected segments
○ When rejoined a few nucleotides are randomly added and/or removed
• Mature cells proliferate, so many of the same
○ Hypersomatic mutation
Function
1
...

2
...
Antibody-dependent cell-mediated cytotoxicity
○ ADCC
○ Antibodies bind non-self antigens on the host cell
○ Immune cell Fc receptors bind antibodies
 Host cell apoptosis triggered
 Perforins etc
...
Activation of Complement
○ Antibodies 1 way of activating the complement cascade
○ Complement is a series of proteins in serum
○ Part of innate immune response
○ 3 pathways

○ Classical Pathway
 Antibody-antigen complexes and some non-specific reacting
 Initiated by C1q
 Binds antibodies or pathogen surface
○ Lectin Pathway
 Lectin (PRR) molecules (not antibodies) bind pathogen surfaces
 Initiated by mannose binding lectin or ficolins
□ Bind carbohydrates on pathogen surface
□ Mannose PAMPs on salmonella, fungi
○ Alternative Pathway
 Spontaneous reactivity at pathogen surfaces
 Initiated by C3
 Blocked on host cells by multiple proteins
□ e
...
CD59
○ Complement functions
 Destruction
□ Polymerisation of terminal proteins to form Membrane Attack Complexes
□ MACs form pores in cell membranes
□ Cell lysis
 Opsonisation
□ C3b and C5a proteins induce phagocytosis
Inflammation

□ C3a, C4a, C5a induce inflammation
Immunology Page 11

□ C3a, C4a, C5a induce inflammation
□ Activation of endothelia
 Allow W-P bodies and erythrocytes into tissue fluid

Immunology Page 12

Antibody Isotypes
05 February 2016

11:10

• Different classes
• 5 main isotypes
○ IgM
○ IgD
○ IgG
○ IgA
○ IgE
• Differing roles
• Different heavy chains - determine properties
○ µ-M
○ δ-D
○ γ-G
○ α-A
○ ε-E
• Antibodies interact with host cells via Fc region
• Range of Fc receptors - specific to isotypes - on immune system cells
• Different isotypes activate different effector immune responses
IgM
• 1st in primary immune response
• Doesn't require class switching
• Can form pentamers
○ Not always

• Low affinity
• Effective in stimulating the complement system
○ Binds antigen, C1q binds to Ig on surface

• In blood and lymph
Immunology Page 13

• In blood and lymph
○ Pentamer too large to enter tissue fluid
IgG
• Most common
• Very effective neutralising antibody in blood and tissue fluid
• Opsonises pathogens and activates complement
IgA
•
•
•
•

Opsonises less well
Weakly activates complement
Dimeric IgA in secretions on epithelia
Dimeric IgA secreted by plasma ells in lamina propria of gut
○ Binds to layer of mucus overlaying gut epithelium
○ Neutralises pathogens and toxins

IgE
•
•
•
•

Acts in conjunction with eosinophils, mast cells, basophils
Very little free IgE in blood
Bound to cells by high affinity Fc receptors
Resting mast cell contains granules of histamines etc
...
TCR interaction with antigen on MHC
2
...
(cytokines affecting differentiation)
Signal 1
• Can only recognise antigens in the context of 'self'
○ Must be presented by the correct MHC
• Binding of antigen to MHC is crucial
• Diversity in MHC genes to allow antigen binding
○ Polygenic
 3 sets of class I and 3 of class I genes on each individual
○ Polymorphic
 Many alleles for each gene
 Point mutation, gene conversion, pseudogenes (provide genetic material)
○ Variation concentrated in peptide binding groove (peptide recognition) or alpha helices
forming walls (TCR recognition)
○ Class Ia and class II highly polymorphic
• Evidence for mate selection based on different MHCs
○ Humans
 Men wore the same shirt for 2 nights
 Women selected most attractive
Immunology Page 17

•

•

•

•

 Women selected most attractive
 Complementary MHCs
Cattle - BoLA alleles associated with resistance/susceptibility to
○ Bovine leukosis
○ Ticks
○ Mastitis
○ Trypanosomiasis
○ Foot and mouth
Horses - ELA
○ Allergic response to Culicoides bites
○ Sarcoid tumours
TCR interacts with MHC
○ MHC class I with CD4 helper
○ MHC class II with CD8 cytotoxic
○ Specificity dependent on CD4/8 molecules
 Strengthen/stabilise interaction and transduce the signal into the T cell
All T cells express a TCR that occurs in a CD3 complex
○ CD3 not involved in antigen binding
○ Crucial to signal transduction

Signal 2
• Costimulation
○ CD40 on APC (such as B cell)
○ CD154 (CD40L) on T cell
○ Activatory signal for APC and T cell
○ CD28 on T cell interacts with CD80/86 on APC
○ Activates T cell
• Without CD28-CD80/86 interaction there is no activation
○ Prevents incorrect immune responses
○ Turns T cell into anergic T cell - cannot create response
• After activation and proliferatio due to signals 1 and 2 immune response must be controlled
○ Proliferating T cells express more CTLA-4 (CD152)
○ CD80/86 interaction with CTLA-4 leads to inhibitory signals
Signal 3
• Determines differentiation
• Mainly cytokines from APC
• Influenced by PRR stimulated
Immunology Page 18

• Influenced by PRR stimulated

Immunology Page 19

T Cell Subsets and Function
10 February 2016

10:58

T Cell Activation
• Proliferation
○ Aided by IL2 - produced by activated T cells
○ Activated T cells express higher affinity IL2 receptor
○ IL2 drives proliferation
○ Clonal expansion of T cells with correct TCR
○ Cytokine can act on cell producing it (autokine)
• After proliferation is differentiation
○ Into effector cells
○ Range of subsets
T Cell Subset
• Th1
• Th2
• Th17
• Treg
• Also Tfh, Th0 and others
• Differentiation depends on signal 3
○ Cytokines produced by innate responses
○ Cytokines produced by APCs
○ Bystander cytokines

Th1
• Origin
○ Promoted by signal containing IFN-γ, IL12
○ STAT1, Tbet transcription factors activated
• Function
○ Secrete IFN-γ, IL2, TNF-α
○ Activate macrophages
○ Class switched to IgG (opsonizing)
○ Increase MHC class I and II expression
○ Promoted NK cell activity
○ T-cell proliferation (autokine)
• Target
○ Activating macrophages to migrate
○ killing infected macrophages
○ increase MHC1
○ Intracellular infections
 e
...
viral diseases
Th2
• Origin
○ Differentiation into Th2 is promoted by IL4
○ STAT6, Gata3 transcription factors
• Function
○ Secrete IL4, IL5, IL13
○ B cell proliferation and antibody production
○ Antibody class switching to IgE (mast cell granulation)
○ Mucus production
• Target
○ IgE
Increase antibody
Immunology Page 20

○ Increase antibody
○ Activated mast cells and eosinophils
○ Helminth worms
Th17
• Origin
○ Il23, Il6, TGF-β
○ RORyT transcription factors
• Function
○ Secrete IL17, IL21, IL22
○ Neutrophil recruitment - activation of fibroblasts and epithelial cells to secrete
chemokines
○ Important at mucosa and epithelia
• Target
○ Proinflammatory
○ Extracellular bacteria, fungi
○ Also related to hypersensitivities e
...
rheumatoid arthritis
Treg
• Origin
○ Varies between cell type
○ IL10 and/or TGF-β
○ Downregulation of Th1 cytokine production
○ Downregulation of MHC class II expression
○ Downregulation of co-stimulatory molecules
• Target
○ Regulatory (immunosuppressive)
○ Dampening immune Reponses
○ Important in response to
 Many infections
 Failure in hypersensitivities (autoimmunity)
• Schistosomiasis
○ Chronic infection
○ ~100 eggs/day lodged in liver per worm pair
○ Without Treg suppression inflammation due to Th1 and Th2 would increase pathology

Immunology Page 21

Innate/Adaptive Link
12 February 2016

11:01

Innate
• Plants, vertebrates, invertebrates
• Non-specific
• No memory
Adaptive
• Vertebrates
• Specific
• Memory
Activating T and B Cells
• Initiating immune response
○ AOCs central in activating Th cells
○ Major APCs
 B cells
 Macrophages
 Dendritic cells
○ Naïve adaptive responses initiated by dendritic cells (sentinels)
 Langerhans cells in skin
 Powerful T cell activator
□ Strong costimulatory signal
 Macrophages and B cells more important during active response
 Dendritic cells migrate from tissues to lymph nodes
□ Present collected antigen to T cells
□ Occurs without stimulation
 "immature" non-activated DCs lead to no immune response
□ Interaction with PRR causes costimulatory expression
□ T cells become inactive/anti-inflammatory if activated by immature DCs
 "mature" activated DCs lead to initiation of immune response
 Co-stimulatory CD80/86 expressed on DCs due to
□ PRR/PAMP interaction - release cytokines (signal 3)
□ Cytokines
□ Other signals (damage related)
○ Immune response created an be
 Specific to type
 Compartmentalised within the body
□ Lamb et al
...

□ L
...
sigmodontis caused increased IL-4 near thoracic cavity
Cell Movement
• During immune response a key role of innate and immune responses is recruiting cells to the
site of infection
○ Initial recruitment reliant on innate cell cytokines
○ Later driven by adaptive lymphocyte cytokines
• Chemokines critical
○ Released by immune cells at the site of infection
○ Chemotaxis along chemical gradient
• Leukocyte Extravasation - out of blood
○ Capture
 TNF-alpha induces upregulation of adhesion molecules on endothelium
Immunology Page 22

 TNF-alpha induces upregulation of adhesion molecules on endothelium
 VCAM-1 on endothelium, VLA-4 on leukocyte
○ Rolling
 P-selectin/E-selectin responsible for slow rolling
 PSGL-1 on leukocyte
○ Firm Adhesion
 ICAM-1 on endothelium, LFA-1 on leukocyte
○ Transendothelial Migration
 Main receptor is PECAM-1 on both

• Critical in bTB testing
○ Swelling due to memory lymphocytes recruiting leukocytes to infection site

Cytotoxic T Cells and NK Cells
• Cytotoxic T Cell
○ Major effector of adaptive IR
○ Requires activation in lymphoid tissues - MHC class I/TCR
○ Migrate to infected site
○ Recognise infected cells via MHC1
• Immune evasion
○ MHC1 crucial
○ Viruses was remove from surface
• Natural Killer Cells
○ Innate cell
○ Lymphoid lineage
○ Recognises infected host cells
○ Not as effective as CF8 Tc cells though
○ Distinguish infected from uninfected using MHC class I
○ Normal MHC1 recognised and inhibits NK
○ Lack of correct MHC1 can lead to NK activation and induction of apoptosis
○ Abnormal MHC class I also observed in cancer cells
○ NK can activate in other ways
 Additional receptors

Immunology Page 23

Cytokines
15 February 2016

11:15

Cytokines
• Small soluble proteins (peptides and glycoproteins)
• Intercellular messengers of the immune system
• Bind specific membrane receptors
Chemokines
• Specialised subset
• Chemoattractants
• Mobilise immune cells
Family Nomenclature
• Chemokines - CXC, CC, CX3C, CX
• Cytokines
○ IL-1
 IL1alpha, IL1beta
○ IL-2
 IL-2
 IL-4
○ Interferon-17
 IL17A
 IL17B
○ Interferon
 IFN-alpha
 IFN-10
○ Transforming Growth Factor
 TGF-Beta
○ Tumour Necrosis Factor
 TNF-alpha
Roles
• IL-1B
○ Fever, inflammation
• IL-2
○ T-cell proliferation
• TFG-B
○ Production of IgA in mucosa
• IL-4
○ Important in B cell activation
• IL-4, TGF-B, IL-12
○ Signal 3 to naïve T cells for differentiation
• IL-8
○ Neutrophil migration

Actions of Cytokines
• Autocrine action
○ Act on the cells that secreted them
○ E
...
IL-2 on T cells

Immunology Page 24

• Paracrine Action
○ Act on nearby cells
○ E
...
g
...
g
...
g
...
High L-selectin/CD62L
 L-selectin and CCR7 are homing receptors for secondary lymphoid tissues
 Circulate through blood and lymph nodes
 Increased proliferate potential upon re-stimulation
□ Don’t become effector cells
 Sentinels
○ Central Memory T Cells (1999)
 CCR7+, CD62L high
 Very similar to Tscm
○ Effector Memory T Cells (1999)
 CCR7-, CD62L low
 Circulate through blood
 Specialised for entering inflamed tissue
 Rapidly develop into effector cells upon re-stimulation
...
2015
○ IL-7
○ Causes TAG storage (fats)
○ Express aquaporin 9
 Glycerol uptake and TAG synthesis
○ Long term persistence

Immunology Page 32

Recap
19 February 2016

•
•
•
•
•

11:06

10 immunological regions
16 cell types
28 receptors/ligands
20 cytokines/chemokines
Complex IS
○ Evolved

Innate
• Phagocytosis
○ Major IS effector mechanism
○ Macrophages
○ Neutrophils 50-75% of WBCs in blood
○ Macrophages have receptors that bind antigens
 Bound material is internalised and digested by lysozymes
○ Respiratory infection
 Inflammation from damage
 Neutrophil migration
 Death of neutrophils releases iron co-factored enzymes
 Green bogeys
• Barriers
○ Skin, gut, lungs, eyes, oral cavity
○ Tight epithelial cell junctions
○ Mucus
○ Cilia
○ Tears
○ pH
○ Enzymes
○ Surfactant
○ Defensins
○ Normal microbiota
○ Schistosome Worms
 Release enzymes to break down epithelia
 IL-10 production to reduce inflammatory responses
 Inhibit Langerhans cell migration
Innate/Adaptive Link
• Adaptive immune response blind to what is a threat
• Innate arm of the immune system directs the adaptive

Immunology Page 33

• Cancer cells induce a poor immune response
○ Dendritic cells critical in activation naïve CD8 Tc cells
○ Cancer studies in dogs and humans targeting DCs
○ Activate DCs and culture with tumour antigen
○ Inject chimeric antigen/vector - targets DCs and delivers antigen
Cellular Adaptive Response
• T cell subsets
• Different immune response

Immunology Page 34

Immunology Page 35

Tolerance
22 February 2016

10:57

Adaptive Immune Response
• Random Ig and TCR genes rearrangement means that any antigen can be recognised
• Battle rapidly evolving pathogens
• Stronger immune response next time
• But
○ Can respond to any protein/glycan
○ Food, gut bacteria, own eye, foetus
• Tolerance prevents incorrect responses
○ Self-antigens
○ Innocuous environmental antigens
Immune Tolerance
• Not a failure to recognise
• Active response to particular antigen
• Specific
• Required for
○ T lymphocytes
○ B lymphocytes
• Failure is rare
○ Autoimmunity
○ Hypersensitivity
• 2 types
○ Central - 1o lymph organs
○ Peripheral - 2o lymph organs
○ Defined by where state of tolerance is induced for the specific cell
Central Tolerance
• T Cells
○ Dendritic cells migrate from tissues to lymph nodes
 Present antigens taken up to T cells
 Occurs without stimulation
 Present self and non-self
○ Occurs in thymus
 Selection process of cells via TCRs
 Early T cell development
 Before they are mature and able to influence the immune systems
○ Bone marrow - pre-T cell
 Migrates through blood stream to thymus
 CD4-, CD8-, double negative for CDs
 TCR TCR rearrangements no happened yet
○ Thymus cortex
 TCR+, TCR gene rearrangement has occurred
 CD8+, CD4+, double+, has both
 Cortical epithelial cells present self-antigen via MHC class I and II to thymocytes
 No interaction leads to apoptosis - 90-96%
 High interaction leads to apoptosis - negative selection, 2-5%
 Low/intermediate affinity interaction - survival signal, positive selection, 2-5%
 If the TCR interacts with class I MHC, CD8+ only
 If TCR interacts with MHC class II, CD4+ only
□ Now single+
○ Thymus medulla
 Medullary epithelial cells and/or dendritic cells interact with thymocytes
 Medullary epithelial cells express proteins from everywhere in the body
□ Brain-, kidney-specific etc
...
Clonal anergy (also B)
 secondary signal leads to no ability to respond, and not be activated even by 1o
signal

 Co-stimulation needed
2
...
Clonal Ignorance
 Self-antigen in too low concentration, sequestered away (CNS), anti-inflammatory
sites (eye)
 Self-antigen not commonly presented
□ Ever activates Tc
Immunology Page 37

□ Ever activates Tc
 Reactive Sympathetic Opthalma
□ Injury exposes previously 'hidden' ocular antigens
□ Autoimmune response damages eyes

Immunology Page 38

Hypersensitivity
24 February 2016

12:02

T Cell Selection
• Naïve T cells have positive and negative selection
○ Weak/intermediate interaction
○ No binding --> apoptosis
○ Host antigen binds --> apoptosis
• Similar process for B cells
Hypersensitivities
• Tolerance process fails
• Immune system reacts to "harmless" antigens
• All autoimmune diseases
• 4 types
○ Type 1
 Antibodies against environmental antigens
○ Type 2
 Abs against cellular antigens
○ Type 3
 Abs in immune complexes
○ Type 4
 Cellular response to chemicals/antigens
Type I Hypersensitivity
• "Immediate"
• Always IgE mediated
○ Atopy = excessive IgE production
• Inflammation due to release of histamine and other molecules
• Excessive Th2 environment
• Symptoms
○ Itching
○ Mucus production
○ During allergic anaphylaxis
 Coughing (horses and ruminants)
 Vomiting (dogs and cats)
• Diseases
○ Canine atopic dermatitis (terriers and dalmations)
○ Sweet itch (horses)
○ Allergies to waddle fly
• Cell types
○ Mast cells
○ Basophils
○ Eosinophils
• Major mediators
○ Histamines
○ Leukotrienes and prostaglandins
○ IL35, IL4, IL5, Il6
○ CCL5 CCL11
○ Serotonin
• Mechanism
○ 1st exposure to allergen
○ Antigen activation of Th2 cells and stimulation of IgE switching
○ Production of IgE
IgE binds FcεRI on mast cells
Immunology Page 39

○ IgE binds FcεRI on mast cells
○ Repeated exposure to allergen
○ Mast cell activates
 Releases cytokines and mediators
○ Mediators lead to
 Vascular leak
 Broncho-constriction
 Intestinal hypermobility
 Inflammation
 Tissue damage

○ Late phase reaction

• Current Treatments
○ Avoidance
○ Immunotherapy/desensitisation
Corticosteroids
Immunology Page 40

○ Corticosteroids
○ Epinephrine
Type II Hypersensitivity
• "Antibody Mediated"
• Antibodies against host cells
• Destroy cells using complement pathway
○ Classical pathway
 C1q interacts with antibodies bound to the surface
 Generate C3 convertase, c3 cleaved, C3b bound to surface, C3a released
• Symptoms
○ Loss of platelets and RBCs
• Diseases
○ Incompatible blood transfusions
○ Myasthenia gravis
 Acetylcholine receptors blocked by antibodies
 Muscle weakness and fatigue
○ Haemolytic disease of the newborn
 Thoroughbred horses (and mules)
 Female animals encounter in utero RBCs via placental leakage
 Mother doesn't possess RBC antigen
 Sensitised animals
□ Previous blood transfusions
□ Previous pregnancies
 Anti-newborn RBC antibodies produced and pass into newborn
 Destroy RBCs
 Haemolytic disease
 Symptoms
□ Weakness, anaemia
□ Jaundice
□ Mucus membrane damage
Type III Hypersensitivity
• "Immune Complex Mediated"
• Immune complexes
○ Antibody antigen complexes
○ Not cleared by innate system
○ Accumulate and cause inflammation

• Reactions against soluble antigens
• Symptoms
○ Complexes caught in capillaries
○ Issue in organs
 Nephritis
 Vasculitis
 arthritis
Immunology Page 41

 arthritis
• Diseases
○ Canine adenovirus
○ Bovine viral diarrhoea
○ African swine fever
○ Equinoviral arteritis
○ Systemic lupus erythromatosus
 Loss of hair in horses
○ Canine lupus
 Middle aged dogs
 Collies

Type IV Hypersensitivity
• "Delayed"
• Cell mediated reactions
• 12-36 hours after exposure
• T cells and/or NK cells
• Causes inflammation
• Symptoms
○ Depends on target
• Diseases
○ Allergic contact dermatitis
 Dogs - carpet dyes
 Cows - rubber from milking machines
 Cats - flea collars
 Horses - dyes on tack, leather additives
 Mechanism
□ Inflammation/damage caused
□ Chemicals bind to skin proteins
□ Langerhands to lymph node
□ Secrete IL12, IL23, activate Th1 and Th17
□ T cells move to skin to respond against skin cells
 Produce IFN-gamma, activate Tc and NK cells
• Tuberculin
○ Extract of mycobacteria proteins
○ Used to test cattle for previous M
...
2 up to 9
...
g
...
Attenuated Vaccine
○ Viable pathogen, reduced pathogenicity
○ Can cause infection but not disease
○ Attenuated by deliberate passage
○ E
...
TB, rotavirus, MMR, strangles
○ Attenuation (Polio-Sabin)
 Poliovirus grown in tissue cultures
 Repeated subpassage mutations accumulate
Immunology Page 44






Repeated subpassage mutations accumulate
Test for paralytic activity
Clinical trials
Mutations sequenced

○ Immune response
 Strong, appropriate response
 Cellular immunity
 Humoral immunity (including secretory IgA)
 Long-lasting memory
○ Advantages
 Multiple antigens
 Few immunisations
 Easy to produce without genome
 May not require adjuvant (modifier)
○ Issues
 Reversion to wild type (polio 2 and 3)
 Persistent infection (varicella-zoster - chickenpox, shingles etc
...
Killed vaccine
○ Killed by heat - can denature too many protein antigens
○ Killed by chemical - formaldehyde (Salk polio)
○ e
...
yearly flu vaccination, hepatitis A
○ Immune response
 Weaker response than live vaccines
 Good serum antibody response, little secretory IgA
 Poor cell-mediated immunity
 Booster shots usually required
○ Advantages
 Multiple antigens
 Stable
 Safer than live vaccine
 No refrigeration (attenuated can need this)
○ Issues
 Vaccines not always killed (polio)
 Lack of understanding about why it protects
 Contamination with animal viruses (polio)
 Initial preparation requires working with pathogen
3
...
g
...
g
...
g
...
Pneumoniae, hepatitis B
○ Immune response
 Weak immune response
 Good serum antibody response
 No cell-mediated immunity
 Booster shots usually required
○ Advantages
 Limited antigens - less chance of cross-reactivity
 Higher levels of specificity and reproducibility
 Safe than live no chance of accidental infection
 No need for refrigeration
○ Issues
 Toxoid vaccine
□ Limited to few bacterial diseases
 Limited number of antigenic targets - evolution
 Difficult to develop
 Ajuvant required

Immunology Page 46

Veterinary Vaccines
02 March 2016

11:12

Adjuvants
• Inject antigen into animal - little effect
• No activation, no costimulation, no response/memory
• Required activation of the innate immune response
• Initiation of immune response
○ Maturation and up-regulation of costimulatory molecules (CD80/86) of dendritic cells
○ Activation of DC PRRs by PAMPs
○ Cytokines
○ Other signals (damage related)
○ https://blackboard
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ac
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%2C%202003-%20adjuvants
...
g
...
g
...
g
...
g
...
burgdorferi
 Expressed in E
...
g
...
g
...
g
...
g
...
of cells
○ Differences and distribution info lost
• Flow cytometry
○ Heterogeneity
○ Speed
○ Quantitative
○ Multiparametric
○ Fluorescence activated cell sorted
 Break up stream with vibrations
 Charge droplets and deflect desirable ones and collect
 e
...
all red cells
Applications in Immunology
• Jaye et al 2012
• Cancer
○ Diagnosis
○ Prognosis
• Diseases of the immune system
○ Diagnosis
○ Monitoring
• Cell therapy
• Transplantation
• Human blood cells
○ Different typed
○ Distinct sizes (forward scatter) and granularity (side scatter)

Immunology Page 51

•

•

•

•

○ Gating
 Fluorescent markers and isolation of subpopulations
 Don't need to physically isolate
Clinical diagnosis
○ e
...
Actute Lymphoblastic Leukemia
○ Bone marrowcells evaluated based on SSC and CD45 expression
○ Raised lymphoblasts (immature WBCs)
HIV
○ Markers on WBCs
○ Number of different classes
○ CD4+ T cell counts
Cytometry for life initiative
○ Field application
○ Isolated places
○ Simpler machines
○ Recycles fluid
○ Runs off battery/solar panel
Flow imaging
○ Not as fast
○ 100s cells per second
○ Multiple parameters

Jaye et al
...
medullary cords (macrophages) paracortical outside (T cells)
□ Lymphocytes largely restricted to bloodstream
 Few in lymph

○ Innate Immune Cells
 Neutrophils largest % in most (58-85% in dogs)
Immunology Page 53

 Neutrophils largest % in most (58-85% in dogs)
□ 15-30% in cows
□ In cows/lymphocytes higher
 62% of WBCs in cattle vs 30% in horses
 Gamma/delta T cells - more in ruminants than horses
 T cell receptor composed of 2 transmembrane chains alpha and beta

 T cells composed of gamma and delta - different genes - limited
diversity
 Innate and adaptive roles, enigmatic functions
 Some interact with stressed cell molecules e
...
infected or cancerous
cells
 No MHC needed - direct antigen interaction
• Antibody Formation and Classes
○ Antibody formation
 Heavy chain locus, multiple Variable, Diversity, and Junction segments
 Light chain locus, multiple V and J segments
 In mammals
□ V 38-46, D 23, J 6
□ Randomly generated to create diversity and tackle new pathogens
□ Somatic recombination
 In birds
□ Very limited diversity
□ V 1, D 15, J 1
□ Somatic recombination not source of diversity - Somatic gene conversion
□ 1 DNA sequence replaces a homologous sequence
...
)
□ Nor T cell receptors
 Gene conversion for diversity 1014 unique possibilities
○ Antibody isotypes

Immunology Page 54

 Non-mammalian tetrapods
□ Amphibians - IgM, IgD, IgY
□ Reptiles - IgM, IgD, IgY
□ Birds - IgA, IgM, IgY
□ All lack E and G, Y likely an ancestor
 Ig-NAR and IgW in sharks, similar to IgD
 IgT in some bony fishes, similar to IgA
 IgX similar to IgA
 Monotremes
□ IgG, IgE, IgA, IgD, IgM
□ Also IgO in platypus
 Unique in mammals
 Lacks hinge
 Related to bird IgY and mammalian IgG
 Marsupials
□ IgG, IgE, IgA, IgM
□ Lack IgD in species examined so far
□ Relatively limited heavy chain V diversity
 More complex in light
 Therian mammals
□ Nearly all IgG, IgM, IgD, IgA, IgE
□ Rabbits - lack IgD
□ Camels - heavy chain IgG
 Potential use in drugs
 Increased flexibility to bind toxins

○ Subclasses of antibodies
 In addition to switching classes
 Also subclass switching
 Horses
□ 7xIgG (Ig1 Ig2 etc
...
Swelling, increased tissue fluid
○ Nitric oxide
○ Chemokines: CXCL1, CXCL8
 Attach to proteoglycans in ECM to form concentration gradient
 Neutrophils grow cell projections at leading edge
○ Acute phase proteins
Activation of Tissue Macrophages
• If pathogen has invaded tissues, macrophage may recognise with specific PRR
• Often not enough macrophages to phagocytose and kill all pathogens
• Initiate response - inflammatory response
○ Recruit more cells

• Prostaglandins, platelet activating factor, cytokines
• TNFa, PGs, PAF act on endothelium to increase vascular permeability
• IL1 and TNF activate endothelium cells to increase adhesion
Neutrophil Chemotaxis
https://www
...
com/watch?v=iFOus8ehxUc
•
Activation of Mast Cells

Immunology Page 57

Activation of Mast Cells

•
•
•
•
•

Mast cells are distributed throughout the body
Large granules
Activated and release contents
Vasodilation and increased vascular permeability
Once activated they synthesise prostaglandins and leukotrines
○ Vasodilation and permeability
○ Attract neutrophils

DAMPs
• Innate cell recognition of damage
• Damage associated molecular patterns
• Host proteins
• Nuclear and cytosolic proteins (usually hidden)
• Innate system should only see them after damage
Pain
•
•
•
•

Damaged endothelial cells and platelets produce pain response mediators
e
...
serotonin
Plasma leaking from blood vessels also contains bradykinin
Serotonin and bradykinin stimulate nerve endings - pain

Acute Phase Response
• Pathogen not eliminated
• Systemic response
• Brain and liver
• IL6 lead to upregulation and downregulation of hundreds of proteins
• Liver
○ Mannan binding lectin
○ C reactive protein
○ Complement
• Complement cascade
• C3b is an opsonin
○ Promotes phagocytosis
• Complement
○ C3a - mast cell activation
○ C4a - mast cell activation
○ C5a - chemotaxis, vasodilation, neutrophil and mast cell activation

Immunology Page 58

Acute Phase Proteins
• Haptoglobin
○ Binds haemoglobin
○ Prevents bacteria gaining iron
• Fibrinogen
○ Potential damage to tissues
○ Generates fibrin threads
○ Clot can block pathogen spread

Resolving Inflammation
• If macrophages are successful inflammatory response ends
Immunology Page 59

• If macrophages are successful inflammatory response ends
• Otherwise
○ Short half-life of inflammatory mediators
○ Macrophages convert from pro-inflammatory to anti-inflammatory
○ Anti-inflammatory lipoxins, resolvins, and protectins released by endothelial cells
Wound Healing

• Platelets and macrophages release growth factors e
...
PDGF
○ Stimulate fibroblasts
• Fibroblasts secrete collagen and other connective tissue components
/

Immunology Page 60

Equine Endometritis
16 March 2016

12:54

Debbie Nash PhD
• Uterine inflammation
• Mating Induced Endometritis MIE
○ Post mating
○ Transient response
○ Spermatozoa
○ Bacterial contamination
• Persistent MIE
○ Up to 40% of mares
• Compromise fertility
Tissue Explant
• Whole animal has ethical issues
• Equine epithelial cells notoriously hard to culture
• Encompasses range of cell types and interactions
• Partially maintains tissue architecture
• Simple and saves time
• Good model species
○ Tissue availability
○ Large endometrial surface area
○ Exemplar for pigs, dogs, humans
• Development
○ Effect of different molecules on prostaglandin F20 secretion
○ Blood sample collected for serum P4 determination
○ Uteri selected for stage of cycle and absence of inflammation
○ Cultured in Williams medium
○ 24hr establishment period
○ Applied treatments
 Sampled at 24hr and 72hr
 Added oxytocin to test for functionality - prostaglandin secreted in response, as
alive
 Sperm added, prostaglandin up, as alive
 E
...
Innate cells - Chediak-Higashi Syndrome
 Defect of phagocytosis
 Autosomal recessive
 Humans, hereford cattle, persian cats, tigers, orcas
 LYST gene
 Critical in lysosome membrane fusion - no fusion occurs






Affects function of neutrophils (mainly) and Tc lymphocytes
Large lysosomes in neutrophils
Can't clear bacterial infections
Also affects melanosome fusion
□ Partial albino traits
□ Photosensitivity
2
...
4% of Arabian horses
□ No functional T or B cells
 NK, neutrophils etc
...
B Cells - X linked Agammaglobulinaemia
 Defect in B lymphocytes maturation
 First ID described
 Defective gene on X chromosome
□ B cell tyrosine kinase Btk gene
 Function related to B cell receptor signalling

□ Critical in B cell development
 6-9 months - loss of maternal Ig
 Sites: mucous membranes, ear, lungs, blood, gut, skin, eyes,
meningitis (brain)
 Repeatedly acquire infections with extracellular organisms
□ Streptococcus, pneumococcus
 Treatment - Intravenous Ig (IVIG)
Secondary Immunodeficiencies
• Cause
○ Environment
Immunology Page 64

○ Environment
 Malnutrition
 Cancer
 Drug treatment
 Organ removal
 Infection
 Stress
 Age
○ Primary = absences, secondary = reduced
 Low T cell count
 Lower B cell proliferation
○ Malnutrition
 Different nutritional deficiencies --> different immunodeficiencies
 Obesity also associated with cancer, inflammation, autoimmune
diseases (rheumatoid arthritis)
 Zinc - critical in T cell activation
□ Pigs with Zinc deficiency: decreased Tc activity, B cell activity,
NK activity, phagocytic activity
 Copper deficiency also has impact
 Vitamin A

l
○ Infection
 Select bacterial, protozoal, and helminth pathogens can impact
immune responses
 Major infections are viral
1
...
FIV
 Feline
 Spread by biting, suckling, mating
 Similar to HIV infection
◊ Initial infection, moves to lymph node
◊ Results in lymphopenia
 Binds CD134 and CXCR4
◊ Present on CD4 Th cells
 1-44% infection rate
 Leads to opportunistic infection progression
◊ FeLV, cryptococcus
 No cross-reacting with FeLV (different virus)
3
...

○ Key components
 Dendritic cells
 M cells in Peyer's patches
 B lymphocytes/antibodies
 Gamma/delta T lymphocytes and Treg lymphocytes
○ Additionally
 Intestinal epithelial cells are key
□ Tight junctions to prevent infections
□ Produce antimicrobial peptides (defensins)
□ Possess TLRs
 Not on lumen side and are intracellular
 Commensal bacteria
□ Exclude pathogenic organisms
□ Activate innate immune response (e
...
AMPs)
□ Can lead to Treg production
Antibody/B Lymphocytes and Dendritic Cells
• Antibodies
○ IgA most abundant in mucosal secretions
○ In pigs 90% of intestinal Ab producing cells make IgA
○ IgA opsonises less well, weak complement activation
○ Dimeric IgA binds to mucus and to pathogens/toxins
○ A percentage of IgA is against commensals - prevents opportunistic
infections
○ Main function is immune exclusion - prevents adhesion to host cells
 IgA (and early IgM)
 IgG and IgE function in the mucosa via immune elimination
○ Isotypes matter
 High IgA critical in protection
 High IgG not protective
 Must be considered in vaccines (oral, intranasal, intrarectal)
○ E
...
Bordetella in dogs
Transmissible gastroenteritis vaccine in piglets (oral live vaccine)
Rotaviruses (neonatal diarrhoea in calves) (oral vaccine in human)
• Dendritic Cells
○ Initiate mucosal immunity
○ Intestinal DCs extend into lumen
○ Capture commensal bacteria/antigens
○ Move to lymph nodes and present to B cells
○ DCs and M cells (macrophages acting as bridges from lumen to cell
propria) don't always degrade antigens - allows BCR B cell activation
○ Important cells in T responses
• B Lymphocytes
○ Activated IgA producing B cells against pathogens not limited to specific
mucosal site of infection
Percentage of B cells move
Immunology Page 67

,

○ Percentage of B cells move
Site --> Regional lymph node --> blood stream --> other mucosal sites
γ/δ T Lymphocytes and Treg Lymphocytes
• Alpha/beta, gamma/delta
• Both types in MALT
• γ/δ mainly present just below epithelial layer
○ IFNγ secreted
• Alpha/beta throughout lamina propria
○ Responses depend on subset - important in B cell activation
○ Include Treg lymphocytes
• Treg Cells
○ Il10 and/or TGFβ signal 3
○ Secrete IL10 and/or TGFβ
○ Downregulate Th1 cytokine production
○ Downreg MHC2 expression
○ Downreg costimulatory molecules
• Changed balance between Treg and proinflammatory cells (Th1,2,17) can lead to
Inflammatory Bowel Disease
○ Boxers - Histiocytic ulcerative colitis
○ Wheaten Terriers - Enteropathy
○ Irish Setter - Gluten sensitivity
https://blackboard
...
ac
...
pdf

Immunology Page 68

Pregnancy and Newborn Immunity
18 April 2016

10:57

Reproduction and the Immune System
• The mammalian immune system will reject non-identical tissues/cells of the same species
• Allograft rejection
• Blood transfusion
• Haemolytic disease of the newborn
• Every foetus is at least 50% maternal derived
○ Up to 50% non-self
• 3 main challenges
1
...
Implantation and development of the foetus
3
...
MHC)
○ Up to 90-95% females in mice and horses produce antibodies against foetal MHC1
 May protect by blocking T cell response (inhibition of complements)
Placental Factors
○ Embryo-derived placental layers influence immune responses
○ Estradiol, progesterone
○ Alpha2 macroglobulin
○ Release of IFN-tau is critical in sheep and cattle for pregnancy recognition
○ IFN-omega in horses and dogs
 Leads to progesterone production (Bazer 2013)
Pregnancy can lead to issues
○ Mild immunosuppression in late pregnancy/early post-partum
○ e
...

 Neutrophil number - dairy cows
 NK cell activity - pigs
 Lymphocyte activity - mares
○ Maternal tolerance of developing foetus but immune systems of mother and foetus can
be challenged by infection
○ Immune tissues develop during gestation
 Blood lymphocytes
□ Horse 120 days (340 day gestation)
□ Calf 45 days (280)
□ Pig 70-80 days (115)
 Major veterinary issue is infections during gestation
 Intra-uterine infections
□ Infections during development that can lead to differing endpoints
 Abortion, persistent infection
□ Include
 Toxoplasmosis
 Bovine Herpes Virus 1 (BHV1)
 Bluetongue
 Bovine Viral Diarrhoea (BVD)
◊ <50 days - abortion
◊ 50-120 days - may develop persistent infection
◊ Seen as "self" - no antibodies

Newborn and Immune System
• Survival after birth
• Immune system present, but not appropriate without assistance
• Passive immunity
Immunology Page 70

• Passive immunity
• Haemochorial - humans, rabbits, rats, mice
○ 3 layers, embryo derived
○ Full antibody transfer
• Endochorial - cats, dogs
○ 4 layers, 1 maternal derived, 3 embryonic
○ 6-10% IgG transfer
• Epitheliochorial - ruminants, horses, whales
○ 6 layers, 3 maternal, 3 embryonic
○ No antibody transfer
• Different levels in colostrum and milk

• Chicken passive immunity
○ IgY (ancestral E, G), IgM, IgA in serum
 Hen sera --> egg yolk (IgY)
 Oviduct --> albumin (IgA, M)
○ Protects 10-20 days
• Maternal Abs impact vaccination of early animals - 1/2 life 5-10 days
○ Inhibits newborn ability to respond
○ No maternal Ab in calves - 1 week to make Abs
○ Maternal Ab in calves - 4 weeks to make Abs
○ Cats and dog vaccinated after 8 weeks

Immunology Page 71



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