Immunology 1 Flashcards
1
Q
List the 4 main functions of macrophages
A
1) Antigen presenting cells (APC) ingest and kill microbes -> PAMPS
2) highly phagocytic
3) clearance of RBC, WBC, apoptotic cells - DAMPS - damaged cells
4) Produce cytokines – signalling and amplification of immune response and colony stimulating factors – haematopoiesis
2
Q
What are the 3 main killing mechanisms of neutrophils and examples of granules
A
3 killing mechanisms 1. phagocytosis 2. degranulation 3. NETs - neutrophil extracellular traps Primary (azurophilic) granules: - Larger, denser - Contain peroxidase, lysozyme, hydrolytic enzymes Secondary (specific) granules: - Smaller - Collagenase, lactoferrin, phospholipase
3
Q
What are the 3 main areas mast cells are found and roles there
A
1) Gastrointestinal tract - important for parasites and worms
2) Airways - decreased diameter - prevent more pathogens from entering and increased mucus secretion - entrap the microbes
3) Blood vessels - as mast cells degranulate increase permeability of the blood - if get systemic infection can cause anaphylactic shock as too much leakage through blood vessels throughout body causing a decrease in blood volume and pressure
4
Q
List 2 mediators from mast cells, basophils and eosinophils and their functions
A
Mast cells and basophils
1) histamine -> increase vascular permeability, stimulate smooth muscle contraction
2) Prostaglandin D2 -> Vasodilation, bronchoconstriction, neutrophil chemotaxis
Eosinophils
1) peroxidase -> degrades helminthic and protoxoan cell walls
2) leukotrienes C4 -> prolonged bronchoconstriction, mucus secretion, increased vascular permeability
5
Q
What are Yδ T cells where found and function
A
mainly present in high levels in ruminants and neonatal animals
Only have a limited amount of molecules it can recognise unlike other T cells
6
Q
List the 4 main anatomical and physiological barriers
A
1) Epithelial Barriers
- Skin and mucosal membranes, Tight junctions, keratin, apid turnover rates
2) Secretions
- Lysozymes, Antimicrobial peptides, oxidative radicals, Intraepithelial lymphocytes
3) Effector cells
- Macrophages, Neutrophils Mast cells, Eosinophils, Natural killer (NK) cells
4) Effector proteins
- Complement proteins, Acute phase protein eg Mannose binding lectin & C-reactive proteins
7
Q
List the 6 features of the gut that acts as a barrier to infection
A
1) intestinal epithelial cells - physical
2) goblet cells -> produce mucus -> trapping
3) paneth cells -produce antimicrobial peptides and alpha defensins when given signals - cytokines -> insert themselves into membranes and form pores in the membranes that destroy the pathogen
4) M cells - sample intestinal lumen and stimulate cells within the peyer’s patches below if foreign material detected
5) Peyer’s patches
- lymphoid aggregates
6) Lamina propria
- Lymphocytes, B cells, dendritic cells, macrophages, plasma cells IgA
8
Q
Describe the two main molecules that are involved with the activation of the innate immune system and give 2 examples
A
- Pattern-recognition receptors (PRR) expressed by cells of innate immune system and detect microbes
- Limited repertoire (recognize 103 molecular patterns) - Pathogen-associated molecular patterns PAMP = unique microbial molecules (PAMP) shared by groups of related microbes not found associated with mammalian cells.
1) Flagella protein
2) peptidoglycan from the cell wall
9
Q
What are the 4 main types of PPR and main types within (example if needed)
A
1) Cell surface
1. Mannose receptor
2. scavenger receptor
3. N-formyl Met receptor
4. opsinon receptor
2) Cell surface signalling
- Toll-like receptors - TLR-4 (LPS)
3) endosomal
- other Toll-like receptors - TLR-3 (double-stranded viral RNA)
4) Cytosolic
- NOD like receptors
10
Q
Cell surface PRR what are the 4 types of receptors and their function
A
1) Mannose receptor - bind lectins and mannose rich glycans
2) scavenger receptor - bind to bacterial cell wall components, LPS
3) N-formyl Met receptor - promotes motility and chemotaxis of phagocytes
4) Opsonin receptors - allows others to bind acute phase proteins, complement pathway, immune complexes
11
Q
What are the functions of the PRR cell surface signallying and endosomal
A
1) Cell surface signalling
- TLR-4 recognise LPS
- Most use a intracellular signalling pathway within the cell that involves gene transcription of inflammatory cytokines and chemokines and antiviral chemicals such as interferons
2) Endosomal
- TLR-3 binds double-stranded viral RNA
- Activates same pathway as TOL-like receptors on the cell membrane
12
Q
Cytosolic PRR what type of receptors where found and function
A
NOD-like receptors
Two main families of PRR recognize PAMps within cytosol.
Family of proteins involved in both PAMP (and DAMP) recognition and in cell signalling.
Include
1. NOD-like receptors (NLR) family of over 20 members) recognize peptidoglycan component of bacterial cell walls
- Regulate pro-inflammatory pathways via NFkB signalling via transcription
2. RIG - like receptors (RLR) family of 3 RNA helicases that detect viral RNA within cytosol.
- bind viral RNA (SS and DS)
- Activate NFkB signaling but also release interferons via transcription
13
Q
What are DAMPs
A
- Associated with cellular and tissue injury and the result of release of cytoplasmic or nuclear molecules.
- DAMPs recognised by receptors against mitochondrial DNA, heat shock proteins ,adenosine metabolites, DNA interacting protein (HMGB1) .
14
Q
What are the 3 functions of natural killer cells
A
1) Killing of injured cells - DAMPS
2) Killing of infected cells - altered MCH1 or altered antibody (binding to Fc receptor)
3) Killing of phagocytosed microbes
15
Q
What are the 2 ways NK cells ill microbes
A
1) Via Fc receptor on the NK cell (Fcƴ RIII) - Antibody dependent cellular cytotoxicity (ADCC)
- Antibody is altered when binds to antigens on the surface of infected cells allowing NK cells to bind via their Fc receptor
2) Inhibitory and activating receptors
16
Q
What is the mechanism for NK cells that allows some cells to be killed and others not
A
1) Inhibition
The cells have activating ligand that binds to the activating receptor on the NK cell
Normal cells have MHC class 1 receptors that bind to the inhibitor receptor on the NK cell which prevents NK cell from destroying healthy host cells
2) Activating
- Infected cells still have the activating ligand that bind to the activating receptor on the NK cell however the MCH class 1 receptor has been modified or downregulated due to viral or bacterial infection therefore inhibition doesn’t occur
- This leads to the activation of the NK cell and therefore lysis of the target cell
17
Q
Structure of antibodies, what is the variable region, what gives functional change and what occurs
A
Basic antibody structure is composed of 2 heavy chains and 2 light chains
- The combination of a light chain with a heavy chain determine the antigen binding site -> variable region
- Once bound structure changes in the tail of the antibody so it can bind to Fc receptors, complements etc. ACTIVATED
Tail is giving the functional difference
18
Q
what are the two main functions of antibodies
A
1) Neuralisation - prevent virus , bacteria and toxins from binding to key molecules on cell surfaces or within the body by blocking the binding region
2) Opsonisation - focus immune cells and complement to pathogens leading to phagocytosis or lysis of the pathogen depending on the immune cell it binds to.
19
Q
List 5 innate immune cells that have Fc receptors and what occurs with opsonisation with Abs
A
1) Neutrophil - degranulation
2) Eosinophils - mainly IgE that binds to mast cell then binds eosinophils and causes degranulation
3) NK cells - leads to apoptosis of the cell via ADCC - Antibody-dependent cellular cytotoxicity
4) Macrophage - phagocytosis
5) Complement - activation causing
1. Lysis of microbes
2. Phagocytosis via opsonisation with complement fragments with C3b production
3. Inflammation
20
Q
What are the 5 antibody isotypes and their general functions and what is the same about them
A
HAVE SAME ANTIGEN SPECIFICITY
Heavy chain determine isotype
1) IgM - pentamer -first response, antibody secretion, opsonisation
2) IgA - dimer - gut, resist pepsin cleavage, neutralisation to prevent movement through mucus membranes
3) IgE - parasites, allergic response (mast cells and eosinophils)
4) IgG - secondary response, opsonisation, neutralisation, can go across placenta
5) IgD - B cell receptor
21
Q
What is different in terms of placental transfer in horses, ruminants and pigs, dogs and cats, primate and rodents and why
A
1) Horses, ruminants & pigs - epitheliochorial placenta - there are 6 barriers
- No transfer of Ab via placenta so colostrum extremely important
2) Dogs & cats - endotheliochorial placenta
- Only -5-10% IgG transferred via placenta
3) Primates, rodents - haemochorial placenta
- IgG transferred
22
Q
What are the 6 barriers in epitheliochorial placenta
A
- Endothelial cells, connective tissue, epithelial - baby
- Epithelial cells, connective tissue, endothelial - mother
23
Q
In what species and why are immunoglobulins in colostrum important, what type and timing that it occurs
A
Horses, ruminants & pigs - epitheliochorial placenta so no transfer during pregnancy
- when born no calf antibodies so need maternal from colostrum until can make their own
- IgM, IgG and IgA - neonates suckle (calf 2L colostrum)
- Absorption maximal in first 24h after birth due to epithelial cells not having tight junctions at that point
- Fc receptor on intestinal epithelial cells allow Ab to be transferred
24
Q
Passive immunisation what does it result in and examples
A
Short-term as injecting the antibodies straight into patients so not creating own antibodies
EG - Snake-bite anti-venoms have concentrated Abs purified from serum of vaccinated horses
25
List 5 features of a secondary immune response compared to a first
1. A much faster reaction time
2. A higher antibody response (typically 2-3 order of magnitude)
3. A qualitatively different antibody response (i.e. different isotypes, generally more IgG)
4. require T cell help while primary response can occur independently of T cells
5. A higher affinity of the antibodies to the antigen
26
Define epitope and paratope
- Area on the antigen that is bound is called the epitope and the area on the antibody that binds to the antigen (combination of the variable region of the heavy and light chain) is the paratope
○ Paratope-epitope interaction through complementary 3 dimensional structure with specific biochemical proper1es (charge, hydrophobicity, conformation, hydrogen bonds) - amino acid sequence gives the specificity
27
What is the structure of the MHC class I and II molecules
- Self-molecule: Major Histocompatibility Complex (MHC)
○ beta pleated sheet with 2 alpha helixes on either side makes up the binding site for the peptide
1) Class I MHC - made of 3 alpha domains and 1 beta domain - the alpha 1 and 2 domains hold the peptide
○ Different for individuals
2) Class II MHC
- Foreign antigen is small peptide derived from antigen (8-12 AA) and sits within the MHC complex
28
NK T cells what type of cell, what bind to and structure
NK T cell - different type of T cell
- Not variable between individuals
- Binds glycolipid on the pathogen - type of antigen
NKT TCR - NKT cell receptor binds to the glycolipid
29
T cell epitopes where found
- Could be exposed but does not have to be (can be within the organism)
- Peptides are selected from anywhere within the protein sequence
- Need to be able to be degraded to peptides of the right size
- Need to be associated with MHC
30
What are the 2 types of epitopes and what is important
1) Class I restricted T cell epitopes
2) Class II restricted T cell epitopes
• How the antigen is presented to the immune system is important because it determines the type of T cell that gets activated
31
What is important with activating B cells in vaccines
Respond to antigens outside the cell
- B cells recognise the 3D structure so needs to be perfect protein folding - Recognises 3 areas on the heavy and 3 on the light
- Can recognise amino acids not consecutive
○ Denaturing can change the folding so need to keep cool
○ Any 3D structure will be recognised including proteins with carbohydrates - glycosylation
Recombinant proteins produced in bacteria do not have carbohydrates
Native viral antigens are produced by mammalian cells, which can glycosylate proteins
32
What is important with activating T cells in vaccines
Repsond to antigen within cells
- T cells recognise the chopped up antigen so doesn't need perfect folding just small epitopes
○ Don't really care about glycosylation just need to cut to the right size - amino acids need to be consecutive
- Carbohydrates can affect how antigens are processed and which peptides are produced
Carbohydrates can interfere with antigen processing enzymes
33
What does CD4 and CD8 bind to
MCH molecules bind to TCR receptor on the T cells
- CD4 binds constant part of MHC II
– CD8 binds constant part of MHC I
Interaction with these molecules stablises the interaction between TCR and MCH molecules
34
What is the function of lymphocyte development and what are the steps
Lymphocyte development is designed to generate functional lymphocytes with useful antigen receptors that are not self-reactive
1) Takes a stem cell 2-3 weeks to development within thymus, cytokines leads to division and development of T cells
2) Selection then takes place to choose T cells with TCR appropriate for antigens, needs to be able to bind properly and not bind to self - apoptosis
35
What are the 2 types of T cell receptors their structure and the types of T cells
1) αβ TCR
- Two polypeptide chains α and β
- Constant and a variable regions.
- Single Ag binding combining site - Ag peptides bound to MHC
- CD4 TH cells bind MHCII
- CD8 TC cells bind MHCI
- Ag binding results in signal transduction
2. ƴδ TCR (5% T cells )
- Recognize antigens without involvement of MHC
- lipid antigens, heat shock proteins
○ Limited repertoire
36
What are the 3 steps that create the diversity within TCRs
1) Combinatorial diversity
- Genes for TCRs are present in pieces that can be combined in many different ways in different lymphocytes
2) Junctional diversity (second event that occurs after the combinational)
- diversity is further increased by adding or deleting nucleotides at the junctions
3) Third level of diversity
Two strands of the TCR receptor - need to make alpha and beta chain separately then join together to create different binding sites
37
What are the 5 steps in organsiation of TCR genes to form diversity of TCRs
1. DNA rearrangement during TCR-α chain formation in the thymus
2. Recombination activating gene (RAG) protein enzymatic complex - COMBINATIONAL DIVERSITY
- Loops out genetic material to be cut
3. DNA- Dependent Protein Kinases - Excises intervening DNA and forms DNA hairpin loop
4. Terminal Deoxynucletidyl transferase (TdT) - randomly add a codons (nucleotides) to join Variable, Joining and Diversity regions to add further diversity in the receptor - JUNCTIONAL DIVERSITY
5) add the alpha and beta chains together to create the receptor - THIRD LEVEL
38
List the 6 steps in the development of the T cell
1. Stem cells in bone marrow enter the outer cortical regions
2. Pro-T cell in the thymus is a double negative no CD4 or CD8
3. Proliferation occurs via IL-7 which is produced by the thymic stromal cells
4. Recombination of the β and α genes leads to expression of TCR and CD4 and CD8 receptors
5. The T cell is now a double positive with CD4 and CD8 - if doesn't form the TCR receptor apoptosis occurs
6) Positive and negative selection of the T cells then occurs
39
Describe the steps of positive and negative selection for T cells within the thymus
1. Thymic epithelial cells (reticular cells) present in the cortex display MCH molecules with different peptides
2. TCR tries to bind and if can't - signal for apoptosis - LACK OF POSITIVE SELECTION
○ If get weak interaction - TCR can progress - POSITIVE SELECTION
§ If it binds to MCH1 then becomes CD8 - CD4 downregulated
§ If it binds to MCH2 then becomes CD4 - CD8 downregulated
3. Dendritic cells or macrophages present antigens if TCR binds with high avidity then those cells again undergo apoptosis or if weakly or strongly bind to self-antigens - NEGATIVE SELECTION
40
How are self antigens presented in the thymus when not all are expressed there
- Autoimmune regulatory gene turned on in the thymus - allow cells to produce antigens that are self and only found in other tissues in the periphery
41
What occurs with the thymus and age and what implications may this have
size diminishes with Age
- Role sex steroid hormones causes decrease in size as when these levels increase probably already interacted many pathogens and building a repertoire
○ Therefore available repertoire is diminished
- This has implications following chemotherapy where lymphocytes are removed and need to be replaced
42
List the steps
1. B cells start life in bone marrow
2. Lymphoid cell -> Rearrangement of Heavy chain - unsure whether B or T cell
-> Pro-B cell -> Expression of heavy chain with surrogate
-> Pre-B cell -> Rearrangement of light chain -> immature B cell -> these cells can now undergo differential mRNA splicing to produce IgM and IgD ->
3. Mature B cell moves to periphery looking for antigen 4. IF ACTIVATED
- Ag stimulation
- Differentiation
○ Memory B cells
○ Ab secreting plasma cells - IgM
43
Why are different isotypes needed
antibody needs to be both soluble (we know they are soluble factors in the serum) and membrane bound (as a receptor to tell the cell that it has been selected)
44
Describe where the germline gene arrangement occurs
```
Light chain - kappa and lambda variable regions
Kappa chain
- Different segments
- V = variable regions
- Gap
- J = joining regions
- C = constant
Heavy chain - one variable region
Also have D = diversity
```
45
What are the 6 phases of antibody DNA
1. Germline DNA
2. Rearranged DNA
3. Transcription - RNA
4. mRNA formed with RNA splicing
5. Create polypeptide
6. Light or heavy chain is produced
46
When does B cell rearrangement occur
IN ONE B CELL DO REARRANGMENT OF DNA ONLY ONCE
| Only rearrangement occurs in one of the chromosomes, when occurs in one blocks rearrangement in the other
47
SCID what does it stand for, how transmitted what results in and why
Severe Combined Immunodeficiency in Arabian foals
- Always fatal as no adaptive immune system present
- Genetically transmitted: Recessive
- Can be tested (PCR)
- If homozygote for the mutation
• Lack of DNA dependent protein kinase or RAG-1 and RAG-2
○ Cannot rearrange Ig or T cell receptor
○ No T or B cells present
48
What makes an antibody membrane-bound and when does it change
Hydrophobic amino acid stretch within the antibody
- When removed then create the soluble antibody from membrane-bound
- B cells express membrane bound antibodies however to produce soluble they need to become activated and differentiate into a blast then plasma cell
49
What level does the change between membrane-bound and soluble Ig occur, where occurs and what
- Works at the level of the RNA as DNA already permanently rearranged
- Within the C-terminus of the heavy chain there is a hydrophobic stretch of amino acids
In order for antibodies to become soluble need to replace the hydrophobic amino acids with hydrophilic amino acids
- Achieved using 2 different polyadenylation sites so that 2 different mRNA transcripts are formed and encode for a soluble and a membrane bound heavy chain
50
List the steps in antibody isotype switching
1. If no T cells as first time encounter antigen will start to produce IgM and that is all
2. As time progresses or more commonly during the secondary immune response T cells become activated and they bind to the B cells
- Provide signals (cytokines) from T helper cells to tell the B cell you need to start making IgG (virus) or IgE (parasite)
3. Those signals go through the B cell to create the different isotypes
- Another genetic rearrangement - loop out DNA (as plasmid and lost) from constant area of the already recombined DNA
51
What is the result of isotype switching and how do you know which isotype is chosen
THEREFORE CAN NO LONGER CREATE IgM as that bit of DNA is lost
- no longer create any new membrane bound antibody IgM (if creating IgD then can become membrane-bound as still has the membrane-bound DNA sequence)
- However can make other isotypes if they are spliced further down then the previous isotype - downstream on the DNA
Which isotype is chosen?
1) Depends on cytokine environment
2) Depends on type of T cell help provided
52
Are there differences in isotypes with different species
- Name given to isotypes in different species is inconsistent
- Not related in terms of DNA sequence
- Not related functionally
- Not related in terms of induction by same cytokines
53
What is the difference between monoclonal and polyclonal antibodies
Polyclonal - a range of antibodies from different B cells -> different antibodies present with different epitopes
Monoclonal - Isolate antibodes from just one type of B cells -> produce one type of antibody with one epitope
- immortalized the B cells by fusion with cancer cells and produce one type of antibody
54
Describe the 3 signals involved with the activation of the naive T cell and what do they lead to
Signal 1: TCR-MHC/peptide > Specificity - first signal
Signal 2: CD28-B7 > Activation
Signal 3: Cytokine receptor-cytokine > Differentiation into different T cells
Dependent on the type of antigen (PAMP)
55
What is a CD8 and CD4 cell and their function
```
CD8 = Cytotoxic T cells
- Kill infected cells
CD4 = T helper response
- Activates other lymphocytes and innate immune system
- Allows differentiation of B cells
```
56
MHC class I where express what proteins does it present to who, and structure of the chains
- Expressed on most nucleated cells as these cells can be infected by viruses and have the machinery to make new viruses
- Presents cytosolic proteins to CD8+ T cells
Structure
- α-chain is variable from one individual to the other and selects the peptides to be presented.
○ The α-chain forms the” BBQ with the 2 sausages” that captures the peptides to be presented
- β2-microglobulin is constant amongst individuals and does not interact with the antigenic peptide that is presented.
57
MHC class II where expressed, what proteins does it present to who and structure of the chains
- Expressed on APCs, Macrophages, B cells, activated T cells, Dendritic Cells
- Presents extra-cellular proteins to CD4+ T cells
○ Extracellular as activating B cells that have antibodies that attack proteins
Structure
- α-chain is variable from one individual to the other and selects the peptides to be presented.
- β-chain is also variable from one individual to the other and selects the peptides to be presented.
- The α- chain and the β-chain together form the” BBQ with the 2 sausages” that captures the peptides to be presented
58
Describe the genes involves with MCH I and II molecules
3 polymorphic MHC class I genes as only alpha domain contributes to variability
- Express 6 different class I molecules (3 on each chain)
• 4 polymorphic MHC II genes
- 3α & 4β chains can associate with other chains
- 12 different class II expressed. (3 X4)
One chain from each parent
NO RECOMBINATION OF THE DNA
59
describe the specificity of MCH molecules and does it change between individuals and what haapens in inbreed populations
- Each MHC molecule is capable of presenting one peptide at a time but can bind to many different peptides.
- The MHC selects the peptide presented (any peptide that can bind could be self or non-self)
- No 2 members of an out bred population have same set MHC molecules (except identical twins), each individual inherits a different molecule from mum and dad - - Exception inbred populations > more susceptible to disease (for example inbred mouse strains)
60
What rejection occurs with MHC molecules
MHC is responsible for graft rejection: prevents grafting an organ from one animal to another unless there is some MHC match or the animal is immuno-suppressed
61
Describe the relationship between MCH molecules and heir affect on disease in an individual and population
Certain alleles of MHC prevent MHC molecules expressing certain peptides that may be involved in certain diseases
• MHC alleles are not distributed evenly in populations
• There could be selection pressure to promote certain types of MHC haplotypes over others
- Certain MHC haplotypes are associated with certain diseases
• Diversity in MHC protects a POPULATION against catastrophic breakthrough of disease not recognised by particular MHC
62
What are the two responses to different MHC molecules in grafts
-the host recognises the difference between MHC molecules and rejects the graft
○ This is called the host-versus-graft response
- Additionally the immune cells in the graft will also recognise the MHC molecules of the host tissue and reject the host
○ This is called the graft-versus-host response
63
How does MHC restricted T cell activation work
1. Whole blood collected from the cow will be incubated at 37°C 5% CO2.
2. Antigen presenting cells take up the antigen and present it to memory T cells in infected animals.
3. These T cells (which are only present in animas previously infected) will secrete IFN-γ, which can be detected in the culture supernatant using a capture (sandwich) ELISA.
64
What produce endogenous and exogenous antigens, what molecules presented on and examples
Intra-cellular pathogens: MHC I
virus, cancers some parasites
Extra-cellular pathogens: MHC II
bacteria and some parasites
65
Describe the 8 steps in MHC 1 processing
1) Initial folding of the alpha part of the heavy chain is aided by chaperone Calnexin
2) The partially folded chain is transferred to a second chaperone Calrecticulun which aids further folding of the receptor and association with β2m (microglobulin)
3) Other proteins associate with the MCH molecule such as Tapasin which then bind to the TAP transporter which forms the peptide loading complex
4) Peptides that bind to this complex are formed within the proteasome in the cytoplasm as the proteasome breaks down proteins from the cytosol
5) The peptides are then transported through TAP transporter in the ER membrane
6) Some peptides don't bind to the MCH molecule, others do but are unstable and are released in a process called - peptide editing
7) Finally a peptide binds to the molecule with high avidity leading to final folding of molecule and dissociation of the peptide loading complex
8) The MCH molecule with the peptide is then transported to the cell membrane via the Golgi apparatus and vesicle
66
List 4 ways viruses interfere with MHC 1 signalling
1. Blocks peptide entry to ER
2. Retention of MHC class 1 in endoplasmic reticulum
3. Degradation of MHC class 1
4. Binds MHC class I at cell surface and blocks binding to other cells of the immune system
67
List the 7 steps in peptide loading in MHC II pathway
1) Two chains of MCH II and the invariant chain form within the ER membrane
2) Invariant chain forms a complex with MHC class II molecule, blocking the binding peptide and misfolded proteins present in the ER from binding
3) It also guides the complex out of ER through golgi apparatus into a vesicle that enters the endocytic pathway where pathogens and foreign proteins are taken into the cell
4) Progressive acidification of the endocytic vesicle activate proteases that cleave the Invariant chain in two places leaving a short peptide fragment CLIP bound to the class II molecules
5) The proteins from the pathogens are also broken up into peptides
6) The removal of the CLIP peptide is the function of the HLADM
7) The MCH class II complex with the peptide is then transported to the cell membrane
68
What is the HLADM molecule and function
```
(MCH class II like molecule)
- It acts as a catalyst for both the release of CLIP and binding of the peptide
```
69
Why is cross-presentation needed
- To activate naïve T cells you need a B7 molecule that is only present in antigen presenting cells therefore they are the only cells that can activate both CD8 and CD4 cells
2. If a pathogen isn't infecting the antigen presenting cells it's peptides won't go through the MHC class I pathway as only internal peptides go through this pathway therefore CD8 cells cannot be activated as they are activated by MCH class I molecules as well.
70
How does cross-presentation work
```
- the antigen presenting cell engulfs the pathogen which would normally lead to the peptides only going through the MCH class II pathway (exogenous) and activating CD4 also leads to the peptide going through the MCH class I pathway due to Sec61 molecule that leads the peptides from the early phagosome into the ER.
THUS CD8 cells are also activated as the peptides are bound to the MCH class I molecule needed to activate CD8
- These cells are important especially in a viral infection
```
71
Describe 5 properties of cytokines
1) Pleitrophy - acting on different cell types to cause proliferation of B cells, thymocytes and mast cells
2) Redundancy - multiple cytokines doing the same function
3) Synergy - two cytokines binding to complementary receptors for a combined effect greater than the sum of the two cytokines
4) Antagonism - cytokines inhibits action of an other
5) Cascade - activate other cells to release of a range cytokines
72
List 3 macrophage cytokines and 5 key functions
IL-1, TNF-alpha, IL-6
1. Upregulate receptors on endothelial cells
2. Increase blood flow
3. Increase vascular permeability
4. Target organs such as the liver for production of acute phase proteins - have immune functions
5. Fever - increase thermoregulatory set-point
73
TNF-alpha what increases production and list 5 functions
LPS increased the secretion of TNF-alpha
1) Activate other macrophages via release of pro-inflammatory cytokines - vasodilation
2) Increases synthesis of acute phase proteins that stimulate non-specific immunity
3) Activates microbicidal activates within neutrophils and monocytes
4) Is a endogenous pyrogens - causes fever
5) Increases the expression of adhesion molecules on vascular endothelium
74
What is some pathology overproduction of TNF-alpha leads to
- If have too much produced can go into septic shock - die of circulatory collapse and disseminated intravascular coagulation
- Particularly with chronic conditions - continually producing TNF-alpha
○ Arthritis in the knee
75
List 3 ways cytokines are involved with movement of leukocytes through endothelial cells
1) endothelial cells are activated and upregulated for their binding receptor
2) slow down leukocytes so they can bind to endothelial cells
3) follow chemokine gradient into the tissues
76
What are positive acute phase proteins, list 4 and their functions
Concentration increases during acute phase response
1) C-reactive protein
- Opsonisation
- Can bind to damaged or dying cells
2) Serum amyloid A
- Cytokine inflammatory
3) Mannose-binding protein
- Binds to mannose within the complement pathway acts as an opsonin
4) Fibrinogen
- Coagulation pathway
77
What are the 2 main types of interferons what cells produced by, what induced by and 4 general functions
```
Interferon-α
- Produced by nucleated cells particularly mononuclear phagocytes
• Interferon β
- Produced by nucleated cells particularly fibroblasts
• Induced by virus
1) Inhibition of viral replication
2) Inhibition of cell growth
3) Regulation of cell differentiation
4) Activation of immune system
```
78
List 2 specific molecules that IFN -alpha or beta interfers with within virus and 3 other specific effects
1. Protein Kinase R- inhibition of viable protein translation - virus has taken over this machinery
2. RNAse antiviral pathway - mRNA degranulation - prevent transcription via cleavage of RNA
Other effects
1. Inhibits viral penetration
2. Inhibits viral uncoating
3. Upregulates MCH class I molecules
79
IL-2 how produced and 4 functions
- Activated T cell has intracellular cascade resulting in the production IL-2 and upregulated IL-2 receptor which acts on itself resulting in proliferation - produced by both CD4 and CD8 cells
Function
1) Proliferation of T cells - main role
2) Autocrine growth factor - acts on the cell that produces it
○ Causes differentiation
3) Stimulates NK cells
4) B cell growth factor
80
What cytokines leads to IgE an IgG isotype switching
- IL-4 - IgE
| - IFN-gama - IgG
81
List 5 CD4 cell types and general functions
1. TH1
• IFN-ƴ - activate macrophages
2. TH2
• helper of B cells to produce Ab and activate eosinophils
3. T regulators (Treg )
• Suppressors - switch off immune systems present in the periphery
4. TH17 Cells
• Promote recruitment of leukocytes and release of pro-inflammatory cytokines from macrophages.
5. TFH (Follicular helper cells)
• Specialized providers of B cell help.
82
Dendritic cell what receptors, what occurs and properties
- Different Tol like receptors that recognise PAMPS
- Once recognised then ingest pathogen and display pathogen antigen on MCH class I and II
- Different antigen
- Has really long tentacles - with MCH and antigen - increase change of T cells binding
83
Describe signal 2 and 3 when activating naive T cells
Signal 2 - CD28-B7 - ACTIVATION
B7 receptor not present on the dendritic cell until been activated by pattern recognition receptor - danger signal (DAMP or PAMP) - upregulates B7 receptor
- Binding stabilises the interaction
- Safeguard
- If doesn't receive this signal then the cell shuts down
○ Anergy - dies via apoptosis even if receive right signal later
Signal 3 - Cytokine receptor-cytokine - DIFFERENTIATION
- So knows what type of cell to become
- TH1, TH2 - etc.
- Dependent on the cytokines present in the environment
84
List 4 main results from activation of naive T cells
1. Stabilizes IL-2 message
2. Increases synthesis IL-2 & IL-2R (IL- 2 receptor)
3. Cells proliferate & differentiate into armed effector cells
- IL - 2 moves T- cell through the cell cycle from G0 to G1 phase by switching off inhibitor preventing movement from one phase to another
- Can also differentiate into memory cells for both CD4 and CD8 - two types of memory T cells
4. Also have increased cell survival - Produce molecules that protect cells from apoptosis
85
Describe the 6 steps in the TH2 pathway
1) Dendritic cell activated by PAMPS on PRC such as TOL-like receptor
2) Activate naïve T cell - described above
3) In presence of IL-4 from other cells types such as mast cells or eosinophils (that themselves have been activated by antigens) CD4 T cell differentiates into TH2 cell
4) Has specific receptors that allow it to communicate with B cells
- Also produce specific cytokines such as IL-4,5,13
- Now an EFFECTOR CELL
5) B cell binds to specific antigen with antibody, breaks down the pathogen and presents the antigen on MCH class II
6) TH2 binds to B cell if it is complemented ACTIVATED - specific cytokines are released from cytoplasm resulting in differentiation
86
Describe the 6 steps in the TH1 pathway
1) Dendritic cell activated by PAMPS
2) Activate naïve T cell - described above
3) In presence of IFN-ƴ (produced by activated NK cells) and IL-12 (produced by activated activated macrophages and dendritic cells) and dendritic cells) CD4 T cell differentiates into TH1 cell
4) Effector molecules are Interferon-gama produced and stored within the cytoplasm
5) Macrophage activated via PAMPS - display antigen on MCH class II
6) Fits the TH1 receptor so release stored cytokines resulting in death of certain infected cells
87
What is important to remember with TH1 and TH2 pathways and cytokines
IMPORTANT TO REMEMBER
| WHEN IL-4 PRESENT INDUCED TH2 PATHWAY BUT ALSO INHIBITS TH1 PATHWAY AND VICE VERSA
88
What are the 3 main functions of C3b
○ lysis
○ phagocytosis or
○ inflammation
89
Complement system what made up of, where synthesised and what occurs
- 30+ soluble & membrane bound components
- Liver main site of synthesis
○ Inflammation increases the synthesis of complement components (IL-1 )
- Protease cascade (cleaving the proteins (complement) into two components (a = smaller, b = larger) this activates them -> start as proenzymes
90
What are the 3 general steps in the complement pathway
1. Antibody/antigen complex activating
2. Protease cascade amplification
3. Cell tagging leading to lysis OR pore formation (MAC) leading to lysis
91
What are the 5 main proteins within the complement pathway and their functions
1) PRR - bind to Fc parts of Ab
2) serine proteases - amplify the cascade
3) cell binding proteins
- Labelling (bind to cell surface of) pathogens for destruction or phagocytosis
○ Activated by early proteases
- Induce inflammation
○ VIA Mast cell degranulation
4) Pore-forming proteins - insert into the membrane
5) regulators of complement activity - inhibit complement from reacting with self
92
List and describe the 4 functions of the complement system
1. It assembles MAC pores on foreign cells which activate complement and cause cell lysis.
2. Opsonization: enhanced phagocytosis of pathogens tagged by C3b, through binding of a specific C3b receptor.
3. It causes increases in vascular permeability and phagocyte activation at sites of infection or injury (anaphylatoxin activity by C3a & C5aC3a)
- C3a and C5a bind to mast cells, eosinophils, platelets and neutrophils which promote degranulation and release histamine, serotonin and prostaglandin
4. It is important in the metabolism of soluble immune complexes which occur within the blood.
93
What are the 3 complement pathways and how are they initiated
1) classical pathway
- activation via Ag-Ab complexes
2) Lectin pathway (fungal cell walls)
- initiated via binding of mannose binding lectin (MBL) to pathogen
3) Alternative pathway
- activated by binding of spontaneously generated C3b to microbial cells
94
What are the 3 stages of complement activation
1) Initiation: Involves 3 different ways in which the complement cascade is started. This stage involves some type of pathogen recognition
2) Early steps: Involves a common node in the cascade namely the conversion of C3 into C3a and C3b by proteolysis. This is a critical step in all 3 pathways of complement activation. C3a and C3b have effector functions of their own (see functions above).
3) Late steps: lead to the formation of the membrane attack complex (MAC) which “drills holes” into the plasma membrane of pathogens
95
List the 6 steps in the classical pathway activation
1) C1 activated by complexes of Antibody (IgM or IgG) bound to Antigen by removal of inhibitor protein during cross-linking with Ab
2) Activated C1 cleaves C2 and C4 (inactive until cleaved)
• Cleaved C4 = C4a + C4b
• Cleaved C2 = C2a + C2b
3) C4 + C2 ->Forms C4b2b complex = C3 convertase (multiple created from one C1)
4) C3 convertase binds, cleaves & activates C3
- generates multiple C3a & C3b (main complement molecules)
○ Amplification
5) Some C3b molecules combine to form C5 convertase while cleaves C5
○ C4bC2bC3b = C5 convertase (splits C5)
6) C3b can also act as opsonin or combine with other complement to form a pore (MAC ATTACK COMPLEX)
96
What makes up C3 and C5 convertase
○ C4b2b complex = C3 convertase
| ○ C4bC2bC3b = C5 convertase (splits C5)
97
What are the 3 steps in the lectin pathway
1. Activated by the binding of mannose binding lectin (MBL) to a pathogen (fungal walls)
2) Once activated MBL binds to two associated serine proteases
○ MASP-1 & MASP-2.
3) These serine proteases then cleave and activate C4 & C2 to produce a C3 convertase (C4bC2b)
Next steps are the same as classical complement pathway
98
What are the similarities and differences of the classical and lectin pathway
Similar in action without the need for C1 or Ab instead use mannose binding lectin
99
List the 3 main functions of C3b
1) Forms C5 convertase (C3bC4bC2b)
• Splits C5 creating MAC ATTACK complex
- Results in osmotic lysis of microbe
2) Some C3b alone binds to pathogen
• Acts as a opsonin - phagocytosis
3) C3a acts as an anaphylatoxin - mast cell degranulation
• C3b binds to microbes resulting in release of C3a and C5a
- Requirement and activation of leukocytes via these molecules
- Results in degranulation of mast cells and destruction of microbes via leukocytes
100
What molecules make up the MAC ATTACK complex and what does it result in
C5b binds C6 C7 C8 C9 initiating membrane attack complex (MAC)
- insertion into lipid bilayers, form a channel resulting in osmotic lysis
101
What are the 6 steps in the alternative complement pathway
1. C3b formed by spontaneous hydrolysis of other proteins or via other pathways
2. C3b binds microbe and is now activated - can act to amplify its own generation
3. Binds factor B - creating C3bB
4. Factor D (serine proteinase) then cleaves factor B --> Bb + Ba that is attached to C3b
- This cleavage results in the activation of C3 convertase activity (C3 convertase (C3bBb) formed)
○ C3 convertase then acts on more C3 resulting in the production of more C3b
5. Another C3b then binds to C3 convertase creating C5 convertase below
6. C5convertase creates MAC attack complex via cleavage of C5 and pathway above -> osmotic lysis
102
List and describe the function of 3 inactivation factors
1) Factor H - removes Bb from the alternative pathway C3 convertase breaking the positive feedback loop
2) Factor I - inactivated C3b and C4b
3) C1 inhibitor (C1INH) - binds to sites on activated C1 resulting in inhibition of proteolytic activity
- Therefore when C1 is activated only brief period in which it can cleave C2 and C4 before it is deactivated by C1INH
103
what is the major regulatory protein on plasma membranes that prevent complement binding self
Sialic acid
- High levels present on mammalian cells (not as much on bacterial or yeast walls or virial envelopes)
- Therefore host cell binding results in rapid inactivation of C3b
104
List one complement binding receptor, where present, what binds and function
CR1 receptors for complement on innate cells
- Neutrophils, monocytes, macrophage, RBCS (function below)
- Can bind C3b and C4b
FUNCTION - clearance of immune complexes and phagocytosis
1. Ag-Ab complexes and complement bind
2. RBCs have CR1 receptor that recognises C3b bound to immune complexes
○ Now goes to the spleen or liver with macrophages
○ More effective clearance mechanism
