DEF Flashcards
What are physical barriers to infection?
- Skin provides a protective cover
- Organisms can overcome this barrier
- Cuts or damaged skin
- Insect bites
- Animal bites
What are factors of the innate immune system?
We have looked at barriers, mucus, cilia, secretions that stop organisms entering or replicating, competition from other organisms.
Phagocytes.
What are the steps of phagocytosis?
- chemotaxis
- attachment
- engulfment
- killing
What are the roles of phagocytosis?
- Protection from pathogens
- Processing and presentation of antigens
- Disposal of damaged/dying (apoptotic) cells
- Activation of adaptive immune system
- Links innate and adaptive immunity
What are PRRs: toll-like receptors (TLRs)?
Human TLRs recognise PAMPs:
- Liposaccaride (gram negative)
- Lipoteichoic acid (gram positive)
- Bacterial DNA sequences (unmethylated CpG)
- Single/double-stranded viral RNA
- Glucans (fungi)
Stimulate production of inflammatory cytokines.
What is opsonisation in phagocytosis?
Coating microbes: targets for phagocytosis
Proteins that coat microbes = opsonins
- Antibodies (IgG)
- Proteins of complement system – C3b, C4b
Facilitates phagocytosis.
Phagocytes have receptors for opsonins.
What else is there to innate immunity except phagocytes?
- This is an infection
- What is our response?
- Acute Phase Proteins
- C Reactive Protein (CRP)
- We have inflammation
What other action is taken in innate immunity except phagocytosis?
- Many immune cells make interferons
- Task – to interfere with viruses infecting other cells
Detailed function will be covered in other lectures
What are natural killer cells?
- surveillance role
- any cell that has changed is a target for killing
What is an antibody?
- Y shaped
- Tetrameric protein:
o
2 identical heavy chains
o 2 identical light chains: held together by non-covalent interactions and by –S-S- crosslinks between cysteine a.a residues
What are the light chains of antibodies?
There are two types of light chain
- Kappa and lambda
- But any B-cell will only make one type
- Any Ig molecules will contain either kappa or lambda, never both
- This phenomenon is called ‘light chain restriction’
What do B lymphocytes do?
- Make antibodies
- Immunoglobulins
- There are two types
- IgM – made first
- IgG – made later
How does antigen recognition by T cells occur?
- T cells recognise antigen processed and presented by APC
- Peptide: MHC complexes are presented on the APC surface
- activation of T cells specific for antigenic peptide
CD4+ helper T cells: antigens (peptides) displayed by MHC class II CD8+ cytotoxic T cells: antigens (peptides) displayed by MHC class I
MHC restriction of Ag recognition by T cells.
What is major histocompatibility complex (MHC)?
MHC molecules display peptides from processes Ag.
Two types: MHC I and MHC II
MHC I: alpha chain and beta2-microglobulin; present peptides to CD8+ T cells
MHC II: alpha chain and beta chain; present peptides to CD4+ T cells
What is the expression of major histocompatibility complex I and II?
- MHC I: all nucleated cells
MHC II: antigen presenting cells: dendritic cells macrophages
What is the innate immune system?
- Born with (natural/native immunity)
- Ancient (plants, insects, all animals)
- Developed by evolution
- In place before infection
- Responds in the same way to repeated infections
What are the functions of the innate immune system?
- Reacts to microbes (and injured cells)
- First line of defence (initial response to microbes)
- Rapid (immediate maximal response in hours)
- Prevents, controls, (sometimes) eliminates infection
Many pathogens evolved to resist/escape IIS
Eliminated by adaptive immune system
IIS keeps infection in check adaptive immunity activation
What are the components of the innate immune system?
Barriers
- Physical
- Chemical
Cells (effector cells)
- Phagocytes (PMN, M)
- NK cells
Soluble molecules
- Effector proteins (complement)
- Mediators of inflammation (cytokines)
PMN = polymorphonuclear neutrophils, M = macrophages, NK = natural killer cells
What is the barrier aspect of the innate immune system?
Epithelial surfaces
- Skin
- Mucosa of GI tract
- Mucosa of respiratory tract
Prevent entry of microbes – physical barrier
What are chemical barriers in the innate immune system?
Antibacterial enzymes (lysozyme – tears, saliva)
Antimicrobial peptides
- Defensins, cathelicidins kill bacteria by damaging bacterial cell membrane
Produced by epithelial cells, PMN, NK cells, CTLs
What are microbial barriers in the innate immune system?
Normal flora non-pathogenic bacteria competition
Clinical note! Antibiotic treatment: kills normal flora replaced with pathogenic organisms
What are examples of the innate immune system in disease?
Loss of integrity predisposes to infection
wounds, burns
Genetic defects: cystic fibrosis
defective mucus production
inhibition of ciliary movements
frequenct lung infections
What are the effector cells in the innate immune system?
Cells (phagocytes)
- Myeloid lineage: PMN, M, dendritic cells
- Identify, ingest, destroy pathogens
Other cells:
- Lymphoid lineage: NK cells
What are natural killer (NK) cells?
- Kill virus-infected cells
- Kill malignantly transformed cells (tumour cells)
- Express cytotoxic enzymes (lyse target cells)
What are characteristics of NK cells?
Kill malignant tumour cells without prior activation.
(CD8+ T cells need to be activated and differentiate into CTL (cytotoxic T lymphocytes) to kill target cells)
- Contain perforin pores in target cells
- Contain cytolytic enzymes (Granzymes A, B)
How do NK cells recognise target cells?
NK cells have Inhibitory and activating receptors.
Inhibitory receptors:
- KIRs (killer inhibitory receptors)
- NKG2A (C type lectin receptors)
- Lecocyte Ig-like receptors (LIRs)
Inhbitory receptors recognise ligands on healthy cells.
Activating receptors:
- NKG2S
- KIRs
- CD16
- Adaptor proteins: DAP10, DAP12
Activating receptors recognise infected/injured cells.
The outcome of NK cell interaction with other cells is determines by integration of signals form inhibitory and activating receptors.
- All healthy autologous nucleated cells have MHC I
Inhibitory receptors recognise MHC class I blockage of signals from activating receptors
NK cells do not attack healthy autologous cells!
- Virus infected cells: downregulate MHC I
- Malignant (tumour) cels: downregulate MHC I
Inhibitory receptors are not ligated by MHC class I signals from activating receptors are not blocked NK cells attack and kill virus infected/tumour cells
- Activating receptors recognise ligands that are induced on unhealthy cells (stressed, infected by microbes, transformed cells)
Signals from activating receptors may overwhelm the signals from inhibitory receptors, especially if MHC I is also reduced or lost in unhealthy cells
NK cells attack and kill virus infected/tumour cells
About NK cell receptors…
ITIM: immunoreceptor tyrosine-based inhibitory motif
ITAM: immunoreceptor tyrosine-based activation motif
- Cytoplasmic tails of inhibitory receptors contain ITIM motif
- ITIMs engage molecules (phosphatases) that block the signalling pathways triggered by activating receptors
- Activating receptors contain ITAM motif
- ITAMs engages in signalling events that promote target cell killing and cytokine secretion by NK cells
- ITAMs are often located not in activating receptors but in cytosolic portion of adaptor molecules (eg DAP 12)
How do NK cels kill target cells?
Perforin: forms pores delivery of granzymes
Granzymes: A,B, C: initiate apoptosis
Delivered at the site of contact between NK cell: target prevents killing of neighbouring healthy cells
In addition to killing target cells NK cels activate macrophages to destroy phagocytosed microbes via production of IFN-γ
Granzymes activate caspases apoptosis
Granzyme B: can trigger mitochondrial apoptotic pathway
Killing of infected cells by NKs eliminates reservoirs of infection
What are defects in NK cells and disease?
Human NK cell deficiencies
- As part of broader immune-deficiencies (eg Chediak-Highashi)
- Complete absence of circulating NK cells
- Functional NK cell deficiencies (normal numbers)
Patients have fatal viral infections (herpes viruses)
What are phagocytes?
Specialised cells:
- Identify, ingest, destroy pathogens
- Neutrophils, Macrophages, dendritic cells
- Belong to the innate immune system
What is phagocytosis?
Cell ‘eating’
- Microorganisms
- Other cells
- Nutrients
Mechanism of innate immune system
What are the roles of phagocytosis?
• Protection from pathogens
• Disposal of damaged/dying (apoptotic) cells
• Processing and presentation of antigens (Ag)
- Activation of adaptive immune system
- Links innate and adaptive immunity
• Main phagocytes: PMG, macropahges, DCs
What are the steps of phagocytosis?
- Phagocyte mobilisation (chemotaxis)
- Recognition and attachment
- Engulfment
- Digestion: pathogen destruction
What are phagocyte defects and disease, and examples?
⎝ Quantitative (decreased number)
⎝ Qualitative (decreased function)
Examples:
- Chronic granulomatous disease
- Chediak-higashi syndrome - defective phagosome-lysosome fusion
- Leucocyte adhesion defects (LADs)
What is chediak-higashi syndrome?
Defective phagosome-lysosome fusion
Phagocytosed microbes can’t be killed recurrent infections
- Rare genetic disease
- Defective gene: LYSosomal Trafficking regulator (LYST)
- Defect in lysosome fusion
Neutrophils have defective phagocytosis.
- Repetitive, severe infections
What are leucocyte adhesion defects?
- Defect in beta-chain integrins
- Defective neutrophil chemotaxis
What are TLRs?
TLRs – recognise pathogens
Present on:
- Phagocytes
- Mucosal epithelial cells
- Endothelial cells
Cellular location:
- Cell surface (TLR1, TLR2, TLR4, TLR5) detect extracellular pathogens
- Inside cells (TLR3, TLR7, TLR8, TLR9) detect microbial nucleic acids
What are defects in TLRs and disease?
Humans lacking TLRs have not been identified!
Polymorphism in TLR genes predisposes to:
- Bacterial infections
- Asthma
- Autoimmunity (lupus)
About the structure of an antibody…
Tetrameric protein
- 2 identical heavy chains
- 2 identical light chains: either kappa or lambda
Each chain has a variable region
- amino acid sequence varies from one Ig molecule to another
- binds antigen (Fab)
And a constant region (Fc)
- responsible for effector functions
- eg activating complement, binding to phagocytes
The complementary determining regions (CDRs) also known as the hypervariable regions, are the parts of the V region that bind the antigen.
CDR3 is the most variable region.
About CDRs in antibodies…
- In the primary structure of the protein the CDRs are separated.
- In the tertiary (3D structure) of the protein the CDRs lie adjacent to each other.
- The rest of the V-region forms a framework, allowing the 3 CDRs to face the antigen.
How are the correct antibodies made in infection?
During an infection a small number of B cells will, by chance, be making an Ig that binds to one of the foreign antigens. These B cells are activated and begin to multiply – ‘clonal selection’. Some of the replications become memory cells and others become plasma cells moving to the bone marrow, where they out out large amounts of Ig.
What does activation of antibodies require?
- Direct involvement of CD4+ T helper cells (Th1)
- Cytokines released by Th1 cells
What is the germline kappa light chain composed of?
- One constant region (C) segment
- 35 variable region (V) segments
- and 5 short joining (J) segments
The J segments are quite close to the C segment, but the V segments are a long way away on the DNA.
There is a binding site for an endonuclease enzyme after each V segment and in front of each J segment. The enzyme will cut randomly at 1V and one J.
The free ends are then ligated together joining, in this case, V24 and J2.
What events occur in V(D)J recombination?
- One V segment and 1J segment are brought together via their recognition sequences
- RAG recombinases cut and remove intervening DNA
- End are processed before rejoining
What do exonuclease do in V(D)J recombination?
An exonuclease will mess around with the free ends, before they are ligated together.
An enzyme called terminal deoxynucleotidyl transferase (TdT) randomly adds a few nucleotides to the free ends, before they are ligated together.
What is junctional diversity?
After the hairpin is cut, or if its in the wrong place, some extra nucleotides are added in and TdT adds in some random ones as well.
The V-J join and this further variation at the joins create the most variable region of the antibody – CDR3
CDR1 and CDR 2 are fairly variable, but CDR3 is by far the most.
What is the importance of TdT?
- For generating Ig and TCR gene diversity
- As a leukaemia marker – disease of cells failing to differentiate, lymphoid blast cells make TdT
- Useful enzyme in genetic engineering/ recombinant DNA work
What is the process of recombination for the heavy chain?
Same process as the light chain, but one V can join with one D segment and one J segment, so giving even more combinations.
Exonuclease and TdT can add further variation both to the V-D and D-J junctions.
What is allelic exclusion?
- any B cell is diploid
- it has two alleles of the Ig heavy chain gene
- in theory it could make two different heavy chain proteins
- this never happens – ‘allelic exclusion’
What is the mechanism of allelic exclusion?
Mechanism: as soon as one allele successfully rearranges and starts making heavy chain protein, the gene rearrangement process for heavy chains is switched off.
For light chains, a B-cell has two alleles of the κ chain gene and two alleles of the λ chain gene.
In theory it could make four different light chain proteins.
Again this never happens- “Allelic exclusion”
What is light chain restriction?
One clone of B-cells only makes κ or λ
Polyclonal B cells will be a mixture of cells making either κ or λ light chain
If B-cells in a patient are only making one kind of chain——-????
About Ig expression during B cell maturation…
- Functional Ig expressed as membrane (cell surface) IgM
- Membrane IgM acts as B cell receptor (together with IgD)
- Ag recognition by membrane IgM activation of signalling pathways B cell activation
What happens in B cell activation?
- Functional Ig is first expressed as IgM on the cell surface (sIgM)
- This acts as a ‘B cell receptor’ in a similar way to a growth factor receptor. The IgM does not have intrinsic tyrosin kinase activity, but associates with SRC family tyrosin kinases, eg LYN and FYN
- Binding of antigen to IgM activated the tyrosine kinases and their signal transduction pathways
What does B cell activation require?
- Antigen binding to the B-cell receptor (sIgM), resulting in stimulation of signal transduction pathways
- Co-stimulatory by T-cells
- Co-stimulation by cytokines
What two forms of antibodies are there?
Two forms:
- Membrane-bound on B cell surface Ag receptor
- Secreted (circulation, tissues, mucosa)
Membrane-bound Igs
Ag recognition B cell activation humoural IR
What happens following B cell activation?
- The activated B-cell begins to secrete soluble IgM
- Mechanism – differential splicing
- Cμ is not coded for by a single exon (as implied in the simplified diagram in the previous lecture)
- There are 4 exons, with two alternative versions of exon four
- Differential splicing gives 2 different mRNAs, coding for 2 proteins which differ at the C terminal end
- Note V region, coded by VDJ complex, is identical
What happens to secreted Igs?
- Circulate in blood
- Access various sites to deal with pathogens
- Effector function
neutralisation of microbes, toxins
opsonisation of microbes to enhance phagocytosis
activation of complement (pathogen killing)
How do antibodies work?
Different classes of Abs work best at certain sites (eg IgM, IgG – blood; IgA – mucosa)
Different classes of Abs work best against certain pathogens: IgE – parasites
Bind to extracellular microbes and toxins
- Neutralise (block adherence/entry)
- Eliminate
Opsonisation = increased phagocytosis
Complement activation = opsonisation, lysis
What is class (isotope) switching of antibodies?
During an immune response B cells become capable to produce Abs of different classes without changing specificity (respond to the same Ag).
IgM switch to IgG, IgA, IgE
IgG switch to IgA, IgE
- Ability to perform different effector functions
- Can deal better with pathogens
- Isotype switch needs signals from helper T cells
Class switching does not alter specificity for Ag!
Class switching does not alter the light chain!
- B cells make many different classes of Ig
- Different classes have different constant (Fc) regions to carry out different functions
- But the first Ig made is always IgM
- Cell needs mechanisms to keep antibody specificity (coded by rearranged VDJ) but add different C regions
What is the mechanism of class switching?
Two mechanisms: Minor - IgD only - By differential splicing - Made at some time as IgM Major - All other classes - By DNA rearrangement
Cμ and Cδ are transcribed as part of a single precursor RNA. Differential splicing can remove the Cμ exons, so the Cδ exons are not used. Result is the same VDJ is now joined to Cδ making an IgD.
- Endonuclease recognition site (switch region) before each CH segment.
- Cut before Cμ and cut before alternative C segment
- Original VDJ now transcribed along with new C region
- Note no change in light chain!
