Immunology Flashcards
What progenitor (and lineage) gives rise to the innate cells of the immune system
myeloid
What progenitor (and lineage) gives rise to the adaptive cells of the immune system
lymphoid
Where does T cell maturation occur
Thymus
Where does B cell maturation occur
Bone marrow
Cells of the innate system and actions
Mast cells - histamine –> inflammation + allergy response
Granulocytes - Basophils, eosinophils and
Describe the 3 growth factors involved in granulocyte and macrophage differentiation
Granulocyte/macrophage colony stimulating factor
Macrophage colony stimulating factor
Granulocyte colony stimulating factor
Cells of the adaptive immune system
T cells
B cells - plasma cells and memory cells
dendritic cells and macrophages
Describe the innate system
1st defence line, fast, non-specific, recognise molecules on pathogens (PAMPs and DAMPs), Fixed response, triggers adaptive system
Describe the adaptive system
2nd defence line, slow, antigen recognition, specific to pathogens, memory
Describe the 3 main components of the innate system in order of defence
- Anatomical and physiological barriers
skin (keratinised), gut epithelial lining, urinary tract
‘Flow’ - ciliated cells and mucosal cells (goblet cells) - Humoral (fluid)
Chemicals - enzymes (lysozyme), stomach acid, antimicrobial peptides (defensins)
Complement system –> inflammation, opsonisation, membrane attack complexes - Cells
Macrophages and Dendritic cells
- phagocytosis and antigen presentation
Natural killer cells - examine host and pathogens for MHC 1, if there is MHC 1 then apoptosis is prevented
no MHC 1 = death
mast cells - histamine for fighting allergy
Neutrophils - phagocytose and die
Describe the complement system
30 complement proteins
Compliment proteins cleave other complement proteins into CXa and CXb via CX convertase
Binding
- Classical pathway
- binds to IgM antibodies on the pathogen - Alternative pathway
- binds directly to the pathogen cell wall - Lectin pathway
- binds to mannose residues on pathogen cell wall
C3a and C5a = inflammation
C3b = opsonisation
C5b = interacts with C6 - C9 to form membrane attack
Describe recognition in innate system (molecules and receptors)
PAMPs - pathogen- associated molecular patterns
- flagellin, bacterial toxins, bacterial lipopolysaccharides
DAMPs - damage-associated molecular patterns
- self-derived host molecules (tissue damage)
Pattern recognition receptors
- -> complement receptors
- ->membrane-bound phagocytic receptors
- ->NLRs (intracellular), TLRs (transmembrane)
Phagocytic receptors
- Complement receptors
- Scavenger receptors
- C-type lectin-like family
Cells in innate immune system
mast cells
Describe the production of polyclonal antibodies?
Immunise an animal (typically rabbit) against collagen for example.
The animal has an adaptive immune response and produces lots of antibodies that specifically recognise the antigen/collagen we have immunised with.
Immunise subcutaneously to prevent clearance.
Initially IgM produced, then starts to produce high affinity antibodies by somatic hypermutation etc.
Give second injection to boost the IgG production.
Harvest the blood - typically 2 weeks post-injection.
Harvested blood is centrifuged and allowed to clot and clotted blood is removed and sera is collected.
What is the problem with polyclonal antibodies?
They recognise many different epitopes.
Antibodies may be made against any impurities in the media that was injected.
Each antisera is different.
Can only be produced in limited volumes - impossible to use the same antisera in multiple experiments/clinical tests.
What kind of technology is involved in the production of monoclonal antibodies?
Hybridoma technology.
What are monoclonal antibodies?
They recognise one specific epitope oon an antigen, perhaps just a few amino acids or 1 particular structure.
Describe the production of monoclonal antibodies?
Immunise a mouse and take the spleen.
By harvesting the spleen we have obtained huge amounts of activated B cells all producing antibodies.
Each B cell makes a specific antibody with slightly different affinity due to somatic hypermutation.
We immortalise these B cells by fusing them with myeloma cells - we fuse using polyethylene glycol.
They are then incubated in a media that encourages grwoth of the fused cell.
These incubated hybrid cells are left to make an antibody and the antibody is tested using an ELISA.
We dilute out the cells so there is 1 cell per well and allow them to proliferate to generate a clone of each cell.
We then test again and look at which clones have the best affinity for the antigen (may be multiple).
We expand that clone, put it in culture flasks and allow that clone to produce lots of antibody which is harvested using chromatography.
We now have a pure monoclonal antibody that binds one particular antigen and only one particular epitope.
This can be stored for years in liquid nitrogen and grwon out again whenever needed.
What is used to fuse the B cells and myeloma cells in the production of monoclonal anitbodies?
Polyethylene glycol.
What are the acitivated B cells harvested from the spleen of an immunised mouse fused with to immortalise them?
Myeloma cells
What are some of the applications of antibodies?
Enzyme-linked immunosorbent assay (ELISA) Immunoblotting Immunocytocehmistry Immunohistochemistry Flow cytometry Therapy Monoclonal antibody treatment Chromatin immunoprecipitation.
How is an indirect ELISA performed?
Coat a plate with antigen.
Monoclonal antibody comes in an binds specifically to the epitope.
Add a second antibody that recognises the first antibody, the secondary antibody is conjugated to a fluorescent signal/colour etc.
This is an indirect ELISA.
What is a sandwhich ELISA?
Coat the plate with the monoclonal antibody.
Bring in test solution with the antigen, the monoclonal antibody binds just one epitope - this is capture.
Then a second antibody can be used that binds the antigen - this can now be a polyclonal antibody because the epitope has already been captured.
Describe the basic steps of immunocytochemistry/immunohistochemistry?
- Tissue or cells attached to glass slide.
- Add specific antibody.
- Add second antibody conjugated to signal.
- Add insoluble enzyme substrate.
- Counterstain e.g. with haemtoxylin/DAPI.
Why is monoclonal antibody staining superior over polyclonal antibody staining?
Monoclonal antibody staining can recognise specific epitopes e.g. can distinguish between a phosphorylated or unphosphoryalted protein or it could recognise a cleavage product etc.
Describe the structure and function of the T cell receptor
1 alpha chain and 1 beta chain
1 variable region and 1 constant region in each chain
2 transmembrane domains
1 disulphide bond between the 2 transmembrane domains
Describe 3 monoclonal antibody treatments?
- Rituxan (Rituximab) targets CD20 on B cells used in Non-Hodgkin’s lymphoma.
- Mylotarg (Gamtuzumab) targets CD33 adhesion molecules on myeloid cells, used in AML.
- Herceptin (Trastuzumab) binds Her2 receptor used to treat Her2+ breast cancer.
Describe leukocyte recruitment
Chemokines
Adhesion molecules
Describe the steps of T cell maturation in the thymus
- lymphoid progenitor migrates to cortex of thymus and interacts with cortical thymic epithelial cell (destined to T cell lineage) and becomes double negative
- VDJ recombination in the beta chain of the TCR
- New beta chain is tested with a test alpha chain
- VJ recombination in alpha chain of the TCR
- Tested with the already made Beta chain
- if they fit together = exported to the cell surface - T cell becomes double positive and expresses both CD4 and CD8
- increase affinity for MHC molecules (CD4 - MHC 2, CD8 - MHC1) - T cell exposed to MHC molecules and whichever they bind best to determines the type of CD molecule they will eventually present = positive selection
- T cell migrates to medulla where it is exposed to APCs and medullary thymic epithelial cells which express auto-immune regulatory gene allowing expression of every peptide possible in the body
The TCR is tested to see if it binds to host antigens (MHC1)
If binding occurs = death (negative selection)
No binding = cells become committed to the CD type that was determined in the cortex
- T cell migrates to secondary lymphoid tissue (spleen, lymph nodes, MALTs)
What CD molecule do t helper cells present?
CD4
What MHC do cytotoxic t cells recognise?
MHC class 1
What MHC do t helper cells recognise?
MHC class 2
What cells do B cells become once activated?
Memory cells or plasma cells
What is an antigen?
molecule specifically recognised by recognition receptors of lymphocytes
It elicits an adaptive immune response
What are MHC molecules?
molecules expressed on the cell surface of antigen presenting cells. They are involved in antigen presentation to other cells such as T cells and B cells
Describe MHC class 1 function and structure
Present host or intracellular pathogen (viral) antigens
Activates NK cells, Activates cytotoxic T cells
Present on all nucleated cells (not erythrocytes)
Structure
3 alpha domains, 1 beta domain with a peptide-binding cleft (self or pathogen peptide)
1 transmembrane domain
Describe the mechanism of antigen presentation using MHC class 1
The MHC molecule is unstable without its peptide therefore its transported to TAP channels in the endoplasmic reticulum where it awaits a host/pathogen peptide
Host peptide
Defective Ribosomal Proteins (dRIPs) are ubiquitinated and degraded by proteasome and transported by TAP proteins to the MHC 1 where the peptide binds to the peptide-binding cleft
Pathogen peptide
Pathogen is engulfed and broken down into peptides in proteasome and peptides are transported via TAP proteins to the MHC 1 and bind to peptide binding cleft
MHC 1 then exported to surface
Describe MHC class 2 function and structure
Activates dendritic cells, macrophages, naive B cells and T helper cells
Presents extracellular pathogen antigens
Structure
2 alpha domains, 2 beta domains with a peptide binding cleft
Describe the mechanism of antigen presentation using MHC class 2
MHC 2 is inside an endosome and the peptide binding cleft is blocked by Li peptide.
Pathogen degraded in endosome by acidification - becomes a phagolysosome
Blockage prevented and pathogen peptide binds to MHC 2 and is exported to the membrane
Describe the structure and function of the B cell receptor
One BCR binds 2 antigens. BCRs (antibodies) coat pathogens and prevent function and trigger the complement system
2 heavy chains, 2 light chains
4 disulphide bonds (2 between heavy and light chains, 2 between the heavy chains)
Heavy chain
- 3 constant regions
- 1 variable region
- genes = mu (IgM), gamma (IgG), alpha (IgA), delta (IgD), epilson (IgE)
Light chain
- 1 constant region
- 1 variable region
- genes = kappa or lamda
Describe the structure and function of the T cell receptor
1 alpha chain and 1 beta chain
1 variable region and 1 constant region in each chain
How is receptor diversity obtained?
Recombination
Describe the recombination process that takes place in TCR and BCR diversity
Recombination occurs between exons with 23 base pair nucleotide spacer (variable exons) and 12 base pair nucleotide spacer (diversity exons). This ensures that recombination only occurs between variable and diversity exons and not between 2 variable exons or 2 diversity exons
- RAG (recombination activating genes) initiate recombination and variable and diversity exons are decided
- Hairpin loop formation of DNA between the V and J exons chosen
- The loop is excised off and TdT adds random nucleotides to increase variation
- DNA ligase joins the ends together and completes recombination
Describe the steps of T cell maturation in the thymus
- lymphoid progenitor migrates to cortex of thymus and interacts with cortical thymic epithelial cell (destined to T cell lineage) and becomes double negative
2.
Describe the activation of the 2 T cell types
Cytotoxic
- APC presents antigen using MHC class 1 and binds to the TCR of a CD8+ T cell
- IL-2 is expressed from the APC and causes the T cell to survive and differentiate into cytotoxic T cells. Clonal expansion occurs
T helper cells
- APC presents antigen using MHC class 2 and binds to the TCR of CD4+ T cell
- CD28 allows cell survival
- T cells differentiate into different T helper cell types depending on what cytokine is released from the APC
eg. IL-6 causes differentiation into T follicular helper cells for B cell activation
Some T helpers help granulocytes, macrophages and mast cells
Describe the steps of B cell maturation in bone marrow
Throughout B maturation, BCR is an IgM antibody and contains the mu chain on the heavy chain
- Lymphoid progenitor induced into B cell maturation by stromal cells
- Heavy chain recombination
- DJ recombination followed by V-DJ recombination - Heavy chain tested using a test light chain
antibody made = survives and proliferates - Light chain VJ recombination and tested using the already made
antibody made = survives and proliferates - B cell tested for self recognition
- binds to APCs in bone marrow = death
- Recombination can occur a number of times so they have a few chances - If heavy and light chains are functional then IgM and IgD are expressed (unknown what IgD does but its marks a naive B cell)
- The naive B cell migrates to lymph nodes for activation
Describe the 2 types of B cell activation
T cell dependent
- B cells recognise polyvalent antigens (have repeating sequences) and causes antibodies to cluster together and activate Ras/MAPK pathway
- TLRs activate signalling pathways
Leads to B cell activation and opsonisation
T cell independent 1. B cells in germinal centre engulf pathogens and present antigens using MHC class 2
- CD4+ T cells are activated by APCs with MHC class 2 and become T follicular helper cells (paracortex)
- The T follicular helper cell binds TCR to the B cell MHC class 2 - CD40 receptor assisted
- TfHC secretes IL-21 causing B cell activation and causes survival
- B cells proliferate by clonal expansion to form a centroblast (dark zone) - Centroblast expresses AID (activation-induced cytidine deaminase) which causes hypermutation in the BCR by point mutations in variable regions –> increases affinity for pathogens
- B cells with increased affinity migrate to the follicular dendritic cell in the light zone of the germinal centre where pathogens are concentrated
- B cells with decreased affinity are killed by T cells - B cells with increased affinity engulf pathogens and express the antigen on their surface MHC 2 molecules
- TfHCs secrete cytokines which cause B cells to change their antibody expression from IgM to IgA, IgG and IgE
- B cells become plasma cells and memory cells
