immuno Flashcards
common techniques used in immunology
ELISA : enzyme-linked immunosorbent assay -> measures antibodies, antigens, proteins and glycoproteins in samples
eg anti- AChR ELISA test: myasthenia gravis diagnosis
flow cytometry: analyse cells in blood
hematopoiesis
formation of blood cellular components
- hematopoietic stem cells are pluripotent and give rise to 2 cell lines
lymphoid: T cell, B cell, NK cell
myeloid: monocyte (fight pathogens), macrophage, basophil , neutrophil, eosinophil
lymphoid organs
primary: bone marrow and thymus (sites of B and T cell maturation)
secondary: spleen, lymph nodes, peyer’s patches in small intestine and mucosa-associated lymphoid tissue (MALT) in submucosal membrane sites
neutrophil
most abundant WBC, first to reach site of infection, high count indicates bacterial infection
express TLR 1,2,4,5
basophil
increase histamine release as part of inflammatory response
basophil circulating in blood while mast cells at tissues
eosinophil
increase allergy reaction and anti-parasitic activity
monocytes
in blood: blood monocyte
in tissue: tissue macrophage
- produce cytokines to initiate and regulate inflammation
- phagocytosis
- clear dead tissue
- tissue repair
natural killer cell
anti-viral defence -> contain granules with perforin, granzymes and granulysin
non specific cytotoxic killing of pathogens
- to protect healthy cells, NK cell shuts off when inhibitory receptors on NK encounter self MHC
NK cell + macrophage
macrophage ingest microbe + secrete IL-12 -> NK cell activated by IL-12 -> NK secrete IFN-gamma -> macrophagte activated to become more effective at killing -> cytokines IL-12, 15, IFN-gamma secreted by macrophage and DC enhance effects of NK cell
(similar process as T cell and macrophage)
surface barriers
physcial: skin, mucosa, bronchial cilia
physiological: diarrhoea, cough, sneeze, etc
chemical: low pH (prevent pathogen growth) eg skin, gastric acid, vagina
innate antimicrobial molecules eg IgA, defensins, complements
biological: normal flora
innate immunity
provides a rapid, non-specific response against pathogens but does not confer long-lasting immunity
triggered by PAMPs and DAMPs
3 main innate immune cells: dendritic cell, macrophage, neutrophils
key molecules involved in innate immunity: complement proteins, lysosomes, type 1 interferons, defensins
PAMPs and DAMPs
recognised by toll-like receptors expressed by innate immune cells
binding to TLR induces signal transduction pathway and triggers pro-inflammation cascade with cytokine production and proliferation (TNF and IL1) which bring leukocytes and plasma proteins to site of infection by binding them to endothelial adhesion molecules
PAMPs: conserved molecular patterns shared by broad classes of pathogens
detected by:
bacteria -> TLR 2,4,5
RNA -> TLR 3,7,8
DNA -> TLR 3,9
DAMPs: originate from host and signals released from damaged tissues
detected by: TLR 4
key molecules in innate immunity
defensins: antibiotic peptides found in tears, sweat and saliva and adheres to bacterial surface to induce osmotic lysis through pore formation
type 1 interferons: produced in response to viral infections and interferes with viral replication, activate NK cell and increase MHC I expression
IFN-a: dc, macrophage
IFN-b: fibroblast
lysosymes: digestive enzymes secreted in tears and saliva that digest cell wall
complement system
complement system
complement proteins mainly synthesised by liver and found circulating in blood
classical activation pathway: binding of IgM/G with antigen to C1
- C3 splits into C3a, C3b and C5
- C3a cause mast cells to release histamine for inflammation to bring leukocytes to site of complement activation
- C3b binds to extracellular microbe, resulting in opsonisation (opsonised with complement proteins)
- C3b splits C5 into C5a and C5b
- C5a cause mast cells to release histamine for inflammation to recruit immune cells with opsosin receptor to recognise opsosin in microbe
- C5b binds to C6-9 to form membrane attack complex which causes inflow of extracellular fluid and cause microbe to lyse
Major histocompatibility complex (MHC)
MHC I (intracelluar + all nucleated cells)
1. ubiquitin bind to misfolded host and viral proteins and protease cleave to peptides
2. MHC complex formed in ER
3. TAP transporter complex pumps peptide into ER to form peptide:MHC complex via chaperone
4. peptide:MHC complex transported to golgi apparatus and expressed on cell surface membrane
interact with cytotocis CD8 T cell
MHC II (extracellular + professional APC -> monocyte, macrophage, DC, B cells)
1. MHC class II form in ER and folds around chaperone, invariant chain Li to prevent peptides in the ER from binding to MHC
2. phagosome fuse with lysosome to form phagolysosome and microbe is cleaved
3. invariant chain-MHC class II transported to endocytic pathway and invariant chain processed into CLIP peptide
3. peptide with higher binding affinity to MHC class II will replace CLIP to form stable peptide MHC complex
interact with CD4 helper T cell
naive T cell migration to lymph node
APC express CCR7 which causes it to be drawn to the lymph node T cell zone for presentation to the T cell. Binding of CCR7 to CCL 19 and 2 at lymph node induced chemotaxis (directed movement of cells along a chemical gradient) -> naive T cells sense chemokine gradient and migrate towards source of CCL 19 and 21
in presence of APC, L selectin on T cell binds to L selectin receptor on high endothelial venule (weak binding). CCR7 sends downstream expression LFA receptor which binds to ICAM on APC and HEV (weak binding)
Upon binding of TCR to peptide:MHC complex, conformational change of LFA-1 to bind with greater affinity to ICAM-1.
There is simultaneous suppression of S1P receptor to keep T cell within lymph node
activated T cell migration to site of injury
within low S1P environment in the lymph node, T cell express more S1P receptors to push the T cell out of the S1P poor lymph node to the blood which is S1P rich and to the site of infection
activated T cell express E/P selectin ligand which binds to the E-/P- selectin receptor at the infection site
activated T cell express receptor LFA-1/VLA-4 which binds to ICAM-1/VCAM-1
T cell activation
requires 2 signals
1st signal: initial interaction
TCR binding to peptide:MHC complex which is mediated by LFA-1
2nd signal: co-stimulation
CD28 on T cell binds to B7-1/B7-2 proteins on APC (microbes stimulate expression of B7 on APC -> ensuring T cells are only activated by foreign antigens
T cell differentiation
activated T cells synthesise IL-2 and high affinity IL-2 receptor. IL-2 binding to receptor serves as signal for T cell to enter cell cycle and triggers T cell clonal expansion.
resting T cells: only moderate affinity IL-2R ( 2 subunits: by)
activated T cells: high affinity IL-2R, IL-2 (3 subunits: aby)
immune tolerance
immunity: triggered during infection in response to antigens derived from pathogens
tolerance: tolerance to self antigens to prevent autoimmune conditions
immune tolerance divided into
- central tolerance: immature lymphocytes undergo selection in primary lymphoid organs (early development of B and T cell maturation)
- peripheral tolerance: mature lymphocytes undergo selection in periphery after leaving primary lymphoid organs (deletion of autoreactive B and T cells to prevent autoimmunity)
central tolerance (T cells)
- positive selection
location: cortex of thymus
ensure production of functional T cells
- test if TCR bind appropriately to MHC, just enough to elicit immune response (if bind too strongly/weakly, apoptosis)
- negative selection
location: medulla of thymus
ensure no self-reacting T cells
- test if TCR bind to tissue-restricted self antigens presented on MHC by thymic medullary cells
- T cells that dont bind receive survival signals while those that bind undergo apoptosis except regulatory T cells
- mediated by autoimmune regulator protein (AIRE)
4 mechanisms of peripheral tolerance
- ignorance -> interaction between TCR and self-peptide MHC too weal so antigen recognition
- anergy -> no costimulatory signal -> inactivation of T cell
cause:
- immature DC =>express low levels of costimulatory molecules
- non-APC => present peptide MHC I complex but does not express/express low levels of costimulatory molecules - deletion -> mature T cells that recognise self antigens undergo apoptosis
major pathways
- mitochondrial pathway => regulated by members of bcl-2 family
- death receptor pathway => mediated by Fas-Fas ligand signalling - regulation -> Tregs inhibit immune response
mechanism: posses TCR that recognise self peptide-self MHC strongly and act on self-reactive immune cells to suppress activity
eg CD4 + CD25+ T cells express transcription factor Foxp3 (constitutive expression of CTLA4, secreting inhibitory cytokines)
T cell immune checkpoints
serve as immune checkpoint by
1. limit T cell mediated tissue damage during chronic infection
2. inhibit autoreactive T cell
CTLA-4
mechansim: cross linking of CD28 to B7-1/B7-2 -> upregulate CTLA-4 expression-> CTLA-4 bind with greater affinity to B7 -> transduction of negative signal -> T cell inactivation
ensure T cell not activated all the time
also constitutively expressed by Tregs
Programmed cell death protein 1 (PD-1)
- expressed by T cells and bind to PD-L1 on APC to inhibit T cell proliferation
- negatively regulates T cell function during chronic antigen stimulation
CD4 T cell subsets
Th1: CD40 ligand and IFN-y activate M1 macrophages
- CD 40 ligand binds to CD 40 receptor on macrophage and activated macrophage becomes highly microbicidal
- CD 40 ligand binds to CD 40 receptor on B cell to stimulate CD 8 T cells
- secrete IFN-y to inhibit Th2 growth
Th2: IL-4,5,13
- 4,13: act on B cells to stimulate M2 (tissue repair, fibrosis) and stimulate IgE production
- 5: act on eosinophil to kill helmiths
- secrete TGF-b and IL-10 to inhibit Th1 growth
Th17
- IL-17: inflammatory cytokines for chemokine production -> recruit neutrophils to site of inflammation
- IL-22: main GIT barrier integrity
Tfh (T follicular helper cells)
- produce Th2 cytokines for B cell activation and maturation
cytotoxic T cells
effector molecules in granules
- perforin: form pore in cell membrane of infected cell
- granzymes: a type of serine protease which activates apoptosis
- granulysin: induce apoptosis
- binding of FasL on CTL to FAS on target triggers apoptosis
process
- presentation of antigen to CD8 T cell -> migration of specific cytotoxic T cell to site which carries out killing
types of cytokines
IL-1: fever, inflammation
2: IFN-gamma production, Th1 differentiation
IFN-gamma: activation of macrophage
10: inhibition of IL-12 production (reduce expression of costimulators and MHC II)
memory T cells
allow for faster and more potent response upon repeat encounter
central memory T cell: stay in lymphoid tissue
effector memory T cell: migrate to peripheral tissue
macrophage activation
M0 (undifferentiated macrophages) require
- cytokines produced by Th1 to become M1 (pro-inflammator -> phagocytosis)
- cytokines produced by Th2 to become M2 (anti-inflammatory -> wound repair, fibrosis)
early B cell development
- maturation of B cell in bone marrow (but not activated)
- involves production and expression of pre-BCR
- positive selection -> select for functional VDJ heavy chain (pre BCR)
- see if it reacts to bad things
- failure to express pre BCR = death - negative selection -> autoreactivity (see if it reacts to self)
- if BCR binds to self antigen on stromal cell in bone marrow, death
final stage of B cell development -> secondary lymphoid organ (spleen and lymph node)
B cell activation
B cell activation can be:
T cell dependent -> CD4 helper T cell
T cell independent -> immediate response to non-protein antigens
types of antibodies
IgM-> first produced ad is evidence of acute infection
structure: pentamieric (5 subunits) and decavalent (10 binding sites)
low affinity antibodies so compensate by having many binding domains
Ig G -> neutralise pathogen (block binding between pathogen and receptor on host cell) + complement activation
structure: tetrameric (4 subunits) and divalent (2 binding sites)
only antibody that can cross the blood-placental barrier -> passive immunity from mother to foetus
IgA -> at mucosal sites
IgE -> mast cell degranulation + defence against parasites (Fc region binds to specific receptors on mast cells at tissues and basophils in plasma, providing immunity agaisnt large parasites
affinity vs avidity
affinity: strength of monovalent binding
avidity: combined strength of simultaneous binding involving more than 1 binding site
isotype swtiching
change in constant region of antibodies -> irreversible process
affintiy maturation
B cells interact with Th cells within germinal centre of lymph nodes to secrete immunoglobins with high affinity for specific antigens
repeated exposure to antigen results in the production of antibodies with increasing affinity
as immune response to atigen develops, amt of antibody produced increase so amt of available antigen decreases. B cells selected must be able to bind to antigen low conc so higher affinity BCR selected
late B cells development in lymph node
somatic hypermutation and affinity maturation
dark zone:
rapidly proliferating b cells (centroblasts) undergoing somatic hypermutation (rapid point mutations in VDJ region which is driven by activation induced deaminase (AID) _> cytosine to uracil
light zone:
centrocytes compete to bind to iccosomes from follicular dendritic cells
mantle zone:
if can bind to antigen, present peptide:MHC complex to helper T cell. if TCR binds to peptide, B cell receive survival and differentiation signal
AFTER: isotype switching trigged by binding of CD40 on B cell and CD40L on T cell
istotype swtiching of B cells at the periphery results in the production of short-lived plasma B cells, some of which would migrate to the germinal centre of lymph node for affinity maturation
antibody functions
agglutination: antibody have multiple binding sites -> form large complex -> easier for phagocytes to destroy pathogen
complement activation: complement bind to Fc region of antibody to initiate complement cascade, resulting in formation of MAC
opsonisation: antibodies bind to pathogen -> Fc region on antibody binds to Fc receptor on phagocyte and phagocyte destorys microbe
neutralisation: antibodies bind to surface of pathogen to block their binding to receptors on host cells
antibody dependent cellular toxicity: antibodu binds to pathogen -> Fc region on antibody to Fc receptor on NK cell -> cross linking trigger degranulation
PANIC (precipitation, agglutination, neutralisation, inflammation, complement)
process for bacteria
bacteria induce APC to secrete IL-12 and native T cell activated. T cell secrete IL-2 and express more IL-2 receptor, Binding triggers release of IFN-gamma to activate M1 macrophage and differentiate into Th1 cell
T cell express CD40 ligand to bind to bind to CD40 receptor on macrophage and activated macrophage become highly microbicidal
process for helmiths
worms induce APC to secrete IL-4 and naive T cell activated in presence of IL-4 to differentiate into Th2 cell. secrete IL-5 to activate eosinophil and stimulate IgE production activates m2 macrophages
cytokines
interluekin -> influence function of other cells
chemokines -> recruit immune cells
interferons -> viral defence
tumour necrosis factor -> regulate inflammation
mechanisms of microbial defence
virus
- innate immunity
-> soluble factors: type 1 interferon
-> cells: NK cell
- adaptive immunity
-> soluble factors: antibodies
-> cells: CD8 T cells helped by CD 4 T cell
extracellular bacteria
- innate immunity
-> soluble factors: complement
-> cells: neutrophils, macrophage
- adaptive immunity
-> soluble factors: antibodies
-> cells: CD4 T cell for activation of B cell
mycobacteria
- innate immunity
-> soluble factors: TNF-alpha, IL-1
-> cells: macrophage
- adaptive immunity
-> soluble factors: IL-12, IFN-gamma
-> cells: CD4 T cell for activation of macrophage
