immunology lab Flashcards
what are the central sites for immune system
- primary lymphoid oragns
- bone marrow, thymus - secondary lymphoid organs
- spleen, lymph nodes, mucosal and cutaneous associated lymphoid tissues
where is periphery sites for immune system
other tissues and systems,
-skin, liver, gut, heart, brain, CNS, muscle, lungs etc
where did all lymphocytes arise
from stem cells in bone marrow
whar does self-renewing haematopoietic stem cell differentiate into
common myeloid progenitor and common lymphoid progenitor
what does myeloid progenitor differentiate in
innate immune system:neutrophi;, basophil, eosinophil and monocyte
what does common lymphoid progenitor differentiate in
adaptive immune system: lymphocytes
what are the hall marks of innate immune system:
1. speed early, rapid 2. duration short lived 3. repetitive -respond the same way each time 4. interactive - with other cells of the innate immune system - with cells of the adaptive immune system 5. non-reactive to the host
what r the component of innate immunity:
- epithelial barriers
- cells in circulation and tissue
- phagocytes: neutrophils and macrophages
- exocytes:eosinophil, mast cells, basophils (release active mediators from granules) - molecules
- cytokines: tumour necrosis factor, interleukin 1 (IL-1)
- plasma proteins: complement proteins, mannose-bidning lectin, C-reactive protein.
what is thymus
site of T-cell maturation
- process of acquiring antigen-specific receptors\
- mature T-lymphoctes leaves the thymus and enter the circulation and the peripheral lymphoid organs
what r the 2 main types of lymphocytes
- B-lymphocytes
- T-lymphocytes
- helper t cell (Th)
- Cytotoxic T lymphoctes (CTL)
where did B lymphoctes derived
from common lymphoid progenitors in bone marrow
what does B lymphocytes form
part of the humoral immune response
what is the main function of B lymphoctes
to secrete antibodies
- antibodies are proteins that binds to EXTRACELLULAR ANTIGENS
what is the character of T help cell
- their surface expression of CD4 MOLECULE
how Th help other cells in immune response
- by release cytokines or
- via membrane bound molecules
what is the character of CTL
their surface expression of CD8 molecules
what is immature lymphocytes
- no not yet have antigen specific recpeotrs
- unable to respond to foreign antigens
- found in primary lymphoid tissues (bone marrow and thymus)
what is mature lymphocytes
- have functional antigen-specific receptors
- can respond to foreign antigens
- found in secondary lymphoid tissues (lymph nodes, spleen)
- found in the peripheral tissues
as the lymph passes through lymph nodes, antigen presenting cells in the nodes are able tosample microbial antigens hat may have entered through epithelia into tissues
true
dendritic cells can also pick up microbial antigens from the epithelial and other tissues and transport these antigens to lymph nodes
T
are B-cell and T cells zone mixed together
F.
B CELL AND T cells home to discrete zones in secondary lymphoid tissues
what is the function of secondary lymphoid tissues
- to drain lymph from the periphery
- to collect or trap antigens and antigen presenting cells (response is very localised)
- to provide a meeting place for
- recirculating T cells and
- DC arriving from the periphery - site of B cell - T cell interations
describe spleen
- blood entering the spleen flows through a network of channels (sinusoids) - blood borne antigens are trapped
- contain abundant phagocytes
- T lymphoctes are concentrated in periarteriolar lymphoid sheaths (PALS), surrounding small arterioles
what does specific mean
-adaptive immunity has exquisite antigen specificity
refers to the capapcity of the immune system to make a response to only 1 particular antigens
- property of B and T lymphocytes, which carry receptors unique for only 1 paricular antigen on their surface
what are antigens
antigens are substances that induce an immune response, such antigens are called immunogenic
what r types of antigens
proteins, carbohydrates, lipids, chemical haptens
what does the antigen specific receptors on lymphoctes binds to which part of antigens
a few amino cids or sugar only, they are called antigenic determinants or EPITOPES
what is naive lymphocytes
- mature lymphocytes that exist BEFOREantigen exposure
- have never engaged with cognate antigen
- the pre-cursor frequency of naive T cells specfic for antigen is very low
- migrate through and recirculate between secondary lymphoid organs
- typically die after 1-3 months if they dont make contact with antigen
- presentation of peptide + MHC molecules by DC activates naive T cells
what are activated lymphocytes
- mature lymphocytes taht have been presented with their foreign cognate antigen and been activated
- stop recirculating between lymphoid tissues
- migrate to periphral tissues and B-cell follicles to help B cells
what is effector lymphocyets
effector lymphocytes are acitivated lymphocytes that have differentiated into cells that produce molecules that function to eliminate antigens
whta does activated B lymphocytes differentiate into
antibody producing cells
antibodies from these called plasma cells, elimitae extracellular antigens
what does CD4+ Th cells do
they express surface molecules and produce cytokines, they help other cells (B-cells, macrophages, CTLs) to become fully activated and differentiate
what does CD8+ CTL cells do
TEHY HAVE MACHINERY TO KILL INFECTED TARGET HOST CELLS
DOES effector T cells have long lived
no. they have short lived and die after the antigen is eliminated
which cell survive longest
memory lymphocytes survive for long periods in the absense of antigen
where does memmory T-cell found
memory T cell found in lymphoid organs, in mucosal tissues and in the circulations
what r the 2 major type of memory T cells
- central memory t cell
2. effector memory t cell
describe central memory T cell (TCM)
- reside in lymphoid tissues
- central memory t cells express the chemokine receptors and selectins that allow them recirculate between lymphoid organs
- TCM have a limited capacity to perform effector function when they encounter antigen
- TCM undergo rapid proliferation to generate many effector cell upon re-exposure to antigen
describe effector memory t cell (TEM)
- reside in periphery (especially mucosal tissues)
- TEM do not express the chemokine receptor CCR& and L-selectin
- TEM are strategically located to rapidly produce effector cytokines upon re-encounter with their cognate antigen
- TEM do not proliferate much and although to mount a rapid response to repeat exposure to a microbe, complete eradication of an infection often requires large numbers of effectors generated from TCM.
on any 1 cell every antigen receptor has the same specificity
T`
immune system must be able to recognise all posible antigenic specificities encountered
T
space constraints dictate that the frequency of cells specific for 1 antigen is very low
T
each lymphoctes has only 1 receptor with 1 specificity
T
what is pre-cursor freqency?
it is estimated that teh total human t lymphoctes repertoire is ~ 10^12 cells. only as few as 1 to 100/10,000 lymphoctes are specific for 1 antigens
where does clones develop
in primary lymphoid tissue, that is thymus and bone marrow before antigen encounter
who overcome the problem with low- precursor frequency
Burnet’s clonal selection hypothesis
- explains how the immune system could response to a large number and variety of antigens
- antigen specific lymphocyte clones develop before and independently of exposure to antigens
- the lymphocyte clones have identical antigen receptors which differ from other clones.
what is the functional significance of speficity in immune response?
ensures that distinct antigens elicit responses that target those antigens
what is the functional significance of diveristy in immune response?
enables immune system to response to a large variety of antigens
what is the functional significance of memory in immune response?
leads to rapid and enhanced responses to repeated exposures to the same antigens
what is the functional significance of clonal expansion ?
increase number of antigen specifc lymphocytes to keep pace with microbes
what is the functional significance of specilaization in immune response?
generates responses that r optimal for defense against different types of microbes
what is the functional significance of contraction and homeostatsis in immune response?
allows immune system to respond to newly encountered antigens
what is the functional significance of nonreactivity to self in immune response?
prevent injury to the host during responses to foreign antigens
what are the key cellular features of adaptve immunity
- MOBILE : cells circulate and search for antigen
- PROLIFERATE: cells are capable of proliferation
- DIFFERENTIATION: cells undergo differentiation and have different effector functions
5 features of immune system
- strategic distribution of peripheral lymphoid organs
- network of lymphatic and BV facilitating the trafficking of antigen and cells
- migration of first responder cells from the periphery transporting antigen and signals to these lymphoid organs
- constant recirculation of naive lymphocytes
- utilisation of the same network of vessels for the carrying out of effector function
- antiodies against extracelullar antigens ( humoral immunity)
- T cells against intracellular antigens (cell mediated immunity)
list the steps that lymph nodes collects antigen from epithelium and CT
- the DC resident on skin: detect the presence of pathogen ad respond to it
- the DC takes the pathogen and process it
- the DC travel to lymph nodes and present antigen to other cell
what cell express pattern recognition receptors
- epithelial cells
- endothelial cells
- resident immune cells
e. g.in the skin (keratinocytes, fibroblasts, endothelial cells, dermal mast cells, langerhans cells, DC)
what does different microbes express?
they express different microbial patterns called PATHOGEN ASSOCIATED MOLECULAR PATTERNS (PAMPs)
what cell recognise damaged or necrotic cells? and what do they call for the molecules?
DC and other cells of the innate immune system also recognise molecules that are release from damaged or necrotic cells called DAMAGE ASSOCIATED MOLECULAR PATTERNS (DAMPs)
what does binding of PAMPs and DAMPs to PRRs trigger?
they trigger a cascade of event
- release of soluble molecules e.g cytokines, IL-1
- recruitment of innate immune cells e,g, neutrophils, monocytes.
who is the first responder?
tissue resident cells
what does binding of PAMPs to specfic cell surface PRRs triggers a cascade of events:
1, release of histamine and inflammatory cytokines e.g. TNF and IL-1
- dialted blood vessel allow for more blood flow to the area (redness) and fluid to come in (swelling)- carrying innate immune cells and plasma proteins, complement and antibodies
- induces the expression of adhesion molecules on endothelial cells lining the BV (veins)
- attract neutrophils and monocytes, perform phagocytosis, secrete more inflammtory cytokines, extend web-like extracellular traps for extracellular bacteria - attract cells adhere to endothelial cells only at sites of inflammation
what happen in antigen processing
- captured proteins need to be broken down into peptides
- the peptide antigens then need to be loaded onto special surface molecules in order for T cells to see them, these molecules are called Major histocompatability complex (MHC) molecules
what happen after resident cells ?
- antigen processing
- DC maturation
- DC migration
what happen in DC maturation
upregulating of molecules that allow DC to fully activate naive T cells
what happen in DC migration
the DC in the periphy need to rendezvous with the naive T cells.
- the DC migrate from periphral where inflammation occur to l lymph nodes (particular T-zone ) via lymph vessels
- the DC present antigen, the T cell inspect
- if T cell see antigen-MHC complex is specific for that peptide, T cell will be activated
what types of antigen does B-lymphocytes
1. whole proteins 2, carbohydrates 3. lipids. 4. nucleic acids on cell membranes or in solutions
what types of antigen does T-lymphocytes
peptides presented in MHC molecules only. T cell are MHC-restricted, they do not see free floating antigens
where did MHC discovered
at the genetic locus that determined graft acceptance or rejection
individual identical at MHC locus can accept graft from 1 another
T
what is the function of MHC protein
is to display peptides to antigen specific T cells
what locus made up human HLA for class 1 MHC
locus A,B and C
what locus made up human HLA for class 2 MHC
locus DP, DQ and DR
what locus made up mouse H-2 for class 1 MHC
locus D, L and K
what locus made up mouse H-2 for class 2 MHC
locus I-A, I-E
what r the general features of MHC molecules and the significan of each feature
- Co-dominant expression )both parental alleles of each MHC gene are expressed)
significance: increase # of different MHC molecules that can present peptides to T cells - polymorphic genes(many different alleles are present in the populations)
significance: ensures that different individuals are able to present and respond to different microbial peptides
what does MHC class 1 molecule express
express by all nucleated cells.
what chain does MHC 1 molecule consist of
alpha chain
- alpha1 and alpha2 where the polymorphic residues are , they form the closed small peptide (8-11 a.a) binding cleft
- alpha 3 domain is invariant, contains the binding site for T cell co-receptor CD8
what is the function of alpha 3 domain in MHC class1 molecules do
- functional it ensures MHC restricted CD8 T cells
2. and it anchors the MHC class1 molecule to the membrane
what chain does MHC 2 molecule consist of
an alpha and beta chain
- alpha1 and beta1 where the polymorphic residues are, they form the open conformation larger peptide (10-30 a.a) binding cleft
- the beta2 domain is invariant contain the binding site for T cell co-receptor CD$
- alpha2 and beta2 chains anchor MHC2 molecules in the membrane of professional antigen presenting cells
what cell express MHC class 2 molecules
professitonal antigen presenting cells: DC, B cells , macrophages
how do APC acquire these antigens and process them? MHC class 1 pathways
- proteins sourced from the cytosol
- protein are unfolded & tagged with ubiquitin for degradation
- the proteasome “shreds” the proteins into peptides (enhanced by inflammatory cytokines TNF, IL-1)
- peptides are actively moved by Transporter associated with Antigen Processing (TAP) in to the ER
- in the ER class 1 molecules are being synthesised
- the newly formed Class 1 molecule binds to TAP via Tapsin
- the peptide loaded MHC class 1 molecules then exit to Golgi apparatus (tobe packed into exocytic vesicles)
- for presentation to CD8 MHC class1 restricted CTL
what proteins are sourced from the cytosol
- virus
- tumour and normal self proteins
- microbial proteins that have been transported out of phagosomes
what happen in the ER whenn class 1 molecule is syntehsis
- the alpha chain (whose correct folding is assisted by chaperone molecules)’
- beta 2 microglobulin help stability on membrane
what happen when the newly formed Class 1 molecule binds to TAP via Tapsin
the peptides are loaded into MHC class1 groove (the eptide binding site)
peripheral tissue contain different types of DC in specific location and with different function
T. DC are strategically located to max. chance of 1st encounter
why DC at periphral site express a range of PRRs
- allow them to detect microbial patterns or PAMPs
- trigger the production of inflammatory cytokines
- trigger the uptake of antigens
only DC can activate naive T cells
T
list steps for phagocytosis
- microbe bidn to phagocyte receptors
- phagocyte membrane zips up around microbe
- microbe ingested in phagosome
- fusion of phagosome with lysosome
- activatio of phagocyte
- killing of microbes by ROS, NO, and lysosomal enzymes in phagolysosomes
MHC calls 2 pathway
- extracelullar antigens are recognised by pattern recognition receptors , these are internalised by phagpcytosis into phagosome. endosomes
- the endosome fuses with the lysosome form phagolysosome
at the samle time - the alpha and beta chain of MHC 2 molecules are being synthesised in the ER, the invariant chain (Ii) with CLIP occupies the peptide binding cleft in these neyly synthesised class 2 molecules
- class 2molecules are transported out of ER via the Golgi in an exocytic vesicle
- the exocytic vesicle fuses with phagolysosome (bring MHC2 molecules +degraded proteins together)
- the enzyme in the late endosome/lysosomes also contain a class 2 MHC-like protein called DM (H2-M in mice)
- MHC class 2 molecules loaded with peptide antigen are then transported to the cell membrane presenting antigen to CD4 MHC class2 restricted T cells
what does lysosome do
it contains proteolytic enzymes and an acidic pH, degrades proteins into peptides
what is CLIP and whats the function
contains a sequence called the class2 invariant chain peptide (CLIP). keeps the MHC molecule stable and block other peptides in the ER from binding newly synthesised MHC class2 molecules
what is the function of DM protein
function to exchange CLIP for higher infinity peptides in this compartment
what composition of stable class2 MHC pathway
polymorphic alpha and beta chain of MHC, peptide.
what composition of stable class1 MHC pathway
polymorphic alpha chain of MHC, beta microglobulin, peptide
what cell express MHC class 2
DC, mononuclear phagoctes, B lymphocytes, endothelial cells, thymic epithelium
what cell express MHC class 1
all nucleated cells
what cell respond to class 2MHC +peptide
CD4+ T cells
what cell respond to class 1MHC +peptide
CD8+ t cells
what source is class 2 MHC molecules
endosomal/lysosomal proteins (mostly internalised from extracellular environment)
what source is class 1 MHC molecules
cytosolic proteins mostly synthesized in the cell, may enter cytosol from phagosomes
what enzyme responsible for peptide generation in class 2 MHC molecule
endosomal and lysosomal proteases (e.g. cathepsins)
what enzyme responsible for peptide generation in class 1 MHC molecule
cytoplasmic proteasome
where is the site of peptide loading of MHC class 2 molecules
endocytic vesicle that has fused with endosomes/lysosomes
where is the site of peptide loading of MHC class 1 molecules
endoplasmic reticulum
what is the molecule involve in transport of peptides and loading of MHC 2 molecules
invariant chain, DM(remove CLIP)
what is the molecule involve in transport of peptides and loading of MHC 1 molecules
TAP
why MHC molecule have broad specificity
many different peptides can bidn to the same MHC molecules
why each MHC molecules display 1 peptides at a time
EACH T CELL RESPOnd to a single peptide bound to an MHC molecule
what is the significance of MHC molecules bind only peptides
MHC restricted t cells respond to protein antigens, and not to other chemicals
what is the significance of peptides are acquired during intracellular assembly
class 1 and class 2 MHC molecules display peptides from different cellular compartments
what is the significance of stable surface expression of MHC molecules requires bound peptide
only peptide loaded MHC molecules are expressed on the cell surface for recognition by t cells
what is the significance of very slow off rate
MHC molecule displays bound peptide for long enough to be located by t cells
why MHC antigen presentation so important
- T cells MHC restricted
- every nucleated cell expresses MHC class 1 molecules, enable CD8 CTL to see infected cells
- phagocytes are experts at sampling the extracellular space, they present them to CD4 help t cells
tese 2 presentation pathways ensure efficient eradication of ALL microbes
once DC process class2 MHC, in response to inflammatory signal, they will down regulate some of the adhering molecules RAMPs and DAMPs , TNF and IL-1. they will start to migrate via lymphatic vessels following chemokines until reach t cell zone
T
different cells express diffferent chmokine receptos. the chmokine receptor on the cell surface follows the chemokine gradient towards the gradient souce
T
how do naive t cell fin their way into lymph nodes?
naive t cells
- express specific homing receptos that allow them to recirculate between lymphoid tissue
- naive t cells are therfore CD4/8+CD3+TCR+CCR7+L-selectin+LFA-1
- naive t cell that have homed into lymph nodes but fail to recognise antigen and to become activated will eventually return to blood stream
what r the homing receptors on naive t cells
- L-selectin, a homing receptor that binds a ligand expressed on high endothelial venules (HEV)
- CCR7: a chemokine receptor that allows naive T cell to home to specific chemokine CCL19/21 expressed by HEV
this permits naive t cell to enter and recirculate through secondary lymphoid tissue
naive t cell recirculate !
T
why naive t cells return to the blood
provides naive t cells with another chance to enter secondary lymphoid tissues and search for the antigens they do recognise
what r the major route of blood re-entry
- through the EFFERENT LYMPHATICS
- through the lymphatic vasclature to the thoracic or right lymphatic duct
- finally into superior vena cava or right subclavian vein
how do naive t cells know when to return to the blood
- naive t cells need to be given time to inspect DC for antigen MHC complexes
- recently arrived naive t cell express low levels of sphingosine 1 phosphate receptor 1 (S1PR1)
- if naive t cell do not engage with their cognate antigens after several hours they will re-express S1PR1 on their surface, allowing then to respond to the S1P gradient and egress from lymph nodes
why recently arrived naive t cell express low levels of sphingosine 1 phosphate receptor 1 (S1PR1)
because engagement with S1P in blood lead to S1PR1 internalization
this means they are unable to respond to high conc. of S1P (a lipid chemoattractant) in the medullary sinus, efferent lymphatics and blood
the S1P gradient is maintained (therefore low in tissue) because S1P-degrading enzyme S1Plyase is ubiquitously present in tissue
if you find the antigen, action of fingolimod downregulated S1PR1
this make naive t cell stay in t cell zone
T
what happen after naive T cell found antigen
t cell and b cell proliferate and differentiate
where is b cell located
b cell strategically located near the site of antigen drainage within the 2nd lymphoid tissue, therefore b cell are one of the first lymphocytes to encounter antigen
B cell require MHC to see their antigen
F. B cell can see antigenss either free floating of delivered by cells
what is immunoglobulins (Ig)
antibodies and BCR
what does naive b cells express
IgM and IgD
what perform effector functions
antibodies which is part of humeral response
where does antibody present
plasma, mucosal secretions , interstitial fluid of tissue
what is the structure of antibody
- variable part
- involved in antigen recognition
- vary between b cells cones - constant part
- involved in effector functions
- relatively conserved between b cell clones
- can change/ switch
what antigen binding feature for Ig
made up of 3 CDRs in Vh and 3 CDRs in VL
what antigen binding feature for TCR
made up 3 CDRs in Valpha and 3 CDRs in Vbeta
is there any changes in constant regions in Ig
heavy chain class switching and change from membran to secretory Ig
is there any changes in constant regions innTCR
none
what is the affinity of antigen binding of feature of Ig
average affinity Igs increase during immune rsponse
what is the affinity of antigen binding of feature of TCR
no change during immune response
what is on rate and oof rate of Ig
rapid on rate. variable off rate
what is on rate and oof rate of TCR
slow on rate and slow off rate
describe antibodies
- four polypeptide chains assembled into Y shape
- two identical light (L) chains (~25kDa)
- two identical heavy (H) chains (~50kDa)
what does lght chain of antibody consist
1 varibale and 1 constant domain
what does heavy chain consist of
1 varibale and 3-4 constant domains
what makes uo the antigen binding site
2 variable regions
what does the hinge region do in antibody
gives flexibility
- allow varibale regions to come close together or further apart to bidn epitopes close together or far apart on an antigen
what does the variable regions of both heavy and light chains contain
they each contain 3 hyper-variable regions called COMPLEMENTARITY DETERMING REGIONS (CDRs), this is where antigen binds, also called antigen binding site)
what r the 2 types of light chain
K and lamba, each b cell exprsses either k or lamba but not bothe, they are differ in constant domain but there are no functional difference
what r they types of heavy chains
delta, u, gamma, abcelong E and alpha. they differ in constant domain and in function
how can antibody class/isotype are named
according totheir heavy chains delta=IgD u=IgM Y=IgG E= IGE alpha=IgA
describe IgD on serum conc, serum T1/2, secreted form and function
serum conc. trace
serum T1/2: 3 days
secreted form: not secreted
function: naive BCR (antigen recognition)
describe IgM on serum conc, serum T1/2, secreted form and function
serum conc. 1.5 mg/mL
serum T1/2: 5 days
secreted form: pentamer
function: naive BCR (antigen recognition+ non-protein Ags)
describe IgG on serum conc, serum T1/2, secreted form and function
serum conc. 13.5 mg/mL
serum T1/2: 23 days
secreted form: monomer
function: antiviral and antibacterial defence+neonatal immunity
describe IgE on serum conc, serum T1/2, secreted form and function
serum conc. 0.05 mg/mL
serum T1/2: 2 days
secreted form: monomer
function: defence against helminths and parasites
describe IgA on serum conc, serum T1/2, secreted form and function
serum conc. 3.5 mg/mL
serum T1/2: 6 days
secreted form: dimer
function: mucosal immunity
how are different epitopes of protein antigens recognised
- the sequence of a stretch of amino acids (linear epitopes)
- the 3D shape of the protein antigen (conformational epitopes)
- some of these epitopes are hidden within antigen molecules and are exposed as a result of a physiochemical change
what binding force between antigen- antibody
weak non-covalent force, binding is reversible
- H bond (shared H ion)
- electrostatic forces (opposite forces)
- Van der waals forces (electron clouds)
- hydrophobic forces (exclude water molecules)
what does high affinity mean in antibody antigen binding
high attraction forces + low repulsion forces , means a good fit
what does low affinity mean
low attraction forces+high repulsion forces , a poor fit
what is affinity
affinity is the strength of binding between one antigen-binding site on an antibody molecule and its corresponding epitope
- expressed as the diassociation constant Kd=the conc. of antigen required to ocupy half the available antibody molecules in solution. the lower the Kd the higher the affinity
in primary immune response, what is the Kd of most antibodes
10^-6 to 10^-9 M
what does repeated stimulation of antigen to affinity of antibodies
the affinity increase to a Kd 10^-8 to 10^-11 M
hat is avidity
overall strength of the interaction between antibody and its antigen. it is much greater than affinity
what doesavidity depend on
- affinity
- valency of the interactions
e. g. IgM have higher avidity than IgG
what is cross rxn
antibodies produced against one antigen may bind to other, structurally similar antigens. such binding to similar epitopes is called a cross rxn.
how does polyclonal antibodies produce
antigen with several epitopes may stimulate serveral B cell clones and induce polyclonal antibodies
give examples of diseases caused by antibody cross reactivity
streptococcal infection,
- after such infection, some individuals produce anti-streptococcal antibodies that cross-react with an antigen in heart muscle, deposition of these antibodies in the heart triggers an inflammatory disease called rheumatic fever
- other individuals make anti-streptococcal antibodies that deposit in kidney glomeruli causing poststreptococcal glomerulonephritis
what is the structure of TCR
- variable domains
- involved in peptide-MHC recognition
- vary between T cell clones - constat domains
- conserved between T cell clones
- does not change / switch
what feature is common in both BCR and TCR
- membrane bound
- constant regions
- variable regions (3 hypervatiable regions in each variable regions)
what is the unique features to the TCR
- two chains alpha and beta chains, each with 2 Ig domains
- no heavy and light chains
- slow on rate and slow off rate
what does TCR recognise
self MHC-foreign peptide complex, each TCR recognises as few as 1-3 residues of the MHC peptide complex
what is immunodominant epitopes
peptides generated within APCs that bind most avidly to MHC molecules, these are the peptides most likely to stimulate T cells
what happen after TCR recognise antigen
TCR cannot transmit signals to the T cell
- CD3 and zeta proteins together with CD4/CD8 and the TCR make up the TCR complex
list the steps in signal transduction in TCR
- TCR recognises peptide-MHC antigen
- TCR has short cytoplasmic tail, with no signal transduction sites
- activation via signalling molecule complex
- CD3 molecules (Y, delta, epsilon) + zeta chain
- always associated with TCR in cell membrane
- provide activation signal transduction via ITAM (immunoreceptor tyrosine based activation motif) sequences - phosphorylation of ITAM leads to the activation of the transcription factors (NTAT, NFkB and AP-1)
where did the receptor diversity come cfrom
-by V(D)J recombination
receptor diversity is generated by randomly (and permanently) combining the V, (D) &J segments.
NO diversity D segments in light chain locus of BCR
T
5 different heavy chain gene segments for BCR
u, delta, Y, E, alpha
no diversity D segents in alpha-chain locus of TCR
T
what initiated the recombination of Ig genes
mediated by a group of enzymes called VDJ recombinase
- these group of enzymes are lymphoid specific
- only expressed by immature T and B cells
what does VDJ recombinase composed of
composed of recombinase-activating gene 1 and 2 (RAG-1 and RAG-2) proteins
- RAG proteins recognise DNA sequences that flank all antigen receptor V, D, J gene segments
1. upon recognition, the recombinase brings the V,D, J segments close together for cleavage
2. the DNA breaks are then repaired by ligases, producing full length recombines V-J or V-D-J exon without the intervening DNA segments
list the stpes for VDJ recombination
- random somatic recombination of D &J
- random somatic recombination of DJ &V
3 transcription of new rearranged gene
what r the 2 types of diversity
- recombinatorial diversity
- due to random recombination of V,D and J segments
- limited by the number of V,D, and J segments - junctional diversity
- extra diversity generated by random addition and subtraction of nucleotides at V-D-J or V-J junctions
- almost limitless possibilitis
what happen to Omenn syndrome
only partial V(D)J recombination activity, cant develop receptor, no receptor present and no T and B cell
what is immunological tolerance
a system for determining which lymphocyte clones will be allowed to survive
what r the types of tolerance
- central tolerance
- occur in the primary lymphoid tissues (bone marrow for B cells and thymus for T cells) - periphral
- occur in secondary lymphoid tissues, mediated primarilt by regulatory cells
what does breakdown in tolerance lead to
autoimmune diseases
where is the positive and nagative selection ocur
in thymus primary lymphoid oragn
what is double positive cell and double negative cell mean
double negative=CD4-CD8- pro-T cell
double positive: CD4+ CD8+ immature T cell
what is positive selection
selecting T cells to differentiate into either CD4+ or CD8+ Y cells. ensure appropriate functions to eliminate all types of pathogens: intracelular (CD*) and extracellular by helping B cells and macrophage (CD4)
what is negative selection
detecting self-reactive T cell clones helps prevent autoimmunity
what is AIRE and what does it do?
autoimmune regulator.
1 AIRE induces medullary epithelial cell (MEC) expression of a broad repertoire of peripheral tissue anigens (PTAs)
2. these PTAs are processed and then presented on surface-displayed MHC/HLA molecules
3. soon after the induction of AIRE &PTAs, MEC apoptose
4. mature thymocytes percolate through the medulla, and if their TCRs recognise an MHC: PTA complex they will be overactivated and deleted from the repertoire
5. thymocytes can recognise MHC:PTA complexes directly on MECs or indirectly on DCs that have engulfed apoptotic MECs or MEC fragments
what does an AIRE deficiency leads to
a failure in central tolerance. humans with AIRE defiency develop autoimmune polyendocrinopathy-candidiases ectodermal dystrophy(APECED)
what does immature B cells do if it recognise self antigens
they undergo receptor editing
- Ig gene recombination machinery is reactivated (i.e. RAG)
- heavy chain is not changed
- associates with previoudly constructed heavy chain
- new receptor is no longer specific for self antigens
- no evidence this occur for the TCR in thymus
how many percent of B cells undergo receptor editing
25-50% of b cells
no known diseases caused by a break down in central B cell tolerance
what does regulatory T cell do
they supress the activation of the bad lymphocytes in peripheral
what r the 3 pathways to periphral tolerance
- Anergy (a state of unresponsiveness)
- deletion
- suppression (regulatory T cell spress the activation)
what is DC doing in the pripheral
- PRRs recognise PAMPs
- high phagocytic capacity
3, process protein antigens onto MHC 1 and MHC 2
4 migrate to T cell zone in response to PAMPs, DAMPs and cytokines
what is DC doing in lymph nodes
- home to T cell zones
2, express the required surface molecules to activate naive T cells -MHC, co-stimulatory molecules and cytokines
what does t cell activation involve
cross talk between t cell and DC, an exchange of signals occurs at the immunological synapse
- th pt of contact between t cell and DC
stpes for t cell activation
- antigen recognition
2 coreceptors
3 adhesion molecules
4 co stimulation
describe step 1 of t cell activation : recognition of peptide +MHC
- TCR recognises peptide+ MHC (antigen specific)
- TCR binding to peptide-MHC is low affinity
- the TCR itself does not transduce activation signals
- two or more TCR must be engaged, for several minutes to commence activation
describe step 2 of t cell activation: co-receptors
- CD4 coreceptor on Y helper cells binds conserved beta2 chain on MHC class II
- CD8 coreceptor on CTL binds conserved alpha3 chain on MHC class I
- stabilises low affinity binding of TCR to peptide-MHC
- ensure appropriate T cell type is activated
- activation occur via signally through TCR complex (CD3 molecules (Y,delta,E)+zeta chain, alway associated with TCR in cell membrane, provide activation signal transduction via immunoreceptor tyrosine- based activation motif (ITAM( sequences
describe step 3 of t cell activation : adhesion molecules
- LFA-11: leukocyte function-associated antigen-1 (CD11a)
- an integrin molecule expressed in the Tcell membrane - binds ICAM-1: intercellular adhesion molecule-1 (CD-45)
- adhesion molecules on APC - adhesion molecules increase binding affinity of TCR to peptide -MHC complex
describe step 4 of t cell activation costimulation
1.T cell require additional signals to achieve full activation
2. two signal hypothesis
Signal 1=antigen recognition
- TCR+MHC+peptide
Signal 2= costimulatory signal from DC
-B7-1 and B7-2 expressed on DC
-expression increased when APC encounters microbial antigen or inflammation
- B7 binds CD28 expressed on all T cells
what happen at immunological synapse
- site of transduction of activation signals
- some effector molecules and cytokines may be secreted through this region ensuring that they do not diffuse away but are targeted to the APC
- enzymes that serves to degrade ot inhibit signally molecules are also recruited to the synapse, so it maybe involved interminating lymphocyte activation
why costimulation is so important
- resting APCs, which has not been exposed to microbes or adjuvants, may present peptide antigens, but they do not express costimulators(B7) and are unable to activate naive T cells
- T cells that recognise antigen without costimulation may become unresponsive to subsequent exposure to antigen
- microbes +cytokines produced during innate immune responses to microbes, induce expression of costimulators (e.g B7 molecules)
- activated APCs also produce cytokines that stimulate the differentiation of naive T cells into effector cells
what happen when microbes +cytokines produced during innate immune responses to microbes, induce expression of costimulators (e.g B7 molecules)
- the B7 costimulators are recognised by the CD28 receptors on naive T cells, providing signal 2.
- in conjunction with antigen recognition (signal 1), this recognition initiate T cell responses
what r the key events occurring in activated t cells
- binding of foreign peptide: self-MHC complex by TCR and a co-receptor (CD4 or CD8) transmits a signal to the T cell that “antigen has been encountered”
- activation of naive T cell requires a second signal
- the co-stimulatory signal delivered by the same DC, CD28 on the T cell encountering B7 molecules on DC delivers signal 2 and leads to increased survival and proliferation of the T cells - different pathways of differentiation produce subsets of effector T cells that carry out different effector responses
- depending on the nature of a 3rd signal delivered by the APC, cytokines are commonly, but not exclusively, involved in directing this differentiation
what is interleukin 2
all naive T cell express the low-moderare affinity interleukin 2 receptor (IL-2Rbeta) on their surface
- an early molecular event occurring in T cells following signally through the TCR complex is up regulation of genes requires for T cell proliferation and differentiation
- the high affinity IL-2 receptor IL-2Ralpha or CD25 and IL-2 itself - binding of IL-2 to CD25 is a major trigger of T cell proliferation
why t cells need to proliferate
they need then differentiate into effector cells CD4 and CD8+ cells
what transcription factor turn on in Th1, and sinature cytokines secreted by Th1 what immune rxns involved, and what host defence and role in disease
T-bet transcription factor
signature cytokines IFNY
immune rxns: macrophage activation; IgG production
Host defense: intracellular microbes
role in diseases: autoimmune diseases, tissue damage associated with chronic infection
what transcription factor turn on in Th2, and sinature cytokines secreted by Th2 what immune rxns involved, and what host defence and role in disease
Gata3 transcription factor signature cytokines: IL-4, IL-5, IL-13 immune rxns: mast cell, eosinophil activation, IgE production, alternative macrophage activation Host defense: Helminthic parasites role in diseases: allergic diseases
what transcription factor turn on in Th17, and sinature cytokines secreted by Th17 what immune rxns involved, and what host defence and role in disease
RORgammat transcription factor
signature cytokines: IL-17A, IL-17F, IL-22
immune rxns: neutrophilic monocytic inflammation
Host defense: extracellular bacteria, fungi
role in diseases: autoimmune and inflammatory disease
what r different subsets of CD4+ helper T lymphocytes
Th1, Th2, Th17
what r they characteristic of subset CD4+ helper t cells
- mature, acticated DC secrete cytokines
- the binding of these cytokines ti their corresponding cytokine receptors on the surface of newly activated T cells activated transcription factors inside the t cells
- the type of cytokine produced determines which transcription factor is activated which in turn influence the t cell differentiation pathway and hense what sort of the effector helper t cell it becomes
- this is important because different helper t cells are required to respond to and eradicate different infections
what do balance between Th1 and Th2 activation
e.g. Th2 cell activation inhibit Th1 cell activation, the IL-4, Il-10 and IL-13 inhibit microbicidal activity of macrophage.
how did CD8 t cell activated via helper t cells
1, DC or tissue cell infected with microbe
- CD8+ and CD4+ T cells recognise antigen presented by APC
- clonal expansion and differentiation of CD8+ T cells
what happen after clonal expansion
contraction (homeostatsis)
describe memory cells
memory cells survive even after the infection is eradicated
- they do not continue to produce cytokines or kill cells
- they r a pool of lymphocytes waiting for the infection to return
- they can be found in lymphoid organs, in mucosal tissues, and in the circulation
- reside in lymphoid tissues are central memory t cells (TCM)
- others that reside in the periphery are called effector memory t cells (TEM)
how did Tcell memory and the effector decline
elimination of antigen deprives cells of survival stimuli and many cells in the expanded antigen-reactive clones die by apoptosis
- important feed back mechanism to limit inflammation and over activation
memory CD4 and CD8 T cells survive as long-lived resting cells in circulation and lymphoid tissue
the outcome of this process determines the success of vaccination programs
how did effector T cells activated
- effector lymphoctes are activated lymphocytes that have differentiated into cells that produce molecules that function to eliminate antigens
- effector CD4 t helper cells express surface molecules and produce cytokines
- effector CD8 CTL have the machinery to kill infected target host cells
- effector t cells are shot lived and die after the antigen is eliminated
what is regulatory t cells do
- they supress rather than activate the immune response, they are CD4+ and may express FoxP3 (Tanscription factor)
what r the 2 main type of regulatory t cells
- naturally occuring Tregs
- produced in the thymus during T cell maturation
- constitutively express high affnity IL-2Ralpha (CD25) - inducible Tregs
- induced from naive CD4+ T cells in the periphery
what does Tregs depend on
depend on IL-2 for their survival but not generation
peripheral T cell tolerance
- naive t cells need at least 2 signals to induce their proliferation and differentiation into effector and memory cells - signal 1 (antigen recognition ) and signal 2 (co-stimulation through CD28)
- T cells that recognise self antigens may receive signal 1 through TCR
- they do not receive strong costimulation because there is no innate immune response - the presence/absence of the costimulation is a major factor determining whether T cells are activated or tolerised
what is anergy
anergy is the long lived functional inactivation that occurs when T cells recognise antigens without adequate level of co-stimulation
- anergy cells survide but are incapable of responding to the antigen
how did anergy occur via
- a blocking signalling by the TCR complex
- when t cells recognise antigens in the absense of costimulation, the TCR complex may loses its ability to transmit activating signals - the delivery of inhibitory signals from receptors other than TCR complex
- on recognition of self antigens, T cells may prederentially engaged one of the inhibitory receptors of CD28 family, CTLA-4(CTL associated antigen 4) or PD-1(programed death protein1)
what does most Tregs express
high affinity IL-2 receptor(CD25) and transcription factor FoxP3
where is Tregs developed in
- thymus (natural Tregs)
2. periphral lymphoid organs (inducible Tregs)
how did Tregs maintain peripheral tolerance
by supression
- self-reactive T cell activation
- self-reactive effector T functions
they release supressive cytokines (IL-10 and TGFbeta) and inhibitory molecules (CTLA-4)
what does recognition self antigens trigger
they trigger pathway of apoptosis that result in elimination of the self reactive lymphocytes
what does recognition of self antigen without a strong costimulation happen
fails to stimulate production of anti-apoptotic proteins- a lack of survival signals induces apoptosis
what does recognition of self antigen may lead to
the coexpression of death receptors (CD95 or Fas) and their ligands (CD95L or FasL)
-culminates in the activation of caspases and apoptosis by what is called the death receptor pathway
effector function of CD4 t helper cells
- macrophage activation
- helping CTL
- helping macrophage eliminate internalised intracellular bacteria
lymphocyte tranffick
- naived T lymphocyte leave the blood stream across high endothelial venules
- in the t-cell area of lymphoid organs, this specialised endothelial expresses a number of molecules involve in lymphocyte homing to the lymph nodes, especially the homing receptor GLYCAM-I, adhesion molecule ICAM-I and chemokines MIP3beta
- the initial binding of naive T cell to the endothelial is mediated by L-selectin binding to GlyCAM-I
- binding of chemokines display extracellular matrix triggers tight adhesion to integrin LFA-Ito its ligand ICAM-I
- the lymphocyte is then able to migrate across endothelial into the T cell area of lymph nodes
- the naive T cells can then inspect DC in the lymph node for the presence of its specific antigen
- if it does not recognise antigen, the T cell is not activated and pass out from the lymph node to the circulation
- tcells do meet specific antigen in the lymph nodes are activated and begin to proliferate into mature effector cells
- eventually after few days, the cell leaves lymph nodes and return to circulation
how to naive T cells find their way
- they express specific homing receptors that allow them to recirculate between lymphoid tissue
why do immature DC stay in peripheral and donot home to lymph nodes
they are CCR7 - , low L-selectin, low MHC-II, CD80/86 low
what happen to mature DC
they have encountered antigen or danger signal
- upregulating MHC II, costimulation molecules as well as CCR7 and L-selectin
- this enables them to home to the T cell zones of the lymph node where antigen presentation occue
they now CCR7+, Lselectin+, MHC II high and CD80/86 high
some effector T cells remain in the lymph node, where they function to eradicate infected cells at that sites or provide signals to B cells that promote antibody responses against the microbe
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other effector T cells leave the lymph node and enter the circulation. they need to find their way to sites of infection to perform their effector function
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how do activate T cells find their way
after activated by DC in the lymph nodes, T cells change their phenotype
- decrease L-selectin, decrease CCR7
enable them to STOP homing to t cell zones of lymphoid tissues
- they increase LFA-1, increase VLA-4, increase CCR6, increase CXCR3, increase ligands for E and or P-selectin
enable them to home to peripheral sites
describe process of rolling
- cytokines are released from site of inflammation
- stimulation of the endothelial cells lies in the BV
- the endothelial cells express selectins
- selectin bind to carbohydrated displayed on the membrane of leucocytes, causing then to stick on the wall of BV
- this binding interaction have low affinity, the leucocytes can roll the BV wall in searching for pts exit the BV. there they adhere tightly and squeeze between endothlial cell without disrupting BV wall
- they crawl out of BV into adjacent CT
- leucocytes only adhere to the surface of veins, they do not crawl out of arteries
does homing of effector t cells to sites of infection dependent on antigen recognition
NO, they are independent, effector t cell homing depends on adhesion molecules and chemokines. any effector T cell present in blood regardless of antigen specificity can enter the site of any infection , this increase the chance of effector cells entering tissue where they may encounter the microbes they recognise
how does t cell retain at sites of infection if they recognise
they will upregulate adhesion molecules e.g. VLA integrins
how to activate macrophage
- activation of effector cell, CD4 effector T cell Th1 cell
contact with macrophage that have ingested bacteria - activation of macrophage by binding of CD40L-CD40 and release of IFN-Y
- the macrophage kill the phagocytosed bacteria
- they increase secretions of cytokines (TNF,IL-1,IL-12,chemokines)
- increased expression of MHC and costimulators
how do effector Th1 cells activate macrophage
- in the periphery, macrophage ingest microbes into phagosomes
- they fuse with lysosome to form phagolysosomes
- the microbial proteins are processed on to class II MHC molecules and presentd on the surface - effector CD4+ T cells specific for these peptides recognise these class II associated peptides
- the macrophages can receive help from the T cells in 2 main ways.
- by expressing CD40 on their surface
- by expressing the receptor for IFNY which is the signature cytokine produced by Th1 cells
how do activated CTLs kill their targets
- antigen recognition and binding of CTL to target cell, -formation of immunological synapse, re-enforcement of CTL activation exquite specificity of killing
- CTL activation and granule exocytosis
- alignment of the cytotoxic granules at secretory zone of the immunological synapse - release of perforin and granzymes (cytokines like IFNY) at immunological synapse,
- perforin facilitates granzymes entry into the cytosol, granzymes activate apoptosis - apoptosis of target cell
- activation of pro-apoptosis caspases - phagocytes clean up the debris and t cell migrate away
how do CD4 helper help regulate VD8 CTL
- CTL T cell contain numbers of cytoplasmic organelles
- cytotoxic granule are found only in CD8 effector and natural killer cells
- the adherence molecule LFA-1 mediated the initial contact with potential target cell. binding to ICAMI
- if T cell found no specific antigen, the t cell is not stimulated
- if T cell found specific antigen, become activated changes occur in cytoskeletal of t cells - the cytoplasmic organelles of t cells move to face target cell. this re organise both causing release new protein syntheiss into interphase between t cell and its target
CD4 T helper eradicate intracellular microbes. if microbes escape from vesibles into the cytoplasm, they must eliminated by CD8 CTLs.
- CTL must produce IFNY that activates macrophages to destroy ingested microbes
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• Humoral Immunity
• Humoral Immunity
- Mediated by B cells producing antibodies - phases of the humoral immune response
• How do B cells become activated
- antigen capture and BCR complex signalling
• Early B cell responses to activation
- divide, secrete IgM, migrate and differentiate
Types of B cell responses
- Different B cell subsets mediate different
responses - T dependent and T-independent antibody responses
Late B cell responses to activation
- migrate and differentiate
Early steps in B cell activation
• BCR recognises and binds to cognate antigen
- free floating (doesn’t need to be “presented” on MHC)
• BCR cross linking triggers a cascade of biochemical signals
- signals transduced by receptor- associated molecules (not BCR)
- Like T cell activation, these signals induce the B cells to…
1. clonally expand
2. start secreting IgM
3. migrate and differentiate
What is the nature of T cell help to B cells?
Proliferation & cytokine/ chemokine receptor upregulation
B cell subsets: Specialised B cells with dedicated immunological functions
follicular B cell
follicular B cell in spleen and other lymphoid organs
- secrete IgM and IgD->protein antigen+helper T cell help:
1. isotype switched, high affinity antibodies, long lived plasma cells e.g. IgA, IgA and IgE
B cell subsets: Specialised B cells with dedicated immunological functions marginal zone B cell
marginal zone B cell secrete IgM, -> lipids, polysaccharides etc -> mainly IgM, short lived plasma cells
B cell subsets: Specialised B cells with dedicated immunological functions
B-1 cells
B-1 cells in muscosal tissues, pritoneal caviry secrete IgM-> lipids, polysaccharides etc-> mainly IgM, short lived plasma cells
T-independent antigens trigger IgM secretion by activated B cells
• IgM secretion is greatest when antigens are multivalent (eg polysaccharides)
- many bacteria have polysaccharide-rich capsules
- early IgM production is very important in immunity to
these microbes
- IgM response to proteins is poor (B cells need “help” - next lecture)
• IgM activates elimination mechanisms eg complement activation (more on this in U4L21)
• e.g. The A-B-O blood group antigens
- A-B-O antigens are Carbohydrate antigens found on
the surface of red blood cells (RBC)
- B cells with BCR specific for the A-B-O antigens will secrete IgM (“natural” antibodies)
- Not proteins = no isotype class switching (more later)
what is the chemical nature of Thymus independent antigen
polysaccharides also glycolipids, nucleic acid
isotype switing for thymus independent antigen
low level switching IgM
antigen maturation for thymus independent antigen
little or no
secondary response (memry B cells) for thymus independent antigen
only seen with some polysaccharide antigens
T-dependent antigens trigger CD4+ T cell help to B cells
• Protein antigens (few repeating epitopes) elicit a poor IgM response
• Protein antigens require T cell ‘help’ to elicit efficient antibody production
• 2 fundamental questions:
1. Where & how do antigen-specific T
helper cells and B cells get together? 2. What is the nature of T cell ‘help’?
chemical structue of thymus dependent antigen
protein
isotype switching for thymus dependent antigen
yes. IgM switch to IgG, IgE and IgA
affinity maturation for thymus dependent antigen
yes
secondary response (memmory B cells) for thymus dependent antigen
yes
naive T cell-> CCR7 for T cell zone to T cell zone migration
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Homing to the T cell Zones
• Naive T cells express CCR7
- allows them to home to CCL19 and CCL21 in the T cell zones
• Activated effector T cells lose CCR7 and up regulate other chemokine receptors that allow them to home to the periphery
- they no longer have the capacity to respond to CCL19 or CCL21
B cell-helping T cells migrate to the B cell follicles
B cell-helping T cells lose CCR7 but up regulate CXCR5
- they stop migrating between T cell zones
- they migrate towards CXCL13 expressed in the B cell follicles ‣ where they help B cells
Newly activated B cells are making their way to the T cell zone
Naive B cells express CXCR5
- allows them to migrate towards CXCL13 expressed in the B cell follicles
• Newly activated B cells lose CXCR5 and up regulate CCR7
- the same chemokine expressed by naive T cells
- this enables B cells to home to the T cell zones by migrating towards CCL19 and CCL21
Receiving T cell “help”
• The T cells and B cells are specific for the same antigen
- If the T cell recognises the peptide MHC II complex it will receive a signal through the immunological synapse
‣ becomes re-activated
‣ sends a signal to the B cell by engaging
its CD40L with CD40 on B cells
The T cell will also produces cytokines
‣ sends additional signals to the B cell
• T cell-B cell interaction is called:
cooperation, collaboration or cross-talk
leads to :
activation of B cells by CD40 ligand and cytokines-> B cell proliferation, initial antibody production, germinal centre reaction
class switching
- In response to non-protein antigens B cells produce IgM antibodies
- Heavy-chain isotype switching is induced by a combination of CD40L-mediated signals and cytokines
B cell switch to IgG subclasses inresponse to IFN-Y
IgG: Fc receptor dependent phagocyte responses, complement activation, neonatal immunity
B cell switch to IgE subclasses inresponse to IL-4
IgE:
immunity against helminths
mast cell degranulation (immeidate hypersensitivity)
B cell switch to IgA subclasses inresponse to cytokines produced in mucosal tissues e.g. TGF-B. BAFF others
IgA: mucosal immunity (transport IgA through epithelia)
Human CD40L “knockouts”?
• In the absence of CD40 or CD40L:
- B cells secrete only IgM
- fail to switch to other isotypes
- cannot become memory cells
• A disease called hyper-IgM syndrome is caused by mutations in CD40L
- mutation is located on the X chromosome (so X-linked),
- leads to production of nonfunctional forms of CD40L.
- much of the serum antibody is IgM, because of defective heavy-chain class switching.
- patients are extremely susceptible to recurrent bacterial infections because they have defective cell-mediated immunity against intracellular microbes
‣ because CD40L is important for T cell–mediated activation of macrophages and ‣ for the amplification of T cell responses by dendritic cells
Mechanism of heavy-chain isotype switching
• In an IgM-secreting B cell the primary transcript of the rearranged VDJ heavy-chain gene is spliced, combining the VDJ exon with the μ exons to produce an mRNA encoding the μ heavy chain - The VDJ joins with the μ exons because the μ gene is closest to the rearranged VDJ unit. - this will form part of an IgM antibody and is why IgM is produced first during an immune response • Signals from helper T cells (CD40 ligand and cytokines) may induce recombination of switch (S) regions • the rearranged VDJ DNA is moved close to a different C gene - Activation-induced deaminase (AID) alters nucleotides in the switch regions so that they can be cleaved by other enzymes and joined to downstream switch regions - When the heavy chain gene is transcribed, the VDJ exon is spliced onto the exons of the downstream C gene • This results in the production of a heavy chain with a new constant region and thus a new class of antibody.
• the cytokine produced by the T cells dictates which switch regions are activated (and hence which class of antibody is produced) • Only one type of T cell response occurs - ensuring that only one type of antibody is produced, and... - that the antibody produced is the most effective class to eradicate the pathogen
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Why class switch?
• Different antibody isotypes perform different functions
- site of antigen exposure can also affect switch recombination (e.g. IgA in mucosal sites)
• Isotype switching broadens the functional capabilities of the humoral immune response
- more on this in the next lecture
• Different cells have different Fc-receptors that bind specific heavy chains
- macrophages express Fcγ receptors that bind IgG
- eosinophils and mast cells express high affinity Fcε
receptors for binding IgE
Affinity Maturation and Somatic Hypermutation
- Due to its pentamer structure and 10 antigen binding sites IgM has a high avidity
- The affinity of IgM (especially for protein antigens) is low
- Necessitates a process by which the affinity of an antibody variable region can be enhanced
- Inducing mutations in the variable genes (somatic hypermutation) increases the affinity of an antibody for its antigen
Affinity increases over time and with repeated antigen exposure
- Point mutations in VDJ gene segment elicits changed binding affinity in antigen- binding site of Ig molecules (CDRs 1, 2 and 3)
- Only occurs in T-dependent responses to protein antigens
- T cell ‘help’ signals somatic hypermutation and frequency of point mutations in Ig genes much greater than in other cells
Follicular Dendritic Cells (FDC)
• FDCs are found only in lymphoid follicles
• FDCs provide a critical source of antigen for GC B cells
- involved in displaying antigens for the selection of germinal center B cells - FDC test the B cells by presenting antigen bound antibody complexes
‣ Furious competition begins!
‣ FDCs will provide survival signals to the
B cells that out compete the others for
binding to the antigen being presented
‣ B cells that receive no signal die
• 2 to 7 days after antigen exposure, some activated helper T cells that migrate to meet activated B cells at the edge of the follicles will be triggered by these antigen presenting B cells to differentiate into follicular helper T cells (TFH cells)
The Final Stages of Humoral Immunity: The Memory Phase
• Some of the B cells activated in the germinal centres acquire the ability to survive for long periods
- don’t require continuing antigenic stimulation.
• Memory B cells make rapid responses to
subsequent antigen re-exposure
• Some memory B cells remain in the lymphoid organ where they were generated. Others exit & recirculate
• Production of large amounts of isotype-switched, high-affinity antibodies is greatly accelerated after secondary exposure to antigens
- this can be attributed to the earlier activation of memory cells in germinal centres
• Effective vaccines against microbes and microbial toxins induce both affinity maturation and memory B cell formation
The Final Stages of Humoral Immunity 4: Plasma Cells
Long-lived plasma cells (antibody secreting cells) are generated in germinal centres
- They enter the circulation & home to the bone marrow
• Typically 2 to 3 weeks after immunisation with a T cell–dependent antigen, the bone marrow becomes a major site of antibody production.
- Plasma cells in the bone marrow may continue to secrete antibodies for months or even years after the antigen is no longer present.
- These antibodies can provide immediate protection if the antigen is encountered later.
- ~half the antibodies in the blood of a healthy adult is produced by long-lived plasma cells and is
specific for antigens that were encountered in
the past.
- Secreted antibodies enter the circulation and mucosal secretions, but mature plasma cells do not recirculate.
• B cells respond to antigen by: - dividing
- secreting antibodies
‣ IgM is first (explains why IgM appears early during an infection)
- migrating
‣ towards the T cell zones to receive T cell help to class switch
‣ into Germinal Centres to undergo affinity maturation and memory development
• CD40-CD40L cross talk is essential for:
- class switching, affinity maturation & memory development
• Without T cell help:
- no class switching occurs
- this explains why IgM is the isotype produced against
the ABO carbohydrate antigens
- no memory develops
ok
• How do B cell-derived antibodies eliminate pathogens?
- The importance of cell surface Fc-receptors
‣ neutralisation, opsonisation
‣ antibody-dependent cell-mediated cytotoxicity (ADCC) ‣ complement activation
What makes IgA special?
What makes IgA special?
- role in mucosal immunity
• The role of IgG in passive
and neonatal immunity
ok
• How IgE works
- to eliminate parasites and helminths
- to mediate allergy (or atopic reactions)
‣ more in our senior immunology electives
Antibodies function to eliminate extracellular microbes and toxins
Antibodies prevent infections by blocking microbes and toxins binding to host cells:
- antibodies that block a virus from entering a host cell prevent virus replication and hence viral infection
- blocking a bacterial exotoxin prevents host-cell damage
• Provoking antibody production is the basis of most currently used (and
successful) vaccines
• Antibodies cannot enter host cells, therefore intracellular microbes resist antibody elimination & require cell-mediated immunity
The importance of the Fc (fragment, crystalline) region
• Antibodies use the variable part to recognise antigen
• Fc describes the corresponding region of an intact immunoglobulin molecule that mediates effector functions
- Fc fragments are so named because they tend to crystallise out of solution.
• Fc (or constant) regions are what determines the effector function of the antibody
- Different antibody isotypes perform different functions
- site of antigen exposure can also affect switch recombination
‣ e.g. IgA in mucosal sites (more later)
• Isotype switching broadens the functional capabilities of the
humoral immune response
• Different cells have different Fc-receptors that bind specific heavy chains
- macrophages express Fcγ receptors that bind IgG
- eosinophils & mast cells express high affinity Fcε receptors for binding IgE
Classes of antibodies that neutralise
• IgG
- all subclasses
- high affinity antibodies provide efficient neutralisation
• IgA
- particularly at mucosal surfaces where a specialised secretion mechanism promotes efficient neutralisation of organisms prior to penetrating these host surfaces
antibody effector mechanism 1 neutralization
the formation of antgen antibody in vivo required for the ability of the cell to see antigen, thus elimination
Antibody effector Mechanism 2. Opsonisation
• Antibodies of IgG isotype coat (opsonise) antigenic particles (microbes, cells) • antigen-antibody complexes promote their phagocytosis by binding high
affinity Fcγ receptors (FcγR1) on phagocytes (macrophages & neutrophils)
Receptors for IgG-antigen complexes (Fcγ Receptors)
• Many different types of FcγR exist
- each have different binding affinities for
IgG-antigen complexes
- FcγR bind Fc of IgG (no other antibody)
- Fcγ receptors only bind IgG when it’s attached to a microbe or cell
‣ not unattached free-floating IgG • As you know…
- Phagocytes also express pattern recognition receptors (PRR) that bind microbes and allow their ingestion without antibodies
‣ Antibody-mediated ingestion is much more efficient
Other important Fc-receptors: FcεR
• Antibody-Dependent Cell-mediated Cytotoxicity (ADCC) 2
- Most helminth worms are too large to be ingested by phagocytic cells
- Helminths induce IgE response that coats (opsonises) worms
• Fcε receptor on eosinophils binds IgE-coated worms
- induces eosinophil activation
- release of cytoplasmic granules
‣ contain proteins that damage integument & kill the parasites
Do you Recycle?
• IgG is taken up inside endocytic vesicles of endothelial cells
• In endosomes, IgG binds FcRn - dependent on a low pH environment
• binding of IgG to FcRN protects the IgG from degradation
• The FcRn-IgG complexes are “recycled” back to the cell surface
- there they are exposed to the neutral pH of the blood, which releases the bound antibody back into the circulation.
Why you should recycle
FcRn-mediated protection of IgG from intracellular
catabolism is why IgG has a half-life of ~3 weeks
- much longer than that of other antibody classes and most other plasma proteins.
• Partly explains why the concentration of IgG is highest in blood
• Switching to IgG prolongs the duration an antibody lasts in the blood
- increases the functional activity of the antibody
Mucosal immunity: a property of IgA
• Dimeric IgA produced by plasma cells in the lamina propria binds to an IgA-Fc receptor, called the poly-Ig receptor
- The poly-Ig receptor also recognises IgM
• The IgA-Receptor complex is endocytosed into the epithelial cell
- this is how IgA is “transported” across the mucosal epithelium
• On the luminal surface, the IgA with a portion of the bound receptor is released.
- this ‘secretory piece’ protects IgA from proteolytic cleavage in the lumen
• IgA binds microbes in lumen & prevents binding & entry into host
- eg. oral poliovirus vaccine induces IgA & prevents poliovirus infection
take home messenge
the constant region of antibody determine what type of antibody it is. the constant region determine the effective function of antibody. different antibody or different classes of antibody form different function (wither to neutralise, opsonise or being involve to Fc receptors, FcY, neotate Fc receptors or FcE receptors)
-IgG and IgA good for neutralisation
- IgG1 best for opsonization
Ig
The Complement System
• What is the complement system
and how is it activated?
• What are the 3 pathways
of complement activation?
- alternative, classical, and lectin
• What are various components of complement?
• What are the consequences and products of
complement activation?
• Why is complement so important?
• How is complement activation regulated?
• What diseases are mediated by defects in
complement?
What is the complement system?
• A collection of circulating and cell membrane proteins that are important in host defence against microbes and in antibody-mediated tissue injury
• Complement refers to the ability of these proteins to assist, or “complement”, the antimicrobial activity of antibodies
• May be activated by microbes in the
absence of antibody
- innate immune response to infection,
• May be activated by antibodies
attached to microbes
- part of adaptive immunity
How is the complement system activated?
Involves sequential proteolytic cleavage of complement proteins
- leads to the generation of effector molecules that participate in eliminating microbes
• This cascade of complement protein activation is capable of achieving tremendous amplification
- molecules produced early in the cascade may generate a large number of effector molecules.
• Activated complement proteins become covalently attached to the cell surfaces where the activation occurs
- ensures that activation is limited to the correct sites.
- The complement system is tightly regulated by molecules present on normal host cell
- this regulation prevents uncontrolled and potentially harmful complement activation.
• The triggers for complement activation may be different, but…
- All 3 Pathways rely on C3 ‣ the most abundant
complement protein in blood
• The molecules involved may be different, but…
- All 3 pathways will proteolytically convert C3 into its active forms
‣ C3b ‣ C3a
ok
The Alternative Complement Pathway
• Microbe-bound C3b becomes a substrate for the binding of another serum protein: factor B
• Factor B is broken down by a plasma protease called Factor D to generate the Bb fragment
- The Bb fragment remains attached to the C3b and is stabilised by the serum protein properdin
• The C3b-Bb complex enzymatically breaks down more C3, functioning as the “alternative pathway C3 convertase.”
• many more C3b and C3bBb molecules are produced and become attached to the microbe.
• Some of the C3bBb molecules bind additional C3b, and the C3b-Bb-C3b complex functions as a C5 convertase
• break down the complement protein C5 and initiate the late steps of complement activation.
Activating the classical complement pathway
• Activation of the classical complement pathway requires antibody bound to antigens
• It begins when the complement protein complex (C1) binds to antigen-antibody complexes
• C1 is made up of C1q + a tetramer composed of two C1r and two C1s molecules (serine proteases)
• The C1 complex must bind to two or more Fc portions to initiate the classical complement cascade
- Only antibodies bound to antigens, and not free circulating antibodies, can initiate classical pathway activation
The Classical Complement Cascade
• Triggered when IgM or subclasses of IgG (IgG1 & IgG3 in humans) bind antigen (not free antibody)
• Antigen-antibody complexes that activate the classical pathway may be (1) soluble, (2) fixed on the surface of cells, or (3) deposited on extracellular matrices
• Activated C1s cleaves the next protein in the cascade, C4 (the 2nd most abundant complement protein in blood generating C4b and C4a
• C4b contains an internal thioester bond, similar to C3b which covalently attaches to the adjacent surface of the cell or antigen complex to which the antibody is bound.
- Attachment of C4b ensures that classical pathway
The Classical Complement Cascade
• C2 complexes with C4b and is cleaved by a nearby C1s molecule to generate a soluble C2b fragment and C2a
• C2a forms a complex with C4b on the cell surface. This C4b-2a complex is the classical pathway C3 convertase
- binds to & proteolytically cleaves C3 into C3b + C3a
• many more C3b molecules are produced and
attach to the microbe (called opsonisation)
• Some of the C4b2a molecules bind C3b
• the resulting C4b-2a-C3b complex functions as a classical pathway C5 convertase
The Lectin pathway of complement activation
- Triggered by a plasma protein called mannose-binding lectin (MBL)
- recognises terminal mannose residues on microbial glycoproteins and glycolipids
- has a hexameric structure similar to the C1q component of the classical complement system
- MASP1 (mannose-associated serine protease 1) and MASP2, have similar functions to C1r and C1s,
- MBL binding to microbes initiates downstream proteolytic steps identical to the classical pathway
Late Steps of Complement Activation
• initiated by the binding of C5 to the C5 convertase generated by the alternative, classical, or lectin pathway
• the subsequent proteolysis of C5, generates C5b and C5a
- the C5b fragment remains bound to the complement proteins deposited on the cell surface
• C5b transiently maintains a conformation capable of binding the next proteins in the cascade, C6 and C7
• The C7 component of the resulting C5b,6,7 complex is hydrophobic, and it inserts into the lipid bilayer of cell membranes,
• here it becomes a high-affinity receptor for the C8 molecule
Late Steps of Complement Activation
• This stably inserted C5b,6,7,8 complex (C5b-8) has a limited ability to lyse cells.
• C9 is a serum protein that polymerizes at the site of the bound C5b-8 to form the Membrane Attack Complex (MAC) pores in
- Pores in plasma membranes that are about 100 Å in diameter,
- they form channels that allow free movement of water and ions.
‣ The entry of water results in osmotic swelling and rupture of the cells on whose surface the MAC is deposited
C3
the most abundant complement protein. plays a central role in all 3 pathways
C3 convertase in all 3 pathway, to convert C3 to active form when microbe present (activate C3a and C3b)
yes
C3a and C3b is common to all 3 pathways
Yes
they all have a C5 convertase 0> convert C5 into active form C5a and C5b
yes
does soluble IgM (planar form) cause complement activation
NO
does antigen bound IgM (stable form) cause complement activation
yes
does soluble IgG (Fc portions not adjacent) cause complement activation
NO
does antigen bound IgG cause complement activation
YES
