Lectures 4-7: Cell biology of the specific immune system Flashcards
How do B cells develop?
From haematopoietic stem cells in bone marrow that express PAX5 transcription factor
Re-arrangement and expression of Ig genes
Removal of self-reactive cells
How do B cells react to antigens?
B-cell precursor rearranges Ig genes
Immature B cells bound to self cell-surface antigen is removed from the repertoire using negative selection
Then bind to foreign antigens activating B cells giving rise to plasma and memory cells
How are pre-B cells produced?
H chain genes rearrange then move to cell surface with Ig alpha and beta and express with surrogate light chain
Produces pre-B cell receptor
Which proteins determine good heavy chain binding for the surrogate light chain?
V preV and λ5
How are immature B cells produced?
Light chains rearrange and displace proteins to produce IgM BCR
What is the function of a pre-BCR?
Delivers a signal to pre-B cell that H chain looks functional
No Ag required yet
What signals are emitted from pre-BCR?
Turns off RAG-1&2
5-6 rounds of cell division
Surrogate light chain expression stops
RAG-1&2 turned on again
L chain rearrangement starts
What is the gene arrangement on the H-chain in a B-cell?
D-J rearrangement on both chromosomes
↓
V-DJ rearrangement on first chromosome
↓ (-)
V-DJ rearrangement on second chromosome
↓ (-)
Cell loss
What is the gene arrangement on the L-chain in a B-cell?
Rearrange Kappa gene on first chromosome
↓(-)
Rearrange Kappa gene on second chromosome
↓(-)
Rearrange Lambda gene on first chromosome
↓(-)
Rearrange Lambda gene on second chromosome
↓(-)
Cell loss
Which part of B-cell development has the largest chance of survival?
Pre-B cell
What are factors of Ig gene re-arrangement?
Error prone
If cell fails to productively re-arrange both H and L genes, it dies
Why does the light Kappa chain have so many chances at rearrangement?
Because there are 5 J Kappa genes on each chromosome
What are the 2 different mechanisms immature B cells do to multivalent self-antigens?
Clonal deletion - cell dies by apoptosis
Receptor editing - further light chain gene rearrangements of variable regions
When does the immature B cell become anergic?
When it binds soluble self antigen
Anergic = becomes unresponsive
What is the process of T cell development?
Originate from bone marrow
Re-arrange receptor genes in thymus
Express pre-T receptor
Eliminate self-reactive T cells via negative selection
Undergo development/selection in thymus
T cells expressing αβ TCR must bind with self MHC expressed in thymus
How are T cells made and activated?
Precursors use Notch signalling to initiate T cell receptor gene rearrangements
Immature T cells recognising self MHC receive signals for survival ones that interact strongly with self antigen are removed from the repertoire
Mature T cells encounter foreign antigens in the peripheral lymphoid organs and are activated
Activated T cells proliferate and eliminate infection
What is the thymus?
Bi-lobed organ in anterior mediastinum
Lobe divided into many lobules
Lobules have outer cortex and inner medulla
Cells - lymphoid cells, epithelial cells, macrophage and dendritic cells
How does the T cell mature in the thymus?
Pro-thermocytes enter cortex via blood vessels from bone marrow
Inside, TCR genes re-arranged (TCRβ first, expressed along with pre-T cell receptor, proliferation then re-arrange TCRα genes)
Express TCR together with CD3 and both CD4 and 8
How is the full TCR complex assembled?
Requires CD3 complex (δ,ε,γ)
CD3 transmits signal to T cell
What are the differences between γδ TCR in comparison to αβ?
Similar structure
γδ do not express CD4 or CD8
γδ have less diversity
γδ expressed on separate T cell population (1-5% in circulation , epithelial cells + mucosal surfaces)
Recognise different antigens
Depends on which genes are rearranged successfully first
What are the disadvantages of cells expressing randomly rearranged αβ?
Recognise self MHC + peptide from foreign antigen (immunity)
Recognise self MHC + peptide from self antigen (autoimmunity)
Not able to recognise self-MHC (useless)
What happens in positive selection of T cells?
Recognise self MHC
Double positive T cells recognise MHC on cortical epithelial cells in thymus - apoptosis if not recognised
Rearrangement gives random TCR repertoire
What happens in negative selection of T cells?
Recognise self MHC on thymus dendritic cells/macrophages with high affinity
TCR binding to MHC/self-peptide with high affinity causes T cell to die (apoptosis)
When does positive and negative selection of T cells occur?
Sequentially, at different regions of the thymus
What is the paradox of positive and negative selection?
T cells positively and negatively selected on self MHC + self peptide
T cells positively selected aren’t always subsequently eliminated by negative selection
What is the basis for selection of TCR affinity for p/MHC?
All T cells recognising self MHC are positively selected
Highest affinity TCR are negatively selected
GOAL: population of T cells with low affinity for self peptide + self MHC
What percentage of T cells survive thymus selection?
<5%
Express TCR capable of binding self MHC
Depleted of self-reactive cells
(CD8+ T cells MHC class I)
(CD4+ T cells MHC class II)
What is T cell mediated immunity?
Naive T cells recirculate via blood/lymphatics through secondary lymphoid tissue
Contact with specific Ag and APC -> clonal proliferation and differentiation
Naive -> effector/memory T cells
CD8+ kill infected cells
CD4+ secrete cytokines
What are naive T cells?
They haven’t yet recognised foreign antigen
What do the lymphoid tissues contain?
T cells that recognise antigen/MHC on antigen presenting cells
Array of APC, some specialised, trap and present antigens
Lymph nodes
Spleen
Where do T cells go one activated?
They leave the lymphoid tissues and migrate to the sites of infection
How do T cells get to where they need to be?
They enter the lymph node from blood via high endothelial venues (HEV)
Move to T cell area rich in dendritic cells and macrophages
APC presents antigen and delivers other activation signals (cytokines)
What happens to T cells that are not activated?
They leave the lymph via cortical sinuses into the lymphatics and re-enter the circulation (re-used)
What are the signals required for T cells to get where they need to be?
Molecules are expressed on the surface of T cells (chemokine receptors) bind ligands (chemokine) expressed/released by other cells
Once close to other cells different molecular sets of cell adhesion molecules mediate cell-cell interactions
What are examples of CAMs mediating cell-cell interactions?
Naive T cells with high endothelial venules (HEV)
T cell with APC
Effector T cell and target cell
What are CAMs?
Cell adhesion molecules
What happens once T cells make contact with the APC?
Contact using CAMs
TCR scans APC peptide-MHC complexes
(no recognition -> disengages)
(recognition -> signal from TCR complex)
- Increased affinity of CAM interactions
- T cell divides
- Progeny differentiate to effector cells, exit lymph
What is LFA-1?
Leukocyte function associated antigen
What is ICAM-1?
Intercellular adhesion molecule
How do T cells initially become activated?
They bind to APC through low affinity LFA-1:ICAM-1 interactions then this binding leads to TCR signalling LFA-1
Conformational change in LFA-1 increases affinity and prolongs cell-cell contact
How are T cells signalled?
They receive a signal from TCR contacting MHC/peptide on APC
Involving CD3 chain
How many signals are required for T cell activation?
3
What is co-stimulation in T cells?
APC express co-stimulatory molecules that bind CD28 expressed by naive T cells and deliver signal 2
APC release cytokines which bind cytokine receptors now up-regulated on naive T cells with deliver signal 3
What are the B7.1/2 molecules?
They are the co-stimulatory molecules required to release the second signal
What is CD28?
It is a molecule required for the release of the second signal to activate T cells
What examples of cytokines are required for the third signal to activate T cells?
IL-6, IL-12, TGF-β and IL-4
Why is the co-stimulatory signal and cytokine signal not required when the effector T cell releases the effect?
Because the signals have already changed the naive T cell to the effector T cells which allows it to have an effect in the secondary lymph tissue
What are the ICOS and CTLA-4 molecules?
ICOS is related to CD28 binding ICOSL (ligand) on the APC to induce cytokine secretion by T cells
CTLA-4 is highly related to CD28, shows stronger binding to B7.1/2 than CD28 (competition)
What happens once CTLA-4 has bound to B7.1/2 on APC?
A negative signal is delivered which deactivates the T cell
Why does the T cell express the ICOS and CTLA-4 molecules?
After 3 signals released and T cells are activated they proliferate and express these molecules
What is the importance of CTLA-4?
Mutations within the molecule are associated with several autoimmune diseases as the T cell cannot become inactivated
Cancer patients are often treated with anti-CTLA-4 which can enhance the immune response to the tumour (melanoma and renal carcinoma)
What is the significance of the co-stimulatory molecules?
B7.1/2 are the most important and contain functional differences
The expression varies - constitutive on mature dendritic cells, inducible on macrophages and B cells
How are APCs activated?
They express receptors for microbial molecules (PRR)
Binding of pathogen-associated molecules activates the APC (danger signal)
Leads to up regulation of MHC and co-stimulatory molecules
Ensuring signal 2 released to activate T-cell mediated response only occurs during infection
What is signal 3?
It is the release of cytokines to dictate the differentiation of activated CD4 cells into different sub-sets of effector cells
What are the different types of cytokines that can be signalled?
TGF-β, IL-6, TGF-β&IL-6, IL-12&IFN-γ, IL-4
What does TGF-β cytokine lead to?
A transcription factor FoxP3 produces TGF-β and IL-10 which leads to Treg cells (regulation)
What does IL-6 cytokine lead to?
A transcription factor Bcl6 expressing IL-21 and ICOS which leads to Tfh cells
What does TGF-β and IL-6 lead to?
A transcription factor RORγT expressing IL-6 and IL-17 which leads to Th17 cells
What does IL-12 and IFN-γ lead to?
A transcription factor T-bet expressing IL-2 and IFN-γ leading to Th1 cells
What does IL-4 lead to?
A transcription factor GATA3 expressing IL-4 and IL-5 leading to Th2 cells
What are the different types of T cells?
Treg cells, Tfh cells, Th17 cells, Th1 cells, Th2 cells
What are the different types of APCs?
Dendritic cells (only present Ags) crucial for activation of naive T cells
Macrophages and B cells that present antigen to receive help from effector cells
What are the different types of DC?
Myeloid conventional DC (DC2,3)
Plasmacytoid DC (pDC, DC6)
What is myeloid DC?
DC2,3 which have potent APC and are involved in activation of naive T cells
What is plasmacytoid DC?
pDC, DC6 have important viral infection and secrete several type 1 α and β interferons and also express TLR 7 and 9
Why are myeloid DCs significant?
It is a key APC that initiates T cell responses
Bone-marrow derived
Immature form found in epithelia
Macropinocytic and phagocytic
Do not express co-stimulatory molecules till activated
Induced to mature and migrate to lymph node following danger signal activation
How do dendritic cells act as efficient activators of naive T cells?
They are found in T cell areas of lymphoid tissue
DC MHC I and II loaded with peptides from pathogens from peripheral tissues
Levels of co-stimulatory molecules will be very high
Express high levels of adhesion molecules
How do immature dendritic cells go to mature dendritic cells?
In peripheral tissues encounter pathogens and are activated by PAMPs (Pathogen-associated molecular patterns)
TLR signalling induces CCR7 and enhances processing of pathogen-derived antigens
CCR7 directs migration into lymphoid tides and augments expression of co-stimulatory molecules and MHC
Mature dendritic cell in T-cell zone primes naive T cells
What is cross presentation?
Specialised DC (DC1) take up and process exogenous Ag and present it via MHC I
Allows these DC to activate CD8+ T cells, CD8 effector cells can kill infected cells that are not APCs so they aren’t expressing co-stim
What is the significance of macrophages?
Function as scavengers of pathogens also important APC for extracellular pathogens
Highly phagocytic
Express MHC II and B7 increasing following T cell help
Resident in many tissues at peripheral sites as well as in lymphoid tissue and other tissues around the body
One activated by T cells secrete many inflammatory cytokines
What is the significance of using B cells as APCs?
Vary poor at phagocytosis (don’t engulf MOs)
Internalise soluble antigens for processing and presentation by BCR
Antigen binding to BCR up-regulates B7 (provide signal 2 to T cell)
Similar to DC, found in lymph nodes presenting to T cells
Role as APC may be more important after initiation of immune response by APC
Use BCR to physically extract antigen from other cells
How is IL-2 important for T cell survival?
It is an important autocrine T cell growth factor (naive T cells have low affinity)
Activated T cells (after signal 1+2+3) have high affinity IL-2R and secrete IL-2
IL-2 binding to IL-2R on activated T cells leads to lots of T cell proliferation
What is the significance of IL-2 in drug development?
Target of immunosuppressive drugs (stopping T cell proliferation)
What are the different T cells once activated?
Effector T cells
CD8+ cytotoxic (kill MHC I/peptide complexes)
CD4+ by secreting cytokines (effects on other T cells)
What is the significance of effector T cells?
Display effector function when TCR engaged
Dont require co-stimulation
Change expression of adhesion molecules
No longer enter lymph nodes
Enter tissues activated by endothelia (sites of infection and inflammation)
Where do B cells move once they have survived in the bone marrow?
Into the blood and lymphatics
What are the different functions B cells can differentiate into?
Neutralisation
Opsonisation
Complement activation
How are B cells activated?
Using several signals
Naive B cells express membrane Ig/BCR
Encounter non-self antigen in secondary lymphoid tissue
Binding of antigen to BCR provides signal 1 to B cell
What is the molecular basis of BCR signal 1?
BCR-associated polypeptides involved (Igα and Igβ)
Cross-linking BCR activates intracellular kinases
What are ITAMs?
Immunoreceptor tyrosine-based activation motif which are involved in signalling (Igα and Igβ)
How is signal 1 increased/enhanced?
When antigen activates complement cascade
Lots of C3b
complement receptor 2 (CR2) on B cell surface (CD21)
CR2/CD19/CD81 form BCR co-receptor complex
Augments the signal
What are the 2 different ways B cells receive signal 2?
Thymus-independent antigen (TI)
Provided by:
antigen itself
extensive cross linking of BCR
Thymus -dependent antigen (TD)
Provided by CD4+ T cells
How does TI-1 antigen signalling work?
IgM production with no T cell involvement
TI-1 antigen: binds BCR and other receptors on all B cells providing signal 2, high concentrations these antigens act as polyclonal activators for B cells
The 2 signals lead to B cell activation proliferation and antibody secretion
How does TI-2 antigen signalling work?
Contain repeated epitopes
- often polysaccharides
- important in some bacterial infections
Cross-link many BCR molecules on the same B cell surface
Take longer to induce B cell activation (more Ag required)
Antibody responses don’t develop until >5 years old in humans
How does TD antigen signalling work?
Antibodies require the presence of CD4+ T cells
Responses are much better compared to TI antigen response
T cells activated by peptide-MHC on APC
BCR binds antigen - signal 1
B cell internalises antigen, processes and presents antigen to CD4+ cells - signal 2 (via CD40/CD40 ligand interaction)
Cytokines are then secreted by T cell
Which classes of antibody can be produced by TD antigen?
ALL!
What is the process of TD antigens recognising a virus?
B cell binds virus through viral protein coat
Virus particle is internalised and degraded (using lower pH)
Peptides from internal proteins of the virus are presented to the T cell (CD154(CD40L)-CD40) which activates the B cell by producing signal 2
Activated B cell produces antibody against viral coat protein
How are epitopes recognised by antibody and T cell physically linked?
From different parts of the same molecule
OR
Different molecules of the complex
How can a vaccine against pathogens be made more efficient?
By converting a TI antigen signalling to TD antigen signalling
What types of vaccines are made when the TI is switched to TD antigen signalling?
Conjugate vaccines
What is an example of a conjugate vaccine?
Haemophilus influenza type B
Protective response requires antibodies to capsular polysaccharide
Coupling this to a protein such as tents toxoid converts it to a TD antigen
So young children can be immunised and protected
(other e.g. MenC(meningitis) and pneumococcal conjugate vaccine)
Which responses are important for good antibody responses?
B-CD4+ T cell interactions
Why are B-CD4+ T cell interactions important for good antibody response?
B cells enter lymph from blood
B cell comes into contact with its specific antigen which becomes activated
If antigen is TD then B cell presents peptide from antigen to CD4+ Th cells at the boundary of T/B areas within the lymph forming B/T cell conjugates
What happens once the T cell expresses CD40 ligand and secretes cytokines?
B cell receives signal 2 from T cell via CD40/CD40L binding and via cytokine from T cells binding receptors -> B cell proliferation
CD40 signal also induces activation induced deaminase (AID) which is required for class-switching and somatic hypermutation (SHM)
How are B cells activated using a TD antigen?
Conjugates of B lymphoblasts and T cells move to primary follicles
Germinal centres (GC) are formed within a B cell follicle in secondary lymphoid tissues
B cells divide rapidly becoming centroblasts and undergo:
- SMH of Ig genes
- isotope switching
Differentiate into non-dividing centrocytes (smaller)
What happens to B cells once they are in the Germinal Centres(GC)?
Either differentiate into plasma cells
- secrete various isotypes
- high affinity antibody, somatically mutated
Or form long-lived memory cells
- recirculate
Or die within lymphoid tissue
- if BCR no longer binds to antigen
What is somatic hypermutation?
It introduces point mutations into V regions of the Ig
Approx one mutation/V region/cell division (10^6 x normal DNA mutation rate)
Enzymes primarily involved includes activation induced deaminase (AID) and DNA repair genes
What other cells are located within GCs?
Follicular dendritic cells (FDCs)
What are follicular dendritic cells(FDCs)?
Not bone-marrow derived dendritic APCs
Cells in primary follicle that capture intact antigen for centrocytes brindles via BCR
Why are follicular dendritic cells important in B cell maturation?
Centrocytes that have undergone SMH express mutated BCR on surface
- centrocytes compete with each other for antigen on FDC and signals from Tfh cell
- if mutated BCR binds antigen on FDC better than the un-mutated, presents more efficiently and receive CD40 signal from Tfh cell (failure = apoptosis/re-cycle to dark zone)
- centrocytes with higher affinity for BCR survive and differentiate into plasma cells
Which B cell interactions are good for antibody responses?
Follicular T helper cells (Tfh)
What is the basis for affinity maturation?
Mutated BCR with low affinity for antigen:
Germinal centre B cell with mutated low-affinity surface Ig
BCR not cross-linked and B cell cannot present antigen to T cell -> apoptosis
Mutated BCR with high affinity for antigen:
Germinal centre B cell with mutated high affinity surface Ig
T-cell help and BCR cross-linking sustain B cell proliferation and maturation -> memory B cell/ plasma cell
What is the role of CD40?
CD40 single using CD40L expressed on Tfh
- protects centrocytes from apoptosis
Induced isotope switching
- different cytokines induce different isotopes to be produced
How is isotope switching controlled?
By different antigens
- polysaccharides: IgM (TI)
- proteins: IgG1 & IgG3 or IgG4 (TD)
Antigen at mucosal surfaces induce IgA
Some antigens elicit IgE
Role of cytokines(Tfh cell):
- e.g. IL-4 important for IgE switch
Why is immunological tolerance needed?
To allow random generation of repertoire of BCR and TCR
Many self-reactive specificities will be produced
No tolerance means auto reactivity would lead to serious pathology
What is T cell tolerance?
Random TCR rearrangements
Leads to T cells expressing TCR that:
-fails to recognise self-MHC (dies by neglect
- recognises self-MHC + peptide generated from antigen present in thymus (potential dangerous)
- recognise self-MHC + any other peptide not present in thymus (potentially useful)
2&3 expanded by positive selection
2 eliminated by negative selection
What is AIRE?
An autoimmune regulator protein
What is the use of AIRE?
Transcription factor
Role in tolerance induction
Allows expression of many tissue-specific antigens(TSA)
Negative selection/deletion of T cells that recognise these antigens
What happens to patients with AIRE deficiency?
They have a major autoimmune syndrome
What is B cell tolerance?
Random Ig gene rearrangement many B cells could express self-reactive BCR
Auto-reactive B cells are negatively selected/deleted
B cells get second chance to re-arrange self-reactive BCR
What is anergy in B cells?
When immature B cells bind to self antigen
What is tolerance via anergy?
Lymphocytes recognise self antigen can become unresponsive
Immature B cells: BCR encounters antigen in bone marrow that is not multivalent, down regulate BCR and leave bone marrow as unresponsive
Many potentially self-reactive B cells but anergia B cells leave bone marrow
How do T cells become anergic?
When T cell only receives signal 1 so no co-stimulatory signal is delivered so T cells have not recognised a nonbacterial antigen
What are other mechanisms of tolerance?
Immunological ignorance: antigens not presented at sufficient levels to activate T cells
Privileged sites: antigens sequestered from immune system
B cell responses that are T cells dependent: if antigen-specific T cells are absent/tolerant no help fro B cell is available = no antibody response
What are regulatory T cells?
They are another CD4+ T cell subset surpassing immune responses
Crucial for autoimmune responses
Arise in thymus from T cells with high affinity TCR for self antigens
Express Fox P3 transcription factor
Natural and induced regulatory T cells
What happens during regulatory T cell deficiency?
Leads to severe autoimmune syndrome IPEX (immune dsyregulation, polyendocrinopathy, enteropathy, X-linked syndrome) very serious autoimmune condition
What are regulatory B cells?
Secrete IL-10 crucial in preventing autoimmunity
Why is regulation of the immune response required?
To ensure responses only continue for as long as they are needed
Minimise collateral damage
Ensure responses are qualitatively appropriate
What are CD4+ Th1 cells required for?
Activation of macrophages, NK cells, cytotoxic T cells
What are CD4+ Th2 cells required for?
Promoting responses mediated by eosinophils and mast cells; role in antibody responses especially IgE
What are CD4+ Th17 cells required for?
Promote responses against fungi
Secrete IL-17
Recruit neutrophils early
Implicated in autoimmune disease
Evolutionary oldest form of acquired immunity
What are CD4+ Treg/Breg cells required for?
Suppress unwanted responses
Mix of CD4+CD25+
CD8+ T cells can have Treg activity
Arise in thymus or circulating T cells in peripheral tissues
What are CD4+ Tfh cells required for?
Specialised Th found in germinal centres to help B cells (produce other T cell subsets)
How do Th1 cells help macrophage function?
They activate macrophages via secretion of cytokines
Express CD40L which binds to CD40 on macrophage
Can kill chronically-infected macrophages
Fas ligand-Fas induce apoptosis
released bacteria destroyed by healthy macrophages
->other cells can then kill the pathogens
What are Treg cells mode of action?
Secretion of suppressive cytokines
TGF-β and IL-10
Can also involve cell-cell contact
IL-10 inhibits APC function
What are the key cytokines involved in naive T cell activation by APC?
IL-12 and IFN-γ play a key role in induction of Th1 responses
IL-4 important for induction of Th2 responses
What is the importance of polarised responses?
Ensures correct responses for different types of pathogens
Can go wrong may lead to allergy
Control of auto-reactivity/pregnancy
What are follicular T helper cells?
Defined as CD4+ Th subset predominantly in B cell follicles of the lymph node
Specialised to provide help to B cell
Secrete either Th1 or Th2 type cytokines
Identified with specific markers that differ from other subsets of CD4 Th cells
