Week 8

Question 1

Effector T cell populations

Answer 1

CD8+ T cells: cytotoxic T lymphocytes
CD4+ T cells: ‘helper T cells’
Th1, Th2, Th17, Tfh, TReg
Functional role in immunity and autoimmunity
Mechanism of action including cytokine profiles and effector functions

Question 2

Generating an “army” of T cells

Answer 2

One T cell in the “army” is not enough to eliminate an infection or control cancer
First requirement is to replicate the cells that constitute the “army”
Second requirement is to “arm” the “Army”- differentiate

Question 3

Naive T cells must proliferate and differentiate

Answer 3

T cells having matured in the thymus enter the circulation in a largely undifferentiated state- they have little effector function
- when they leave they are either CD4, CD8 or T regulatory cells
At this stage they’re known as naive T cells (not encountered pathogen)
During this time the frequency of T cells for any given peptide/MHC complex is very low
-proliferation of antigen-specific cells
-differentiation to provide effector function
To initiate proliferation and differentiation we need to present the antigen via the MHC complex located on the surface of an APC to the T cell
In this case the APC is a dendritic cell
Naive T cells activated by antigen presentation in secondary lymphoid organs like lymph nodes

Question 4

Antigen presenting cells

Answer 4

Dendritic cells
Macrophages
B cells
There are many similarities between them: they’re efficient at taking up antigens and processing it, they then upregulate the level of MHC expression ( B cell already high) and also upregulate co-stimulation molecule activity
Differences:
-dendritic cell: found throughout the body, result in activation of naive T cells
-macrophages: found in lymphoid and connective tissue and in body cavities, result in activation of macrophages
-B cells- found in lymphoid tissue and peripheral blood, result in delivery of help to B cells

Question 5

Migration of dendritic cells

Answer 5

Tissue dendritic cells migrate to the draining lymph nodes when activated
Langerhans cells: dendritic cells found in epidermis of skin in a resting state, they are activated by presence of pathogen of the products of inflammation due to presence of pathogen, these dendritic cells will alter molecules on their surface which allows them to migrate into draining lymph nodes, this is where they activate T cells

Question 6

Molecules on dendritic cells for migration

Answer 6

An immature dendritic cell has a number of receptors on its surface e.g patterns recognition receptors
It also has chemokines receptors
Chemokines receptors allows the cells to migrate from one place to another via chemokines
Chemokine receptors 1,2,5,6 are inflammatory chemokines receptors, so inflammatory chemokines made in tissue allow dendritic cell to migrate to site of inflammation/infection
However once the dendritic cell has encountered a pathogen it needs to migrate into draining lymph node
To do this once the dendritic cell is activated by PAMPS due to the presence of a pathogen, it processes pathogen derived antigen, down regulates expression of inflammatory chemokine receptors and then switches on another chemokine receptor CCR7 which allows it migrate to lymphoid organs
Once the dendritic cells are in the lymph node, they need to alter the way they can interact with T cells:
-any antigen that the dendritic cell has taken up needs to be processed and attached to MHC to be presented on cell surface
-it also needs to further increase expression of MHC and costimulatory molecules B7

Question 7

Naive T cell activation

Answer 7

Naive T cells are present in the T cell zone of secondary lymphoid organs such as lymph nodes
They migrate around and if they come into contact with a dendritic cell that presents the peptide MHC complex to which its T cell receptor has high affinity for the T cell is activated
It then proliferates to make more copies of itself then some of those T cells exit lymph node to populate periphery

Question 8

Molecular interactions between dendritic cells and T cells

Answer 8

The T cell receptor binds to the peptide MHC complex
CD4 T cell binds to MHC class 2
T cell receptor binds to combination of peptide and MHC
This produces signal one- primary signal for activation
However for a naive T cell signal one is not enough
Additionally B7.1 and B7.2 (costimulatory molecules) will bind to CD28 on the T cell- this produces signal 2
-CD28 on T cell is a co-stimulatory molecule that induces a very strong and prolonged activation signal that aids cell survival
Signal 3- for T cell differentiation. T cell have receptors for many molecules in the environment eg cytokines, the binding of specific molecules drives differentiation cells into specific effector T cells. After the body has dealt with the invading pathogen, it begins reduce effector T cell number by apoptosis, this helps reduce swelling of lymph nodes. However some of the effector T cells are retained as memory cells to hep with the returning of same pathogen

Question 9

CD8+ cytotoxic T cells

Answer 9

Two major killing mechanisms:
-granules contain perforin and granzymes
-Fas ligand (FasL) on T cell binds to Fas on the target cell triggering apoptosis
Kills virus infected cells
Pathogens targeted: viruses, some intracellular bacteria

Question 10

CD4 Th1 cells

Answer 10

Activate infected macrophages, provide help to B cells for antibody production
Pathogens targeted: microbes that persist in macrophage vesicles, extracellular bacteria
Differentiation:
-differentiate in the presence of IL-12 and secrete IFN gamma when stimulated by antigen
IFN gamma acts as a positive feedback loop to further enhance differentiation to Th1 phenotype
Key functions:
-IFN gammas stimulates infected macrophages to help control infected by increasing:
-MHC expression
-costimulatory molecule expression
-nitric oxide (NO) production
-phagolysosome maturation
-TNF-alpha production
M.tuberculosis is controlled by Th1 cells activating infected macrophages
Granuloma can form with a sheath of T cells surrounding a collection of infected multi-nucleated giant cells (fused macrophages)
Deficiencies in Th1 cells results in reactivation of latent infections or inadequate control of de novo infection

Question 11

CD4+ Th1 cells in pathology

Answer 11

Th1 cells are also found at affected sites in autoimmune and inflammatory disease:
-multiple sclerosis
-autoimmune thyroiditis
-rheumatoid arthritis
-type 1 diabetes
-psoriasis
-Crohn’s disease
-allograft rejection

Question 12

CD4 Th2 cells

Answer 12

Functions: provide help to B cells for antibody production, especially switching to IgE
Pathogens targeted: helminth parasites, large extracellular organisms
Differentiation: to a Th2 phenotype is stimulated by Il-4
Key functions:
-Th2 cells secrete IL-4, IL-5 and IL-13
-these act on effector cells including basophils, eosinophils and mast cells, which promote resistance to large extracellular helminth parasites
-IL-4 from Th2 cells promotes B cell class switching to IgE

Question 13

CD4+Th2 cells in pathology

Answer 13

Th2 cells implicated in allergic and asthmatic disease
IL-4: smooth muscle spasm
IL-13: mucus hypersecretion, goblet cell hyperplasia
IL-5: eosinophilic inflammation
Unwanted effects of Th2-derived cytokines on innate immune effectors in the airway

Question 14

CD4Th17 cells

Answer 14

Enhance neutrophil response, promote barrier integrity (skin, intestine)
Pathogen targeted: fungal infection
Differentiation and function:
-differentiation to a Th17 phenotype is stimulated by IL-1b, IL-6, IL-21, TGF-B and maintained by IL-23
-Th17 express the transcription factor ROR-gammat and IL-17A/F, IL-22, CCL20
-protect against fungal infection and some bacteria
-increases neutrophil recruitment and controls epithelial barrier function
-also implicated in autoimmunity, for example multiple sclerosis, Crohn’s disease, rheumatoid arthritis

Question 15

Tfh cells Follicular helper

Answer 15

B cell help isotype switching, antibody production
Pathogen: all types
Tfh act within the secondary lymphoid compartment to help B cells
Increases Tfh differentiation by IL-21
Express the transcription factor Bcl-6
Express the chemokine receptor CXCR5 to allow migration towards B cell areas
co-express a wide range of Th phenotypes to allow appropriate B cell help

Question 16

CD4 regulatory T cells (various types)

Answer 16

Suppress T cell responses
Derived from 2 major sources
Some thymic derived TReg binding self antigen
Peripheral derived Treg (pTreg/iTreg) generated as part of any peripheral immune response
pTreg differentiate in the presence of TGF-B and retinoic acid
Express the transcription factor FoxP3
Function to restrict immune responses
Treg suppress immune responses through:
-cytokine consumption
-suppressive cytokine release (eg TGF-beta, IL-10, IL-35)
-cytotoxicity
-suppression of antigen presenting cell function-inhibitory signalling, removal of co-stimulatory molecules, cytotoxicity

Question 17

Mature B cells

Answer 17

Mature B cells can become plasma cells and plasma cells secrete antibody
Mature B cells can become memory b cells and memory b cells do not secrete antibody unless antigen is reencountered

Question 18

The adaptive immune system is incredibly specific which creates a problem

Answer 18

Each lymphocyte (T and B cells) only recognises a specific portion (epitope) of an antigen
Also the pathogen/antigen could be present anywhere in the body (enter via lungs, skin, gut)
This means the chances of an individual lymphocyte from the billions you have randomly encountering its target is very small
Therefore to improve this likelihood lymphocytes travel around the body
B cells need signals from CD4 T cells to enable them to differentiate into plasma cells and make antibody. T dependent
Some of the ways this process is made efficient is by bringing antigen presenting cells eg dendritic cells into the same place where T and B cells can be found (lymph nodes and spleen)
This is like an immunological party where DC talk to T cells and T cells talk to B cells if successful leads to productive and helpful long lasting immune responses
But this process needs to be very carefully controlled to prevent autoimmunity and cancer developing
Done through making sure it’s tightly regulated

Question 19

Antibody responses develop through two major pathways

Answer 19

T-independent antibody response:
-IgM, some IgG, low affinity Ab
T-dependent antibody response:
-IgM, IgG, IgA, IgE, high affinity Ab, memory, long lived

IgM is the first antibody secreted

Question 20

The induction of T dependent and T independent antibody responses

Answer 20

T cells are essential for switching to IgG, IgA and IgE
T cells are essential for high affinity antibody production
T cells are essential for the longest lived antibody responses
T cells are essential for (nearly all) B cell memory
This is because T and B cells are both needed for productive germinal centres
T-independent antibody responses are shorter lived, less IgG, virtually no memory, no affinity maturation- because there are no GC

T cells are needed because they enable control of the B cell response and help prevent inappropriate antibody responses, the development of autoimmunity and B cell cancers
The induction of T-dependent responses enables higher levels of antibody to be produced for longer and this can be highly beneficial

Question 21

Primary and secondary antibody responses to T-dependent (protein) antigens

Answer 21

Primary response:
-there is a lag phase after vaccination
-this is where you cant detect antibody in blood
-this is because there are not many B cells that recognise the antigen and so produce antibodies
-over a few weeks you can start to detect antibody in blood
-this plateau before it goes down but not down to zero
Secondary response:
-gets a booster
-the response is much faster
-the amplitude is way higher than achieved by primary response
-this is due to having T cells engaged in the response (T dependent response)

Question 22

After immunisation the nature of the antibody response to that antigen is changed

Answer 22

Unimmunised donor:
-primary response
-frequency of antigen-specific cells is low
-isotype of antibody produced IgM>IgG
-affinity of antibody is low
-somatic hypermutation is low; the mutation occurring in the variable regions of the B cell genes

Immunised donor:
-secondary response
-the frequency of antigen specific B cell is high
-isotype of antibody produced more IgG and IgA
-affinity of antibody is high
-somatic hypermutation is high

Question 23

Responses to T-independent antigens like capsular polysaccharide vaccines- also risk of hyporesponsiveness

Answer 23

An individual is given a capsular polysaccharide vaccine (absence of proteins)-> T independent response:
Primary response:
-you get a rapid increase in antibody levels
-this then plateaus very quickly at modest level
-crashes back to zero
Patient given second dose of vaccine:
Secondary response:
-response identical to primary response
Some individuals are hyporesponsive once they have second vaccine the response does not go up to same level
The longevity of this protection is modest (couple years) whereas longevity we get from T-dependent antigen provides lifelong protection

Question 24

The immune system is organised

Answer 24

Lymphocytes move between sites to increase the chance of encountering antigen and increase efficiency
Lymphocytes can be found in high numbers in the gut
Only a small % are in the blood at any one time
Most lymphocytes are distributed between the lymph nodes (LN) and spleen 20%
Without LN/spleen most lymphocytes are concentrated within distinct sites
Individual lymphocytes will only remain in a LN for 12-24 hours before moving on
Lymphocytes enter via vessels (HEV) and leave via the efferent lymphatics
Lymphocytes rejoin the bloodstream via the thoracic duct which empties into the venous circulation

Question 25

Secondary lymphoid tissues

Answer 25

Spread around the body in strategic locations to help try to protect mucosal sites (eg Lung, Gut and MALT) and systemic sites Examples: -lymph nodes that drain the gut -lymph nodes that drain the lungs This means if theres a pathogen at a specific site of the body the responses can be very local and can be faster The blood and lymph act as motorways for immune cells including lymphocytes to help them get to where they reside (lymph nodes, spleen)

Question 26

Lymph nodes are organised and lymphocytes show a “preference” for different regions

Answer 26

Paracortex (T zone): the adaptive immune responses start here. T cells, dendritic cells meet here and communicate. B cells transit through here Medulla: plasma cells and macrophages reside here. Extrafollicular region, antibody responses occur here Cortex, Follicles: germinal centres form here, B cell and follicular dendritic cells found here High endothelial vessels (HEV): T and B cells enter here Afferent lymph vessels: antigen, dendritic cells enter

Question 27

How do antibody responses to protein antigens begin

Answer 27

Antigen-specific B cell binds antigen Specific antigen efficiently internalised by receptor-mediated endocytosis High density of specific antigen fragments presented Increased MHCII, CD80, CD86, CD40

Question 28

The key difference between DC and B cell uptake of antigen is their specificity- B cells are highly specific (BCR), DC “sample” antigen continuously

Answer 28

DC: not specific in what antigen they take up . DC prime T cells -bacterium infects macrophage and enters vesicle producing peptide fragments -bacterial fragments bound by MHC class II in vesicles -bound peptides transported by MHC class II to cell surface B cells: highly specific in what antigen they take up, T cells “licence” B cells -antigen bound by B cell surface receptor -antigen internalised and degrade to peptide fragments -fragments bind to MHC class II and are transported to cell surface

Question 29

T dependent antibody responses require 2 signals

Answer 29

Signal 1- physical interaction between specialised T cells and B cells Signal 2- soluble messengers (cytokines) to reinforce these interactions This requires intimate cell to cell contact (+cytokines) initiated by B cell binding ag via BCR then processing and presenting to Th cell TCR i.e “cognate interaction”

Question 30

A specialised type of CD4 T cells are vital for the generation of T-dependent antibody responses

Answer 30

CD4 T follicular helper (Tfh) cells Tfh recognise antigen (peptides) presented through MHC class II To be primed CD4 T cells recognise peptide presented through MHCII by DC To positively select B cells they recognise peptide presented through MHCII by B cells This positive selection signal to B cells is therefore through cognate interaction Also vital are additional molecules including CD40 (B cells) and CD40L (Tcells) Soluble messengers called cytokines help reinforce the signals from cognate interaction Important cytokines include IL-4 and IL-21

Question 31

Tfh cells “licence” B cells to differentiate into plasma cells through contact-dependent mechanisms- MHCII: TCR and CD40:CD40L interactions and cytokines (IL-21 and IL-4)

Answer 31

B cell binds virus through viral coat protein Virus particle is internalised and degraded Peptides from internal proteins of the virus are presented to the T cell, which activates the B cell Activated B cell produces antibody against viral coat protein These processes come together in the germinal centre response

Question 32

Round 1 and 2 of making plasma cells

Answer 32

Short lived plasma cells derive from extrafollicular responses Long lived plasma cells derive from germinal centre responses

Question 33

Long lived plasma cell and memory B cells derive from germinal centres

Answer 33

GC are necessary for long lived antibody production and nearly all B cell memory The antibody that is produced is of high affinity and class switched to IgG or IgA To achieve this requires B cells to undergo further recombination of their Ig V genes Control and check to ensure the same antigen is recognised after GC B cells have mutated Control is needed to exclude B cells that bind self antigens and prevent auto-immunity Control is needed to ensure cells leave cell cycle and B cell cancers do not develop This has resulted in the GC being a complex and organised process

Question 34

The germinal centre response plays a critical role in generating antibody responses: i) Ig class switching, II) high affinity antibody iii) memory B cells Iv) long lived plasma cells

Answer 34

1. GC b cells mutate IgV genes 2. GC B cells compete for antigen on FDC 3. Winners get survival signals from GC T cells 4. GC B cells differentiate into plasma cells and memB cells

Question 35

Ig switching

Answer 35

Involves changing Ig heavy chain Somatic hypermutation and affinity maturation are about making antibody hold on better- affinity and avidity Somatic hypermutation: process that results in mutations in the immunoglobulin gene variable regions that can change the affinity of the BCR for its cognate antigen affinity maturation: increased affinity for the target antigen resulting from the germinal centre response IgM has high avidity because it has 10 arms but each Fab may only bind weakly IgG the GC results in IgG with higher affinity since the Fab binds its targets strongly

Question 36

GC are complex environments that involve multiple cell types

Answer 36

Centroblasts Cb: are proliferating GC B cells that undergo somatic hypermutation- dark zone Centrocytes Cc: are GC B cells that have undergone affinity maturation and are out of cell cycle- light zone Follicular dendritic cells FDC- these are different to iDC they have intact antigen in its native conformation bound to their surface and Cc compete to bind this antigen- light zone Follicular T helper cells- these are GC T cells that give survival signals to Cc after they come out of dark zone- light zone

Question 37

Some important molecules involved in GC responses

Answer 37

Activation-induced cytidine deaminase AID: when expressed in B cells and is essential for class switch recombination and IgV hypermutation B cell lymphoma-6 (BCL-6): is the master transcription factor for commitment of GC B cells and is required for the generation of Tfh cells CD40 on B cells and CD40L on T cells IL21 is important for the generation of Tfh cells Blimp-1 is a transcription factor required for the plasma cell programme

Question 38

Germinal centre zones

Answer 38

Dark zone: -in this site centroblasts are undergoing mutation and proliferation -however a consequence of random mutations is that there will be a lot of failed attempts to increase affinity and avidity -we need a way to check for that by performing selection Light zone: -all selection goes on in light zone -this allows us to see whether the mutations are effective or not -to do that you need an antigen to see if they bind to their target better or worse -antigen is held on FDC

Question 39

The germinal centre reaction

Answer 39

b cells entering the GC response first form rapidly dividing centroblasts in the dark zone Each centroblast undergoes random point mutations within its Ig V (variable) region genes These cell then exit cycle become centrocytes and compete for antigen held in the form of immune complexes on follicular dendritic cells in the GC light zone -most B cell epitopes are conformational (3D shape) rather than linear -therefore you need FDC to present antigens in their natural conformation rather than being processed -antigen is on the surface of FDC as immune complex (stuck on surface in normal shape) Those centrocytes who have mutated to high affinity antigen receptors compete most successfully and can then seek out antigen-specific Th cells (t follicular helper cells) for cognate interaction in order to receive rescue signals Those failing to do so (vast majority) die by apoptosis Positively selected centrocytes then exit the GC either as plasma cells secreting high affinity Ab or as memory B cells ready for a rapid response on any subsequent antigen re challenge