Lecture 11 - B cell encounter with antigen for activation

Question 1

BcR: what is it, what does it detect, how does it convey its signal, and what does it do?

Answer 1

B cell receptor - membrane-tethered antibody

Unprocessed antigens

Igα and Igβ subunits of the complex which contain ITAMs

• Release its BcR as a functional antibody which interacts with FcR using its Fc region to mediate effector mechanisms
• Skews to make an appropriate response by class-switching its receptors to mediate correct effector mechanisms against pathogens

Question 2

Antibodies: what are they and what do they do?

Answer 2

Secreted BcRs

Recruits effector functions through its Fc region

Question 3

CLP: what is it and how does B/T-cell differentiation work?

Answer 3

The common lymphoid progenitor

CLP is produced in the bone marrow:
* CLP moving to the Thymus receives Notch signals to differentiate into a ProT-cell
* CLP staying in the bone marrow receives signals from the stroma to differentiate into a pro-B-cell

Question 4

Germinal centre: what immunoglobulin is present and what is occurring with it?

Answer 4

Non-specific antibody - IgM

Ensuring no self-reactivity before moving to the periphery

Question 5

B cells: how do they survive in the germinal centre?

Answer 5

Germinal centre B cells are prone to dying, they require antigen from follicular dendritic cells and T cell interactions (Tfh cells)

Question 6

B cells entering the periphery: what are they called, how do they enter the periphery, how long do they survive without aid, what is the aid, and what complications do they have?

Answer 6

Transitional/immature B cells

Using the S1PR, promoting movement towards sphingosine-1-phosphate

~3 days

Survival signals from lymphoid follicles

They only enter if they had managed to pass positive selection (self-antigen recognition) well enough and they also must compete against circulating B cells which naturally have a higher affinity for entering them

Question 7

B cell maturation: once entering into lymphoid follicles, what is their maturation and development?

Answer 7

B cell enters lymphoid follicle (ie spleen):
* Receive survival signals from BAFF and associate with antigens held on follicular dendritic cells
* Begin to display CD21 (transitional B-2 B-cell)
* Differentiate into either a follicular B-2 B-cell or a marginal zone B-2 B-cell

Question 8

Follicular B-2 B-cells: what are they, what molecules do they express on their surface, and how long do they survive?

Answer 8

Long-lived mature B-cells - the majority of circulating B cells

• CD21
• IgM
• IgD

~3-8 weeks without returning back to follicles for BAFF and BcR tonic-signalling for survival

Question 9

Marginal zone B-2 B-cell: what are they, where are they located, and what do they do?

Answer 9

Minor population of B cells that migrate to the spleen marginal zone and undergo rapid response to blood-borne pathogens in a T-independent manner

They have a higher expression of CD21 on their surface (CR2) which can interact with circulating complement proteins and release antibodies

Question 10

B-1 B-cells: what are they and what do they do?

Answer 10

Similar to early γδ T-cells - produced early in development (fetal liver)

Role in first-line defence:
* Limited receptor diversity
* Found in peritoneal and pleural cavities
* Source of ‘natural’ antibodies
* Repetitive antigens
* T-cell independent
* Rapid peripheral response to viral/bacterial infection

Question 11

Antigens: what are the types, what do they mean, and what are their characteristics?

Answer 11

Thymus-dependent antigens - antigens not able to activate B-cells without the help of T-cells:
* Immune synapse formation, caused by rearrangement of actin filaments, results in signalling and antigen internalisation
* Interacts with activated CD4+ Tfh cell for additional costimulatory molecules for activation to occur

Thymus-independent antigens - antigens able to activate B-cells directly without the help of T-cells:
* Highly repetitive, polyvalent microbial antigens
* May require a secondary signal provided by TLR recognition for example
* Facilitates fast responses
* No affinity maturation or memory
* Short-lived responses

Question 12

Linked recognition: what is it and what can it medically be used for?

Answer 12

The need for a B-cell to interact with a T-cell that has reacted to the same pathogen to become activated (antigens from the same pathogen, don’t explicitly need to be the same direct antigen)

The Hapten carrier effect - Haptens (small molecules that elicit an immune response only when attached to a large carrier such as a protein) are used in conjugate vaccines by adding peptides from a desired pathogen onto a bigger peptide and allowing the body to produce B cells for that compound

Question 13

BCR co-receptor: what is it, what is it affected by, and what signalling occurs?

Answer 13

CD21/19/81 complex (CD21 = CR2, involved in reacting to complement proteins used to opsonise cells)

Greatly enhanced when the antigen has been ‘opsonised’ by complement - 1/10000 dose needed in mice to activate B-cells for a hen egg white lysosine (HEL) antigen if opsonised

• Binding of both BCR and BCR co-receptors causes phosphorylation of the cytoplasmic tail of Igα/Igβ and CD19 by BCR-associated Src-family kinases
• Additional pathways (ER or look at lecture image or tb)

Question 14

Inhibitory molecules for B cells: what are they, how do they inhibit B cells, and what is the mechanism behind it?

Answer 14

FcγRIIb, CD22, etc

Have ITIMs - recruit phosphatases which dephosphorylate proteins that are phosphorylated and cause B-cell activation

• Recognition of antibody (by FcγRIIb) and antigen (by the BCR) together in an immune complex indicates sufficient circulating antibody is available and BCR signalling is ‘tuned down’ by recruitment of a phosphatase
• CD22 recognises sialic acid-modified glycoproteins common to mammalian, but not microbial surfaces, and desensitises activation against self by raising the signalling threshold

Question 15

Antigen binding: how does it cause B cell activation?

Answer 15

• Conformational change model
• Kinetic segregation model

Question 16

Conformational change model

Answer 16

Receptor-binding causes a conformational change in signalling chains allowing kinase phosphorylation and forming a signalling zone complex

Question 17

Kinetic segregation model

Answer 17

• Kinases and phosphatases phosphorylate and dephosphorylate proteins in the signalling pathway, resulting in no overall action
• Antigen binding causes cell surface contact sites to near and then these contact sites get stabilised by integrin
• Receptor gets anchored in the contact zone
• Kinases continue to enter into the contact zone and phosphate proteins while the larger phosphatases are too big to dephosphorylate them
• This results in overall phosphorylation of the receptor and a signal being produced

Question 18

IgM and IgD on B-cell membranes: in what form do they exist, how do they change as B-cells become activated, and what does this result in?

Answer 18

Nanoclusters, most likely of mixes with IgM and IgD

Once activated, cytoskeleton actin changes facilitate the joining of nanoclusters to form larger signalling microclusters including signalling molecules like CD19

Question 19

Antigen uptake by B cells: what is the process?

Answer 19

• B-cell contacts APC with antigen tethered at its surface
• Engagement causes lead edge activation causing actin polymerisation
• Microsignalosomes associate at the contact area at a much higher frequency than before
• Integrins in the pSMAC stabilises the binding
• Microsignalosomes at the cSMAC have a much higher stabilisation which allows antigen uptake

Question 20

Immune synapse: what cellular changes occur to help extract antigens?

Answer 20

• Centrosome/MTOC and Golgi apparatus reposition at the immune synapse
• Lysosomes are recruited to the contact site causing acidification and release of proteases into the synaptic cleft for efficient extraction of immobilised antigen
• A myosin II-mediated ‘pulling’ force triggers invagination and endocytosis of antigen-containing membrane (contributes to affinity discrimination)
• Endosomes fuse with lysosomes - processing and presentation of acquired antigen on MHC II to recruit T cell help

Question 21

Secondary lymphoid organs: how do they sample antigens as efficiently as they do?

Answer 21

• T-cells in paracortex areas
• B-cells in cortex areas, form primary follicles

Arrival of lymphocytes/DCs:
* Entering from the periphery (blood) scan for a while then move through the medullary sinus to the outgoing lymph
* DCs enter lymph nodes through incoming lymph and drain through to the outgoing lymph but has to find a way through the marginal sinus

Question 22

Lymph nodes: how are antigens brought in?

Answer 22

Many different mechanisms:
* Particulate opsonised antigen - antigens are taken up by the subcapsular sinus macrophages and transported to the other side then cognate B-cells cargo antigens to FDCs who then hold antigens for B-cell scanning

• Small, soluble or proteolysed antigens - antigens (<70KDa) move through conduits in the marginal sinus and travel through the B-cell to the T-cell area, B-cells reach processes into this area and interact with antigens
• Large, cell-borne antigen - DCs use receptors to take up antigen and recycle it back to the surface for presentation to B-cells
• Marginal zone B-2 B-cells - pick up antigens in the marginal zone, reducing S1PR presentation, causing them to be attracted to high expression S1PR areas and deliver antigens to FDCs before changing expression back and localising back to the marginal zone

Question 23

Primary follicles: what are they?

Answer 23

Islands of FDCs in the lymph node cortex where the FDCs display antigens for B-cell to interact with displayed antigens

Question 24

Bringing antigens into lymph nodes - particulate opsonised antigens: what is the process?

Answer 24

• Antigens are taken up by the subcapsular sinus macrophages and transported to the other side, with specificity of transport made by Fc, complement, and lectin receptors
• Cognate B-cells (B-cells that recognise the antigen) use their own receptors to cargo antigens to FDCs
• FDCs then hold antigens for B-cell scanning

Question 25

Bringing antigens into lymph nodes - small, soluble, or proteolysed antigens: what is the process?

Answer 25

• Antigens <70KDa, move through conduits in the marginal sinus formed by fibroblastic reticular cells with collagen bundles inside
• Antigens then travel through the B-cell to the T-cell area
• B-cells are suggested to be able to reach processes in this area and interact with antigens

Question 26

Bringing antigens into lymph nodes - large cell-borne antigens: what is the process?

Answer 26

• DCs use receptors (ie FcγRIIb and DC-sign) to take up antigen and recycle it back to the surface for presentation to B-cells

Question 27

Bringing antigens into lymph nodes - Marginal zone B-2 B-cells: what is the process?

Answer 27

• Marginal zone B-2 B-cells pick up antigens in the marginal zone
• They reduce S1PR presentation, causing them to be attracted to high-expression S1PR areas
• They deliver antigens to FDCs
• They change expression back and localise back to the marginal zone

Question 28

B-cell activation: how does it move to associate with the T-cells needed for full activation?

Answer 28

FDC antigen recognition leads to B-cells to modulate their chemokine receptors and begin to express CCR7, associating them with the T/B-cell area boundaries and allowing interaction with T-cells that have also been activated in their zones and are associating with the boundary as they express the CXCR5 chemokine receptor