B cell Activation and Antibodies

Question 1

Where does B cell development start and where is it completed?

Answer 1

-It begins in the bone marrow and continues in the spleen.

Question 2

Why is there no positive selection for BCRs?

Answer 2

Because BCRs don’t bind to MHC, they bind free-flowing antigens, hence they don’t need to undergo positive selection to test whether they bind to MHC or not

So how do they bind to TFH then??

Question 3

What is the purpose of the second round of negative selection in the spleen?

Answer 3

-To present more self-antigens to B-cells

Question 4

What happens to self-reactive B-cells?

Answer 4

-Receptor editing in light chains (toleranceqq)
or
-Cell death

Question 5

IN CLASS QUIZ1: Which enzyme is not involved in the light chain recombination?

Answer 5

Tdt gets turned off in recombination of the BCR light chain
-Others involved: Artemis, RAG1/2, Exonucleases

Question 6

What does the development of BCRs in the spleen look like?

Answer 6

T1 subset:
-Since the spleen filters the blood, it can expose blood-borne antigens to B-cells

-Recombination enzymes like RAG1/2 are turned off at this stage, so no light chain editing is possible

T2 subset:
-creates IgM and IgD antibodies and upregulates receptors for BAFF (B-cell activating factor) that upon binding create a survival signal -> if it doesn’t the cell will undergo APOPTOSIS

Question 7

At which part of the antibodies does recombination occur?

Answer 7

VDJ recombination including P and N nucleotides at the variable region on the top of the heavy chain:

-The lower part reflects the isotypes: IgM, IgD, IgG, IgA, IgE

Question 8

Which Ab isotypes are expressed by mature B-cells?

Answer 8

-IgM and IgD

-This is achieved by splicing different products from one single mRNA + removing unused J regions

-Note that IgD and IgM still bind the same antigen bc the VDJ region at the binding site is the same, only the c region is changed -> in case of isotype switching to give the antibody different abilities

Question 9

How is the membrane and secreted form of antibodies generated?

Answer 9

-Splicing that determines if the membrane-bound (BCR) or secreted form (Antibody) is produced by removing the transmembrane domain

-Once the B-cell finds an antigen, it will start creating the secreted version which is the antibody

Question 10

How does the Ig isotype switch occur?

Answer 10

-Depending on the pathogen and cytokines secreted by the T helper cell (stimulated by DC)

-genetic DNA recombination:
VDJ M D -> cutting off M or D segment for A,G or E

Question 11

What are Marginal zone B-cells and B-1 B cells?

Answer 11

The normal B cell is a B-2 B-cell !!

-Marginal zone B-cells derives from T2 subset B-cell during development in the spleen -> the ones that are too sensitive to self-antigens are converted into marginal zone B-cells, which are specialized to recognize carbohydrate Ag
-> they have IgM Ab and cant switch their Isotype, innate type of B-cells

-B1 B cells derived from the stem cells from the gut
limited receptor repertoire Tdt is minimally expressed in the precursor of B1 cells
-they also bin carbohydrates
-they need interaction with self-carbohydrates first, and never get support from T cells

Question 12

What are T-Independent B Cell Responses?

Answer 12

-Response of B-cell without the help of T cells
-Therefore it needs a strong signal to get activated, one simple antigen is not enough (we do not want a response to that without T cell help anyway bc it could be self)

TI-1 antigens: bacterial cell wall components and LPS -> bind through PRRs and BCRs

TI-2 antigens: polymeric proteins or polysaccharides -> bind BCRs with the help of complement

Question 13

What is the T-dependent B Cell Response?

Answer 13

1. B-Cell binds Anitigen
   2.CD40 (B-cell) interact with CD40L
2. Cytokines -> helps turn B-cell on and start maturation process
   this is something T independent dont get, they can secrete out IgM and IgD but nothing else

Question 14

How do B cells get exposed to antigens?

Answer 14

B-cells don’t have Antigen-presenting cells, instead they are waiting in the secondary lymph tissues for antigens (from dead pathogens) to come along through the lymph system

Question 15

How do antigens reach the B-cells in the secondary lymph tissues?

Answer 15

There is a barrier bc we don’t want every Ag to be in the lymph node and cause an infection

-Small Ag though can pass directly through the gaps and reach the B-cells

-Larger Ags are captured by subcapsular sinus macrophages (SCSMs) and handed off to B cells in the follicles

-Follicular dendritic cells collect Ags and present them to B-cells

Question 16

Once B-cells have bind to antigens, what does the B-cell do with it?

Answer 16

-It will present the Ag through exogenous pathway through MHC-II
-B-cells position themselves from follicles into T cell-rich zone to find a T-cell that help them to develop
-Once they find one they will create the 3 signals:
on the B-cell side: 1. Antigen binding -> 2. CD40-CD40L and CD80/86, 3. Cytokines

on T-cell side: MHC-II with peptide to TCR 2. CD28 to CD 80 on B -cell and 3. cytokines

Question 17

Which type of T cell is involved in B cell development in the germinal zone?

Answer 17

-Development of T-cells and B-cells goes sidewise
-T-cells need antigen-presenting cells to expose them to antigens for the development

-When B-cells find antigens there are no T1 or T2 cells at that point, only naive T cells-> so if B-cells find antigens they present it to TFH that direct the B-cell differentiation process

Question 18

Which development steps occur in the germinal center?

Answer 18

-Some of the naive B cells will start making antibodies (IgM, IgD), some will turn into plasma cells, some will turn into memory cells

-Other will undergo further development through somatic Hypermutation and Class-switching recombination

Question 19

What is Somatic Hypermutation?

Answer 19

individual point mutations in Ig heavy- and light-chain rearrangements at the top of the antibody on the variable regions

Follicle DC presents Ag: B-cells that don’t bind to Ag well enough after mutation die

-The purpose is the selection of B-cells with higher affinity to a particular Ag (these ones will receive the survival signal)

Question 20

Explain Class switch Recombination:

Answer 20

IgM and IgD are the first isotypes created, they are broad
IgA (mucosal), G (viral), and E (helminth) are more targeted to a specific type of antigen

-The cytokine signal (PAMP, PRR, cytokine, Th) determines which isotype is to be produced

-The enzyme used is the activation-induced deaminase (AID), also used in SHM

Question 21

How does the Class switch Recombination work?

Answer 21

-It happens at the lower end of the antibody -> constant region
AID induces double-strand breaks by a mutation in switch regions, gets rid of the M/D segment, and they are looped together with the new class (IgG, E, A) -> the variable binding site on top (somatic hypermutated) stays the same!

Question 22

What are the plasma cells?

Answer 22

-They are activated and class-switched and ready to produce antibodies (secreting version)
-They lose their external BCR

-found within 5-6 days of immune responses in lymph nodes
-long-lived in bone marrow (but not able to class-switch)

Question 23

How are Memory cells created?

Answer 23

-Some B-cells that have been activated by T helper cells (3 signals) don’t convert into plasma cells but they are kept as memory cells

-bc plasma cells don’t have BCR anymore, memory cells do have BCRs and can interact with antigens that are coming back and turn into plasma cells or create new memory cells

Two types:
-IgG bearing: have undergone SHM producing higher-affinity antibody -> EARLY MEMORY RESPONSE

-IgM bearing: generated prior SHM (survive longer): not differentiated but can undergo differentiation in case the pathogen has changed and form new plasma and memory cells -> LATE MEMORY RESPONSE

Question 24

What advantages do IgM memory cells have?

Answer 24

-They might have lower affinity but:
-capable of class-switching
-Further SHM
-in case that pathogen has changed, IgM memory cells can create new and better plasma cells based on that new antigen, and memory cells

Question 25

How do Antibodies fight against pathogens?

Answer 25

-Neutralization: protects against bacterial and viral infection or toxins (all classes) -> Ab blocks spike protein -> no viral entry; neutralizes active site of endotoxins

-Agglutination: multiple Ab acting like a net -> enhance neutralization and helps with clearance of pathogens through Granulocytes (all classes, especially IgM)

-Opsonization: can be used in the classic complement system (C1q, C1) -> MAC or it helps in phagocytosis

Question 26

How do Antibodies fight against pathogens? part 2

Answer 26

-Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC): sometimes viruses present their spike proteins to the outer membrane to make it ready for virus release -> these spikes can be targeted by antibodies (IgG and IgA)

-Antibody-Dependent Degranulation and Mediator Release: Antibodies used to target pathogens like helminths to induce Granule and mediator release of Granulocytes

Question 27

Functions of Antibodies: IgM

First one produced

Answer 27

-As a BCR in monomer form, once secreted it polymerizes to a pentamer (5)

-Forms dense Ab-pathogen complexes -> engulfed by macrophages
or
-Complement fixation -> MAF formation

-low-affinity bc did not go through SHM -> instead it uses higher numbers of Abs (Agglutination)

Question 28

Functions of Antibodies: IgG

most prominent

Answer 28

-have subclasses, human IgG1/IgG3 are good at complement fixation
-IgG1 is good at mediating ADCC by NK cells
-Opsonization of pathogens -> bind to Fc receptors of Macrophages for phagocytosis

Question 29

Functions of Antibodies: IgE

Answer 29

-Binding and Degranulation of eosinophils/basophils, release molecules like histamine to damage large pathogens

-protection against helminths (worms), protozoa
play a role in asthma and allergy

Question 30

Functions of Antibodies: IgD

Answer 30

-also bind to basophils/mast cells
-expressed on naive B cells
-receptor not well characterized

Question 31

Functions of Antibodies: IgA

Answer 31

-mostly found in secretions of Mucus in the gut, Milk, Tears, Saliva

-Dimer in secretions, Monomer in serum

-Effective in neutralizing toxins
-does not fix complement -> does not drive inflammation (C3a/5a)

Question 32

How are IgAs transported outside of epithelial cells?

Answer 32

-PolyIgR (for IgM or IgA) initiates transport from blood to the lumen of multiple tissues: GI tract, respiratory tract, reproductive tract

-responsible for carrying Ab into tears and milk and populating gut mucosa with IgA Ab

Question 33

Why is Isotype switching important?

Answer 33

Because different immune cells have different receptors for specific isotypes: Granulocytes have IgE receptors, phagocytes, and NK cells have IgG receptors