B cell activation (humoral immunity I)

Question 1

The two branches of adaptive immunity are:

Answer 1

1) Humoral immuntiy (B cells)
2) Cell-mediated immunity (T cells)

Question 2

There are three different subsets of mature naive B cells that respond to specific types of antigens to become activated.

1) What are the different B cell subtypes?

2) What type of antigen does each subtype recognize?

Answer 2

1) Most B cells are FOLLICULAR B CELLS, derived from the bone marrow precursors after birth with highly diverse receptors.

MARGINAL ZONE B CELLS are abundant in the spleen/lymph node, derived from bone marrow precursors with LIMITED receptor diversity (mainly IgM).

B-1 CELLS derived from FETAL liver with LIMITED receptor diversity (mainly IgM).

2) Follicular B cells recognize protein antigens, while marginal and B-1 cells recognize polysaccharides and lipids.

Question 3

While follicular B cells are T-_______ and are _____-_____ plasma cells, marginal B and B-1 Cells are T- _________ and are ____-______ plasma cells.

Answer 3

Dependent; long-lived

Independent; short-lived

Question 4

Compare and contrast Thymus (T)-dependent antigens to Thymus-independent antigens.

Answer 4

T-dependent (TD antigens):
- PROTEIN antigens
- recognized by FOLLICULAR B cells

T-independent (TI antigens):
- POLYVALENT antigens (polysaccharides)
- recognized by MARGINAL ZONE B CELLS and B-1 CELLS

Question 5

(T/F) TI antigens are generally non-protein antigens that can cross-link multiple membrane Ig molecules thereby activating B cells without help from CD4+ T cells.

Answer 5

True!

Question 6

(T/F) While T-Dependent cells do not require signals from helper T cells for activation, T-Independent cells require help from helper T cells.

Answer 6

False!

T-dependent cells (follicular B cells) require signals from helper T cells for activation while T-independent cells (marginal zone B cells & B-1 cells) DO NOT require signals from helper T cells for activation.

Question 7

How are Follicular B cells activated in response to TD antigens?

Answer 7

B cell activation requires multiple signals in response to either TD or TI antigens.

For follicular B cells with TD antigen, there needs to be TWO signals.

A signal is generated by BCR interacting with the antigen.

The second signal from follicular helper T cell is generated when BCR and antigen complex is ingested and processed by the B cell to present to T cells (recall B cell = APC).

Proliferation, differentiation and survival are a result of the two signals.

Question 8

Fill in the blanks regarding the multiple signals required for B cell activation in response to TD antigens.

Signal 1 is the antigen being recognized by BCR and BCR _______ resulting in the nuclear localization of ____ and ____ TFs which promote B cell ___________ and __________.

Signal 2 is the helper T cell recognizing the peptide-MHC II complex presented by the B cell. Antigen recognition combined with _____-_______ (co-stimulatory molecules) interaction promotes signalling via the _____ kinase, promoting binding of TFs that induces expression of pro-_____ genes (____).

Answer 8

Coreceptor; NFAT; AP-1; proliferation; differentiation

CD40-CD40L (costimulatory molecules); NIK; survival; Bcl-2

Question 9

Follicular helper T cells provide signals to B cells to promote ______ ______ _______ which ultimately leads to the production of _______ that are effective in eliminating pathogens.

Answer 9

germinal center responses; antibodies

*thus after being activated by helper T cells, B cells form germinal centers in lymphoid follicles.

Question 10

Naive B cells travel to the lymph node via the ______.

B cells that encounter antigens in the follicle form a ______ _____. Some _______ B cells migrate into the follicle to form a _______ ______.

______ cells are generated and migrate to the medullary cords or leave via the ______ lymphatics.

Answer 10

bloodstream

primary focus; proliferating; germinal center

plasma; efferent

Question 11

Germinal centers are organized into light and dark zones.

Light zone contains ______ ____ cells, while dark zone contains _______ ___ cells.

Answer 11

follicular dendritic; proliferating B

Question 12

Germinal center B cells undergo processes that produce high-affinity antibodies.

What are the three steps that lead to this?

Answer 12

1) Activated B cells migrate into the follicle and proliferate, forming the dark zone of the germinal centre.

2) B cells migrate to the light zone where they undergo germinal centre reactions (isotype switching and affinity maturation).

3) B cells with the highest affinity Ig receptors are selected to survive and differentiate into memory B cells and antibody-secreting plasma cells.

Question 13

What is the primary antibody repertoire composed of?

Answer 13

The primary antibody repertoire is composed of IgM that contains variable regions produced by V(D)J recombination in the bone marrow.

This is also known as somatic recombination.

Question 14

The antibody repertoire is then diversified from the primary antibody repertoire by two major processes.

Briefly state and describe them.

Answer 14

1) Somatic hypermutation: introduction of mutations in heavy and light chain variable regions that can alter the affinity of the antibody for its antigen.

2) Class switch recombination: involves the replacement of the μ heavy chain constant region for heavy chain constant regions of other isotypes.

Question 15

What are the four main mechanisms that generate the variable regions of the immunoglobulin receptor?

Answer 15

1) Combinatorial diversity
2) Junctional diversity
3) Joining of light and heavy chains
4) Somatic hypermutation

*1-3 occur in the bone marrow during B cell development, while 4 occurs in the peripheral lymphoid organs once development is complete.

Question 16

(T/F) Somatic hypermutation requires antigen binding.

Answer 16

True!

Somatic hypermutation is the diversity created due to high level of mutations introduced in the DNA sequence of the rearranged variable region in mature ACTIVATED B cells!

Question 17

1) Which enzyme is responsible for somatic hypermutation?

2) Why does somatic hypermutation not occur in naive B cells?

3) How does the enzyme generate mutations?

Answer 17

1) Somatic hypermutation is mediated by Activation-Induced-Cytidine-Deaminase (AID).

2) AID is expressed in germinal centre B cells as its expression is induced by CD40 signals from T(fh) cells during B cell activation.

3) AID binds to a cytidine side chain of SINGLE-STRANDED DNA molecule of actively transcribe Ig gene. AID promotes a NUCLEOPHILIC attack on the cytidine RING, DEAMINATING the cytidine to form URIDINE. Uridine is foreign to DNA!

Question 18

Which process does the somatic hypermutation enable? What does the process allow for?

Answer 18

Somatic hypermutation enables a process called AFFINITY MATURATION which allows for the SELECTION for the survival of mutated B cells that have exceptionally high affinity for antigen.

Question 19

Briefly describe the process of selection for affinity maturation.

Answer 19

B cells MUTATE their antibody genes in the DARK zone of the germinal centre (somatic hypermutation).

B cells with HIGH affinity for antigen can capture and process it for presentation by MHCII molecules. B cells that present antigens to T(FH) cells will receive survival and mitogenic signals via CD40 and cytokines.

B cells that receive help from T(FH) cells can reenter the dark zone to undergo additional mutations and REPEAT this process, generating higher and higher affinity immunoglobulin.

Question 20

Which statement is false?

1) Somatic hypermutation and affinity maturation occur in the BCR of B cells and not of plasma cells.

2) Somatic hypermutation generates only the mutations that result in a higher affinity of the B cell for its antigen.

Answer 20

2!

Somatic hypermutation can generate mutations that result in a LOWER affinity as well.

Question 21

The result of selection for enhanced binding to antigen is that the mutations tend to cluster within the ________ _____.

Answer 21

hypervariable regions (CDRs)

*this makes sense because this is the part of the immunoglobulin that recognizes antigen!

Question 22

The first Ig receptors expressed by B cells are ____ and ____.

The first antibody produced in an immune response is ____.

Answer 22

IgM; IgD

IgM

Question 23

1) Which region of the immunoglobulin are the isotypes distinguished by?

2) What does class switching allow for?

Answer 23

1) The constant region of the heavy chain!

2) Originally, Ig receptors expressed are IgM and IgD. Later in the immune response, THE SAME VARIABLE REGION presented in IgM and IgD can be expressed in IgG, IgA or IgE antibodies (recognizes same antigen but with different effector functions). This is done by class switching!

*the work done by somatic hypermutation remains as variable region is not changed!

Question 24

What are switch regions?

Answer 24

Stretches of repetitive DNA that lie in the INTRON upstream of genes encoding each heavy chain constant region gene.

Question 25

Match the steps of isotype switching (class switching) to the right order.

1) Step 1
2) Step 2
3) Step 3
4) Step 4
5) Step 5

A) RNA polymerase stalls within the switch region due to the repetitive sequences, allowing for regions to serve as substrates for AID (followed by UNG, APE1).

B) The intervening DNA is excised, generating a new constant region that is located adjacent to to variable CDJ exon.

C) The switch regions are then brought into proximity by the DNA repair machinery.

D) switching is guided by the initiation of transcription by RNA polymerase through specific promoter regions upstream of the switch regions (two RNA polymerases - one/switch region)

E) AID, UNG and APE1 introduce nicks on BOTH strands of the DNA, recruiting the ds break repair machinery.

Answer 25

Step 1: switching is guided by the initiation of transcription by RNA polymerase through specific promoter regions upstream of the switch regions (two RNA polymerases - one/switch region)

Step 2: RNA polymerase stalls within the switch region due to the repetitive sequences, allowing for regions to serve as substrates for AID (followed by UNG, APE1).

Step 3: AID, UNG and APE1 introduce nicks on BOTH strands of the DNA, recruiting the ds break repair machinery.

Step 4: The switch regions are then brought into proximity by the DNA repair machinery.

Step 5: The intervening DNA is excised, generating a new constant region that is located adjacent to to variable CDJ exon.

Question 26

Which constant gene does not have a corresponding switch region?

Answer 26

The delta constant gene for IgD.

Question 27

Why can mature B cells co-express IgD and IgM?

Answer 27

The delta constant gene for IgD does not have a corresponding switch region.

In mature B cells, transcription extends through both Cμ (IgM) and Cδ (IgD) exons. This long primary transcript is then processed by cleavage, polyadenylation (AAA) and splicing, generating two transcripts.

This is why mature B cells can co-express IgD and IgM because they are constructed by ALTERNATIVE SPLICING of the same RNA transcript.

Question 28

Cleavage and polyadenylation at the __ site and splicing between __ exons yields an mRNA encoding the μ heavy chain.

Cleavage and polyadenylation at the __ site and splicing between __ exons yields an mRNA encoding the δ heavy chain.

Answer 28

μ; Cμ

δ; Cδ

Question 29

Immunoglobulins can serve as the antigen receptor on the surface of B cells. They can also be secreted by plasma cells.
What dictates if an immunoglobulin is membrane-bound or secreted?

Answer 29

Transmembrane and secreted forms of immunoglobulins are derived from the same heavy chain sequence by ALTERNATIVE RNA PROCESSING.

Each heavy chain C gene has two exons: membrane coding (MC) and secreting coding (SC). Each heavy chain C gene has two polyadenylation sites: pAs and pAm.

Cleavage and polyadenylation at the pAm followed by splicing results in the removal of SC, generating a transmembrane Ig.

Cleavage and polyadenylation at the pAs eliminates the MC exon and gives rise to the secreted Ig.