L6: B-Cells / Humoral Immunity

Question 1

Discuss two types of antibody responses

Answer 1

1. ) Primary immune response: occurs 7-10 days after first infection, smaller peak response, antibody isotype primarily IgM, lower antibody affinity, induced by all immunogens
2. ) Secondary immune response: occurs 2-3 days after repeat infection, larger peak, antibody isotype shows relative increase in IgG in comparison to first response, but IgA and E also seen in certain situations, higher antibody affinity as affinity maturation has occurred, induced by mainly protein antigens

Question 2

Discuss B cell maturation in terms of DNA, Ig expression, anatomic site and response to antigen

Answer 2

1. ) Stem cell: no Ig expression, bone marrow, no response to antigen
2. ) Pre-B cell: expresses cytoplasmic mu and pre-B receptor-associated mu, bone marrow, no response to antigen
3. ) Immature B cell: membrane IgM expressed (no IgD), released from bone marrow into periphery, undergoes negative selection and receptor editing in response to antigen
4. ) Mature B cell: membrane IgM and IgD seen, in periphery, undergoes activation (proliferation and differentiation) in response to antigen

Question 3

After activation of a mature B cell, what are its options?

Answer 3

1. ) Differentiate into a plasma cell
2. ) Undergo isotype switching
3. ) Undergo affinity maturation
4. ) Differentiate into a memory B cell

Question 4

Mature B cells express IgM and IgD. What are the specificities of each of these Igs?

Answer 4

• Specificity for same antigen/epitope

Question 5

Discuss how B and T-cells interact? How is this process advantageous? Result?

Answer 5

• B-cells bind epitope of microbial antigen using their BCR and internalize antigen via receptor-mediated endocytosis
• Antigen is processed and presented onto class II MHC
• B-cell presents processed antigen in MHC II to CD4+ T-cell’s TCR. B cell’s B7 interacts with T-cell’s CD28, B cell’s CD40 binds T-cell’s CD40L
• Result: T-cell releases cytokines, B-cell is activated and differentiates into a plasma cell. If T-cell wasn’t active, it will also now be.
• The part of the antigen that the T-cell sees is likely different to what the B-cell saw

Question 6

Why are B cells less efficient at processing antigen?

Answer 6

• Fewer lysosomes

Question 7

Outcomes of antibody release by B cell

Answer 7

1. ) neutralization of microbes and toxins
2. ) opsonization and phagocytosis of microbes
3. ) ADCC (NK cells)
4. ) Complement activation (phagocytosis of microbes opsonized with complement fragments, inflammation, lysis of microbes)

Question 8

Distinguish and differentiate between TD and TI antigens that bind B cells

Answer 8

1. ) TD antigens are protein antigens that are thymus-dependenent, in other words, are TH cell dependent. These bind B cell Ig receptors. Activation of B cells with TD antigens allows isotype switching, high affinity maturation, plasma cell differentiation, memory B cell differentiation
2. ) TI antigens are polysaccharides, nucleic acids and lipids that are thymus-independent, in other words, are TH cell independent. These antigens provide all necessary B cell signals to activate them. Activation of B cells with TI antigens prevents B cells from isotype switching, undergoing high affinity maturation, differentiating into memory B cells
   a. ) TI-1 antigens: bind non-Ig receptors, such as TLR4 (LPS)
   b. ) TI-2 antigens: bind B cell Ig, such as polysaccharide antigens with repeated epitopes

Question 9

How is signal transduction for B cells made possible as their plasma membrane bound Igs have short cytoplasmic tails?

Answer 9

• They require Ig-alpha / Ig-beta heterodimer

Question 10

Genes that make up heavy chain of antibody? Light chain?

Answer 10

• V(ariable), D(iversity), J(oining) and C(onstant) genes make up heavy chain
• V, J and C genes make up light chain. There are kappa and lambda chain loci for each of these genes on different chromosomes

Question 11

What mechanisms account for vast diversity of immunoglobulins?

Answer 11

1. ) Pairing of heavy and light chains
2. ) Combinatorial diversity: different gene segments rearranged via recombinases (RAG1/2), heavy chain first then light chain genes
3. ) Junctional diversity: NT addition / removal occurs at joints between gene segments via TdT
4. ) Somatic mutation: point mutations in variable regions of heavy and light chain gene segments

Question 12

SCID results from deficiency in what genes?

Answer 12

• RAG1 and RAG2 genes

Question 13

Describe Ig gene rearrangement

Answer 13

Heavy chain:
- Random D and J segments are brought together, intervening sequences are deleted
- V gene segment randomly selected and placed 5’ to DJ segment to form VDJ segment. VDJ and C region gene separated by intron
- Introns removed during RNA processing
Light chain:
- Random V and J segments are brought together, intervening sequences are deleted. VJ and C region gene separated by intron
- Introns removed during RNA processing

Question 14

What is allelic exclusion?

Answer 14

• We inherit two sets of heavy chain genes, four sets of light chain genes (2 lambda and 2 kappa)
• If rearrangement of 1st heavy chain gene set is non-productive, heavy chain genes from 2nd heavy chain gene set is attempted. Allelic exclusion refers successful rearrangement of 1st set of genes.
• Same thing occurs with light chains with exception that there can be a total of 4 attempts at rearrangement

Question 15

Naïve B Cells express IgM and IgD that are specific for same antigens. Explain how this occurs from a molecular standpoint.

Answer 15

• Primary RNA transcript contains VDJ region coupled to both constant-mu and constant-delta genes
• Alternative mRNA splicing encodes IgM and IgD, but VDJ region used is the same

Question 16

What is the molecular determinant of membrane vs secreted antibody?

Answer 16

• Primary RNA transcript codes for membrane-anchored and secreted forms. If secreted form is required, alternative splicing removes membrane-anchoring sequence from the heavy chain

Question 17

Molecular mechanisms that account for isotype switching

Answer 17

1. ) Long primary RNA transcript containing VDJ region and many/all of constant regions. Alternative splicing sets constant region
2. ) Deletion of intervening constant region genes (DNA) to align VDJ regions with new constant gene region

Question 18

What cytokines determine isotype switching to IgG, IgE and IgA?

Answer 18

• IgG (1 and 3): IFN-gamma
• IgE, IgG4: IL-4
• IgA: TGF-beta, APRIL, BAFF

Question 19

What is affinity maturation? Describe the process

Answer 19

• Affinity maturation refers to process that creates higher affinity antibodies in secondary immune responses in comparison to the primary immune response
  Process: (called somatic hypermutation)
• VDJ regions are allowed to accumulate point mutations. Activation-induced deaminase aids this process
• Highest affinity antibodies are selected for by virtue of increased ability to bind antigen and be stimulated by Tfh cells. Inability to do so leads cells to apoptosis.