Lecture 2 - B cells

Question 1

Development and activation of B cells

Answer 1

• The development of B-cells begins in the bone marrow and is driven by IL-7 produced by marrow stromal cells.
• Cells are activated by encountering their cognate antigen
• Activated B cells secrete antibodies
• Activated B-cells develop into long-lived plasma and memory cells which persist in lymph nodes or return to the bone marrow

Question 2

Precursors of B1 and B2 cells

Answer 2

• Fetal liver–derived HSCs are the precursors of B-1 cells.
• These respond to T-independent antigens and make natural antibodies reactive with polysaccharides.
• Bone marrow–derived HSCs give rise to the majority of B cells (B-2).
• These cells develop into marginal zone B cells or into follicular B cells

Question 3

Stem cell (development and response to antigen)

Answer 3

Anatomic site: Bone marrow

Ig DNA, RNA: Unrecombined/germline DNA

Ig expression: None

Surface markers: CD43+

Response to antigen: None

Question 4

Pro-B cell (development and response to antigen)

Answer 4

Anatomic site: Bone marrow

Ig DNA, RNA: Unrecombined/germline DNA

Ig expression: None

Surface markers: CD43+, CD19+, CD10+

Response to antigen: None

Question 5

Pre-B cell (development and response to antigen)

Answer 5

Anatomic site: Bone marrow

Ig DNA, RNA: Recombined H chain gene (VDJ); u mRNA

Ig expression: Cytoplasmic u and pre B-receptor associated u

Surface markers: B220lo, CD43+

Response to antigen: None

Question 6

Immature B cell (development and response to antigen)

Answer 6

Anatomic site: Periphery

Ig DNA, RNA: Recombined H chain gene (VDJ), k or λ chain genes, u or k or λ mRNA

Ig expression: Membrane IgM (u + k or λ light chain)

Surface markers: IgMlo, CD43-

Response to antigen: Negative selection (depletion), receptor editing

Question 7

Mature B cell (development and response to antigen)

Answer 7

Anatomic site: Periphery

Ig DNA, RNA: Alternative splicing of VDJ-C RNA (primary transcript) to form Cu and Cδ mRNA

Ig expression: Membrane IgM and IgD

Surface markers: IgMhi

Response to antigen: Activation (proliferation and differentiation)

Question 8

Functional changes after B cell activation

Answer 8

• Clonal expansion
• Antibody secretion
• Isotype switching
• Affinity maturation
• Memory B cells forming

Question 9

Role of heavy chains in antibodies

Answer 9

• Regulate the formation of pentamers (IgM) and dimers (IgA)
• Determine binding to cells via Fc receptors.
• Determine functional properties (eg: C’ fixation )

Question 10

Role of light chains in antibodies

Answer 10

• Important for antigen specificity and binding

- labelled as either k or λ

Question 11

Weight of heavy and light chains

Answer 11

Heavy chain: 50kDa
Light chain: 25 kDa

Full Ig: 150kDa

Question 12

Role of IgA

Answer 12

• Found of mucosal surfaces and in many bodily secretions
• Important first line of defence against antigens
• about 13% of total serum
• are usually monomers or dimer structures

Question 13

Role of IgG

Answer 13

• Important for memory/ long term immune response
• Binds many pathogens to control infections
• about 80% of total serum
• monomer structure

Question 14

Role of IgM

Answer 14

• Important for initial response to a pathogen/infection (short term immune response
• controls B cell activation
• about 6% of total serum
• pentamer structure

Question 15

Role of IgE

Answer 15

• Important for allergic responses and responding to intestinal parasites
• less than 0.1% of total serum
• monomer structure

Question 16

Role of IgD

Answer 16

• Important as a B cell surface receptor
• Signals B cells to be activated and then secrete antibodies
• about 0.2% of total serum
• monomer structure

Question 17

Variable and constant regions

Answer 17

• The genes encoding heavy and light chains have variable and constant regions, which are shuffled (somatic recombination) to yield multiple antigen specificities
• This occurs in the bone marrow & is followed by a phase in which autoreactive cells are purged.

Question 18

Which immunoglobulins serve as antigen receptors on B cells?

Answer 18

Membrane IgM (u chain) and IgD (𝛿 chain)

Question 19

Isotope switching (B and T cells)

Answer 19

At the primary follicle (spleen) there is:

• Activation and migration of T & B cells in LN
• T-B cell interactions
• B cell differentiation, Ig secretion, isotype switching
• Germinal Centre reaction (contains follicular dendtritic cells), affinity maturation, memory B cells

Question 20

What happens (with regards to immunoglobulin) when B cells are activated?

Answer 20

IgM: complement activation

IgG subclasses: Fc receptor-dependent phagocyte responses; complement activation; neonatal immunity (placental transfer of IgG)

IgE: Immunity against helminths; mast cell degranulation (immediate hypersensitivity)

IgA: Musocal immunity (transfer of IgA through epithelium)

Question 21

Main enzyme needed for isotope switching

Answer 21

The key enzyme required for isotype switching and affinity maturation is activation-induced deaminase (AID) - activated mainly by CD40 signals from TFH cells

Question 22

How isotope switching works

Answer 22

• AID converts cytosine (C) residues to uracil (U) residues.
• Switch regions are rich in G and C bases, and switch region transcripts tend to form stable DNA-RNA
  hybrids involving the coding strand of DNA
• this frees up the bottom or non-template strand, which forms an open single stranded DNA loop called an R-loop.
• The double-stranded breaks in the two switch regions are joined together
• DNA between the two switch regions is deleted,
• the original rearranged V region becomes adjacent to a new constant region.

Question 23

How affinity maturation works

Answer 23

• A B cell is activated by a protein antigen and helper T cells
• The B cell migrates into the germinal centre
• In the germinal centre there are B cells with somatically mutated Ig V genes and Igs with varing affinities for the antigen
• Only B cells with high affinity membrane Ig bind the antigen on follicular dendritic cells
• B cells that encounter the antigen on follicular dendritic cells are selected to survive, and other B cells die

Question 24

Cellular components of the germinal center

Answer 24

• Mantle zone (outermost)
• Light zone (middle)
• Dark zone (innermost)
• After activation by helper T cells at the edge of a primary follicle, B-cells migrate into the follicle
  and proliferate, forming the dark zone.
• Somatic mutations of Ig V genes occur in these B cells, and they migrate into the light zone where they encounter follicular dendritic cells displaying
  antigen.
• B-cells with the highest affinity Ig receptors are selected to survive - they differentiate
  into antibody-secreting or memory B cells.

Question 25

Histology of a secondary follicle with a germinal center in a lymph node

Answer 25

The germinal center is contained within the follicle and includes a basal dark zone and an adjacent light zone. The mantle zone is the parent follicle within which the germinal center has formed

Question 26

FDCs with immunohistochemistry

Answer 26

- Immunohistochemical stain shows follicular dendritic cells (FDC, stained brown with a specific enzyme-labeled antibody) in a follicle of the spleen. B-cells in the follicle are stained blue. - FDC are HLA class II-negative and do not arise in the bone marrow.

Question 27

Role of Fc receptors

Answer 27

involved in B-cell regulation and antibody function

Question 28

Opsonisation and phagocytosis steps

Answer 28

- Opsonistaion of the microbe by IgG - Binding of opsonised microbes to phagocyte Fc receptors (FcγRI) - Fc receptor signals activate phagocyte - Phagocytosis and killing of ingested microbe