Chapter 6

Question 1

What happens in pro-B-cells?

Answer 1

• earliest identifiable cells of B-cell lineage
• rearrangement of heavy-chain genes
• early pro-B-cell stage
  
  • joining of D and J segments
• late stage
  
  • V with DJ
• VDJ upstream of C-region genes (closest to Cμ)

Question 2

When does pro-B-cell become a pre-B-cell?

Answer 2

• on expression of the μ chain

Question 3

What are two stages in pre-B-cell development and what happens during them?

Answer 3

• immature large pre-B-cell
  
  • no recombination
  • production of μ heavy chain
    
    • checkpoint: surrogate light chain (to see if produced heavy chain can bind correctly) and Igα and Igβ → forming pre-B-cell receptor
    • if well, signal to start recombination of light chain
    • if not, cell apoptosis
• mature small pre-B-cell
  
  • recombination of light chain

Question 4

How is light chain assembled?

Answer 4

• κ light-chain genes rearrange first
• only if unsuccessful, λ light-chain genes rearrange
• successful → light + heavy chain moved to ER and assembled with Igα and Igβ
  
  • chaperones: calnexin, calreticulin (also used in MHC class I and II) and heavy-chain binding protein (Bip)

Question 5

When does small pre-B-cell become immature B cell?

Answer 5

• surface expression of B-cell receptors

Question 6

How do stromal cells stimulate B-cell development?

Answer 6

• contact by adhesion molecules attaching to ligands on B cells
  
  • growth factors produced
• as B-cells develop they move from subendosteum (interior surface of the bone) → centre
  
  • less dependent on stromal cells, eventually detach

Question 7

What happens to immature B-cells?

Answer 7

• maturation in secondary lymphoid tissues (spleen, lymph node, etc)

Question 8

What are the 2 major transcription factors for development of pro-B cell?

Answer 8

• E2A
  
  • induces expression of EBF
• together induce synthesis of proteins for rearrangement of heavy chain genes
  
  • including RAG-1 and RAG-2
• induce expression of Pax-5
  
  • switched on expression of proteins such as Igα, Igβ, and CD19
  • CD19 = cell-surface protein + component of B-cell co-receptor (mature B cells can respond to antigen)

Question 9

Why is heavy chain gene rearrangement inefficient?

Answer 9

• addition of N and P nucleotides (random) → can be nonsense
  
  • no translation ⇒ nonproductive rearrangements

Question 10

How does rearrangement of immunoglobulin heavy-chain genes in pro-B cells give rise to productive and nonproductive rearrangements?

Answer 10

• D-J rearrangements (both chromosomes) almost always successful → three reading frames of D gene → functional protein sequence
• V-DJ rearrangement on first chromosome → only 1 of 3 frames encodes V region (1/3 of pro-B cells successful) → transcription increases → IgM mde
  
  • if unsuccessful (2/3 pro-B cells) → moves onto 2nd chromosome
    
    • again only 1/3 successful
• unsuccessful ⇒ apoptosis

Question 11

What are survival signals?

Answer 11

• generally refers to the signals that developing and naive mature B cells and T cells must receive from other cells if they are to survive

Question 12

How exactly is quality of heavy chain checked?

Answer 12

• pro-B cells produce VpreB (V-region resemblance) and λ5 (C-region) proteins
  
  • bind μ heavy chains ⇒ surrogate light chain
• transcription controlled by E2A and EBF
• μ chains form disulphide-bonded homodimers → with VpreB, λ5, Igα, and Igβ form pre-B cell receptor
  
  • assembled correctly in ER → sends signals (thanks to extensions on VpreB and λ5)
  • no more heavy chain rearrangement → pro-B cells divide → pre-B cells
• pre-B cell receptor ensures no B cell rearranges both copies of heavy-chain locus
  
  • signal to terminate transcription of RAG genes → reorganisation of chromatin so no more rearrangement takes place

Question 13

What happens in pre-B cell?

Answer 13

• pre-B cell divides
  
  • new copies don’t produce surrogate light chain
• light chain genes rearranged
  
  • RAG reactivated
• V-J arrangement needed only for light chain
  
  • several attempts to rearrange the same light-chain gene by using V and J gene segments not involved in previous arrangements
  • 4 light chain loci (κ λ on each chromosome)
  • 85% pre-B cells successful
• membrane-bound IgM formed (in ER)
  
  • once on membrane signal to stop light chain rearrangements (same as heavy chain)
  • becomes immature B cell

Question 14

What are benefits of clonal expansion of large pre-B cells?

Answer 14

• functional heavy chain obtained is not lost if light chain isn’t formed
• variety in B cell population
  
  • same μ chain but different light chains

Question 15

What is X-linked agammaglobulinemia?

Answer 15

• immunodeficiency of B cells (and antibodies)
• caused by lack of Bruton’s tyrosine kinase (BTK)
  
  • essential for B-cell development
• recurrent infections
• X-linked disease

Question 16

What is translocation and does it occur in B-cells?

Answer 16

• fusion of one chromosome’s part with another
• often in B cells during gene rearrangement
• immunoglobulin gene fuses with cell growth gene which can result in formation of tumor cells
  
  • fusion with proto-oncogenes

Question 17

What are B-1 cells?

Also known as CD5 B cells

Answer 17

• B cells arising in embryonic development (even before conventional B cells)
• express CD5 (normally found in T cells)
• low expression of IgD
• most active prenatally
• in heavy chain gene rearrangement V_h segment closest to D used + no TdT -> small diversity
  
  • unspecific, low affinity binding (polyspecificity) -> useful in bind of polysaccharides antigens
• gradually less production -> live only through proliferation of existing cells (self-renewal abilities)

Question 18

What are self-reactive B cells?

Answer 18

• B cells which receptors bind to self antigens
• cannot be let to mature
• negative intracellular signal generated when they bind to antigen
• die by apoptosis

Question 19

How does the body recognise and stop self-reactive B cells?

Answer 19

• first immature B cells are in bone marrow
• if B cell binds to stromal cell / hematopoietic cells (or other self cells), it won’t leave the bone marrow
  
  • development is suspended
  • altering B cell receptors -> second chance (so that they don’t bind self)

This is the mechanism for multivalent self antigens

Question 20

How does receptor editing occur?

Answer 20

• immature B cell is self-reactive
• IgM on the surface reduced
• RAG complex is still expressed
  
  • rearrangment of light-chain loci
  • old VJ discarded (no self binding)
• mechanism repeats if new sequence is also self-reactive
  
  • if all options are exhausted, apoptosis = clonal deletion

This is the mechanism for multivalent self antigens

Question 21

What happens when B cell is specific to a monovalent self antibody?

Answer 21

• no apoptosis nor gene rearrangement
• developmental arrest = anergy
• make both IgM and IgD but IgM prevented from assembling B-cell receptor
  
  • IgM retained in cell
  • IgD on cell surface but doesn’t activate B cell when binding to antigen
• anergic B cells can enter peripheral circulation -> shorter lifespan

Question 22

What is the difference between central and peripheral tolerance?

Answer 22

• central acquired in bone marrow
  
  • immature B cells prevented from binding self antigens found there
• peripheral induced outside bone marrow (ex. blood)
  
  • if B cell binds self antigens there -> apoptosis
  • gene rearrangement mechanism shut down already
• stress / damage may expose B cells to new areas (where they can bind self antigens)
  
  • autoimmune response

Question 23

What happens after B cells leave the bone marrow?

Answer 23

• enter secondary lymphoid tissues to further mature
• ex. spleen, lymph nodes, Peyer’s patches
• enter through high endothelial venules (HEV)
• stromal cells secrete CCL21
  
  • chemokine recognised by receptor CCR7 on B cells
• cell-adhesion molecules and cytokines allow homing (attract B cells to HEV)
• dendritic cells in lymph node secrete CCL19 -> further attraction
• chemokine gradient attracts B cells -> squeeze through HEV to enter

Question 24

What happens once B cells enter secondary lymphoid tissue?

Answer 24

• chemokines guide B cells to form primary lymphoid follicles
  
  • B cells in a network of folliular dendritic cells (FDCs)
  • chemokine: CXCL13
• B cells help preserving FDCs -> protein on their surface lymphotoxi (LT)
  
  • relative of tumor necrosis factor-α (TNF-α)
  • in turn BAFF (TNF family) maintains B cells and is secreted by secondary lymphoid organs
• after this when B cell encounters its specific antigen -> becomes plasma cell

Question 25

What are mature B cells that haven’t encountered antigen yet called?

Answer 25

• naive B cells

Question 26

How do naive B cells survive in circulation?

Answer 26

• regularly pass through primary follicles of secondary lymphoid tissue
• competition with immature B cells
  
  • mature are favoured

Question 27

What happens when naive B cell encounters antigen?

Answer 27

• in primary follicle antigens for B cell to encounter
• if successful, moves to boundary between follicle and T cell area -> CD4 helper T cell (activated by antigen-bearing dendritic cells)
• once T cell binds -> B cell activated (prolif + differentiation)
  
  • moves to medullary cords or nearby primary follicles
  • change in heavy-chain mRNA -> secreted immunoglobulin made (IgM) instead of membrane-bound

Question 28

What are plasma cells?

Answer 28

• B cells synthesising antibodies for secretion
• secretory organs developed (+ there’s many)
• don’t
  
  • divide
  • express cell-surface antibodies or MHC class II
  • interact with antigen or T cells
• T cell B cell pairs that moved to medullary cords become plasma cells immeditely
  
  • secrete IgM

Question 29

What happens with T cell B cell pairs that move to nearby primary follicle?

Answer 29

• change in morphology of follicle to become secondary lymphoid follicle
  
  • has germinal center
• B cells -> lymphoblasts (large and proliferating)
  
  • this specific kind is called centroblasts -> mature into slowly dividing B cells = centrocytes
  • centrocytes have undergone isotype switching and somatic hypermutation
  • centrocytes selected by affinity maturation
• move to other sites to become plasma cells (high affinity, isotype-switched antibodies)

Question 30

How are memory cells created?

Answer 30

• after primary immune response (naive B cells -> plasma cells)
• germinal-center B cells differentiate into resting memory B cells
  
  • activation = secondary immune response
  • antibodies other than IgM are predominant