02212013 - 1 - B-Cell Development

Front 1

B-cell functionality

Back 1

- Respond to Ag's
- Produce Ab's

Front 2

What is an Antigen?

Back 2

- Any chemical structure body recognizes as "not self"

Front 3

Epitopes

Back 3

- Small segments of larger Ag's that are actually recognized by immune system
- Part that actually interacts with Ab

Front 4

Linear vs. discontinuous epitopes

Back 4

- Linear = several AA peptides in a row that are recognized
- Discontinuous = 2 different parts of protein that simulataneously hit the receptor
- If protein denatured, immune system wouldn't "see" it

Front 5

Epitopes needed to generate Immunogenicity

Back 5

- Ability of Ag to ilicit an immune response
- Generally, single epitope is not enough to generate a response
- Need several repeated or several different epitopes to get solid response
- Also, certain minimal dose

Front 6

Haptens

Back 6

- Small molecules - not immunogenic by themselves
- Bind to something else (cell, etc.) - act as epitopes!

Front 7

B-cell uniqueness

Back 7

- Each is a unique clone - makes a unique Ab

Front 8

Antibody structure

Back 8

- Y-shaped glycoprotein
- 2 heavy chains, 2 light chains
- Light and heavy chains are identical!

Front 9

Antibody variable/constant regions for heavy and light chains

Back 9

- Light chains = 1/2 variable, 1/2 constant
- Heavy chains = 1/4 variable, 3/4 constant

Front 10

Isotypes of Antibodies

Back 10

5 types, 9 total isotypes
- 4 IgG
- 2 IgA
- 1 IgD
- 1 IgM
- 1 IgE
*** M and E are physically larger...extra segment

Front 11

2 Ig polymers

Back 11

- IgM can be pentameric in serum
- IgA can be dimer in mucosa

Front 12

J-chain

Back 12

- Joins Ig's to make polymer structures

Front 13

Ig properties

Back 13

- IgM, IgG1, and IgG3 = good at activating complement
- IgA - doesn't activate complement at all...
- IgE primarily binds mast cells
- IgG1 is most abundant, longest 1/2-life
- IgE is least abundant, shortest 1/2-life

Front 14

Antibody segment diversity - V(D)J recombo

Back 14

- Each B-cell undergoes unique recombination during development

Front 15

Light vs. heavy segments

Back 15

- Light chain variable regions = V and J segments
- Heavy chain variable regions = V, D, and J segments

Front 16

Antibody diversity - how many combinations

Back 16

- About 2 million total combinations (from segment types alone)
- Technically an infinite number
- Can make Ab's to literally any Ag in body

Front 17

3 Additional diversity mechanisms

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1) Sloppy splicing when joining segments
2) Random addition of nucleotides when joining segments
3) Somatic hypermutation of B-cells during response

Front 18

Rag protein function

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Rag 1 and 2 = bind recognition (heptamer, nonamer) sequences in V,D,J segments - facilitate recombination

Front 19

Heptamer, nonamer sequence function

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- Signal sequences bound by Rag proteins
- Help facilitate recombination

Front 20

B-cell development location, process

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- Primarily in the bone marrow
- Develop IgM, IgD receptors
- Migrate to peripheral blood, lymphoid tissues

Front 21

B-cell and stromal cell interaction

Back 21

- Stromal cells assist B-cell development
- B-cells attach to stromal cells as they develop
- Cytokines (IL-7, c-kit) and growth factors from stromal cells initiate maturation responses

Front 22

B-cell development stages and rearrangement steps

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- Stem cell = germline heavy and light chain genes
- Early pro-B-cell = Heavy D-J rearrangement
- Late pro-B-cell = Heavy V-DJ rearrangement
- Large pre-B-cell = Heavy VDJ complete, expresses pre-B-cell receptor
- Small pre-B-cell = Light V-J rearrangement
- Immature B-cell = Rearrangements complete, IgM on surface

Front 23

B-cell turnover from random failures

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- Only about 1/3 of heavy chain rearrangements are "in frame"
- 2/3 fail, large B-cell turnover during development
- Light chains don't really fail as often
- Many more chances to successfully recombine - more copies on more chromosomes
*** Heavy chain success is really the rate-limiting factor

Front 24

Heavy vs. light chain rearrangement

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- Heavy chain = 2 strikes -> Fail!
- Light chains have 2 chances on 2 different chromosomes
- Also, no D element in the way
- Can actually keep recombining V and J segments until they get an in-frame match

Front 25

Allelic exclusion purpose

Back 25

- B-cells do these rearrangements stepwise to ensure they only have one heavy and one light chain
- Don't want activated B-cells to make more than one specific type of Ab

Front 26

Surrogate light chain importance

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- When heavy chain successfully made, needs to go to surface
- This tells the cell to stop doing heavy chains and move on to light chain rearrangement

Front 27

BTK tyrosine kinase

Back 27

- Heavy chain/surrogate light chain signal to B-cell through BTK
- Tell the cell that heavy chain is functional

Front 28

Negative selection process purpose

Back 28

- Eliminate B-cells that produce "self"-binding Ig

Front 29

2 different Negative selection processes

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1) Deletion = Strong, multivalent self response - stimulated apoptosis
2) Anergy = relatively weak response
- Cell IgM strongly down-regulated
- Remains alive, but is functionally non-specific

Front 30

6 Mature B-cell surface markers

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- IgM and IgD (both have same variable region)
- CR1 and CR2 - complement receptors
- Class II MHC
- Presents Ag to T-cells
- Fc receptor
- CD 19

Front 31

Two B-Cell types

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- B-1 cells (non-conventional) and B-2 cells (conventional)

Front 32

B-1 cells features

Back 32

- Actually considered part of innate response
- Around from early development
- Self-renewing out in the body in non-lymphoid areas
- Pre-programmed to respond to bacterial carbohydrates, etc.
- Spontaneously make Ab's
- Make lots of IgM - good for activating complement, lysing bacteria

Front 33

B-2 cell features

Back 33

- "Normal" B-cells formed in marrow
- Don't produce Ab's unless activated

Front 34

Differential RNA processing - IgM vs. IgD

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- Allows IgM and IgD on a given B-cell to have IDENTICAL variable regions
- Variable regions encoded first - same no matter what
- Poly AAA addition placement determines splicing to make either IgM or IgD

Front 35

Differential RNA processing - Membrane vs. Secreted

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- Poly AAA region at end - get transmembrane domain
- Poly AAA added at middle - get excretion nub
*** All 5 isotypes can be membrane or secreted like this!

Front 36

Isotype class switching

Back 36

- B-cell normally expresses IgM and IgD, but changes during response via AID enzyme
- Will express IgE, IgA, IgG as well
- Genome is too big to simply differential RNA splice
- Intervening DNA actually loops out, is lost

Front 37

Average B-cell life cycle

Back 37

- Formation, development of IgM
- Negative selection
- Migration to tissues
- DON'T ever see Ag's...
- Death via "neglect"

Front 38

Major steps in life-cycle of activated B-cell (Big Picture)

Back 38

- Pre-B-cell - still developing light chains
- Immature B-cell = has IgM, negative selection process
- Mature B-cell = has IgM, IgD, resting and naive
*** Ag Exposure ***
- Germinal center B-cell = rapidly proliferating, isotype switching, many die
- Plasma cell = non-proliferating, secrete massive amount of Ab, go to marrow, live a long time
- Memory cell = resting, waiting to see same Ag again, very long-lived

Front 39

Cancer correlation to B-cell development - locations

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- Different cancers develop based on stage of B-cell lifetime
- Immature stages (lymphoblastic, pre-B) - bone marrow
- Multiple myeloma - from plasma cells - marrow
- All others = out in the periphery of the body!