m121 lec 7: B cell development pt 2 - somatic hypermutation + affinity maturation + Ab application

immune cells of the [1] system

the phagocytes

[10] — circulating precursor of the

1. innate

2. myeloid

3. lymphoid

4. megakaryocyte-erythroid precursor cell

5. erythroblast

6. megakaryocyte

7. erythrocyte

8. platelets

9. granulocyte

10. monocyte

11. neutrophil

12. eosinophil

13. basophil

14. macrophage

15. dendritic cell

16. mast cell

immune cells of the [1] system

one [1] cell goes to one [2] (use abbrev) specificity. makes millions of copies of this one specific [2].

mechanism: copy and paste bits of [3], put together in different ways to get unique [2] specificities → [4] [5]

structure of [2]: its binding site needs extra [6], particularly at the [7]-[7]-[7] (CDR), H1-H3, where the protein Ag binds

selection – if [2] recognizes self-Ag, the [1] cell will die because you don’t want [1] cells making [2] that recognize yourself ([8])

1. B

2. Ab

3. DNA

4. RNA

5. splicing

6. diversity

7. complementarity-determining region

8. autoimmunity

genomic organization

the [3] and [4] regions stick together and always have the most exons

DNA [1] chain (ch [2]) – [3] + [4] + [5] + [6] + [7] regions

light chains

• [8] on ch [9]

• [10] on ch [10]

1. heavy

2. 14

3. leader

4. variable

5. diversity

6. joining

7. constant

8. kappa

9. 2

10. lambda

11. 22

[2] & [7] chain recombination

every [1]’s [2] chain has VDJC. 

• DJ first, then VDJ, then connect to C via [3]

start with [4] DNA → [5] DNA

• [6] recombination

  • label D-J joining step

  • label V-DJ joining step

remember [7] chains have no D segment! otherwise, same process

1. Ab

2. heavy

3. splicing

4. germline

5. recombined

6. somatic

7. light

[1] & [2] chain recombination CONT’D

mechanism using [3] [3] [3] (RSSs) and the [4]/[4] rule. need the [5] [5]/[5] enzyme complex to chop off V & J segments and loop up the rest of the [6] DNA that isn’t wanted

• [4]/[4] rule ensures proper V-D-J joining

1. heavy

2. light

3. recombination signal sequences

4. 12/23

5. RAG 1/2

6. germline

[1] & [2] chain recombination CONT’D

[3] diversity: each [1] chain can combine with any [2] chain, and these combinations of kappa & lamba [2] chain + [1] chain creates sooo many [4] specificities…. but there are even MORE than listed here thanks to [5] diversity

1. heavy

2. light

3. combinatorial

4. Ab

5. junctional

[1] & [2] chain recombination CONT’D

after [3] of [4] DNA and adding [5] diversity to the [6] DNA…

• [7] with the primary [8] transcript, which is an exact copy of the DNA

• [8] [3] to cut out the parts that we don’t want. the stuff we want stays as mRNA

• [9]! making the protein that forms the [10] regions

• then, this peanut-shaped protein folds so that the diverse region sticks out at the Ag-binding site for efficient binding through its CDRs

1. heavy

2. light

3. splicing

4. germline

5. combinatorial

6. recombined

7. transcription

8. RNA

9. translation

10. Ab

[1] & [2] chain recombination CONT’D

[3] diversity: V-D-J recombination also involves adding/subtracting any number of [4], but only in multiples of 3 (since codons have 3 NTs) do you get extra/fewer aa (not very efficient process, but still generates much diversity).

thus, you can get 1e11 (even greater than 1.6e6) different types of [5] from [3] diversity compared to [6] diversity

1. heavy

2. light

3. junctional

4. nucleotides

5. Ab

6. combinatorial

[3] cell development (in bone marrow)

name / Ig DNA / Ig expression

1. [1] cell / [2] DNA / no

2. early [11] [3] cell / [2] DNA, D-J rearranging / no

3. late [11] [3] cell / [2] DNA, V-DJ rearranging / no

4. large [4] [3] cell / recombined [5] chain / μ + surrogate light chain ([4] [3] cell receptor)

5. small [4] [3] cell / recombined [5] chain, V-J of [7] chain rearranging / [12] μ chain

6. [6] [3] cell / recombined [5] and [7] chain / membrane [8]

7. [9] [3] cell / alternative RNA splicing of [5] chain / membrane [8] and [10]

1. stem

2. germline

3. B

4. pre

5. heavy

6. immature

7. light

8. IgM

9. mature

10. IgD

11. pro

12. intracellular

[3] cell development (in [5] [5]) CONT’D

[1] selection occurring in the [5] [5]

[2] [3] receptor — if successful [4] chain rearrangement occurs, it sends out survival signal and shuts off [4] chain recombination. then, it turns on [6] chain recombination (kappa first → lambda)

• if no signaling from [2] [3] receptor, cell dies by [7] (form of [11] tolerance, meaning we build tolerance from the primary immune site)

• otherwise, cell survives and proliferates (clones itself) to make lots of [8]

[1] selection (occurring after [9] [3] cell has formed, becoming a [10] [3] cell)

• takes out self-reactive [3] cells that have strong reactions in bone marrow

• [12] tolerance: some [9] [3] cells are released into circulation before they’re actually [10], in order to sample Ag in the periphery

  • mechanism: [13]

what remains after [1] selection: [10] [3] cells that recognize [14]-[14] Ag

1. negative

2. pre

3. B

4. heavy

5. bone marrow

6. light

7. apoptosis

8. Ab

9. immature

10. mature

11. central

12. peripheral

13. anergy

14. non-self

GENERATION OF DIVERSITY AFTER EXPOSURE TO Ag/PATHOGEN

occurs in all 6 CDRs within a BCR

[4] [5] (3rd mechanism for generation of diversity): when B cells divide, there are two mechanisms working together to increase (by 1,000-fold) the point mutations in Ig genes. occurs at a rate of 1 mutation per cell division.

• [1] [2] (occurs in [3] B cells): some surface Ig (BCR) will have increased [1]

• [10] [10] via [6]-[6] [6] [6] (AID) enzyme that induces a bunch of mutations intentionally to create new protein sequences that are selected for higher [1]. makes sure to keep the useful B cells by having clones with the highest [1] dominate the population.

  • functions by converting cytosine into [7] in DNA, which triggers the nucleotide/base excision repair (NER/BER) mechanisms that recruit low-fidelity polymerases to fix because these polymerases have higher error rates

  • B cells with highest [1] [8] survive!

• [9] cell dependent process, needs helper [9] cells to become [3] differentiated B cells (memory or plasma)

  • cross-linking of B cell with helper [9] cell

1. affinity

2. maturation

3. mature

4. somatic

5. hypermutation

6. activation-induced cytidine deaminase

7. uracil

8. Ab

9. T

10. clonal expansion

pt 1: in order for B cells to [1] and survive, they must enter [2] [3] tissue

1. entrance through [4] [4] [4] (HEV) first to get into the lymph node

2. reach [5] cell area to be recruited by chemokines

3. then, B cells enter the [6] [3] follicle

4. if B cells detect Ag, they stay here and multiply into germinal centers in the [6] [3] follicle

1. mature

2. secondary

3. lymphoid

4. high endothelial venule

5. T

6. primary

pt 2: in order for B cells to [1] and survive, they must enter [2] [3] tissue

this is the how:

[4] attract B cells with signals so that the B cells know where to go

• propagation of [4] creates a chemical gradient that promotes [5], a process that makes a cell move down a gradient

• [6] (the bouncers that let of-age ppl into party): secreted by [7] cells within lymph node

  • B cells follow the [6] gradient to enter the [8] [8] [8]

• [9] & [6]: attracts B cells into the lymph node’s [11] cell area

• [12]: secreted by [13] [14] cells (which are not actually [14] cells, but a [7] cell type) to attract B cells into the B cell area (the [10] [10])

  • [13] [14] cells help to show more Ag to B cells and give it signals

signals sent to the B cell to survive/[1]/differentiate:

• B cell [15] [15] (BAFF): helps keep cell alive

• [11] cell help

[1] B cells recirculate between the lymph, blood, and [2] [3] tissues

1. mature

2. secondary

3. lymphoid

4. chemokines

5. chemotaxis

6. CCL21

7. stromal

8. high endothelial venule

9. CCL19

10. primary follicle

11. T

12. CXCL13

13. follicular

14. dendritic

15. activation factor

pt 3: in order for B cells to [1] and survive, they must enter [2] [3] tissue

what happens when a B cell sees Ag?

[4] cell help directs three things:

• [5] [5]

• [6] [6] recombination

• differentiation

1. mature

2. secondary

3. lymphoid

4. T

5. affinity maturation

6. class switch

[1] cell [2] dictate B cell fate (partially what we mean by [1] cell help)

together, [3]-[4] [5] is an interaction between [1] cell [2] and B cells

• [3] is expressed on [1] cells to provide the [5] signal that activates the B cell [6] and makes the signaling inside the B cell even [7]

• [4] is expressed on B cells

[8]: a [2] expressed by [9] [1] cells to help B cells differentiate into [10] cells

[11]: a [2] that helps B cells differentiate into [12] cells

1. T

2. cytokines

3. CD40L

4. CD40

5. costimulation

6. receptor

7. stronger

8. IL-10

9. regulatory

10. plasma

11. IL-4

12. memory

defining [1] for clinical and research use

• [2] [1]: there are many specificities/clones of [1] that attack a variety of [3]/targets on the same Ag

  • option 1 is an [5]: take a rabbit and inject it with protein, wait for it to make [1] against this protein (via immune response), then take the [5] which will have mix of [1]

  • option 2 is an [6] purified [2] [1] where you take an Ag and purify [1] based off of its reactivity, that way you can get just multiple clones of [1] against that Ag without the extra stuff in an [5]

• [4] [1]: it’s a single clone/specificity for one [3] on an Ag

  • one very high [6] SINGLE TYPE of [1]

1. Ab

2. polyclonal

3. epitopes

4. monoclonal

5. antiserum

6. affinity