Lecture 7 - Ab diversity and B cell development

Question 1

Describe B cell development

Answer 1

-B cells start out in bone marrow and fetal liver by rearranging their chromosomal DNA to create exon encoding the Ag binding pocket –> both light and heavy chain undero process

Question 2

What is the only isotype generated at this stage of B cell development?

Answer 2

IgM (and a little IgD)

Question 3

What happens after cells commit to being B cells?

Answer 3

Ab genes start to rearrange

Question 4

B cells leave bone marrow with what bound?

Answer 4

IgM and maybe a little IgD

Question 5

Describe 4 basic steps of B cell development described

Answer 5

1) B cell precursor rearranges its Ab genes –> generation of B cell receptors in bone marrow
2) immature B cell bound to self cell-surface Ag is removed –> negative selection in bone marrow
3) mature B cell bound to foreign Ag is activated –> migration of B cells to peripheral lymphoid organs and activation
4) activated B cells give rise to plasma cells and memory cells –> Ab secretion and memory cells in bone marrow and lymphoid tissue

Question 6

What do the heavy and light chain genes consist of?

Answer 6

large numbers of variable (V) domain genes, joining (J) genes and (+/-) diversity (D) regions

Question 7

How are variable region genes constructed?

Answer 7

by joining one of gene segments from each region (1 V seg + 1 D seg + 1 J seg) to form a continuous piece of DNA

Question 8

What must occur to form a joined gene?

Answer 8

somatic rearrangement

Question 9

What is somatic rearrangement?

Answer 9

random event which occurs only on one chromosome for each light or heavy chain genes (allelic exclusion)

Question 10

What is the constant region encoded by?

Answer 10

separate exons

Question 11

How is the constant and variable region exons joined?

Answer 11

by splicing of the primary RNA transcript

Question 12

When can isotype switching occur?

Answer 12

following VDJ region recombination

Question 13

What genes does the light chain region not have?

Answer 13

diversity region

Question 14

True or False: each b cell develops receptors that can bind to a single epitope that is different than the epitope recognized by other B cells

Answer 14

True; does this during development in the bone marrow by splicing its chromosomal DNA to create new coding sequences for the Ag binding pocket exons

Question 15

What part is most variable within Ab

Answer 15

AAs that contact the epitope

Question 16

The immune system has the capacity to recognize what?

Answer 16

approx 10^10 different antigenic epitopes; each B cell produces Ab of a single specificity

Question 17

The pool of B cells within an individual provides what?

Answer 17

universal recognition

Question 18

What 5 processes aid in Ab diversity?

Answer 18

1) multiple germline gene exons
2) H and L chain combination
3) Imprecise joining of segments
4) Random insertion of bases
5) somatic hypermutation

Question 19

During B cell development, what do stromal cells secrete?

Answer 19

B cell growth factors

Question 20

As a pre-B cell develops, what happens?

Answer 20

Ab genes begin to rearrange –> heavy chain first, then light chain

Question 21

Which cells survive and leave bone marrow?

Answer 21

those that successfully rearrange both heavy and light chain genes

Question 22

What is “multiple germline genes”?

Answer 22

genomic organization of Ab genes has many different gene segments and permits rearrangement.

• using multiple gene segments, great diversity can be ahcieved
• B cells have different VDJ and VJ heavy and light chain rearrangements;
• B cells also have different combinations of VDJ and/or VJ segments

Question 23

What is “H-L chain combinations”?

Answer 23

• heavy chain rearranges independently of light chain
• by combining different H and L chains, each with their own variable region rearrangements –> generate Ag-binding pockets of various specificty

Question 24

How are the V, D or J regions selected?

Answer 24

randomly

Question 25

What is "imprecise joining (or junctional flexibility)"

Answer 25

by arbitrarily splicing nucleic acids together within the region, different reading frames are generated leading to different AA structure at the Ag binding pocket - occurs at all V-D-J junctions during recombination - what is randomly spliced out changes the nucleic acids spliced together --> changes AA structure

Question 26

What is "random base insertion (or P nucleotide addition)"?

Answer 26

- during recombination of V-D-J segments, nucleic acids can be randomly inserted into the junction - generates a different AA structure at Ag binding pocket

Question 27

Do most B cells make it out of bone marrow?

Answer 27

no

Question 28

What is allelic exclusion?

Answer 28

rearrangement of Ab gene DNA occurring only on one chromosome for each light and heavy chain genes

Question 29

What does allelic exclusion assure?

Answer 29

assures that each individual B cell generates only a single Ab mol rather than expressing genes from both chromosomes -every Ab that B cell produces is identical

Question 30

Describe an example

Answer 30

an animal with two different alleles for the heavy chain gene loci produces Abs using both chromosomes. -for each individual B cell, only the blue or yellow chromosome is expressed, not both

Question 31

What does allelic exclusion prevent?

Answer 31

B cells from having low avidity receptor interactions with Ag -ensures each B cell produces identical Ab --> every Ab will bind to target Ag

Question 32

What happens without allelic exclusion?

Answer 32

produces heterogeneous B cell receptors (Abs) with low avidity binding

Question 33

In rearrangement of Ab variable region genes, heavy chain gene rearranges first (then light). what happens if first splice event fails?

Answer 33

chromosome may attempt to splice further down the gene cluster

Question 34

Does choice of heavy chain VDJ regions influence which light chain V and J regions selected?

Answer 34

no, they are independent events

Question 35

What are RAG proteins?

Answer 35

recombination activating gene

Question 36

What do RAG proteins/enzymes do?

Answer 36

recognize conserved sequences flanking each gene segment, bend the DNA strand such that proteins interact and cleave out the intervening DNA sequences

Question 37

What happens to animals who are deficient in RAG activity?

Answer 37

they are severely immunocompromised

Question 38

B cells develop in bone marrow and are released with what?

Answer 38

rearranged Ig genes

Question 39

What is found on surface of mature B cells released from marrow?

Answer 39

IgM +/- IgD

Question 40

Rearrangement occurs when?

Answer 40

in absence of Ag

Question 41

When does heavy chain VDJ region genes start rearranging?

Answer 41

Early pro-B cell --> large pre-B cell

Question 42

When does light chain VJ region genes rearrange?

Answer 42

small pre-B cell --> immature B cell

Question 43

At what stage of B cell development are surface Ig expressed?

Answer 43

Immature B cells

Question 44

Why is IgM the first Ab produced by B cells?

Answer 44

it's encoded immediately adjacent to VDJ genes

Question 45

When is IgD also found on B cell surface?

Answer 45

on surface of newly formed B cells due to mRNA splicing artifact

Question 46

After B cells are released from bone marrow, where do they go?

Answer 46

circulate through secondary lymphoid tissues - lymph nodes, tonsils, spleens and peyer's patches

Question 47

B cells that encounter Ag (and T cell help) form what?

Answer 47

foci called germinal centers that contain proliferating B cells

Question 48

What happens to some of plasma cells?

Answer 48

some leave the germinal center and reenter circulation | -some take up residence in bone marrow and spleen

Question 49

What happens in the periphery for B cells?

Answer 49

- maturation to an Ab secreting cell (plasma cell) - isotype switching - somatic hypermutation * **these events occur following specific antigenic stimulation

Question 50

If mature B cell fails to access secondary lymphoid tissue what happens?

Answer 50

cell dies

Question 51

If B cell makes it to secondary lymphoid tissue, but doesn't interact with Ag, what happens?

Answer 51

half life of 3-8 wks

Question 52

What happens if B cell encounters Ag?

Answer 52

-longer lived some mature into plasma cells --> Ab secretion (IgM) -some form germinal centers

Question 53

B cells start to mature in bone marrow, then move to peripheral tissues ONLY if what happens?

Answer 53

light and heavy chains rearrange successfully

Question 54

What happens in periphery?

Answer 54

B cell encounters its Ag and receives T cell help (cytokines) --> convert to plasma cells that secrete IgM -further activation results in isotype switching to secrete IgG or IgA

Question 55

Exposure to Ag in periphery results in what?

Answer 55

- activation and cell division - differentiation into plasma cells (Ab) - class switching and somatic hypermutation (introduce AA changes) - selection for receptor specificity with greater affinity (affinity maturation) - differentiation into memory B cells

Question 56

Which processes require exposure to Ag in periphery?

Answer 56

all activation of B cells and subsequent changes in DNA (somatic hypermutation and isotype switching) or RNA processing

Question 57

What doesn't require exposure to Ag?

Answer 57

gene conversion events

Question 58

What are germinal centers?

Answer 58

specialized areas of secondary lymphoid organs -if stimulated by antigen-specific T cells, B cells will multiply and start to secrete IgM --> undergo somatic hypermutation and selection for Ag binding in these sites

Question 59

Where do B cells interact with T cells?

Answer 59

in secondary lymphoid organs

Question 60

What is the first thing to happen following B cell activation?

Answer 60

proliferation and conversion to secretory IgM (plasma cell) --> then somatic hypermutation --> affects exon encoding the Ag binding site