lec 6- antibody diversity and B cell development

Question 1

what three mechanisms lead to diversity in the antigen binding sites (paratrope)?

Answer 1

-somatic recombination
-junctional diversity
-combinatorial diversity

Question 2

do B-cells exit the bone marrow expressing IgM and IgD receptors?

Answer 2

yes, antigen binding sites is identical, just the constant regions are different

Question 3

what is somatic recombination?

Answer 3

the rearranging of DNA segments to create diversity, more diversity allows for a larger variety of antibodies that can recognize more antigens

Question 4

how is the variable region generated in a light chain and heavy chain during somatic recombination done during the germ line level?

Answer 4

light: one V segment and one J segment are joined
heavy: one V, D, and J segment are joined

Question 5

what is junctional diversity?

Answer 5

the diversity that occurs when DNA segments are joined

Question 6

what is combinatorial diversity?

Answer 6

the diversity that occurs when individual heavy chains are joined to random light chains

Question 7

what is made first in somatic recombination?

Answer 7

the heavy chain

Question 8

what happens during the junctional diversity of heavy chains?

Answer 8

-D and J join first, then D and V, create the diversity of CDR3
-CDR1 and CDR2 are from the random V region

Question 9

after the variable domain is complete what is added?

Answer 9

the micro constant region

Question 10

what happens during junctional diversity of light chains?

Answer 10

-V to J joining, create the diversity of CDR3
-the random V region contains CDR1 and CDR2
-after variable domain is complete the C region is added

Question 11

recombination of V (D) J segments is directed by what?

Answer 11

recombination signal sequences (RSSs)

Question 12

what are the two types of RSSs?

Answer 12

Nonomer (9 base pairs) and Heptamer (7 base pairs)

Question 13

what separates the RSSs?

Answer 13

a 12 and 23 base pair spacer

Question 14

what is the 23/12 rule of recombination?

Answer 14

-recombination must occur between RSSs with spacers different in lengths
-a heavy chain V region (23) cannot join J region (23) directly

Question 15

where are recombination activating genes expressed?

Answer 15

in developing B-cell and T cells

Question 16

what do recombination activating genes (RAG-1 and RAG-2) produce?

Answer 16

RAG-1 and RAG-2 proteins that interact with each other to form the RAG complex

Question 17

what does the RAG complex do?

Answer 17

-each RAG-1 subunit has binding sites for the nonamer and Heptamer nucleotide motifs and brings together the V and J gene segments
-different conformations of RAG-1 subunits account for the ability to bind either nonomer or heptamer
-the two gene segments are brought together, followed by cutting and splicing

Question 17

what does the RAG complex do to heavy chains?

Answer 17

-RAG complex cleaves the Heptamer RSSs from D and J gene segments to yield DNA hairpins
-RAG complex opens hair pins by nicking one strand of the DNA, generating P-nucleotides

Question 18

what modifies the open ends of the DNA hairpins?

Answer 18

-Terminal deoxynucleotidyl transferase (TdT), which randomly adds nucleotides
-these are N-nucleotides (non-templated) not encoded by germ line

Question 19

what removes the unpaired germ line encoded nucleases?

Answer 19

exonucleases (can also remove nucleotides before TdT adds nucleotides)

Question 20

after pairing what is filled in the DNA strand?

Answer 20

missing nucleotides

Question 21

how does generations of junctional diversity result in non-productive rearrangements?

Answer 21

because the nucleotides can be out of frame

Question 22

what is the light and heavy chain CDR3 segments?

Answer 22

light: V and J joining
heavy: V, D, and J joining

Question 23

what is expressed in the B cell to generate the BCR?

Answer 23

-one light chain (kappa or lambda) and one heavy chain

Question 24

is each B cell specific for one epitope on an antigen?

Answer 24

yes

Question 25

what subclass of receptors do immature B cells express?

Answer 25

IgM on the cell surface

Question 26

what subclass of receptors do mature B cells express?

Answer 26

IgM and IgD on the cell surface

Question 27

what two other proteins are membrane bound B cell receptors always associated to?

Answer 27

IgB (beta) and Iga (alpha), together they form the B cell receptor

Question 28

when must the IgB and Iga associate with the BCR to get it onto the cell membrane?

Answer 28

when CBR is at the ER

Question 29

why does the BCR associate with IgB and Iga?

Answer 29

because the BCRs cytoplasmic tail is too small for signal transduction, but the cytoplasmic tail soft IgB and LIga are long enough for it

Question 30

do B cells leave the bone marrow to go to secondary lymphoid organs in which they can encounter antigens and then differentiate into plasma cells to secrete IgM?

Answer 30

yes

Question 31

what do B cells undergo in germinal centers of lymphoid organs?

Answer 31

-somatic hypermutation -affinity maturation -class switching

Question 32

further diversification in V-gene sequences occurs by?

Answer 32

somatic hypermutation

Question 33

what occurs in somatic hypermutation? what drives it?

Answer 33

single nucleotide substitutions (point mutations) driven by cytidine deaminase (AID) and are targeted to CDRs

Question 34

what is the goal of somatic hypermutation?

Answer 34

to increase the affinity of antibody for its antigen

Question 35

what are the stage of development in B cells?

Answer 35

Stem cell --> early pro B cell --> late pro B cell --> large pre B cell --> small pre B cell --> immature B cell

Question 36

throughout most of their development, B cells are associating with?

Answer 36

stromal cells which provide a microenvironment of adhesion and growth factors for development

Question 37

B cells at different stages of development are identified by different combinations of what?

Answer 37

CD proteins on the cell surface

Question 38

what is allelic exclusion?

Answer 38

only one loci from heavy chains and one loci from light chain are expressed at the end of development

Question 39

what happens in pro B cells?

Answer 39

D-J rearrangements on both chromosomes, but is inefficient

Question 40

because B cell gene rearrangement is so inefficient light chain gene rearrangment doe snot occur until ?

Answer 40

a productive heavy chain rearrangment has occured

Question 41

what happens first in large pre B cells?

Answer 41

large pre B cells synthesize an invariant surrogate light chain of two proteins that can pair with the micro chain to form the pre B cell receptor

Question 42

what is the pre B cell receptor made of?

Answer 42

-VpreB (similar to V region of a light chain) -lambda5 (similar to constant region of light chain)

Question 43

what are the two things surrogate light chains test of large pre B cell heavy chains?

Answer 43

-they test if they can bind light chains -they test to see if the pre B cell receptor can get to the cell surface

Question 44

is the pre B cell receptor complex expressed transiently on the cell surface and does it generate signals to indicate a functional heavy chain that can bind to a real light chain and get to the cell surface?

Answer 44

yes

Question 45

once a functional heavy chain that can bind a light chain and get to the cell surface is established, is there any use for a surrogate light chain?

Answer 45

no

Question 46

what happens second in large pre B cells?

Answer 46

the B cell expressing just the functional heavy chains clones itself 100 times

Question 47

what happens in small pre B cells?

Answer 47

combinatorial diversity, each of the heavy chain B cell clones has a unique light chain to have unique antigen specificity

Question 48

how is combinatorial diversity done in small pre B cells?

Answer 48

-RAG genes are reactivated -kappa locus begins rearrangement first -several attempts to generate a productive joining -if kappa not successful, then lambda begins rearranging -no difference between kappa and lambda, both types together increase success of functional light chain -85% of small pre-B cells clones generate a productive light chain

Question 49

does light chain rearrangment occur separately in each of the 100 pre B cells that all express the same micro chains which leads to diversity in antigen binding sites?

Answer 49

yes

Question 50

what are ethe 2 checkpoints developing B cells pass through?

Answer 50

-for functional heavy chain -for functional light chain

Question 51

what are the two types of B cells?

Answer 51

B-1 and B-2 cells

Question 52

are B cells that recognize self antigen arrested?

Answer 52

yes, either eliminated or inactivated, but may be given a chance to alter light chain

Question 53

what is receptor editing of a B cell?

Answer 53

-when receptors are constantly edited to get rid of self recognizing capabilities, once it is different, it leaves the bone marrow -if it still cant get rid of self reacting capabilities, it dies

Question 54

what happens to a B cell that binds monovalent self?

Answer 54

-it becomes unresponsive = anergy -enter circulation but don't last long

Question 55

what are the three fates of self reactive B cells?

Answer 55

-receptor editing -apoptosis -anergy

Question 56

what is central tolerance?

Answer 56

the getting rid of self reacting B cells in the bone marrow

Question 57

what is peripheral tolerance?

Answer 57

the getting rid of self reacting B cells from outside of the bone marrow